AJP: Heart and Circulatory Physiology recent issues

JAK redux: a second look at the regulation and role of JAKs in the heart

Kurdi, M., Booz, G. W. — Mon, 26 Oct 2009 13:43:28 PDT

A number of type 1 receptor cytokine family members protect the heart from acute and chronic oxidative stress. This protection involves activation of two intracellular signaling cascades: the reperfusion injury salvage kinase (RISK) pathway, which entails activation of phosphatidylinositol 3-kinase (PI3-kinase) and ERK1/2, and JAK-STAT signaling, which involves activation of transcription factor signal transducer and activator of transcription 3 (STAT3). Obligatory for activation of both RISK and STAT3 by nearly all of these cytokines are the kinases JAK1 and JAK2. Yet surprisingly little is known about how JAK1 and JAK2 are regulated in the heart or how they couple to PI3-kinase activation. Although the JAKs are linked to antioxidative stress programs in the heart, we recently reported that these kinases are inhibited by oxidative stress in cardiac myocytes. In contrast, others have reported that cardiac JAK2 is activated by acute oxidative stress by an undefined process. Here we summarize recent insights into the regulation of JAK1 and JAK2. Besides oxidative stress, inhibitory regulation involves phosphorylation, nitration, and intramolecular restraints. Stimulatory regulation involves phosphorylation and adaptor proteins. The net effect of stress on JAK activity in the heart likely represents the sum of both inhibitory and stimulatory processes, along with their dynamic interaction. Thus the regulation of JAKs in the heart, once touted as the paragon of simplicity, is proving rather complicated indeed, requiring a second look. It is our contention that a better understanding of the regulation of this kinase family that is implicated in cardiac protection could translate into effective therapeutic strategies for preventing myocardial damage or repairing the injured heart.

Regulation of central angiotensin type 1 receptors and sympathetic outflow in heart failure

Zucker, I. H., Schultz, H. D., Patel, K. P., Wang, W., Gao, L. — Mon, 26 Oct 2009 13:43:28 PDT

Angiotensin type 1 receptors (AT₁Rs) play a critical role in a variety of physiological functions and pathophysiological states. They have been strongly implicated in the modulation of sympathetic outflow in the brain. An understanding of the mechanisms by which AT₁Rs are regulated in a variety of disease states that are characterized by sympathoexcitation is pivotal in development of new strategies for the treatment of these disorders. This review concentrates on several aspects of AT₁R regulation in the setting of chronic heart failure (CHF). There is now good evidence that AT₁R expression in neurons is mediated by activation of the transcription factor activator protein 1 (AP-1). This transcription factor and its component proteins are upregulated in the rostral ventrolateral medulla of animals with CHF. Because the increase in AT₁R expression and transcription factor activation can be blocked by the AT₁R antagonist losartan, a positive feedback mechanism of AT₁R expression in CHF is suggested. Oxidative stress has also been implicated in the regulation of receptor expression. Recent data suggest that the newly discovered catabolic enzyme angiotensin-converting enzyme 2 (ACE2) may play a role in the modulation of AT₁R expression by altering the balance between the octapeptide ANG II and ANG- (1–7). Finally, exercise training reduces both central oxidative stress and AT₁R expression in animals with CHF. These data strongly suggest that multiple central and peripheral influences dynamically alter AT₁R expression in CHF.

Angiotensin II, mechanotransduction, and pulsatile arterial hemodynamics in hypertension

Lacolley, P., Safar, M. E., Regnault, V., Frohlich, E. D. — Mon, 26 Oct 2009 13:43:28 PDT

The aortic blood pressure curve involves two components: a steady component, the mean arterial pressure (MAP), which is dependent on cardiac output and vascular resistance, and a pulsatile component pulse pressure (PP), which is dependent on arterial stiffness and pulse wave reflections. The transduction mechanisms of MAP and PP differ markedly, involving focal adhesion kinase for MAP and oxygen free radicals for PP. Angiotensin II (ANG II) and its blockade are associated with changed vascular resistance and MAP; however, their effects on PP (peripheral and mostly central PP) have been inadequately investigated. In hypertensive rats, when compared with their normotensive controls, ANG II blockade normalizes central PP (<50 mmHg) but not MAP when the same drug dosage is used for each. In hypertensive patients, ANG II blockade reduces arterial stiffness and pulse wave reflections, but with the same reduction in MAP, there is a greater reduction in central than peripheral PP, thereby increasing carotid-brachial PP amplification. With long-term ANG II blockade, the hypertensive arteriolar hypertrophy observed at baseline is corrected in association with reduced arteriolar reflection coefficients, reduced carotid arterial attachments linking ₅-integrin to its ligand fibronectin, and decreased circulating C-reactive protein. When given a normal salt diet, each of these factors contributes separately in reducing arterial stiffness and wave reflections. These responses disappear with a high-salt diet, a condition that usually involves the activation of the local vascular renin-angiotensin-aldosterone system and can be prevented by its selective blockade. Thus ANG II inhibition seems to contribute independently in reducing central PP and aortic stiffness.

Calcineurin and cardiac function: is more or less better for the heart?

Wolska, B. M. — Mon, 26 Oct 2009 13:43:28 PDT

Pathways to embryonic heart failure

Sedmera, D. — Mon, 26 Oct 2009 13:43:28 PDT

BK channels in cardiovascular disease: a complex story of channel dysregulation

Rusch, N. J. — Mon, 26 Oct 2009 13:43:28 PDT

A new piece in the hypertension puzzle: central blood pressure regulation by sex steroids

Yanes, L. L., Reckelhoff, J. F. — Mon, 26 Oct 2009 13:43:28 PDT

A high-fat diet increases adiposity but maintains mitochondrial oxidative enzymes without affecting development of heart failure with pressure overload

Chess, D. J., Khairallah, R. J., O'Shea, K. M., Xu, W., Stanley, W. C. — Mon, 26 Oct 2009 13:43:28 PDT

A high-fat diet can increase adiposity, leptin secretion, and plasma fatty acid concentration. In hypertension, this scenario may accelerate cardiac hypertrophy and development of heart failure but could be protective by activating peroxisome proliferator-activated receptors and expression of mitochondrial oxidative enzymes. We assessed the effects of a high-fat diet on the development of left ventricular hypertrophy, remodeling, contractile dysfunction, and the activity of mitochondrial oxidative enzymes. Mice (n = 10–12/group) underwent transverse aortic constriction (TAC) or sham surgery and were fed either a low-fat diet (10% of energy intake as fat) or a high-fat diet (45% fat) for 6 wk. The high-fat diet increased adipose tissue mass and plasma leptin and insulin. Left ventricular mass and chamber size were unaffected by diet in sham animals. TAC increased left ventricular mass (~70%) and end-systolic and end-diastolic areas (~100% and ~45%, respectively) to the same extent in both dietary groups. The high-fat diet increased plasma free fatty acid concentration and prevented the decline in the activity of the mitochondrial enzymes medium chain acyl-coenzyme A dehydrogenase (MCAD) and citrate synthase that was observed with TAC animals on a low-fat diet. In conclusion, a high-fat diet did not worsen cardiac hypertrophy or left ventricular chamber enlargement despite increases in fat mass and insulin and leptin concentrations. Furthermore, a high-fat diet preserved MCAD and citrate synthase activities during pressure overload, suggesting that it may help maintain mitochondrial oxidative capacity in failing myocardium.

Increased susceptibility of aged hearts to ventricular fibrillation during oxidative stress

Mon, 26 Oct 2009 13:43:28 PDT

Oxidative stress with hydrogen peroxide (H₂O₂) readily promotes early afterdepolarizations (EADs) and triggered activity (TA) in isolated rat and rabbit ventricular myocytes. Here we examined the effects of H₂O₂ on arrhythmias in intact Langendorff rat and rabbit hearts using dual-membrane voltage and intracellular calcium optical mapping and glass microelectrode recordings. Young adult rat (3–5 mo, N = 25) and rabbit (3–5 mo, N = 6) hearts exhibited no arrhythmias when perfused with H₂O₂ (0.1–2 mM) for up to 3 h. However, in 33 out of 35 (94%) aged (24–26 mo) rat hearts, 0.1 mM H₂O₂ caused EAD-mediated TA, leading to ventricular tachycardia (VT) and fibrillation (VF). Aged rabbits (life span, 8–12 yr) were not available, but 4 of 10 middle-aged rabbits (3–5 yr) developed EADs, TA, VT, and VF. These arrhythmias were suppressed by the reducing agent N-acetylcysteine (2 mM) and CaMKII inhibitor KN-93 (1 µM) but not by its inactive form (KN-92, 1 µM). There were no significant differences between action potential duration (APD) or APD restitution slope before or after H₂O₂ in aged or young adult rat hearts. In histological sections, however, trichrome staining revealed that aged rat hearts exhibited extensive fibrosis, ranging from 10–90%; middle-aged rabbit hearts had less fibrosis (5–35%), whereas young adult rat and rabbit hearts had <4% fibrosis. In aged rat hearts, EADs and TA arose most frequently (70%) from the left ventricular base where fibrosis was intermediate (~30%). Computer simulations in two-dimensional tissue incorporating variable degrees of fibrosis showed that intermediate (but not mild or severe) fibrosis promoted EADs and TA. We conclude that in aged ventricles exposed to oxidative stress, fibrosis facilitates the ability of cellular EADs to emerge and generate TA, VT, and VF at the tissue level.

Allogenic stem cell therapy improves right ventricular function by improving lung pathology in rats with pulmonary hypertension

Mon, 26 Oct 2009 13:43:28 PDT

Pulmonary arterial hypertension (PAH) is a chronic lung disease that leads to right ventricular (RV) hypertrophy (RVH), remodeling, and failure. We tested treatment with bone marrow-derived mesenchymal stem cells (MSCs) obtained from donor rats with monocrotaline (MCT)-induced PAH to recipient rats with MCT-induced PAH on pulmonary artery pressure, lung pathology, and RV function. This model was chosen to mimic autologous MSC therapy. On day 1, PAH was induced by MCT (60 mg/kg) in 20 female Wistar rats. On day 14, rats were treated with 10⁶ MSCs intravenously (MCT + MSC) or with saline (MCT60). MSCs were obtained from donor rats with PAH at 28 days after MCT. A control group received saline on days 1 and 14. On day 28, the RV function of recipient rats was assessed, followed by isolation of the lungs and heart. RVH was quantified by the weight ratio of the RV/(left ventricle + interventricular septum). MCT induced an increase of RV peak pressure (from 27 ± 5 to 42 ± 17 mmHg) and RVH (from 0.25 ± 0.04 to 0.47 ± 0.12), depressed the RV ejection fraction (from 56 ± 11 to 43 ± 6%), and increased lung weight (from 0.96 ± 0.15 to 1.66 ± 0.32 g), including thickening of the arteriolar walls and alveolar septa. MSC treatment attenuated PAH (31 ± 4 mmHg) and RVH (0.32 ± 0.07), normalized the RV ejection fraction (52 ± 5%), reduced lung weight (1.16 ± 0.24 g), and inhibited the thickening of the arterioles and alveolar septa. We conclude that the application of MSCs from donor rats with PAH reduces RV pressure overload, RV dysfunction, and lung pathology in recipient rats with PAH. These results suggest that autologous MSC therapy may alleviate cardiac and pulmonary symptoms in PAH patients.

Outflow tract cushions perform a critical valve-like function in the early embryonic heart requiring BMPRIA-mediated signaling in cardiac neural crest

Mon, 26 Oct 2009 13:43:28 PDT

Neural crest-specific ablation of BMP type IA receptor (BMPRIA) causes embryonic lethality by embryonic day (E) 12.5, and this was previously postulated to arise from a myocardial defect related to signaling by a small population of cardiac neural crest cells (cNCC) in the epicardium. However, as BMP signaling via cNCC is also required for proper development of the outflow tract cushions, precursors to the semilunar valves, a plausible alternate or additional hypothesis is that heart failure may result from an outflow tract cushion defect. To investigate whether the outflow tract cushions may serve as dynamic valves in regulating hemodynamic function in the early embryo, in this study we used noninvasive ultrasound biomicroscopy-Doppler imaging to quantitatively assess hemodynamic function in mouse embryos with P0-Cre transgene mediated neural crest ablation of Bmpr1a (P0 mutants). Similar to previous studies, the neural crest-deleted Bmpr1a P0 mutants died at ~E12.5, exhibiting persistent truncus arteriosus, thinned myocardium, and congestive heart failure. Surprisingly, our ultrasound analyses showed normal contractile indices, heart rate, and atrioventricular conduction in the P0 mutants. However, reversed diastolic arterial blood flow was detected as early as E11.5, with cardiovascular insufficiency and death rapidly ensuing by E12.5. Quantitative computed tomography showed thinning of the outflow cushions, and this was associated with a marked reduction in cell proliferation. These results suggest BMP signaling to cNCC is required for growth of the outflow tract cushions. This study provides definitive evidence that the outflow cushions perform a valve-like function critical for survival of the early mouse embryo.

Impaired function of coronary BKCa channels in metabolic syndrome

Mon, 26 Oct 2009 13:43:28 PDT

The role of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in regulation of coronary microvascular function is widely appreciated, but molecular and functional changes underlying the deleterious influence of metabolic syndrome (MetS) have not been determined. Male Ossabaw miniature swine consumed for 3–6 mo a normal diet (11% kcal from fat) or an excess-calorie atherogenic diet that induces MetS (45% kcal from fat, 2% cholesterol, 20% kcal from fructose). MetS significantly impaired coronary vasodilation to the BK_Ca opener NS-1619 in vivo (30–100 µg) and reduced the contribution of these channels to adenosine-induced microvascular vasodilation in vitro (1–100 µM). MetS reduced whole cell penitrem A (1 µM)-sensitive K⁺ current and NS-1619-activated (10 µM) current in isolated coronary vascular smooth muscle cells. MetS increased the concentration of free intracellular Ca²⁺ and augmented coronary vasoconstriction to the L-type Ca²⁺ channel agonist BAY K 8644 (10 pM–10 nM). BK_Ca channel and β₁ protein expression was increased in coronary arteries from MetS swine. Coronary vascular dysfunction in MetS is related to impaired BK_Ca channel function and is accompanied by significant increases in L-type Ca²⁺ channel-mediated coronary vasoconstriction.

Protective actions of estrogen on angiotensin II-induced hypertension: role of central nitric oxide

Xue, B., Singh, M., Guo, F., Hay, M., Johnson, A. K. — Mon, 26 Oct 2009 13:43:28 PDT

The present study tested the hypotheses that 1) nitric oxide (NO) is involved in attenuated responses to ANG II in female mice, and 2) there is differential expression of neuronal NO synthase (nNOS) in the subfornical organ (SFO) and paraventricular nucleus (PVN) in response to systemic infusions of ANG II in males vs. females. Aortic blood pressure (BP) was measured in conscious mice with telemetry implants. N^G-nitro-l-arginine methyl ester (l-NAME; 100 µg·kg^·–1day^–1), an inhibitor of NOS, was administrated into the lateral cerebral ventricle for 14 days before and during ANG II pump implantation. Central infusion of l-NAME augmented the pressor effects of systemic ANG II in females (21.5 ± 2.2 vs. 9.2 ± 1.5 mmHg) but not in males (29.4 ± 2.5 vs. 30.1 ± 2.5 mmHg). Central administration of N⁵-(1-imino-3-butenyl)-l-ornithine (l-VNIO), a selective nNOS inhibitor, also significantly potentiated the increase in BP induced by ANG II in females (17.5 ± 3.2 vs. 9.2 ± 1.5 mmHg). In gonadectomized mice, central l-NAME infusion did not affect the pressor response to ANG II in either males or females. Ganglionic blockade after ANG II infusion resulted in a greater reduction in BP in central l-NAME- or l-VNIO-treated females compared with control females. Western blot analysis of nNOS protein expression indicated that levels were ~12-fold higher in both the SFO and PVN of intact females compared with those in intact males. Seven days of ANG II treatment resulted in a further increase in nNOS protein expression only in intact females (PVN, to ~51-fold). Immunohistochemical studies revealed colocalization of nNOS and estrogen receptors in the SFO and PVN. These results suggest that NO attenuates the increase in BP induced by ANG II through reduced sympathetic outflow in females and that increased nNOS protein expression associated with the presence of female sex hormones plays a protective role against ANG II-induced hypertension in female mice.

Disruption of endothelial peroxisome proliferator-activated receptor-{gamma} reduces vascular nitric oxide production

Mon, 26 Oct 2009 13:43:28 PDT

Vascular endothelial cells express the ligand-activated transcription factor, peroxisome proliferator-activated receptor- (PPAR), which participates in the regulation of metabolism, cell proliferation, and inflammation. PPAR ligands attenuate, whereas the loss of function mutations in PPAR stimulate, endothelial dysfunction, suggesting that PPAR may regulate vascular endothelial nitric oxide production. To explore the role of endothelial PPAR in the regulation of vascular nitric oxide production in vivo, mice expressing Cre recombinase driven by an endothelial-specific promoter were crossed with mice carrying a floxed PPAR gene to produce endothelial PPAR null mice (ePPAR^–/–). When compared with littermate controls, ePPAR^–/– animals were hypertensive at baseline and demonstrated comparable increases in systolic blood pressure in response to angiotensin II infusion. When compared with those of control animals, aortic ring relaxation responses to acetylcholine were impaired, whereas relaxation responses to sodium nitroprusside were unaffected in ePPAR^–/– mice. Similarly, intact aortic segments from ePPAR^–/– mice released less nitric oxide than those from controls, whereas endothelial nitric oxide synthase expression was similar in control and ePPAR^–/– aortas. Reduced nitric oxide production in ePPAR^–/– aortas was associated with an increase in the parameters of oxidative stress in the blood and the activation of nuclear factor-B in aortic homogenates. These findings demonstrate that endothelial PPAR regulates vascular nitric oxide production and that the disruption of endothelial PPAR contributes to endothelial dysfunction in vivo.

Absence of adenosine-mediated aortic relaxation in A2A adenosine receptor knockout mice

Ponnoth, D. S., Sanjani, M. S., Ledent, C., Roush, K., Krahn, T., Mustafa, S. J. — Mon, 26 Oct 2009 13:43:28 PDT

Adenosine mediates vascular responses through four receptor subtypes: A₁, A_2A, A_2B, and A₃. The role of A_2A receptors in aortic vascular tone was investigated using A_2A adenosine receptor (AR) knockout (A_2AKO) and corresponding wild-type (A_2AWT) mice. Isolated aortic rings from A_2AWT and A_2AKO mice were precontracted with phenylephrine (10^–7 M), and concentration responses for adenosine analogs and selective agonists/antagonists were obtained. Nonselective adenosine analog (NECA; EC₅₀ = 6.78 µM) and CGS-21680 (A_2AAR selective agonist; EC₅₀ = 0.013 µM) produced concentration-dependent relaxation (maximum of 25% and 28% relaxation at 10^–5 M NECA and CGS-21680, respectively) in A_2AWT aorta. In A_2AKO aorta, NECA (EC₅₀ = 0.075 µM) induced concentration-dependent contraction (maximum contraction of 47% at 10^–6 M; P < 0.05 compared with A_2AWT), whereas CGS-21680 produced no response. SCH-58261 (10^–6 M; A_2AAR selective antagonist) abolished both NECA- and CGS-21680-mediated vasorelaxation in A_2AWT (P < 0.05), whereas no change was observed in A_2AKO. When DPCPX (10^–5 M; A₁ selective antagonist) was used in NECA concentration response, greater vasorelaxation was observed in A_2AWT (50% vs. 25% in controls at 10^–5 M; P < 0.05), whereas lower contraction was seen in A_2AKO tissues (5% vs. 47% in controls at 10^–6 M; P < 0.05). Aortic endothelial function, determined by response to acetylcholine, was significantly higher in WT compared with KO (66% vs. 51%; P < 0.05). BAY 60–6583 (A_2B selective agonist) produced similar relaxation in both KO and WT tissues. In conclusion, A_2AAR KO mice had significantly lower aortic relaxation and endothelial function, suggesting that the A_2AAR plays an important role in vasorelaxation, probably through an endothelium-dependent mechanism.

Vasopressin is a major vasoconstrictor involved in hindlimb vascular responses to stimulation of adenosine A1 receptors in the nucleus of the solitary tract

McClure, J. M., Rossi, N. F., Chen, H., O'Leary, D. S., Scislo, T. J. — Mon, 26 Oct 2009 13:43:28 PDT

Our previous study showed that stimulation of adenosine A₁ receptors located in the nucleus of the solitary tract (NTS) exerts counteracting effects on the iliac vascular bed: activation of the adrenal medulla and β-adrenergic vasodilation versus vasoconstriction mediated by neural and unknown humoral factors. In the present study we investigated the relative contribution of three major potential humoral vasoconstrictors: vasopressin, angiotensin II, and norepinephrine in this response. In urethane-chloralose anesthetized rats we compared the integral changes in iliac vascular conductance evoked by microinjections into the NTS of the selective A₁ receptor agonist N⁶-cyclopentyladenosine (CPA; 330 pmol in 50 nl) in intact (Int) animals and following: V₁ vasopressin receptor blockade (VX), angiotensin II AT₁ receptor blockade (ATX), bilateral adrenalectomy + ganglionic blockade (ADX + GX; which eliminated the potential increases in circulating norepinephrine and epinephrine), ADX + GX + VX and ADX + GX + VX + ATX. In Int animals, stimulation of NTS A₁ adenosine receptors evoked typical variable responses with prevailing pressor and vasoconstrictor effects. VX reversed the responses to depressor ones. ATX did not significantly alter the responses. ADX + GX accentuated pressor and vasoconstrictor responses, whereas ADX + GX + VX and ADX + GX + VX + ATX virtually abolished the responses. Stimulation of NTS A₁ adenosine receptors increased circulating vasopressin over fourfold (26.4 ± 10.4 vs. 117.0 ± 19 pg/ml). These data strongly suggest that vasopressin is a major vasoconstrictor factor opposing β-adrenergic vasodilation in iliac vascular responses triggered by stimulation of NTS A₁ adenosine receptors, whereas angiotensin II and norepinephrine do not contribute significantly to the vasoconstrictor responses.

Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition

Mon, 26 Oct 2009 13:43:29 PDT

Medial artery calcification, which does not accompany lipid or cholesterol deposit, preferentially occurs in elderly population, but its underlying mechanisms remain unclear. In the present study, we investigated the potential role of senescent vascular smooth muscle cells (VSMCs) in the formation of senescence-associated medial calcification. Replicative senescence was induced by the extended passages (until passages 11–13) in human primary VSMCs, and cells in early passage (passage 6) were used as control young cells. VSMC calcification was markedly enhanced in the senescent cells compared with that in the control young cells. We identified that genes highly expressed in osteoblasts, such as alkaline phosphatase (ALP) and type I collagen, were significantly upregulated in the senescent VSMCs, suggesting their osteoblastic transition during the senescence. Knockdown of either ALP or type I collagen significantly reduced the calcification in the senescent VSMCs. Of note, runt-related transcription factor-2 (RUNX-2), a core transcriptional factor that initiates the osteoblastic differentiation, was also upregulated in the senescent VSMCs. Knockdown of RUNX-2 significantly reduced the ALP expression and calcification in the senescent VSMCs, suggesting that RUNX-2 is involved in the senescence-mediated osteoblastic transition. Furthermore, immunohistochemistry of aorta from the klotho^–/– aging mouse model demonstrated in vivo emergence of osteoblast-like cells expressing RUNX-2 exclusively in the calcified media. We also found that statin and Rho-kinase inhibitor effectively reduced the VSMC calcification by inhibiting P_i-induced apoptosis and potentially enhancing matrix Gla protein expression in the senescent VSMCs. These findings strongly suggest an important role of senescent VSMCs in the pathophysiology of senescence-associated medial calcification, and the inhibition of osteoblastic transition could be a new therapeutic approach for the prevention of senescence-associated medial calcification.

VEGF-C regulates lymphangiogenesis and capillary stability by regulation of PDGF-B

Mon, 26 Oct 2009 13:43:29 PDT

Emerging evidence indicates that the tight communication between vascular endothelial cells and mural cells using platelet-derived growth factor (PDGF)-BB is essential for capillary stabilization during the angiogenic process. However, little is known about the related regulator that determines PDGF-BB expression. Using murine models of therapeutic neovascularization, we here show that a typical lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, is an essential regulator determining PDGF-BB expression for vascular stabilization via a paracrine mode of action. The blockade of VEGF type 3 receptor (VEGFR3) using neutralizing antibody AFL-4 abrogated FGF-2-mediated limb salvage and blood flow recovery in severely ischemic hindlimb. Interestingly, inhibition of VEGFR3 activity not only diminished lymphangiogenesis, but induced marked dilatation of capillary vessels, showing mural cell dissociation. In these mice, VEGF-C and PDGF-B were upregulated in the later phase after induced ischemia, on day 7, when exogenous FGF-2 expression had already declined, and blockade of VEGFR3 or PDGF-BB activities diminished PDGF-B or VEGF-C expression, respectively. These results clearly indicate that VEGF-C is a critical mediator, not only for lymphangiogenesis, but also for capillary stabilization, the essential molecular mechanism of communication between endothelial cells and mural cells during neovascularization.

Regulation of VASP phosphorylation in cardiac myocytes: differential regulation by cyclic nucleotides and modulation of protein expression in diabetic and hypertrophic heart

Sartoretto, J. L., Jin, B. Y., Bauer, M., Gertler, F. B., Liao, R., Michel, T. — Mon, 26 Oct 2009 13:43:29 PDT

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases that has been implicated in cardiac pathology, yet many aspects of VASP's molecular regulation in cardiomyocytes are incompletely understood. In these studies, we explored the role of VASP, both in signaling pathways in isolated murine myocytes, as well as in a model of cardiac hypertrophy in VASP^null mice. We found that the β-adrenergic agonist isoproterenol promotes the rapid and reversible phosphorylation of VASP at Ser157 and Ser239. Forskolin and the cAMP analog 8-(4-chlorophenylthio)-cAMP promote a similar pattern of VASP phosphorylation at both sites. The effects of isoproterenol are blocked by atenolol and by compound H-89, an inhibitor of the cAMP-dependent protein kinase. By contrast, phosphorylation of VASP only at Ser239 is seen following activation of particulate guanylate cyclase by atrial natriuretic peptide, or following activation of soluble guanylate cyclase by sodium nitroprusside, or following treatment of myocytes with cGMP analog. We found that basal and isoproterenol-induced VASP phosphorylation is entirely unchanged in cardiomyocytes isolated from either endothelial or neuronal nitric oxide synthase knockout mice. In cardiomyocytes isolated from diabetic mice, only basal VASP phosphorylation is increased, whereas, in cells isolated from mice subjected to ascending aortic constriction (AAC), we found a significant increase in basal VASP expression, along with an increase in VASP phosphorylation, compared with cardiac myocytes isolated from sham-operated mice. Moreover, there is further increase in VASP phosphorylation in cells isolated from hypertrophic hearts following isoproterenol treatment. Finally, we found that VASP^null mice subjected to transverse aortic constriction develop cardiac hypertrophy with a pattern similar to VASP^+/+ mice. Our findings establish differential receptor-modulated regulation of VASP phosphorylation in cardiomyocytes by cyclic nucleotides. Furthermore, these studies demonstrate for the first time that VASP expression is upregulated in hypertrophied heart.

O-GlcNAc signaling attenuates ER stress-induced cardiomyocyte death

Ngoh, G. A., Hamid, T., Prabhu, S. D., Jones, S. P. — Mon, 26 Oct 2009 13:43:29 PDT

We previously demonstrated that the O-linked β-N-acetylglucosamine (O-GlcNAc) posttranslational modification confers cardioprotection at least partially through mitochondrial-dependent mechanisms, but it remained unclear if O-GlcNAc signaling interfered with other mechanisms of cell death. Because ischemia/hypoxia causes endoplasmic reticulum (ER) stress, we ascertained whether O-GlcNAc signaling could attenuate ER stress-induced cell death per se. Before induction of ER stress (with tunicamycin or brefeldin A), we adenovirally overexpressed O-GlcNAc transferase (AdOGT) or pharmacologically inhibited O-GlcNAcase [via O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate] to augment O-GlcNAc levels or adenovirally overexpressed O-GlcNAcase to reduce O-GlcNAc levels. AdOGT significantly (P < 0.05) attenuated the activation of the maladaptive arm of the unfolded protein response [according to C/EBP homologous protein (CHOP) activation] and cardiomyocyte death (reflected by percent propidium iodide positivity). Moreover, pharmacological inhibition of O-GlcNAcase significantly (P < 0.05) mitigated ER stress-induced CHOP activation and cardiac myocyte death. Interestingly, overexpression of GCA did not alter ER stress markers but exacerbated brefeldin A-induced cardiomyocyte death. We conclude that enhanced O-GlcNAc signaling represents a partially proadaptive response to reduce ER stress-induced cell death. These results provide new insights into a possible interaction between O-GlcNAc signaling and ER stress and may partially explain a mechanism of O-GlcNAc-mediated cardioprotection.

Microvascular oxygen delivery-to-utilization mismatch at the onset of heavy-intensity exercise in optimally treated patients with CHF

Mon, 26 Oct 2009 13:43:29 PDT

Impaired muscle blood flow at the onset of heavy-intensity exercise may transiently reduce microvascular O₂ pressure and decrease the rate of O₂ transfer from capillary to mitochondria in chronic heart failure (CHF). However, advances in the pharmacological treatment of CHF (e.g., angiotensin-converting enzyme inhibitors and third-generation β-blockers) may have improved microvascular O₂ delivery to an extent that intramyocyte metabolic inertia might become the main locus of limitation of O₂ uptake (Vo₂) kinetics. We assessed the rate of change of pulmonary Vo₂ (Vo₂_p), (estimated) fractional O₂ extraction in the vastus lateralis (~[deoxy-Hb+Mb] by near-infrared spectroscopy), and cardiac output (Qt) during high-intensity exercise performed to the limit of tolerance (Tlim) in 10 optimally treated sedentary patients (ejection fraction = 29 ± 8%) and 11 controls. Sluggish Vo₂_p and Qt kinetics in patients were significantly related to lower Tlim values (P < 0.05). The dynamics of [deoxy-Hb+Mb], however, were faster in patients than controls [mean response time (MRT) = 15.9 ± 2.0 s vs. 19.0 ± 2.9 s; P < 0.05] with a subsequent response "overshoot" being found only in patients (7/10). Moreover, Vo₂/MRT-[deoxy-Hb+Mb] ratio was greater in patients (4.69 ± 1.42 s vs. 2.25 ± 0.77 s; P < 0.05) and related to Qt kinetics and Tlim (R = 0.89 and –0.78, respectively; P < 0.01). We conclude that despite the advances in the pharmacological treatment of CHF, disturbances in "central" and "peripheral" circulatory adjustments still play a prominent role in limiting Vo₂_p kinetics and tolerance to heavy-intensity exercise in nontrained patients.

Cholesterol diet-induced hyperlipidemia impairs the cardioprotective effect of postconditioning: role of peroxynitrite

Mon, 26 Oct 2009 13:43:29 PDT

The aim of the present study was to investigate if hyperlipidemia interferes with the infarct size-limiting effect of postconditioning and to study the involvement of peroxynitrite in this phenomenon. Rats were fed a 2% cholesterol-enriched or normal diet for 12 wk. Infarct size by triphenyltetrazolium chloride staining was measured in hearts isolated from both groups and subjected to 30 min coronary occlusion followed by 120 min reperfusion with or without the postconditioning protocol induced by six cycles of 10 s coronary occlusion and 10 s reperfusion at the onset of the reperfusion. Postconditioning significantly decreased infarct size in the normolipidemic but not in the hyperlipidemic group. Postconditioning increased cardiac 3-nitrotyrosine concentration (a marker for peroxynitrite formation) in the normal but not in the cholesterol-fed group when measured at the 5th min of reperfusion. Next, we tested if the postconditioning-induced acute increase in peroxynitrite is involved in the cardioprotection in normolipidemic animals in separate experiments. Postconditioning failed to decrease infarct size in the presence of the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron [III] (20 mg/l) in normolipidemic animals. We conclude that an early increase in peroxynitrite after postconditioning plays a role in cardioprotection. Furthermore, hyperlipidemia blocks the cardioprotective effect of postconditioning at least in part via deterioration of the postconditioning-induced early increase in peroxynitrite formation.

Left ventricular function of isoproterenol-induced hypertrophied rat hearts perfused with blood: mechanical work and energetics

Mon, 26 Oct 2009 13:43:29 PDT

We investigated left ventricular (LV) mechanical work and energetics in the cross-circulated (blood-perfused) isoproterenol [Iso 1.2 mg·kg^–1·day^–1 for 3 days (Iso3) or 7 days (Iso7)]-induced hypertrophied rat heart preparation under isovolumic contraction-relaxation. We evaluated pressure-time curves per beat, end-systolic pressure-volume and end-diastolic pressure-volume relations, and myocardial O₂ consumption per beat (Vo₂)-systolic pressure-volume area (PVA; a total mechanical energy per beat) linear relations at 240 beats/min, because Iso-induced hypertrophied hearts failed to completely relax at 300 beats/min. The LV relaxation rate at 240 beats/min in Iso-induced hypertrophied hearts was significantly slower than that in control hearts [saline 24 µl/day for 3 and 7 days (Sa)] with unchanged contraction rate. The Vo₂-intercepts (composed of basal metabolism and Ca²⁺ cycling energy consumption in excitation-contraction coupling) of Vo₂-PVA linear relations were unchanged associated with their unchanged slopes in Sa, Iso3, and Iso7 groups. The oxygen costs of LV contractility were also unchanged in all three groups. The amounts of expression of sarcoplasmic reticulum Ca²⁺-ATPase, phospholamban (PLB), phosphorylated-Ser¹⁶ PLB, phospholemman, and Na⁺-K⁺-ATPase are significantly decreased in Iso3 and Iso7 groups, although the amount of expression of NCX1 is unchanged in all three groups. Furthermore, the marked collagen production (types I and III) was observed in Iso3 and Iso7 groups. These results suggested the possibility that lowering the heart rate was beneficial to improve mechanical work and energetics in isoproterenol-induced hypertrophied rat hearts, although LV relaxation rate was slower than in normal hearts.

Calpain inhibition preserves myocardial structure and function following myocardial infarction

Mon, 26 Oct 2009 13:43:29 PDT

Cardiac pathology, such as myocardial infarction (MI), activates intracellular proteases that often trigger programmed cell death and contribute to maladaptive changes in myocardial structure and function. To test whether inhibition of calpain, a Ca²⁺-dependent cysteine protease, would prevent these changes, we used a mouse MI model. Calpeptin, an aldehydic inhibitor of calpain, was intravenously administered at 0.5 mg/kg body wt before MI induction and then at the same dose subcutaneously once per day. Both calpeptin-treated (n = 6) and untreated (n = 6) MI mice were used to study changes in myocardial structure and function after 4 days of MI, where end-diastolic volume (EDV) and left ventricular ejection fraction (EF) were measured by echocardiography. Calpain activation and programmed cell death were measured by immunohistochemistry, Western blotting, and TdT-mediated dUTP nick-end labeling (TUNEL). In MI mice, calpeptin treatment resulted in a significant improvement in EF [EF decreased from 67 ± 2% pre-MI to 30 ± 4% with MI only vs. 41 ± 2% with MI + calpeptin] and attenuated the increase in EDV [EDV increased from 42 ± 2 µl pre-MI to 73 ± 4 µl with MI only vs. 55 ± 4 µl with MI + calpeptin]. Furthermore, calpeptin treatment resulted in marked reduction in calpain- and caspase-3-associated changes and TUNEL staining. These studies indicate that calpain contributes to MI-induced alterations in myocardial structure and function and that it could be a potential therapeutic target in treating MI patients.

Right ventricular pacing improves right heart function in experimental pulmonary arterial hypertension: a study in the isolated heart

Mon, 26 Oct 2009 13:43:29 PDT

Right heart failure in pulmonary arterial hypertension (PH) is associated with mechanical ventricular dyssynchrony, which leads to impaired right ventricular (RV) function and, by adverse diastolic interaction, to impaired left ventricular (LV) function as well. However, therapies aiming to restore synchrony by pacing are currently not available. In this proof-of-principle study, we determined the acute effects of RV pacing on ventricular dyssynchrony in PH. Chronic PH with right heart failure was induced in rats by injection of monocrotaline (80 mg/kg). To validate for PH-related ventricular dyssynchrony, rats (6 PH, 6 controls) were examined by cardiac magnetic resonance imaging (9.4 T), 23 days after monocrotaline or sham injection. In a second group (10 PH, 4 controls), the effects of RV pacing were studied in detail, using Langendorff-perfused heart preparations. In PH, septum bulging was observed, coinciding with a reversal of the transseptal pressure gradient, as observed in clinical PH. RV pacing improved RV systolic function, compared with unpaced condition (maximal first derivative of RV pressure: +8.5 ± 1.3%, P < 0.001). In addition, RV pacing markedly decreased the pressure-time integral of the transseptal pressure gradient when RV pressure exceeds LV pressure, an index of adverse diastolic interaction (–24 ± 9%, P < 0.01), and RV pacing was able to resynchronize time of RV and LV peak pressure (unpaced: 9.8 ± 1.2 ms vs. paced: 1.7 ± 2.0 ms, P < 0.001). Finally, RV pacing had no detrimental effects on LV function or coronary perfusion, and no LV preexcitation occurred. Taken together, we demonstrate that, in experimental PH, RV pacing improves RV function and diminishes adverse diastolic interaction. These findings provide a strong rationale for further in vivo explorations.

Dissociation between reflex sympathetic and forearm vascular responses to lower body negative pressure in heart failure patients with coronary artery disease

Notarius, C. F., Morris, B. L., Floras, J. S. — Mon, 26 Oct 2009 13:43:29 PDT

Many heart failure (HF) patients exhibit paradoxical forearm vasodilation when central blood volume is reduced by lower body negative pressure (LBNP). We tested the hypothesis that this response results from reflex sympathetic withdrawal. We recorded simultaneously forearm blood flow, muscle sympathetic nerve activity (MSNA), and plasma norepinephrine (PNE) during four random applications of LBNP, –5, –10, –20, and –40 mmHg, in 12 men with HF (mean left ventricular ejection fraction = 24 ± 2%) and 10 healthy, normal, age-matched men (N). Compared with N, MSNA burst frequency (P = 0.001) and PNE (P = 0.005) were significantly higher in the HF group, both at rest and during LBNP. As anticipated in N, LBNP –40 mmHg significantly increased MSNA (+14.2 ± 2.5 bursts/min; P < 0.05) and PNE (+0.83 ± 0.22 nmol/l; P < 0.05) and decreased forearm vascular conductance (FVC) (–11.7 ± 3.2 ml·min^–1·mmHg^–1; P < 0.05). In the HF group, LBNP elicited similar increases in MSNA (+11.5 ± 2.0; P < 0.05) and PNE (+0.85 ± 0.12; P < 0.05), without affecting FVC significantly (–4.1 ± 2.4; P = 0.01 vs. N, interaction P = 0.03). However, within the HF group, responses were bimodal: LBNP –40 mmHg increased MSNA in all subjects (P < 0.001), yet the six patients with nonischemic or dilated cardiomyopathy (DCM) exhibited significant vasoconstriction (decrease in FVC; P = 0.001), whereas the six patients with ischemic cardiomyopathy (ICM) exhibited significant vasodilation (increase in FVC; P < 0.02 vs. DCM and N; interaction P = 0.02). Cold pressor testing increased MSNA and decreased FVC in ICM (n = 4). Thus paradoxical forearm vasodilator responses to LBNP in HF are not mediated by reflex sympathetic withdrawal. ICM and DCM patients differ qualitatively in their vascular responses to hypotensive LBNP.

Activation of the PDK-1/Akt/eNOS pathway involved in aortic endothelial function differs between hyperinsulinemic and insulin-deficient diabetic rats

Kobayashi, T., Taguchi, K., Nemoto, S., Nogami, T., Matsumoto, T., Kamata, K. — Mon, 26 Oct 2009 13:43:29 PDT

In diabetic states, altered plasma insulin is likely to play key roles in 3-phosphoinositide-dependent protein kinase (PDK)/Akt pathway activation, in insulin resistance and in endothelial dysfunction. Since the molecular mechanism(s) remains unclear, we examined the relationship between the PDK/Akt/endothelial nitric oxide synthase (NOS) pathway and endothelial function in aortas from diabetic rats that were either insulin deficient or hyperinsulinemic. Untreated diabetic (diabetic) rats exhibited hyperglycemia and hypoinsulinemia, whereas high-insulin-treated diabetic (HI-diabetic) rats exhibited hyperinsulinemia. Aortas from the diabetic group displayed impaired endothelium-dependent relaxation in response to ACh, whereas the insulin-induced relaxation was increased. In HI-diabetic aortas, the ACh-induced relaxation was normal, but that induced by insulin was impaired. The insulin-induced relaxation was inhibited by treatment with an Akt inhibitor in control and diabetic aortas, but not in the HI-diabetic aorta. This inhibitory effect on insulin-induced relaxation was greater in diabetic aortas than in control aortas. In all groups, ACh-induced relaxation was unaffected by the above inhibitor. In the diabetic group, various insulin-stimulated levels (nitric oxide production, phosphorylation of endothelial NOS at Ser¹¹⁷⁷, of Akt at Thr³⁰⁸, and of PDK-1 at Ser²⁴¹) were significantly increased, whereas, in the HI-diabetic group, these levels were all decreased (vs. control aortas). These results suggest that the plasma insulin level has a close relation to the level of aortic PDK-1/Akt (at Thr³⁰⁸)/NOS activities, and that reduced actions of the PDK-1/Akt (at Thr³⁰⁸) signal pathway may contribute to the impairments of insulin-induced endothelial functions seen in hyperinsulinemic diabetes.

Adenylyl cyclase type 5 protein expression during cardiac development and stress

Mon, 26 Oct 2009 13:43:29 PDT

Adenylyl cyclase (AC) types 5 and 6 (AC5 and AC6) are the two major AC isoforms expressed in the mammalian heart that mediate signals from β-adrenergic receptor stimulation. Because of the unavailability of isoform-specific antibodies, it is difficult to ascertain the expression levels of AC5 protein in the heart. Here we demonstrated the successful generation of an AC5 isoform-specific mouse monoclonal antibody and studied the expression of AC5 protein during cardiac development in different mammalian species. The specificity of the antibody was confirmed using heart and brain tissues from AC5 knockout mice and from transgenic mice overexpressing AC5. In mice, the AC5 protein was highest in the brain but was also detectable in all organs studied, including the heart, brain, lung, liver, stomach, kidney, skeletal muscle, and vascular tissues. Western blot analysis showed that AC5 was most abundant in the neonatal heart and declined to basal levels in the adult heart. AC5 protein increased in the heart with pressure-overload left ventricular hypertrophy. Thus this new AC5 antibody demonstrated that this AC isoform behaves similarly to fetal type genes, such as atrial natriuretic peptide; i.e., it declines with development and increases with pressure-overload hypertrophy.

Role of tetrahydrobiopterin in resistance to myocardial ischemia in Brown Norway and Dahl S rats

An, J., Du, J., Wei, N., Xu, H., Pritchard, K. A., Shi, Y. — Mon, 26 Oct 2009 13:43:29 PDT

Previously we showed that Brown Norway (BN/Mcw) rats are more resistant to myocardial ischemia-reperfusion (I/R) injury than Dahl S (SS/Mcw) rats due to increased nitric oxide (·NO) generation secondary to increased heat shock protein 90 (HSP90) association with endothelial nitric oxide synthase (NOS3). Here we determined whether increased resistance to I/R injury in BN/Mcw hearts is also related to tetrahydrobiopterin (BH₄) and GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for BH₄ synthesis. We observed that BH₄ supplementation via sepiapterin (SP) and inhibition of GCH-1 via 2,4-diamino-6-hydroxypyrimidine (DAHP) differentially modulate cardioprotection and that SP alters the association of HSP90 with NOS3. BH₄ levels were significantly higher and 7,8-dihydrobiopterin (BH₂) levels were significantly lower in BN/Mcw than in SS/Mcw hearts. The BH₄-to-BH₂ ratio in BN/Mcw was more than two times that in SS/Mcw hearts. After I/R, BH₄ decreased and BH₂ increased in hearts from both strains compared with their preischemia levels. However, the increase in BH₂ in SS/Mcw hearts was significantly higher than in BN/Mcw hearts. Real-time PCR revealed that BN/Mcw hearts contained more GCH-1 transcripts than SS/Mcw hearts. SP increased recovery of left ventricular developed pressure (rLVDP) following I/R as well as decreased superoxide (O₂^•–) and increased·NO in SS/Mcw hearts but not in BN/Mcw hearts. DAHP decreased rLVDP as well as increased O₂^•– and decreased·NO in BN/Mcw hearts compared with controls but not in SS/Mcw hearts. SP increased the association of HSP90 with NOS3. These data indicate that BH₄ mediates resistance to I/R by acting as a cofactor and enhancing HSP90-NOS3 association.

Reflex vasoconstriction in aged human skin increasingly relies on Rho kinase-dependent mechanisms during whole body cooling

Lang, J. A., Jennings, J. D., Holowatz, L. A., Kenney, W. L. — Mon, 26 Oct 2009 13:43:29 PDT

Primary human aging may be associated with augmented Rho kinase (ROCK)-mediated contraction of vascular smooth muscle and ROCK-mediated inhibition of nitric oxide synthase (NOS). We hypothesized that the contribution of ROCK to reflex vasoconstriction (VC) is greater in aged skin. Cutaneous VC was elicited by 1) whole body cooling [mean skin temperature (T_sk) = 30.5°C] and 2) local norepinephrine (NE) infusion (1 x 10^–6 M). Four microdialysis fibers were placed in the forearm skin of eight young (Y) and eight older (O) subjects for infusion of 1) Ringer solution (control), 2) 3 mM fasudil (ROCK inhibition), 3) 20 mM N^G-nitro-l-arginine methyl ester (NOS inhibition), and 4) both ROCK + NOS inhibitors. Red cell flux was measured by laser-Doppler flowmetry over each site. Cutaneous vascular conductance (CVC) was calculated as flux/mean arterial pressure and normalized to baseline CVC (%CVC_baseline). VC was reduced at the control site in O during cooling (Y, –34 ± 3; and O, –18 ± 3%CVC_baseline; P < 0.001) and NE infusion (Y, –53 ± 4, and O, –41 ± 9%CVC_baseline; P = 0.006). Fasudil attenuated VC in both age groups during mild cooling; however, this reduction remained only in O but not in Y skin during moderate cooling (Y, –30 ± 5; and O, –7 ± 1%CVC_baseline; P = 0.016) and was not altered by NOS inhibition. Fasudil blunted NE-mediated VC in both age groups (Y, –23 ± 4; and O, –7 ± 3%CVC_baseline; P < 0.01). Cumulatively, these data indicate that reflex VC is more reliant on ROCK in aged skin such that approximately half of the total VC response to whole body cooling is ROCK dependent.

Effects of acute and chronic endurance exercise on intracellular nitric oxide in putative endothelial progenitor cells: role of NAPDH oxidase

Jenkins, N. T., Witkowski, S., Spangenburg, E. E., Hagberg, J. M. — Mon, 26 Oct 2009 13:43:29 PDT

We sought to delineate the effects of acute and chronic exercise on the regulation of intracellular nitric oxide (NO_i) production in putative endothelial progenitor cells (EPCs). Putative EPC colony-forming units (CFU-EC) were cultured from blood drawn before and after 30 min of treadmill exercise at 75% of maximal oxygen uptake in active (n = 8) and inactive (n = 8) men. CFU-EC were similar between groups at baseline, but increased after exercise in active men only (P = 0.04). CFU-EC expressed lower NADPH oxidase subunit gp91^phox mRNA and elevated endothelial nitric oxide synthase mRNA in active relative to inactive men at baseline (P < 0.05). Acute exercise reduced gp91^phox mRNA in CFU-EC of both groups (P < 0.05), whereas p47^phox mRNA levels were reduced in the inactive group only (P = 0.02). There were no differences between groups or with acute exercise in xanthine oxidase, superoxide dismutase isoforms, or gluthathione peroxidase-1 mRNA levels. NO_i was significantly greater in CFU-EC of active men at baseline (P = 0.004). NO_i increased in CFU-EC of inactive men with acute exercise, and in vitro experiments with apocynin indicated the increased NO_i production was caused by suppression of NADPH oxidase. However, the increases in NO_i with the different treatments in the inactive group did not reach the baseline levels in the active group (P < 0.05). We conclude that acute exercise increases NO_i in cells generated by the CFU-EC assay through an NADPH oxidase-inhibition mechanism in sedentary men. However, differences due to chronic exercise must involve additional factors. Our findings support exercise as a means to improve putative EPC function and suggest a novel mechanism that may explain this effect.

Activation of a novel estrogen receptor, GPER, is cardioprotective in male and female rats

Deschamps, A. M., Murphy, E. — Mon, 26 Oct 2009 13:43:29 PDT

Premenopausal females have a lower incidence of cardiovascular disease than their male counterparts, but the mechanism is unclear. Estrogen has been thought to signal through two nuclear receptors: estrogen receptor- or estrogen receptor-β; however, a third, membrane-bound receptor G protein-coupled estrogen receptor (GPER), has been identified and shown to bind estrogen with high affinity. To date, there is little information on GPER in the heart and no study has looked at the effect of GPER activation during myocardial ischemia-reperfusion (I/R). Therefore, the goal of this study was to determine whether activation of GPER is cardioprotective in rats. A highly specific GPER agonist, G-1, was administered to Sprague-Dawley (200–350 g) rat hearts 10 min before 20 min of ischemic followed by 120 min of reperfusion using a Langendorff model. Similar levels of GPER were found in both male and female rat hearts. With administration of 110 nM of G-1, postischemic contractile dysfunction was significantly reduced compared with untreated controls (43.8 ± 4.3% vs. 26.9 ± 2.1% of preischemic rate pressure product; P < 0.05). Additionally, infarct size was reduced in the G-1-treated animals when compared with control (18.8 ± 2.7% vs. 32.4 ± 2.1%; P < 0.05). These observations were demonstrated in both male and intact female rat hearts. Through Western blot analysis, it was demonstrated that G-1 induces the activation of both Akt and ERK1/2. Furthermore, the protection afforded by G-1 was blocked by coadministration of a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin, 100 nM). Taken together, the data show that G-1 activation of GPER improves functional recovery and reduces infarct size in isolated rat hearts following I/R through a PI3K-dependent, gender-independent mechanism.

Genetic inhibition of calcineurin induces diastolic dysfunction in mice with chronic pressure overload

Mon, 26 Oct 2009 13:43:29 PDT

Calcineurin is a Ca²⁺/calmodulin-dependent protein phosphatase that induces myocardial growth in response to several physiological and pathological stimuli. Calcineurin inhibition, induced either via cyclosporine or genetically, can decrease myocardial hypertrophy secondary to pressure overload without affecting left ventricular (LV) systolic function. Since hypertrophy can also affect LV diastolic function, the goal of this study was to examine the effects of chronic pressure overload (2 wk aortic banding) in transgenic (Tg) mice overexpressing Zaki-4β (TgZ), a specific endogenous inhibitor of calcineurin, on LV diastolic function. As expected, in the TgZ mice with calcineurin inhibitor overexpression, aortic banding reduced the degree of LV hypertrophy, as assessed by LV weight-to-body weight ratio (3.5 ± 0.1) compared with that in non-Tg mice (4.6 ± 0.2). LV systolic function remained compensated in both groups with pressure overload. However, the LV end-diastolic stress-to-LV end-diastolic dimension ratio, an index of diastolic stiffness and LV pressure half-time and isovolumic relaxation time, two indexes of isovolumic relaxation, increased significantly more in TgZ mice with aortic banding. Protein levels of phosphorylated phospholamban (PS16), sarco(endo)plasmic reticulum Ca²⁺-ATPase 2a, phosphorylated ryanodine receptor, and the Na⁺/Ca²⁺ exchanger were also reduced significantly (P < 0.05) in the banded TgZ mice. As expected, genetic calcineurin inhibition inhibited the development of LV hypertrophy with chronic pressure overload but also induced LV diastolic dysfunction, as reflected by both impaired isovolumic relaxation and increased myocardial stiffness. Thus genetic calcineurin inhibition reveals a new mechanism regulating LV diastolic function.

Cav1.2 splice variant with exon 9* is critical for regulation of cerebral artery diameter

Nystoriak, M. A., Murakami, K., Penar, P. L., Wellman, G. C. — Mon, 26 Oct 2009 13:43:30 PDT

L-type voltage-dependent Ca²⁺ channels (VDCCs) are essential for numerous processes in the cardiovascular and nervous systems. Alternative splicing modulates proteomic composition of Ca_v1.2 to generate functional variation between channel isoforms. Here, we describe expression and function of Ca_v1.2 channels containing alternatively spliced exon 9* in cerebral artery myocytes. RT-PCR showed expression of Ca_v1.2 splice variants both containing (₁C_9/9*/10) and lacking (₁C_9/10) exon 9* in intact rabbit and human cerebral arteries. With the use of laser capture microdissection and RT-PCR, expression of mRNA for both ₁C_9/9*/10 and ₁C_9/10 was demonstrated in isolated cerebral artery myocytes. Quantitative real-time PCR revealed significantly greater ₁C_9/9*/10 expression relative to ₁C_9/10 in intact rabbit cerebral arteries compared with cardiac tissue and cerebral cortex. To demonstrate a functional role for ₁C_9/9*/10, smooth muscle of intact cerebral arteries was treated with antisense oligonucleotides targeting ₁C_9/9*/10 (₁C_9/9*/10-AS) or exon 9 (₁C-AS), expressed in all Ca_v1.2 splice variants, by reversible permeabilization and organ cultured for 1–4 days. Treatment with ₁C_9/9*/10-AS reduced maximal constriction induced by elevated extracellular K⁺ ([K⁺]_o) by ~75% compared with ₁C_9/9*/10-sense-treated arteries. Maximal constriction in response to the Ca²⁺ ionophore ionomycin and [K⁺]_o EC₅₀ values were not altered by antisense treatment. Decreases in maximal [K⁺]_o-induced constriction were similar between ₁C_9/9*/10-AS and ₁C-AS groups (22.7 ± 9% and 25.6 ± 4% constriction, respectively). We conclude that although cerebral artery myocytes express both ₁C_9/9*/10 and ₁C_9/10 VDCC splice variants, ₁C_9/9*/10 is functionally dominant in the control of cerebral artery diameter.

eNOS uncoupling and endothelial dysfunction in aged vessels

Yang, Y.-M., Huang, A., Kaley, G., Sun, D. — Mon, 26 Oct 2009 13:43:30 PDT

Endothelial nitric oxide synthase (eNOS) uncoupling is a mechanism that leads to endothelial dysfunction. Previously, we reported that shear stress-induced release of nitric oxide in vessels of aged rats was significantly reduced and was accompanied by increased production of superoxide (18, 27). In the present study, we investigated the influence of aging on eNOS uncoupling. Mesenteric arteries were isolated from young (3 mo) and aged (24 mo) C57 BL/6J mice. The expression of eNOS protein in young vs. aged mice was not significantly different. However, the aged mice had remarkable increases in the ratio of eNOS monomers to dimers and N-nitro-l-arginine methyl ester-inhibitable superoxide formation. The level of nitrotyrosine in the total protein and precipitated eNOS of aged vessels was increased compared with that in young vessels. HPLC analysis indicated a reduced level of tetrahydrobiopterin (BH4), an essential cofactor for eNOS, in the mesenteric arteries of aged mice. Quantitative PCR results implied that the diminished BH4 may result from the decreased expressions of GTP cyclohydrolase I and sepiapterin reductase, enzymes involved in BH4 biosynthesis. When isolated and cannulated second-order mesenteric arteries (~150 µm) from aged mice were treated with sepiapterin, acetylcholine-induced, endothelium-dependent vasodilation improved significantly, which was accompanied by stabilization of the eNOS dimer. These data suggest that eNOS uncoupling and increased nitrosylation of eNOS, decreased expressions of GTP cyclohydrolase I and sepiapterin reductase, and subsequent reduced BH4 bioavailability may be important contributors of endothelial dysfunction in aged vessels.

Angiotensin II enhances GABAB receptor-mediated responses and expression in nucleus tractus solitarii of rats

Zhang, Q., Yao, F., O'Rourke, S. T., Qian, S. Y., Sun, C. — Mon, 26 Oct 2009 13:43:30 PDT

Angiotensin II (ANG II) increases GABA_B receptor expression in neuronal cultures from the nucleus tractus solitarii (NTS). In the present study, the chronic effects of ANG II on GABA_B receptor expression and activity were examined in the NTS of Sprague-Dawley rats. Intracerebroventricular infusion of ANG II caused a significant elevation in blood pressure (BP) and an increase in GABA_B receptor expression in the NTS. Conversely, chronic N^G-nitro-l-arginine methyl ester (l-NAME) treatment also increased BP, but had no effect on GABA_B receptor expression in the NTS. Next, we examined the BP response to the GABA_B receptor agonist baclofen microinjected into the NTS of ANG II- or artificial cerebrospinal fluid (aCSF)-infused rats. NTS microinjection of baclofen increased BP in both groups of rats. However, the pressor response to baclofen was enhanced in ANG II-infused rats compared with aCSF-infused rats. In addition, bilateral microinjection of the GABA_B receptor antagonist CGP-35348 into the NTS evoked a decrease in BP in both group of rats, and the depressor responses to CGP-35348 were enhanced in the ANG II-infused rats. In contrast, the pressor responses to the GABA_A receptor agonist muscimol and the depressor responses to the GABA_A receptor antagonist bicuculline were comparable between aCSF- and ANG II-infused rats. These results indicate that chronic ANG II infusion stimulates GABA_B receptor expression and augments GABA_B receptor-mediated responses in the NTS. This effect could contribute to the central nervous system actions of ANG II that result in dampening of baroreflexes and elevation in arterial BP.

Lack of cardiac fibrosis in a new model of high prorenin hyperaldosteronism

Mon, 26 Oct 2009 13:43:30 PDT

The aim of the present study was to test the hypothesis that elevation of prorenin in plasma is sufficient to induce cardiac fibrosis. Normotensive cyp1a1ren-2 transgenic rats with normal plasma prorenin and aldosterone levels were given 0.125% indole-3-carbinol (I3C) orally for a period of 12 wk. Plasma prorenin and aldosterone levels were determined in 4-wk intervals, and cardiac marker enzymes for hypertrophy, fibrosis, and oxidative stress as well as cardiac pathology were investigated. In I3C-treated cyp1a1 ren-2 transgenic rats, plasma prorenin concentrations were >100-fold elevated (≥7.1 ± 2.6 µg ANG I·ml^–1·h^–1 vs. ≤0.07 ± 0.1; P < 0.001), whereas active renin levels were suppressed (0.09 ± 0.02 vs. 0.2 ± 0.1; P < 0.05). Aldosterone concentrations were elevated three- to fourfold for a period of >4 wk (574 ± 51 vs. 160 ± 68 pg/ml; P < 0.01). After 12 wk of I3C, rats exhibited moderate cardiac hypertrophy (heart weight/body weight 2.5 ± 0.04 vs. 3.1 ± 0.1 mg/g; P < 0.01). There was a slight increase in mRNA contents of endothelin 1 (1.21 ± 0.08 vs. 0.75 ± 0.007; P < 0.001), NADP oxidase-2 (1.03 ± 0.006 vs. 0.76 ± 0.04; P < 0.001), transforming growth factor-β (0.99 ± 0.06 vs. 0.84 ± 0.04; P < 0.05), collagen type I (1.32 ± 0.32 vs. 0.94 ± 0.18; P < 0.05), and intercellular adhesion molecule-1 (1.12 ± 0.12 vs. 0.84 ± 0.08; P < 0.05). These genes are known to be stimulated by the renin-angiotensin system. There were no histological signs of fibrosis in the heart. We found that prorenin and aldosterone alone are not sufficient to induce considerable cardiac fibrosis in the absence of sodium load.

Myocardial ischemia and reperfusion injury is dependent on both IgM and mannose-binding lectin

Busche, M. N., Pavlov, V., Takahashi, K., Stahl, G. L. — Mon, 26 Oct 2009 13:43:30 PDT

Complement activation has been shown to play an important role in the inflammation and tissue injury following myocardial ischemia and reperfusion (MI/R). Several recent studies from our laboratory demonstrated the importance of mannose-binding lectin (MBL) as the initiation pathway for complement activation and the resulting pathological effects following MI/R. However, other studies from the past suggest an important role of the classical pathway and perhaps natural antibodies. In the present study, we used newly generated genetically modified mice that lack secreted IgM (sIgM), MBL-A, and MBL-C (sIgM/MBL null) in a plasma reconstitution mouse model of MI/R. Following 30 min of ischemia and 4 h of reperfusion, left ventricular ejection fractions were significantly higher in sIgM/MBL null mice reconstituted with MBL null or sIgM/MBL null plasma compared with reconstitution with wild-type (WT) plasma or WT mice reconstituted with WT plasma following MI/R. Serum troponin I concentration, myocardial polymorphonuclear leukocyte infiltration, and C3 deposition were dependent on the combined presence of sIgM and MBL. These results demonstrate that MI/R-induced complement activation, inflammation, and subsequent tissue injury require both IgM and MBL. Thus MBL-dependent activation of the lectin pathway may not be completely antibody independent in I/R models.

Pharmacological modifications of the stretch-induced effects on ventricular fibrillation in perfused rabbit hearts

Mon, 26 Oct 2009 13:43:30 PDT

Stretch induces modifications in myocardial electrical and mechanical activity. Besides the effects of substances that block the stretch-activated channels, other substances could modulate the effects of stretch through different mechanisms that affect Ca²⁺ handling by myocytes. Thirty-six Langendorff-perfused rabbit hearts were used to analyze the effects of the Na⁺/Ca²⁺ exchanger blocker KB-R7943, propranolol, and the adenosine A₂ receptor antagonist SCH-58261 on the acceleration of ventricular fibrillation (VF) produced by acute myocardial stretching. VF recordings were obtained with two epicardial multiple electrodes before, during, and after local stretching in four experimental series: control (n = 9), KB-R7943 (1 µM, n = 9), propranolol (1 µM, n = 9), and SCH-58261 (1 µM, n = 9). Both the Na⁺/Ca²⁺ exchanger blocker KB-R7943 and propranolol induced a significant reduction (P < 0.001 and P < 0.05, respectively) in the dominant frequency increments produced by stretching with respect to the control and SCH-58261 series (control = 49.9%, SCH-58261 = 52.1%, KB-R7943 = 9.5%, and propranolol = 12.5%). The median of the activation intervals, the functional refractory period, and the wavelength of the activation process during VF decreased significantly under stretch in the control and SCH-58261 series, whereas no significant variations were observed in the propranolol and KB-R7943 series, with the exception of a slight but significant decrease in the median of the fibrillation intervals in the KB-R7943 series. KB-R7943 and propranolol induced a significant reduction in the activation maps complexity increment produced by stretch with respect to the control and SCH-58261 series. In conclusion, the electrophysiological effects responsible for stretch-induced VF acceleration in the rabbit heart are reduced by the Na⁺/Ca²⁺ exchanger blocker KB-R7943 and by propranolol but not by the adenosine A₂ receptor antagonist SCH-58261.

Antioxidants and aging: NMR-based evidence of improved skeletal muscle perfusion and energetics

Mon, 26 Oct 2009 13:43:30 PDT

We sought to examine the potential role of oxidative stress on skeletal muscle function with advancing age. Nuclear magnetic resonance (NMR) was employed to simultaneously assess muscle perfusion (arterial spin labeling) and energetics (³¹P NMR spectroscopy) in the lower leg of young (26 ± 5 yr, n = 6) and older (70 ± 5 yr, n = 6) healthy volunteers following the consumption of either placebo (PL) or an oral antioxidant (AO) cocktail (vitamins C and E and -lipoic acid), previously documented to decrease plasma free radical concentration. NMR measurements were made during and after 5 min of moderate intensity (5 W) plantar flexion exercise. AO administration significantly improved end-exercise perfusion (AO, 50 ± 5, and PL, 43 ± 4 ml·100 g^–1·min^–1) and postexercise perfusion area under the curve (AO, 1,286 ± 236, and PL, 866 ± 144 ml/100 g) in older subjects, whereas AO administration did not alter hemodynamics in the young group. Concomitantly, muscle oxidative capacity (time constant of phosphocreatine recovery, ) was improved following AO in the older (AO, 43 ± 1, and PL, 51 ± 7 s) but not the young (AO, 54 ± 5, and PL, 48 ± 7 s) group. These findings support the concept that oxidative stress may be partially responsible for the age-related decline in skeletal muscle perfusion during physical activity and reveal a muscle metabolic reserve capacity in the elderly that is accessible under conditions of improved perfusion.

Resveratrol attenuates mitochondrial oxidative stress in coronary arterial endothelial cells

Mon, 26 Oct 2009 13:43:30 PDT

The production of hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetic complications. Because resveratrol, a naturally occurring polyphenol, has been reported to confer vasoprotection, improving endothelial function and preventing complications of diabetes, we investigated the effect of resveratrol on mtROS production in cultured human coronary arterial endothelial cells (CAECs). The measurement of MitoSox fluorescence showed that resveratrol attenuates both steady-state and high glucose (30 mM)-induced mtROS production in CAECs, an effect that was prevented by the knockdown of the protein deacetylase silent information regulator 2/sirtuin 1 (SIRT1), an intracellular target of resveratrol. An overexpression of SIRT1 mimicked the effects of resveratrol, attenuating mtROS production. Similar results were obtained in CAECs transfected with mitochondria-targeted H₂O₂-sensitive HyPer-Mito fluorescent sensor. Amplex red assay showed that resveratrol and SIRT1 overexpression significantly reduced cellular H₂O₂ levels as well. Resveratrol upregulated MnSOD expression and increased cellular GSH content in a concentration-dependent manner (measured by HPLC coulometric analysis). These effects were attenuated by SIRT1 knockdown and mimicked by SIRT1 overexpression. We propose that resveratrol, via a pathway that involves the activation of SIRT1 and the upregulation of antioxidant defense mechanisms, attenuates mtROS production, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Altered reactivity to norepinephrine through COX-2 induction by vascular injury in hypercholesterolemic rabbits

Mon, 26 Oct 2009 13:43:30 PDT

Although long-term use of cyclooxygenase (COX)-2 inhibitors may be associated with increased cardiovascular risk, their effects on vascular reactivity in atherosclerosis has remained largely unexplored. The aim of the present study was to evaluate the role of COX-2 induced by an atherosclerotic process, in the local control of vascular tone. New Zealand White rabbits were fed 0.3% cholesterol and subjected to balloon injury of the abdominal aorta. After 2 wk, the aorta was removed and used for organ bath experiments and immunohistochemistry, and the prostaglandins released were measured using enzyme immunoassays. Hypercholesterolemia and vascular injury significantly increased the thickness of the intimal layer, which was associated with an induction of COX-2 immunoreactivity throughout the aortic wall. In these preparations, a significant decrease of the maximal contractions induced by norepinephrine was observed. The norepinephrine-induced contractions of atherosclerotic preparations were restored by the COX inhibitors DuP-697 (0.5 µmol/l) and indomethacin (1.7 µmol/l), to similar contractions as was observed in aortic preparations derived from healthy rabbits. Norepinephrine stimulation of the abdominal aorta was accompanied by increased levels of prostaglandin I₂ but not of prostaglandin E₂, prostaglandin D₂, or thromboxane A₂ in atherosclerotic compared with normal aorta. Selective COX-2 inhibition significantly decreased the prostaglandin I₂ release from atherosclerotic aorta but had no effect on the prostaglandin release from aortic preparations derived from normal rabbits. These observations suggest that the local induction of COX-2 during atherosclerosis decreased the sensitivity to norepinephrine and that COX-2 inhibitors may increase vascular reactivity at sites of atherosclerotic lesions.

Angiotensin II effects on ischemic focal ventricular tachycardia are predominantly mediated through myocardial AT2 receptor

Mon, 26 Oct 2009 13:43:30 PDT

Ischemic focal ventricular tachycardia (VT) occurs in animals and humans. Angiotensin-converting enzyme inhibitors and receptor blockers reduce sudden death in patients with ischemic heart disease. In our dog model of coronary artery occlusion (CAO), we tested the hypothesis that angiotensin II (AGII) will selectively promote focal VT and that the specific AT₂ blocker PD-123319 (PD), or AT₁ blocker losartan, will affect this VT. Anesthetized dogs (n = 90) underwent CAO, followed by three-dimensional activation mapping of inducible VT. Dogs without VT in 1–3 h after CAO received AGII, and those with VT received either PD or losartan. Focal endocardium excised from ischemic sites was studied in vitro with standard microelectrode. Of 33 dogs with no inducible VT, AGII infusion resulted in sustained VT of only focal Purkinje origin in 13 (39%) compared with 0 of 20 dogs with saline. Of 26 dogs with inducible VT at baseline, given PD, reinduction was blocked in 8 of 10 (P < 0.05) focal VT, but only 1 of 15 with reentry. In contrast, of 11 dogs given losartan, reinduction of either mechanism was not blocked. In vitro triggered activity in Purkinje was blocked by PD in 13 of 19 (P < 0.05), but not by losartan in 8. Also, triggered activity was promoted by AGII, losartan, or the combination in 9 of 12 tissues. AGII promotes only focal, mainly Purkinje ischemic VT. PD, but not losartan, preferentially blocked focal VT, which is likely due to triggered activity due to delayed afterdepolarizations in Purkinje.

Additive beneficial effects of lactotripeptides and aerobic exercise on arterial compliance in postmenopausal women

Mon, 26 Oct 2009 13:43:30 PDT

Central arterial compliance plays an important role in the functional abilities of the vasculature. Two active tripeptides, valine-proline-proline and isoleucine-proline-proline, were isolated from sour milk and were referred to as lactotripeptides (LTP). Because LTP appears to act as an angiotensin-converting enzyme inhibitor, it is plausible to hypothesize that LTP improves arterial compliance. We determined the effects of LTP ingestion alone or in combination with regular aerobic exercise on arterial compliance. A total of 55 postmenopausal women (50–65 yr old) were randomly divided into four groups: placebo, LTP, exercise and placebo (Ex + placebo), or exercise and LTP (Ex + LTP). LTP or placebo was administered orally for 8 wk. The exercise groups completed an 8-wk moderate aerobic exercise intervention. There were no differences in baseline arterial compliance and most other key dependent variables among the groups. Carotid arterial compliance increased significantly in the LTP (0.93 ± 0.07 vs. 0.99 ± 0.08 mm²/mmHg·10^–1), Ex + placebo (0.92 ± 0.04 vs. 1.00 ± 0.05 mm²/mmHg·10^–1), and Ex + LTP groups (0.86 ± 0.06 vs. 1.00 ± 0.06 mm²/mmHg·10^–1), whereas no such changes were observed in the placebo control group (0.86 ± 0.06 vs. 0.85 ± 0.07 mm²/mmHg·10^–1). The magnitude of increases in carotid arterial compliance was significantly greater in the Ex + LTP group (19 ± 4%) than in other groups. The improvements in arterial compliance with LTP were associated with the corresponding reductions in arterial blood pressure and plasma angiotensin II concentrations. We concluded that LTP ingestion improves carotid arterial compliance and that the combination of LTP ingestion and regular exercise is additive and synergistic in improving arterial compliance in postmenopausal women.

Endogenous regulation of cardiovascular function by apelin-APJ

Mon, 26 Oct 2009 13:43:30 PDT

Studies have shown significant cardiovascular effects of exogenous apelin administration, including the potent activation of cardiac contraction. However, the role of the endogenous apelin-APJ pathway is less clear. To study the loss of endogenous apelin-APJ signaling, we generated mice lacking either the ligand (apelin) or the receptor (APJ). Apelin-deficient mice were viable, fertile, and showed normal development. In contrast, APJ-deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects. Under basal conditions, both apelin and APJ null mice that survived to adulthood manifested modest decrements in contractile function. However, with exercise stress both mutant lines demonstrated consistent and striking decreases in exercise capacity. To explain these findings, we explored the role of autocrine signaling in vitro using field stimulation of isolated left ventricular cardiomyocytes lacking either apelin or APJ. Both groups manifested less sarcomeric shortening and impaired velocity of contraction and relaxation with no difference in calcium transient. Taken together, these results demonstrate that endogenous apelin-APJ signaling plays a modest role in maintaining basal cardiac function in adult mice with a more substantive role during conditions of stress. In addition, an autocrine pathway seems to exist in myocardial cells, the ablation of which reduces cellular contraction without change in calcium transient. Finally, differences in the developmental phenotype between apelin and APJ null mice suggest the possibility of undiscovered APJ ligands or ligand-independent effects of APJ.

Pyruvate-fortified cardioplegia evokes myocardial erythropoietin signaling in swine undergoing cardiopulmonary bypass

Mon, 26 Oct 2009 13:43:30 PDT

Pyruvate-fortified cardioplegia protects myocardium and hastens postsurgical recovery of patients undergoing cardiopulmonary bypass (CPB). Pyruvate reportedly suppresses degradation of the -subunit of hypoxia-inducible factor-1 (HIF-1), an activator of the gene encoding the cardioprotective cytokine erythropoietin (EPO). This study tested the hypothesis that pyruvate-enriched cardioplegia evoked EPO expression and mobilized EPO signaling mechanisms in myocardium. Hearts of pigs maintained on CPB were arrested for 60 min with 4:1 blood-crystalloid cardioplegia. The crystalloid component contained 188 mM glucose ± 24 mM pyruvate. After 30-min cardiac reperfusion with cardioplegia-free blood, the pigs were weaned from CPB. Left ventricular myocardium was sampled 4 h after CPB for immunoblot assessment of HIF-1, EPO and its receptor, the signaling kinases Akt and ERK, and endothelial nitric oxide synthase (eNOS), an effector of EPO signaling. Pyruvate-fortified cardioplegia stabilized arterial pressure post-CPB, induced myocardial EPO mRNA expression, and increased HIF-1, EPO, and EPO-R protein contents by 60, 58, and 123%, respectively, vs. control cardioplegia (P < 0.05). Pyruvate cardioplegia also increased ERK phosphorylation by 61 and 118%, respectively, vs. control cardioplegia-treated and non-CPB sham myocardium (P < 0.01), but did not alter Akt phosphorylation. Nitric oxide synthase (NOS) activity and eNOS content fell 32% following control CPB vs. sham, but pyruvate cardioplegia prevented these declines, yielding 49 and 80% greater NOS activity and eNOS content vs. respective control values (P < 0.01). Pyruvate-fortified cardioplegia induced myocardial EPO expression and mobilized the EPO-ERK-eNOS mechanism. By stabilizing HIF-1, pyruvate-fortified cardioplegia may evoke sustained activation of EPO's cardioprotective signaling cascade in myocardium.

Ranolazine, an antianginal agent, markedly reduces ventricular arrhythmias induced by ischemia and ischemia-reperfusion

Mon, 26 Oct 2009 13:43:30 PDT

We tested the effect of the antianginal agent ranolazine on ventricular arrhythmias in an ischemic model using two protocols. In protocol 1, anesthetized rats received either vehicle or ranolazine (10 mg/kg, iv bolus) and were subjected to 5 min of left coronary artery (LCA) occlusion and 5 min of reperfusion with electrocardiogram and blood pressure monitoring. In protocol 2, rats received either vehicle or three doses of ranolazine (iv bolus followed by infusion) and 20 min of LCA occlusion. With protocol 1, ventricular tachycardia (VT) occurred in 9/12 (75%) vehicle-treated rats and 1/11 (9%) ranolazine-treated rats during reperfusion (P = 0.003). Sustained VT occurred in 5/12 (42%) vehicle-treated but 0/11 in ranolazine-treated rats (P = 0.037). The median number of episodes of VT during reperfusion in vehicle and ranolazine groups was 5.5 and 0, respectively (P = 0.0006); median duration of VT was 22.2 and 0 s in vehicle and ranolazine rats, respectively (P = 0.0006). With protocol 2, mortality in the vehicle group was 42 vs. 17% (P = 0.371), 10% (P = 0.162) and 0% (P = 0.0373) with ranolazine at plasma concentrations of 2, 4, and 8 µM, respectively. Ranolazine significantly reduced the incidence of ventricular fibrillation [67% in controls vs. 42% (P = 0.414), 30% (P = 0.198) and 8% (P = 0.0094) in ranolazine at 2, 4, and 8 µM, respectively]. Median number (2.5 vs. 0; P = 0.0431) of sustained VT episodes, incidence of sustained VT (83 vs. 33%, P = 0.0361), and the duration of VT per animal (159 vs. 19 s; P = 0.0410) were also significantly reduced by ranolazine at 8 µM. Ranolazine markedly reduced ischemia-reperfusion induced ventricular arrhythmias. Ranolazine demonstrated promising anti-arrhythmic properties that warrant further investigation.

siRNA-mediated knockdown of h-caldesmon in vascular smooth muscle

Mon, 26 Oct 2009 13:43:30 PDT

Smooth muscle contraction involves phosphorylation of the regulatory myosin light chain. However, this thick-filament system of regulation cannot account for all aspects of a smooth muscle contraction. An alternate site of contractile regulation may be in the thin-filament-associated proteins, in particular caldesmon. Caldesmon has been proposed to be an inhibitory protein that acts either as a brake to stop any increase in resting or basal tone, or as a modulatory protein during contraction. The goal of this study was to use short interfering RNA technology to decrease the levels of the smooth muscle-specific isoform of caldesmon in intact vascular smooth muscle tissue to determine more carefully what role(s) caldesmon has in smooth muscle regulation. Intact strips of vascular tissue depleted of caldesmon produced significant levels of shortening velocity, indicative of cross-bridge cycling, in the unstimulated tissue and exhibited lower levels of contractile force to histamine. Our results also suggest that caldesmon does not play a role in the cooperative activation of unphosphorylated cross bridges by phosphorylated cross bridges. The velocity of shortening of the constitutively active tissue and the high basal values of myosin light chain phosphorylation suggest that h-caldesmon in vivo acts as a brake against contractions due to basally phosphorylated myosin. It is also possible that phosphorylation of h-caldesmon alone in the resting state may be a mechanism to produce increases in force without stimulation and increases in calcium. Disinhibition of h-caldesmon by phosphorylation would then allow force to be developed by activated myosin in the resting state.

A random cycle length approach for assessment of myocardial contraction in isolated rabbit myocardium

Varian, K. D., Xu, Y., Torres, C. A. A., Monasky, M. M., Janssen, P. M. L. — Mon, 26 Oct 2009 13:43:30 PDT

It is well known that the strength of cardiac contraction is dependent on the cycle length, evidenced by the force-frequency relationship (FFR) and the existence of postrest potentiation (PRP). Because the contractile strength of the steady-state FFR and force-interval relationship involve instant intrinsic responses to cycle length as well as slower acting components such as posttranslational modification-based mechanisms, it remains unclear how cycle length intrinsically affects cardiac contraction and relaxation. To dissect the impact of cycle length changes from slower acting signaling components associated with persisting changes in cycle length, we developed a novel technique/protocol to study cycle length-dependent effects on cardiac function; twitch contractions of right ventricular rabbit trabeculae at different cycle lengths were randomized around a steady-state frequency. Patterns of cycle lengths that resulted in changes in force and/or relaxation times can now be identified and analyzed. Using this novel protocol, taking under 10 min to complete, we found that the duration of the cycle length before a twitch contraction ("primary" cycle length) positively correlated with force. In sharp contrast, the cycle length one ("secondary") or two ("tertiary") beats before the analyzed twitch correlated negatively with force. Using this protocol, we can quantify the intrinsic effect of cycle length on contractile strength while avoiding rundown and lengthiness that are often complications of FFR and PRP assessments. The data show that the history of up to three cycle lengths before a contraction influences myocardial contractility and that primary cycle length affects cardiac twitch dynamics in the opposite direction from secondary/tertiary cycle lengths.

Extraction of morphometry and branching angles of porcine coronary arterial tree from CT images

Wischgoll, T., Choy, J. S., Kassab, G. S. — Mon, 26 Oct 2009 13:43:30 PDT

The morphometry (diameters, length, and angles) of coronary arteries is related to their function. A simple, easy, and accurate image-based method to seamlessly extract the morphometry for coronary arteries is of significant value for understanding the structure-function relation. Here, the morphometry of large (≥1 mm in diameter) coronary arteries was extracted from computed tomography (CT) images using a recently validated segmentation algorithm. The coronary arteries of seven pigs were filled with Microfil, and the cast hearts were imaged with CT. The centerlines of the extracted vessels, the vessel radii, and the vessel lengths were identified for over 700 vessel segments. The extraction algorithm was based on a topological analysis of a vector field generated by normal vectors of the extracted vessel wall. The diameters, lengths, and angles of the right coronary artery, left anterior descending coronary artery, and left circumflex artery of all vessels ≥1 mm in diameter were tabulated for the respective orders. It was found that bifurcations at orders 9–11 are planar (~90%). The relations between volume and length and area and length were also examined and found to scale as power laws. Furthermore, the bifurcation angles follow the minimum energy hypothesis but with significant scatter. Some of the applications of the semiautomated extraction of morphometric data in applications to coronary physiology and pathophysiology are highlighted.

An adaptive transfer function for deriving the aortic pressure waveform from a peripheral artery pressure waveform

Swamy, G., Xu, D., Olivier, N. B., Mukkamala, R. — Mon, 26 Oct 2009 13:43:30 PDT

We developed a new technique to mathematically transform a peripheral artery pressure (PAP) waveform distorted by wave reflections into the physiologically more relevant aortic pressure (AP) waveform. First, a transfer function relating PAP to AP is defined in terms of the unknown parameters of a parallel tube model of pressure and flow in the arterial tree. The parameters are then estimated from the measured PAP waveform along with a one-time measurement of the wave propagation delay time between the aorta and peripheral artery measurement site (which may be accomplished noninvasively) by exploiting preknowledge of aortic flow. Finally, the transfer function with its estimated parameters is applied to the measured waveform so as to derive the AP waveform. Thus, in contrast to the conventional generalized transfer function, the transfer function is able to adapt to the intersubject and temporal variability of the arterial tree. To demonstrate the feasibility of this adaptive transfer function technique, we performed experiments in 6 healthy dogs in which PAP and reference AP waveforms were simultaneously recorded during 12 different hemodynamic interventions. The AP waveforms derived by the technique showed agreement with the measured AP waveforms (overall total waveform, systolic pressure, and pulse pressure root mean square errors of 3.7, 4.3, and 3.4 mmHg, respectively) statistically superior to the unprocessed PAP waveforms (corresponding errors of 8.6, 17.1, and 20.3 mmHg) and the AP waveforms derived by two previously proposed transfer functions developed with a subset of the same canine data (corresponding errors of, on average, 5.0, 6.3, and 6.7 mmHg).

Cardiac autonomic neural remodeling and susceptibility to sudden cardiac death: effect of endurance exercise training

Billman, G. E. — Thu, 24 Sep 2009 16:56:17 PDT

Sudden cardiac death resulting from ventricular tachyarrhythmias remains the leading cause of death in industrially developed countries, accounting for between 300,000 and 500,000 deaths each year in the United States. Yet, despite the enormity of this problem, both the identification of factors contributing to ventricular fibrillation as well as the development of safe and effective antiarrhythmic agents remain elusive. Subnormal cardiac parasympathetic regulation coupled with an elevated cardiac sympathetic activation may allow for the formation of malignant ventricular arrhythmias. In particular, myocardial infarction can reduce cardiac parasympathetic regulation and alter β-adrenoceptor subtype expression enhancing β₂-adrenoceptor sensitivity that can lead to intracellular calcium dysregulation and arrhythmias. As such, myocardial infarction can induce a remodeling of cardiac autonomic regulation that may be required to maintain cardiac pump function. If alterations in cardiac autonomic regulation play an important role in the genesis of life-threatening arrhythmias, then one would predict that interventions designed to either augment parasympathetic activity and/or reduce cardiac adrenergic activity would also protect against ventricular fibrillation. Recently, studies using a canine model of sudden death demonstrate that endurance exercise training (treadmill running) enhanced cardiac parasympathetic regulation (increased heart rate variability), restored a more normal β-adrenoceptor balance (i.e., reduced β₂-adrenoceptor sensitivity and expression), and protected against ventricular fibrillation induced by acute myocardial ischemia. Thus exercise training may reverse the autonomic neural remodeling induced by myocardial infarction and thereby enhance the electrical stability of the heart in individuals shown to be at an increased risk for sudden cardiac death.

Neointimal hyperplasia, vein graft remodeling, and long-term patency

Wu, J., Zhang, C. — Thu, 24 Sep 2009 16:56:17 PDT

Enhanced interstitial flow as a contributing factor in neointima formation: (shear) stressing vascular wall cell types other than the endothelium

Rizzo, V. — Thu, 24 Sep 2009 16:56:17 PDT

The failing ventricle: what initiates the complex ventricular arrhythmias?

Boyden, P. A. — Thu, 24 Sep 2009 16:56:17 PDT

Established neointimal hyperplasia in vein grafts expands via TGF-{beta}-mediated progressive fibrosis

Jiang, Z., Tao, M., Omalley, K. A., Wang, D., Ozaki, C. K., Berceli, S. A. — Thu, 24 Sep 2009 16:56:17 PDT

In weeks to months following implantation, neointimal hyperplasia (NIH) in vein grafts (VGs) transitions from a cellularized to a decellularized phenotype. The inhibition of early cellular proliferation failed to improve long-term VG patency. We have previously demonstrated that transforming growth factor-β₁ (TGF-β₁)/connective tissue growth factor (CTGF) pathways mediate a conversion of fibroblasts to myofibroblasts in the early VG (<2 wk). We hypothesize that these similar pathways drive fibrosis observed in the late VG lesion. Within rabbit VGs, real-time RT-PCR, Western blot analysis, ELISA, and immunohistochemistry were used to examine TGF-β/CTGF pathways in late (1–6 mo) NIH. All VGs exhibited a steady NIH growth (P = 0.006) with significant reduction in cellularity (P = 0.01) over time. Substantial TGF-β profibrotic activities, as evidenced by enhanced TGF-β₁ activation, TGF-β receptor types I (activin receptor-like kinase 5)-to-II receptor ratio, SMAD2/3 phosphorylation, and CTGF production, persisted throughout the observation period. An increased matrix synthesis was accompanied by a temporal reduction of matrix metalloproteinase-2 (P = 0.001) and -9 (P < 0.001) activity. VG NIH is characterized by a conversion from a proproliferative to a profibrotic morphology. An enhanced signaling via TGF-β/CTGF coupled with reduced matrix metalloproteinase activities promotes progressive fibrotic NIH expansion. The modulation of late TGF-β/CTGF signaling may offer a novel therapeutic strategy to improve the long-term VG durability.

Role of MCP-1 in tumor necrosis factor-{alpha}-induced endothelial dysfunction in type 2 diabetic mice

Thu, 24 Sep 2009 16:56:17 PDT

Tumor necrosis factor- (TNF-) upregulates the expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in type 2 diabetes. We hypothesized that TNF- and MCP-1 may interact to contribute to the evolution of vascular inflammation and endothelial dysfunction in coronary arterioles in type 2 diabetes. To test this hypothesis, we administered anti-MCP-1 to block MCP-1 signaling in genetically modified mice with type 2 diabetes (Lepr^db) and in heterozygote (m Lepr^db) lean control. Anti-MCP-1 partially restored vasodilation to the endothelium-dependent vasodilator acetylcholine in isolated, cannulated, and pressurized coronary arterioles in Lepr^db mice but did not affect vasodilation in m Lepr^db mice. Anti-MCP-1 attenuated superoxide production and the protein expression of nitrotyrosine, which is an indicator of peroxynitrite production, in isolated coronary arterioles of Lepr^db mice. Immunostaining results showed that the expression of MCP-1 and vascular cellular adhesion molecule-1 is colocalized with endothelial cells and macrophages. Anti-TNF- or anti-MCP-1 markedly reduced macrophage infiltration and the number of MCP-1-positive endothelium in Lepr^db mice. The neutralization of TNF- or anti-MCP-1 reduced the expression of adhesion molecules, suggesting that proinflammatory cytokines interact to amplify the signaling process that leads to vascular dysfunction. These findings demonstrate that the endothelial dysfunction occurring in type 2 diabetes is the result of the effects of the inflammatory cytokine TNF- and TNF--related signaling, including the expression of MCP-1 and adhesion molecules, which further exacerbates vessel inflammation and oxidative stress.

Sodium tanshinone IIA sulfonate increased intestinal hemodynamics without systemic circulatory changes in healthy newborn piglets

Thu, 24 Sep 2009 16:56:17 PDT

In traditional Chinese medicine, tanshinone IIA is a lipid-soluble component of Danshen that has been widely used for various cardiovascular and cerebrovascular disorders, including neonatal asphyxia. Despite promising effects, little is known regarding the hemodynamic effects of tanshinone IIA in newborn subjects. To examine the dose-response effects of sodium tanshinone IIA sulfonate (STS) on systemic and regional hemodynamics and oxygen transport, 12 newborn piglets were anesthetized and acutely instrumented for the placement of femoral arterial and venous, pulmonary arterial catheters to measure mean arterial, central venous, and pulmonary arterial pressures, respectively. The blood flow at the common carotid, renal, pulmonary, and superior mesenteric (SMA) arteries were continuously monitored after treating the piglets with either STS (0.1–30 mg/kg iv) or saline treatment (n = 6/group). To further delineate the underlying mechanisms for vasorelaxant effects of STS, in vitro vascular myography was carried out to compare its effect on rat mesenteric and carotid arteries (n = 4–5/group). STS dose-dependently increased the SMA blood flow and the corresponding oxygen delivery with no significant effect on systemic and pulmonary, carotid and renal hemodynamic parameters. In vitro studies also demonstrated that STS selectively dilated rat mesenteric but not carotid arteries. Vasodilation in mesenteric arteries was inhibited by apamin and TRAM-34 (calcium-activated potassium channel inhibitors) but not by meclofenamate (cyclooxygenase inhibitor) or N-nitro-l-arginine methyl ester hydrochloride (nitric oxide synthase inhibitor). In summary, without significant hemodynamic effects on newborn piglets, intravenous infusion of STS selectively increased mesenteric perfusion in a dose-dependent manner, possibly via an endothelium-derived hyperpolarizing factor vasodilating pathway.

Interstitial flow promotes vascular fibroblast, myofibroblast, and smooth muscle cell motility in 3-D collagen I via upregulation of MMP-1

Shi, Z.-D., Ji, X.-Y., Qazi, H., Tarbell, J. M. — Thu, 24 Sep 2009 16:56:17 PDT

Neointima formation often occurs in regions where the endothelium has been damaged and the transmural interstitial flow is elevated. Vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) contribute to intimal thickening by migrating from the media and adventitia into the site of injury. In this study, for the first time, the direct effects of interstitial flow on SMC and FB/MFB migration were investigated in an in vitro three-dimensional system. Collagen I gels were used to mimic three-dimensional extracellular matrix (ECM) for rat aortic SMCs and FBs/MFBs. Exposure to interstitial flow induced by 1 cmH₂O pressure differential (shear stress, ~0.05 dyn/cm²; flow velocity, ~0.5 µm/s; and Darcy permeability, ~10^–11 cm²) substantially enhanced cell motility. Matrix metalloproteinase (MMP) inhibitor (GM-6001) abolished flow-induced migration augmentation, which suggested that the enhanced motility was MMP dependent. The upregulation of MMP-1 played a critical role for the flow-enhanced motility, which was further confirmed by silencing MMP-1 gene expression. Longer exposures to higher flows suppressed the number of migrated cells, although MMP-1 gene expression remained high. This suppression was a result of both flow-induced tissue inhibitor of metalloproteinase-1 upregulation and increased apoptotic and necrotic cell death. Interstitial flow did not affect MMP-2 gene expression or activity in the collagen I gel for any cell type. Our findings shed light on the mechanism by which vascular SMCs and FBs/MFBs contribute to intimal thickening in regions of vascular injury where interstitial flow is elevated.

Spontaneous calcium release in tissue from the failing canine heart

Hoeker, G. S., Katra, R. P., Wilson, L. D., Plummer, B. N., Laurita, K. R. — Thu, 24 Sep 2009 16:56:17 PDT

Abnormalities in calcium handling have been implicated as a significant source of electrical instability in heart failure (HF). While these abnormalities have been investigated extensively in isolated myocytes, how they manifest at the tissue level and trigger arrhythmias is not clear. We hypothesize that in HF, triggered activity (TA) is due to spontaneous calcium release from the sarcoplasmic reticulum that occurs in an aggregate of myocardial cells (an SRC) and that peak SCR amplitude is what determines whether TA will occur. Calcium and voltage optical mapping was performed in ventricular wedge preparations from canines with and without tachycardia-induced HF. In HF, steady-state calcium transients have reduced amplitude [135 vs. 170 ratiometric units (RU), P < 0.05] and increased duration (252 vs. 229 s, P < 0.05) compared with those of normal. Under control conditions and during β-adrenergic stimulation, TA was more frequent in HF (53% and 93%, respectively) compared with normal (0% and 55%, respectively, P < 0.025). The mechanism of arrhythmias was SCRs, leading to delayed afterdepolarization-mediated triggered beats. Interestingly, the rate of SCR rise was greater for events that triggered a beat (0.41 RU/ms) compared with those that did not (0.18 RU/ms, P < 0.001). In contrast, there was no difference in SCR amplitude between the two groups. In conclusion, TA in HF tissue is associated with abnormal calcium regulation and mediated by the spontaneous release of calcium from the sarcoplasmic reticulum in aggregates of myocardial cells (i.e., an SCR), but importantly, it is the rate of SCR rise rather than amplitude that was associated with TA.

Inhaled carbon monoxide reduces leukocytosis in a murine model of sickle cell disease

Thu, 24 Sep 2009 16:56:17 PDT

Carbon monoxide (CO) has anti-inflammatory properties. We previously reported that acute treatments with inhaled CO inhibit vascular inflammation and hypoxia-induced vasoocclusion in sickle cell disease mouse models. Therefore, we hypothesized that chronic CO inhalation would decrease vascular inflammation and organ pathology in a sickle cell disease mouse model. The treatment of sickle cell disease mice with 25 or 250 parts/million inhaled CO for 1 h/day, 3 days/wk for 8–10 wk significantly decreased the total mean white blood cell, neutrophil, and lymphocyte counts in peripheral blood. Eight weeks of 250 parts/million CO treatments reduced staining for myeloid and lymphoid markers in the bone marrow of sickle mice. Bone marrow from treated sickle mice exhibited a significant decrease in colony-forming unit granulocyte-macrophage during colony-forming cell assays. Anti-inflammatory signaling pathways phospho-Akt and phospho-p38 MAPK were markedly increased in CO-treated sickle livers. Importantly, CO-treated sickle mice had a significant reduction in liver parenchymal necrosis, reflecting the anti-inflammatory benefits of CO. We conclude that inhaled CO may be a beneficial anti-inflammatory therapy for sickle cell disease.

A slowly inactivating sodium current contributes to spontaneous diastolic depolarization of atrial myocytes

Song, Y., Shryock, J. C., Belardinelli, L. — Thu, 24 Sep 2009 16:56:17 PDT

Diastolic depolarization (DD) of atrial myocytes can lead to spontaneous action potentials (APs) and, potentially, atrial tachyarrhythmias. This study examined the hypotheses that 1) a slowly inactivating component of the Na⁺ current (referred to as late I_Na) may contribute to DD and initiate AP firing and that 2) blocking late I_Na will reduce spontaneous and induced firing of APs by atrial myocytes. Guinea pig atrial myocytes without or with DD and spontaneous AP firing were studied using the whole cell patch-clamp technique. In experiments using cells with a stable resting membrane potential (no spontaneous DD or firing), hydrogen peroxide (H₂O₂, 50 µmol/l) caused DD and AP firing. The H₂O₂-induced activity was suppressed by the late I_Na inhibitors tetrodotoxin (TTX, 1 µmol/l) and ranolazine (5 µmol/l). In cells with DD but no spontaneous APs, the late I_Na enhancer anemone toxin II (ATX-II, 10 nmol/l) accelerated DD and induced APs. In cells with DD and spontaneous AP firing, TTX and ranolazine (both, 1 µmol/l) significantly reduced the slope of DD by 81 ± 12% and 75 ± 11% and the frequency of spontaneous firing by 70 ± 15% and 74 ± 9%, respectively. Ramp voltage-clamp simulating DD elicited a slow inward current. TTX at 1, 3, and 10 µmol/l inhibited this current by 41 ± 4%, 73 ± 2%, and 91 ± 1%, respectively, suggesting that a slowly inactivating I_Na underlies the DD. ATX-II and H₂O₂ increased the amplitude of this current, and the effects of ATX-II and H₂O₂ were attenuated by ranolazine or TTX. In conclusion, late I_Na can contribute to the DD of atrial myocytes and the inhibition of this current suppresses atrial DD and spontaneous APs.

Impaired contractile function and calcium handling in hearts of cardiac-specific calcineurin b1-deficient mice

Thu, 24 Sep 2009 16:56:17 PDT

To define the necessity of calcineurin (Cn) signaling for cardiac maturation and function, the postnatal phenotype of mice with cardiac-specific targeted ablation of the Cn B1 regulatory subunit (Ppp3r1) gene (csCnb1^–/– mice) was characterized. csCnb1^–/– mice develop a lethal cardiomyopathy, characterized by impaired postnatal growth of the heart and combined systolic and diastolic relaxation abnormalities, despite a lack of structural derangements. Notably, the csCnb1^–/– hearts did not exhibit diastolic dilatation, despite the severe functional phenotype. Myocytes isolated from the mutant mice exhibited reduced rates of contraction/relaxation and abnormalities in calcium transients, consistent with altered sarcoplasmic reticulum loading. Levels of sarco(endo) plasmic reticulum Ca-ATPase 2a (Atp2a2) and phospholamban were normal, but phospholamban phosphorylation was markedly reduced at Ser¹⁶ and Thr¹⁷. In addition, levels of the Na/Ca exchanger (Slc8a1) were modestly reduced. These results define a novel mouse model of cardiac-specific Cn deficiency and demonstrate novel links between Cn signaling, postnatal growth of the heart, pathological ventricular remodeling, and excitation-contraction coupling.

Pharmacological stimulation of soluble guanylate cyclase modulates hypoxia-inducible factor-1{alpha} in rat heart

Thu, 24 Sep 2009 16:56:17 PDT

Mechanical load and ischemia induce a series of adaptive physiological responses by activating the expression of O₂-regulated genes, such as hypoxia inducible factor-1 (HIF-1). The aim of this study was to explore the interaction between HIF-1 and soluble guanylate cyclase (sGC) and its second messenger cGMP in cultured cardiomyocytes exposed to hypoxia and in pressure-overloaded heart. In cultured cardiomyocytes of neonatal rats, either sGC stimulator BAY 41-2272 or cGMP analog 8-bromo-cGMP decreased the hypoxia (1% O₂/5% CO₂)-induced HIF-1 expression, whereas the inhibition of protein kinase G by KT-5823 reversed the effect of BAY 41-2272 on the expression under hypoxic conditions. In pressure-overloaded heart induced by suprarenal aortic constriction (AC) in 7-wk-old male Wistar rats, the administration of BAY 41-2272 (2 mg·kg^–1·day^–1) for 14 days significantly suppressed the protein expression of HIF-1 (P < 0.05), vascular endothelial growth factor (P < 0.01), and the number of capillary vessels (P < 0.01) induced by pressure overload. This study suggests that the pharmacological sGC-cGMP stimulation modulates the HIF-1 expression in response to hypoxia or mechanical load in the heart.

Role of sarcoplasmic reticulum in mitochondrial permeability transition and cardiomyocyte death during reperfusion

Ruiz-Meana, M., Abellan, A., Miro-Casas, E., Agullo, E., Garcia-Dorado, D. — Thu, 24 Sep 2009 16:56:17 PDT

There is solid evidence that a sudden change in mitochondrial membrane permeability (mitochondrial permeability transition, MPT) plays a critical role in reperfusion-induced myocardial necrosis. We hypothesized that sarcoplasmic reticulum (SR) Ca²⁺ cycling may induce partial MPT in microdomains of close anatomic proximity between mitochondria and SR, resulting in hypercontracture and cell death. MPT (mitochondrial calcein release), cell length, and sarcolemmal rupture (Trypan blue and lactate dehydrogenase release) were measured in adult rat cardiomyocytes submitted to simulated ischemia (NaCN/2-deoxyglucose, pH 6.4) and reperfusion. On simulated reperfusion, 83 ± 2% of myocytes developed hypercontracture. In 22 ± 6% of cases, hypercontracture was associated with sarcolemmal disruption [Trypan blue(+)]. During simulated reperfusion there was a 25% release of cyclosporin A-sensitive mitochondrial calcein (with respect to total mitochondrial calcein content). Simultaneous blockade of SR Ca²⁺ uptake and release with thapsigargin and ryanodine, respectively, significantly reduced mitochondrial calcein release, hypercontracture, and cell death during simulated reperfusion. SR Ca²⁺ blockers delayed mitochondrial Ca²⁺ uptake in digitonin-permeabilized cardiomyocytes but did not have any effect on isolated mitochondria. Pretreatment with colchicine to disrupt microtubule network reduced the degree of fluorescent overlap between SR and mitochondria and abolished the protective effect of SR Ca²⁺ blockers on MPT, hypercontracture, and cell death during reperfusion. We conclude that SR Ca²⁺ cycling during reperfusion facilitates partial mitochondrial permeabilization due to the close anatomic proximity between both organelles, favoring hypercontracture and cell death.

In vivo assessment of blood flow patterns in abdominal aorta of mice with MRI: implications for AAA localization

Thu, 24 Sep 2009 16:56:17 PDT

Abdominal aortic aneurysms (AAA) localize in the infrarenal aorta in humans, while they are found in the suprarenal aorta in mouse models. It has been shown previously that humans experience a reversal of flow during early diastole in the infrarenal aorta during each cardiac cycle. This flow reversal causes oscillatory wall shear stress (OWSS) to be present in the infrarenal aorta of humans. OWSS has been linked to a variety of proatherogenic and proinflammatory factors. The presence of reverse flow in the mouse aorta is unknown. In this study we investigated blood flow in mice, using phase-contrast magnetic resonance (PCMR) imaging. We measured blood flow in the suprarenal and infrarenal abdominal aorta of 18 wild-type C57BL/6J mice and 15 apolipoprotein E (apoE)–/– mice. Although OWSS was not directly evaluated, results indicate that, unlike humans, there is no reversal of flow in the infrarenal aorta of wild-type or apoE–/– mice. Distensibility of the mouse aortic wall in both the suprarenal and infrarenal segments is higher than reported values for the human aorta. We conclude that normal mice do not experience the reverse flow in the infrarenal aorta that is observed in humans.

Angiotensin II maintains cerebral vascular relaxation via EGF receptor transactivation and ERK1/2

McEwen, S. T., Balus, S. F., Durand, M. J., Lombard, J. H. — Thu, 24 Sep 2009 16:56:17 PDT

This study identified, on the integrative level, two components of the ANG II signaling pathway that lay downstream from the ANG II type 1 (AT₁) receptor and are critically involved in maintaining vascular relaxation in cerebral resistance arteries. In these experiments, the relaxation of isolated middle cerebral arteries (MCA) in response to ACh (10^–9-10^–5 M), iloprost (10^–16-10^–11 g/ml), and reduced PO₂ was lost and the ratio of phospho-ERK/ERK1/2 was significantly reduced in aortas of male Sprague-Dawley rats fed a high-salt (HS; 4% NaCl) diet to suppress plasma ANG II levels. In salt-fed rats, relaxation of MCA in response to these vasodilator stimuli was restored by chronic (3 days) intravenous infusion of either ANG II (5 ng·kg^–1·min^–1) or epidermal growth factor (EGF; 2 µg/h). The protective effect of ANG II infusion to restore vascular relaxation was eliminated by coinfusion of either the EGF receptor kinase inhibitor AG-1478 (20 µg/h), the ERK1/2 inhibitor PD-98059 (10 µg/h), or the protein synthesis inhibitor cycloheximide (5 µg/h). In rats fed a low-salt (0.4% NaCl) diet, MCA relaxation in response to ACh, reduced PO₂, and iloprost was eliminated by intravenous infusion of AG-1478, PD-98059, or cycloheximide. In ANG II-infused rats fed HS diet, and in rats fed LS diet, vasodilator responses to reduced PO₂ and iloprost were unaffected by the p38 MAP kinase inhibitor SB-203580 and the phosphatidylinositol 3-kinase inhibitor wortmannin. These findings indicate that maintenance of normal vascular relaxation mechanisms by ANG II in rat MCA requires activation of the EGF receptor kinase and ERK1/2.

T-type calcium channels are regulated by hypoxia/reoxygenation in ventricular myocytes

Pluteanu, F., Cribbs, L. L. — Thu, 24 Sep 2009 16:56:17 PDT

Low-voltage-activated calcium channels are reexpressed in ventricular myocytes in pathological conditions associated with hypoxic episodes, but a direct relation between oxidative stress and T-type channel function and regulation in cardiomyocytes has not been established. We aimed to investigate low-voltage-activated channel regulation under oxidative stress in neonatal rat ventricular myocytes. RT-PCR measurements of voltage-gated Ca²⁺ (Ca_v)3.1 and Ca_v3.2 mRNA levels in oxidative stress were compared with whole cell patch-clamp recordings of T-type calcium current. The results indicate that hypoxia reduces T-type current density at –30 mV (the hallmark of this channel) based on the shift of the voltage dependence of activation to more depolarized values and downregulation of Ca_v3.1 at the mRNA level. Upon reoxygenation, both Ca_v3.1 mRNA levels and the voltage dependence of total T-type current are restored, although differently for activation and inactivation. Using Ni²⁺, we distinguished different effects of hypoxia/reoxygenation on the two current components. Long-term incubation in the presence of 100 µM CoCl₂ reproduced the effects of hypoxia on T-type current activation and inactivation, indicating that the chemically induced oxidative state is sufficient to alter T-type calcium current activity, and that hypoxia-inducible factor-1 is involved in Ca_v3.1 downregulation. Our results demonstrate that Ca_v3.1 and Ca_v3.2 T-type calcium channels are differentially regulated by hypoxia/reoxygenation injury, and, therefore, they may serve different functions in the myocyte in response to hypoxic injury.

Poor trunk flexibility is associated with arterial stiffening

Thu, 24 Sep 2009 16:56:17 PDT

Flexibility is one of the components of physical fitness as well as cardiorespiratory fitness and muscular strength and endurance. Flexibility has long been considered a major component in the preventive treatment of musculotendinous strains. The present study investigated a new aspect of flexibility. Using a cross-sectional study design, we tested the hypothesis that a less flexible body would have arterial stiffening. A total of 526 adults, 20 to 39 yr of age (young), 40 to 59 yr of age (middle-aged), and 60 to 83 yr of age (older), participated in this study. Subjects in each age category were divided into either poor- or high-flexibility groups on the basis of a sit-and-reach test. Arterial stiffness was assessed by brachial-ankle pulse wave velocity (baPWV). Two-way ANOVA indicated a significant interaction between age and flexibility in determining baPWV (P < 0.01). In middle-aged and older subjects, baPWV was higher in poor-flexibility than in high-flexibility groups (middle-aged, 1,260 ± 141 vs. 1,200 ± 124 cm/s, P < 0.01; and older, 1,485 ± 224 vs. 1,384 ± 199 cm/s, P < 0.01). In young subjects, there was no significant difference between the two flexibility groups. A stepwise multiple-regression analysis (n = 316) revealed that among the components of fitness (cardiorespiratory fitness, muscular strength, and flexibility) and age, all components and age were independent correlates of baPWV. These findings suggest that flexibility may be a predictor of arterial stiffening, independent of other components of fitness.

Phasic contractions of rat mesenteric lymphatics increase basal and phasic nitric oxide generation in vivo

Bohlen, H. G., Wang, W., Gashev, A., Gasheva, O., Zawieja, D. — Thu, 24 Sep 2009 16:56:17 PDT

Multiple investigators have shown interdependence of lymphatic contractions on nitric oxide (NO) activity by pharmacological and traumatic suppression of endothelial NO synthase (eNOS). We demonstrated that lymphatic diastolic relaxation is particularly sensitive to NO from the lymphatic endothelium. The predicted mechanism is shear forces produced by the lymph flow during phasic pumping, activating eNOS in the lymphatic endothelium to produce NO. We measured [NO] during phasic contractions using microelectrodes on in situ mesenteric lymphatics in anesthetized rats under basal conditions and with an intravenous saline bolus (0.5 ml/100 g) or infusion (0.5 ml·100 g^–1·h^–1). Under basal conditions, [NO] measured on the tubular portions of the lymphatics was ~200–250 nM, slightly higher than in the adjacent adipocyte microvasculature, whereas [NO] measured on the lymphatic bulb surface was ~400 nM. Immunohistochemistry of eNOS in isolated lympathics indicated a much greater expression in the lymph valves and surrounding bulb area than in the tubular regions. During phasic lymphatic contractions, the valve and tubular [NO] increased with each contraction, and during intravenous saline infusion, [NO] increased in proportion to the contraction frequency and, presumably, lymph flow. The partial blockade of eNOS over ~1 cm length with N-nitro-l-arginine methyl ester lowered the [NO]. These in vivo data document for the first time that both valvular and tubular lymphatic segments increase NO generation during each phasic contraction and that [NO] summated with increased contraction frequency. The combined data predict regional variations in eNOS and [NO] in the tubular and valve areas, plus the summated NO responses dependent on contraction frequency provide for a complex relaxation mechanism involving NO.

Synergistic effects of autologous cell and hepatocyte growth factor gene therapy for neovascularization in a murine model of hindlimb ischemia

Thu, 24 Sep 2009 16:56:17 PDT

Autologous cell implantation and angiogenic gene therapy have been evaluated in critical limb ischemic patients. Here, we compared the features of these strategies individually and in combination. C57BL/6J mice with ischemic hindlimbs were injected with adherent mononuclear cells (aMNCs) from bone marrow or adenovirus encoding the hepatocyte growth factor (HGF) gene (Ad-HGF). Under comparable angiogenic conditions, 10 x 10⁵ aMNCs produced significantly higher amounts of VEGF and FGF-2 and stimulated the number of arterioles in ischemic muscle compared with 1 x 10⁸ plaque-forming units (pfu) of Ad-HGF. Ad-HGF produced 10 times more HGF in ischemic muscle compared with aMNCs. Injection of 0.3 x 10⁵ aMNCs previously transfected with Ad-HGF (aMNC/Ad-HGF) increased blood flow and elevated the numbers of capillaries and arterioles to levels comparable with that seen with 10 x 10⁵ aMNCs or 1 x 10⁸ pfu of Ad-HGF. Hypoxic conditions induced the apoptotic death of aMNCs. However, coincubation with HGF or aMNC/Ad-HGF protected cells against apoptosis. HGF stimulated the migration of aMNCs, and the migration capacity of the aMNC/Ad-HGF group was significantly higher than that in the aMNC/Ad-LacZ group. In conclusion, cell-based HGF gene therapy decreased the number of cells required for neovascularization. This strategy can be an effective angiogenic therapy.

Transfer of nitric oxide by blood from upstream to downstream resistance vessels causes microvascular dilation

Bohlen, H. G., Zhou, X., Unthank, J. L., Miller, S. J., Bills, R. — Thu, 24 Sep 2009 16:56:17 PDT

The discovery that hemoglobin, albumin, and glutathione carry and release nitric oxide (NO) may have consequences for movement of NO by blood within microvessels. We hypothesize that NO in plasma or bound to proteins likely survives to downstream locations. To confirm this hypothesis, there must be a finite NO concentration ([NO]) in arteriolar blood, and upstream resistance vessels must be able to increase the vessel wall [NO] of downstream arterioles. Arteriolar blood NO was measured with NO-sensitive microelectrodes, and vessel wall [NO] was consistently 25–40% higher than blood [NO]. Localized suppression of NO production in large arterioles over 500–1,000 µm with l-nitroarginine reduced the [NO] ~40%, indicating as much as 60% of the wall NO was from blood transfer. Flow in mesenteric arteries was elevated by occlusion of adjacent arteries to induce a flow-mediated increase in arterial NO production. Both arterial wall and downstream arteriolar [NO] increased and the arterioles dilated as the blood [NO] was increased. To study receptor-mediated NO generation, bradykinin was locally applied to upstream large arterioles and NO measured there and in downstream arterioles. At both sites, [NO] increased and both sets of vessels dilated. When isoproterenol was applied to the upstream vessels, they dilated, but neither the [NO] or diameter downstream arterioles increased. These observations indicate that NO can move in blood from upstream to downstream resistance vessels. This mechanism allows larger vessels that generate large [NO] to influence vascular tone in downstream vessels in response to both flow and receptor stimuli.

PPAR-{gamma} agonist rosiglitazone reverses increased cerebral venous hydraulic conductivity during hypertension

Roberts, T. J. M., Chapman, A. C., Cipolla, M. J. — Thu, 24 Sep 2009 16:56:17 PDT

Peroxisome proliferator-activated receptor- (PPAR-) agonists have been shown to protect the cerebral vasculature, including the blood-brain barrier. In the present study, we investigated the effect of the PPAR- agonist rosiglitazone on changes in venous permeability during chronic hypertension induced by nitric oxide synthase inhibition. Female Sprague-Dawley rats were either treated with N^G-nitro-l-arginine methyl ester (l-NAME; 0.5 g/l in drinking water) for 5 wk (HTN; n = 8), l-NAME for 5 wk plus the PPAR- agonist rosiglitazone (20 mg/kg in food) for the last 3 wk (HTN + Rosi; n = 5), l-NAME for 5 wk plus the superoxide dismutase mimetic Tempol (1 mmol/l in drinking water) for the last 3 wk (HTN + Tempol; n = 8), or were untreated controls (n = 9). Fluid filtration (J_v/S) and hydraulic conductivity (L_p) of cerebral veins were compared in vitro between groups after a step increase in pressure from 10 to 25 mmHg to mimic the change in hydrostatic pressure during acute hypertension. Hypertension increased J_v/S by 2.2-fold and L_p by 3.2-fold. Rosiglitazone treatment after 2 wk of hypertension completely reversed the increased J_v/S and L_p that occurred during hypertension, whereas Tempol had no effect. These results demonstrate that rosiglitazone was effective at reversing changes in venous permeability that occurred during chronic hypertension, an effect that does not appear to be related to its antioxidant properties. Our findings suggest that PPAR- may be a key regulator of blood-brain barrier permeability and a potential therapeutic target during hypertension.

Serotonin produces monoamine oxidase-dependent oxidative stress in human heart valves

Pena-Silva, R. A., Miller, J. D., Chu, Y., Heistad, D. D. — Thu, 24 Sep 2009 16:56:17 PDT

Heart valve disease and pulmonary hypertension, in patients with carcinoid tumors and people who used the fenfluramine-phentermine combination for weight control, have been associated with high levels of serotonin in blood. The mechanism by which serotonin induces valvular changes is not well understood. We recently reported that increased oxidative stress is associated with valvular changes in aortic valve stenosis in humans and mice. In this study, we tested the hypothesis that serotonin induces oxidative stress in human heart valves, and examined mechanisms by which serotonin may increase reactive oxygen species. Superoxide (O₂^·–) was measured in heart valves from explanted human hearts that were not used for transplantation. O₂^·– levels (lucigenin-enhanced chemoluminescence) were increased in homogenates of cardiac valves and blood vessels after incubation with serotonin. A nonspecific inhibitor of flavin-oxidases (diphenyliodonium), or inhibitors of monoamine oxidase [MAO (tranylcypromine and clorgyline)], prevented the serotonin-induced increase in O₂^·–. Dopamine, another MAO substrate that is increased in patients with carcinoid syndrome, also increased O₂^·– levels in heart valves, and this effect was attenuated by clorgyline. Apocynin [an inhibitor of NAD(P)H oxidase] did not prevent increases in O₂^·– during serotonin treatment. Addition of serotonin to recombinant human MAO-A generated O₂^·–, and this effect was prevented by an MAO inhibitor. In conclusion, we have identified a novel mechanism whereby MAO-A can contribute to increased oxidative stress in human heart valves and pulmonary artery exposed to serotonin and dopamine.

Left ventricular remodeling with exercise in hypertension

Thu, 24 Sep 2009 16:56:18 PDT

We investigated how exercise training superimposed on chronic hypertension impacted left ventricular remodeling. Cardiomyocyte hypertrophy, apoptosis, and proliferation in hearts from female spontaneously hypertensive rats (SHRs) were examined. Four-month-old SHR animals were placed into a sedentary group (SHR-SED; n = 18) or a treadmill running group (SHR-TRD, 20 m/min, 1 h/day, 5 days/wk, 12 wk; n = 18). Age-matched, sedentary Wistar Kyoto (WKY) rats were controls (n = 18). Heart weight was greater in SHR-TRD vs. both WKY (P < 0.01) and SHR-SED (P < 0.05). Morphometric-derived left ventricular anterior, posterior, and septal wall thickness were increased in SHR-SED relative to WKY and augmented in SHR-TRD. Cardiomyocyte surface area, length, and width were increased in SHR-SED relative to WKY and further increased in SHR-TRD. Calcineurin abundance was increased in SHR-SED vs. WKY (P < 0.001) and attenuated in SHR-TRD relative to SHR-SED (P < 0.05). Protein abundance and mRNA of Akt was not different among groups. The rate of apoptosis was increased in SHR-SED relative to WKY and mitigated in SHR-TRD. The abundance of Ki-67⁺ cells across groups was not statistically different across groups. The abundance of cardiac progenitor cells (c-Kit⁺ cells) was increased in SHR-TRD relative to WKY. These data suggest that exercise training superimposed on hypertension augmented cardiomyocyte hypertrophy, despite attenuating calcineurin abundance. Exercise training also mitigated apoptosis in hypertension and showed a tendency to enhance the abundance of cardiac progenitor cells, resulting in a more favorable cardiomyocyte number in the exercise-trained hypertensive heart.

Myocardial interstitial fluid inhibits proliferation and cardiomyocyte differentiation in pluripotent embryonic stem cells

Van Orman, J. R., Weihrauch, D., Warltier, D. C., Lough, J. — Thu, 24 Sep 2009 16:56:18 PDT

Several recent studies have demonstrated that the transplantation of pluripotent murine embryonic stem cells (mESCs) can improve or restore the function of infarcted myocardium. Although the extent of remuscularization and its contribution to the restoration of function are unclear, these outcomes are likely strongly influenced by factors in the infarcted and/or ischemic environment. As an initial step toward understanding how the ischemic environment of host myocardium affects transplanted pluripotent cells, we have taken a reductionist approach wherein mESCs are cultured in medium containing ischemic myocardial interstitial fluid (iMIF). iMIF is generated in canine myocardium during eight hourly episodes of transient ischemia and collected on a daily basis, over a 24-day collection period. iMIF strongly reduced the numbers of pluripotent mESCs after 11 days in culture. This inhibitory effect, which was most pronounced for iMIF pools from early time points of the 24-day collection period, resulted from an inhibition of cell proliferation. iMIF also inhibited the differentiation of pluripotent mESCs into cardiomyocytes. By contrast, the expression of vascular smooth muscle and endothelial cell markers was relatively unaffected, consistent with previous findings that iMIF promotes angiogenesis. Taken together, these results suggest that whereas the ischemic/infarcted environment is favorable to stem cell-mediated angiogenesis, it is hostile to cardiac myogenesis. These findings also imply that observations of mESC-mediated improvement of cardiac function after transplantation of pluripotent cells do not reflect remuscularization.

{beta}1-Adrenergic receptors stimulate cardiac contractility and CaMKII activation in vivo and enhance cardiac dysfunction following myocardial infarction

Thu, 24 Sep 2009 16:56:18 PDT

The β-adrenergic receptor (βAR) signaling system is one of the most powerful regulators of cardiac function and a key regulator of Ca²⁺ homeostasis. We investigated the role of βAR stimulation in augmenting cardiac function and its role in the activation of Ca²⁺/calmodulin-dependent kinase II (CaMKII) using various βAR knockouts (KO) including β₁ARKO, β₂ARKO, and β₁/β₂AR double-KO (DKO) mice. We employed a murine model of left anterior descending coronary artery ligation to examine the differential contributions of specific βAR subtypes in the activation of CaMKII in vivo in failing myocardium. Cardiac inotropy, chronotropy, and CaMKII activity following short-term isoproterenol stimulation were significantly attenuated in β₁ARKO and DKO compared with either the β₂ARKO or wild-type (WT) mice, indicating that β₁ARs are required for catecholamine-induced increases in contractility and CaMKII activity. Eight weeks after myocardial infarction (MI), β₁ARKO and DKO mice showed a significant attenuation in fractional shortening compared with either the β₂ARKO or WT mice. CaMKII activity after MI was significantly increased only in the β₂ARKO and WT hearts and not in the β₁ARKO and DKO hearts. The border zone of the infarct in the β₂ARKO and WT hearts demonstrated significantly increased apoptosis by TUNEL staining compared with the β₁ARKO and DKO hearts. Taken together, these data show that cardiac function and CaMKII activity are mediated almost exclusively by the β₁AR. Moreover, it appears that β₁AR signaling is detrimental to cardiac function following MI, possibly through activation of CaMKII.

SAP97 regulates Kir2.3 channels by multiple mechanisms

Thu, 24 Sep 2009 16:56:18 PDT

We examined the impact of coexpressing the inwardly rectifying potassium channel, Kir2.3, with the scaffolding protein, synapse-associated protein (SAP) 97, and determined that coexpression of these proteins caused an approximately twofold increase in current density. A combination of techniques was used to determine if the SAP97-induced increase in Kir2.3 whole cell currents resulted from changes in the number of channels in the cell membrane, unitary channel conductance, or channel open probability. In the absence of SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicular compartment with relatively little Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3, leading to prominent colocalization of Kir2.3 and SAP97 and a modest increase in cell surface Kir2.3. The median Kir2.3 single channel conductance in the absence of SAP97 was ~13 pS, whereas coexpression of SAP97 led to a wide distribution of channel events with three distinct peaks centered at 16, 29, and 42 pS. These changes occurred without altering channel open probability, current rectification properties, or pH sensitivity. Thus association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance. However, changes in single channel conductance play the major role in determining whole cell currents in this model system. We further suggest that the SAP97 effect results from SAP97 binding to the Kir2.3 COOH-terminal domain and altering channel conformation.

K+ current changes account for the rate dependence of the action potential in the human atrial myocyte

Maleckar, M. M., Greenstein, J. L., Giles, W. R., Trayanova, N. A. — Thu, 24 Sep 2009 16:56:18 PDT

Ongoing investigation of the electrophysiology and pathophysiology of the human atria requires an accurate representation of the membrane dynamics of the human atrial myocyte. However, existing models of the human atrial myocyte action potential do not accurately reproduce experimental observations with respect to the kinetics of key repolarizing currents or rate dependence of the action potential and fail to properly enforce charge conservation, an essential characteristic in any model of the cardiac membrane. In addition, recent advances in experimental methods have resulted in new data regarding the kinetics of repolarizing currents in the human atria. The goal of this study was to develop a new model of the human atrial action potential, based on the Nygren et al. model of the human atrial myocyte and newly available experimental data, that ensures an accurate representation of repolarization processes and reproduction of action potential rate dependence and enforces charge conservation. Specifically, the transient outward K⁺ current (I_t) and ultrarapid rectifier K⁺ current (I_Kur) were newly formulated. The inwardly recitifying K⁺ current (I_K1) was also reanalyzed and implemented appropriately. Simulations of the human atrial myocyte action potential with this new model demonstrated that early repolarization is dependent on the relative conductances of I_t and I_Kur, whereas densities of both I_Kur and I_K1 underlie later repolarization. In addition, this model reproduces experimental measurements of rate dependence of I_t, I_Kur, and action potential duration. This new model constitutes an improved representation of excitability and repolarization reserve in the human atrial myocyte and, therefore, provides a useful computational tool for future studies involving the human atrium in both health and disease.

Dimethylthiourea normalizes velocity-dependent, but not force-dependent, index of ventricular performance in diabetic rats: role of myosin heavy chain isozyme

Thu, 24 Sep 2009 16:56:18 PDT

Hydroxyl radicals and hydrogen peroxide are involved in the pathogenesis of systolic dysfunction in diabetic rats, but the precise mechanisms and the effect of antioxidant therapy in diabetic subjects have not been elucidated. We aimed to evaluate the effects of dimethylthiourea (DMTU), a potent hydroxyl radical scavenger, on both force-dependent and velocity-dependent indexes of cardiac contractility in streptozotocin (STZ)-induced early and chronic diabetic rats. Seventy-two hours and 8 wk after STZ (55 mg/kg) injection, diabetic rats were randomized to either DMTU (50 mg·kg^–1·day^–1 ip) or vehicle treatment for 6 and 12 wk, respectively. All rats were then subjected to invasive hemodynamic studies. Maximal systolic elastance (E_max) and maximum theoretical flow (Q_max) were assessed by curve-fitting techniques in terms of the elastance-resistance model. Both normalized E_max (E_maxn) and afterload-adjusted Q_max (Q_maxad) were depressed in diabetic rats, concomitant with altered myosin heavy chain (MHC) isoform composition and its upstream regulators, such as myocyte enhancer factor-2 (MEF-2) and heart autonomic nervous system and neural crest derivatives (HAND). In chronic diabetic rats, DMTU markedly attenuated the impairment in Q_maxad and normalized the expression of MEF-2 and eHAND and MHC isoform composition but exerted an insignificant benefit on E_maxn. Regarding preventive treatment, DMTU significantly ameliorated both E_maxn and Q_maxad in early diabetic rats. In conclusion, our study shows that DMTU has disparate effects on Q_maxad and E_maxn in chronic diabetic rats. The advantage of DMTU in chronic diabetic rats might involve normalization of MEF-2 and eHAND, as well as reversal of MHC isoform switch.

Autonomic effects on the spectral analysis of heart rate variability after exercise

Ng, J., Sundaram, S., Kadish, A. H., Goldberger, J. J. — Thu, 24 Sep 2009 16:56:18 PDT

Although frequency-domain analysis of heart rate variability (HRV) has been performed in the setting of exercise and recovery from exercise, the relationship of specific frequency components to sympathetic and parasympathetic inputs has not been validated in this setting. The aim of this study is to evaluate the relationship of frequency components of HRV to sympathetic and parasympathetic modulation in the setting of recovery after exercise using selective autonomic blockade. Normal subjects (n = 27, 17 men, 53 ± 7 yr old) underwent bicycle stress testing on four separate days. On day 1, a baseline study without autonomic blockade was performed. On days 2 through 4, either β-adrenergic, parasympathetic, or double blockade was administered during exercise and completed 3 min before recovery. Continuous ECG was recorded for 5 min starting from the end of exercise. Time- and frequency-domain measures of HRV were computed for each of the five 1-min segments of RR intervals. Parasympathetic blockade significantly decreased all the HRV measures compared with baseline (P < 0.02 for all). Root mean square of successive differences of RR intervals (rMSSD) was increased by β-adrenergic blockade (P < 0.0002). All the HRV measures except rMSSD showed increases with time after the first minute of recovery. The low frequency-to-high frequency ratio did not respond to autonomic blockade or to recovery time, consistent with the expected changes in sympathovagal influence. Root mean square (detrended SD) and rMSSD were highly correlated with the square root of the total power (r = 0.96) and high-frequency power (r = 0.95), respectively. Although there are marked reductions in the frequency-domain measures in recovery versus rest, the fluctuations in the low- and high-frequency bands respond to autonomic blockade in the expected fashion. Time-domain measures of HRV were highly correlated with frequency-domain measures and therefore provide a computationally more efficient assessment of autonomic influences during recovery from exercise that is less susceptible to anomalies of frequency-domain analysis.

Sphingosine 1-phosphate is an important endogenous cardioprotectant released by ischemic pre- and postconditioning

Vessey, D. A., Li, L., Honbo, N., Karliner, J. S. — Thu, 24 Sep 2009 16:56:18 PDT

Exogenous sphingosine 1-phosphate (S1P) is an effective cardioprotectant against ischemic injury. We have investigated the hypothesis that S1P is also an important endogenous cardioprotectant released during both ischemic preconditioning (IPC) and ischemic postconditioning (IPOST). IPC of ex vivo rat hearts was instituted by two cycles of 3 min ischemia-5 min reperfusion prior to 40 min of index ischemia and then 40 min of reperfusion. IPC resulted in 70% recovery of left ventricular developed pressure (LVDP) upon reperfusion and a small infarct size (10%). VPC23019 (VPC), a specific antagonist of S1P_{1 and 3} G protein-coupled receptors (GPCRs), when present during preconditioning blocked protection afforded by two cycles of IPC. VPC also blocked preconditioning of isolated rat cardiac myocytes subjected to hypoxia-reoxygenation injury. Increased release of S1P from myocytes in response to IPC was also demonstrated. These data indicate that S1P is released from myocytes in response to IPC and protects by binding to S1P GPCRs. In the ex vivo heart, if a third cycle of IPC was added to increase release of endogenous mediators, then the need for any individual mediator (e.g., S1P) was diminished and VPC had little effect. The adenosine antagonist 8-(p-sulfophenyl)-theophylline (8-SPT) likewise inhibited protection by two cycles but not three cycles of IPC, but VPC plus 8-SPT inhibited protection by three cycles of IPC. Similar to IPC, IPOST induced by four postindex ischemia cycles of 15 s reperfusion-15 s ischemia resulted in 66% recovery of LVDP and a 7% infarct size. When VPC was present during postconditioning and reperfusion, LVDP only recovered by 26% and the infarct size increased to 27%. Adding an additional cycle of IPOST reduced the inhibitory effect of VPC and 8-SPT individually, but not their combined effect. These studies reveal that S1P is an important mediator of both IPC and IPOST that is released along with adenosine during each cycle of IPC or IPOST.

Impact of ionic current variability on human ventricular cellular electrophysiology

Romero, L., Pueyo, E., Fink, M., Rodriguez, B. — Thu, 24 Sep 2009 16:56:18 PDT

Abnormalities in repolarization and its rate dependence are known to be related to increased proarrhythmic risk. A number of repolarization-related electrophysiological properties are commonly used as preclinical biomarkers of arrhythmic risk. However, the variability and complexity of repolarization mechanisms make the use of cellular biomarkers to predict arrhythmic risk preclinically challenging. Our goal is to investigate the role of ionic current properties and their variability in modulating cellular biomarkers of arrhythmic risk to improve risk stratification and identification in humans. A systematic investigation into the sensitivity of the main preclinical biomarkers of arrhythmic risk to changes in ionic current conductances and kinetics was performed using computer simulations. Four stimulation protocols were applied to the ten Tusscher and Panfilov human ventricular model to quantify the impact of ±15 and ±30% variations in key model parameters on action potential (AP) properties, Ca²⁺ and Na⁺ dynamics, and their rate dependence. Simulations show that, in humans, AP duration is moderately sensitive to changes in all repolarization current conductances and in L-type Ca²⁺ current (I_CaL) and slow component of the delayed rectifier current (I_Ks) inactivation kinetics. AP triangulation, however, is strongly dependent only on inward rectifier K⁺ current (I_K1) and delayed rectifier current (I_Kr) conductances. Furthermore, AP rate dependence (i.e., AP duration rate adaptation and restitution properties) and intracellular Ca²⁺ and Na⁺ levels are highly sensitive to both I_CaL and Na⁺/K⁺ pump current (I_NaK) properties. This study provides quantitative insights into the sensitivity of preclinical biomarkers of arrhythmic risk to variations in ionic current properties in humans. The results show the importance of sensitivity analysis as a powerful method for the in-depth validation of mathematical models in cardiac electrophysiology.

GPD1L links redox state to cardiac excitability by PKC-dependent phosphorylation of the sodium channel SCN5A

Valdivia, C. R., Ueda, K., Ackerman, M. J., Makielski, J. C. — Thu, 24 Sep 2009 16:56:18 PDT

The SCN5A-encoded cardiac sodium channel underlies excitability in the heart, and dysfunction of sodium current (I_Na) can cause fatal ventricular arrhythmia in maladies such as long QT syndrome, Brugada syndrome (BrS), and sudden infant death syndrome (SIDS). The gene GPD1L encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1), but the function for this enzyme is unknown. Mutations in GPD1L have been associated with BrS and SIDS and decrease I_Na through an unknown mechanism. Using a heterologous expression system, we show that GPD1L associated with SCN5A and that the BrS- and SIDS-related mutations in GPD1L caused a loss of enzymatic function resulting in glycerol-3-phosphate PKC-dependent phosphorylation of SCN5A at serine 1503 (S1503) through a GPD1L-dependent pathway. The direct phosphorylation of S1503 markedly decreased I_Na. These results show a function for GPD1L in cell physiology and a mechanism linking mutations in GPD1L to sudden cardiac arrest. Because the enzymatic step catalyzed by GPD1L depends upon nicotinamide adenine dinucleotide, this GPD1L pathway links the metabolic state of the cell to I_Na and excitability and may be important more generally in cardiac ischemia and heart failure.

Heme oxygenase-1 induction modulates hypoxic pulmonary vasoconstriction through upregulation of ecSOD

Thu, 24 Sep 2009 16:56:18 PDT

Endothelium-denuded bovine pulmonary arteries (BPA) contract to hypoxia through a mechanism potentially involving removing a superoxide-derived hydrogen peroxide-mediated relaxation. BPA organ cultured for 24 h with 0.1 mM cobalt chloride (CoCl₂) to increase the expression and activity of heme oxygenase-1 (HO-1) is accompanied by a decrease in 5 µM lucigenin-detectable superoxide and an increase in horseradish peroxidase-luminol detectable peroxide levels. Force development to KCl in BPA was not affected by increases in HO-1, but the hypoxic pulmonary vasoconstriction (HPV) response was decreased. Organ culture with a HO-1 inhibitor (10 µM chromium mesoporphyrin) reversed the effects of HO-1 on HPV and peroxide. Treatment of HO-1-induced BPA with extracellular catalase resulted in reversal of the attenuation of HPV without affecting the force development to KCl. Increasing intracellular peroxide scavenging with 0.1 mM ebselen increased force development to KCl and partially reversed the decrease in HPV seen on induction of HO-1. HO-1 induction increases extracellular (ec) superoxide dismutase (SOD) expression without changing Cu,Zn-SOD and Mn-SOD levels. HO-1-induced BPA rings treated with the copper chelator 10 mM diethyldithiocarbamate to inactivate ecSOD and Cu,Zn-SOD showed increased superoxide and decreased peroxide to levels equal to non-HO-1-induced rings, whereas the addition of SOD to freshly isolated BPA rings attenuated HPV similar to HO-1 induction with CoCl₂. Therefore, HO-1 induction in BPA increases ecSOD expression associated with enhanced generation of peroxide in amounts that may not be adequately removed during hypoxia, leading to an attenuation of HPV.

TNF-{alpha} inhibition attenuates adverse myocardial remodeling in a rat model of volume overload

Thu, 24 Sep 2009 16:56:18 PDT

Tumor necrosis factor (TNF)- is a proinflammatory cytokine that has been implicated in the pathogenesis of heart failure. In contrast, we have recently shown that myocardial levels of TNF- are acutely elevated in the aortocaval (AV) fistula model of heart failure. Based on these observations, we hypothesized that progression of adverse myocardial remodeling secondary to volume overload would be prevented by inhibition of TNF- with etanercept. Furthermore, a principal objective of this study was to elucidate the effect of TNF- inhibition during different phases of the myocardial remodeling process. Eight-week-old male Sprague-Dawley rats were randomly divided into the following three groups: sham-operated controls, untreated AV fistulas, and etanercept-treated AV fistulas. Each group was further subdivided to study three different time points consisting of 3 days, 3 wk, and 8 wk postfistula. Etanercept was administered subcutaneously at 1 mg/kg body wt. Etanercept prevented collagen degradation at 3 days and significantly attenuated the decrease in collagen at 8 wk postfistula. Although TNF- antagonism did not prevent the initial ventricular dilatation at 3 wk postfistula, etanercept was effective at significantly attenuating the subsequent ventricular hypertrophy, dilatation, and increased compliance at 8 wk postfistula. These positive adaptations achieved with etanercept administration translated into significant functional improvements. At a cellular level, etanercept also markedly attenuated increases in cardiomyocyte length, width, and area at 8 wk postfistula. These observations demonstrate that TNF- has a pivotal role in adverse myocardial remodeling and that treatment with etanercept can attenuate the progression to heart failure.

Sex-dependent differences in Rho activation contribute to contractile dysfunction in type 2 diabetic mice

Nuno, D. W., Harrod, J. S., Lamping, K. G. — Thu, 24 Sep 2009 16:56:18 PDT

The objective of this study was to determine if mechanisms involved in vascular dysfunction in type 2 diabetes differ with sex. Vascular reactivity, expression, and activation of rhoA and rho kinase were measured in aorta from male and female nondiabetic C57BLKS/J and diabetic BKS.Cg-m^+/+ Lepr^db/J (db/db) mice, a model of type 2 diabetes. Relaxation to acetylcholine and nitroprusside was similar in aorta from nondiabetic male and female mice. Relaxation to acetylcholine was reduced ~50% in both male and female diabetic mice. Although inhibition of rho kinase with H-1152 increased relaxation to acetylcholine and nitroprusside in nondiabetic males, it had no effect on the response in either nondiabetic or diabetic females or diabetic males. Contraction to serotonin was increased similarly in male and female diabetic mice compared with nondiabetic mice and was reduced following inhibition of rho kinase with either fasudil or H-1152. Activation of rhoA and its downstream effector, rho kinase, was greater in aorta from diabetic males compared with nondiabetic males. In contrast, there were no differences in vascular activation of rhoA or rho kinase in diabetic females. The increased activity of rhoA and rho kinase in diabetic mice was not due to a change in protein expression of rhoA or rho kinase (ROCK1 and ROCK2) in vessels from either males or females. Although contractile dysfunction in vessels occurs in both male and female diabetic mice, the dysfunction in diabetic males is dependent upon activation of rhoA and rho kinase. Alternative mechanisms affecting rho kinase activation may be involved in females.

Nitric oxide in rostral ventrolateral medulla regulates cardiac-sympathetic reflexes: role of synthase isoforms

Guo, Z.-L., Tjen-A-Looi, S. C., Fu, L.-W., Longhurst, J. C. — Thu, 24 Sep 2009 16:56:18 PDT

Our previous studies have shown that nitric oxide (NO) synthase (NOS)-containing neurons in the rostral ventrolateral medulla (rVLM) are activated during cardiac sympathoexcitatory reflexes (Refs. 12 and 13). However, the precise function of NO in the rVLM in regulation of these reflexes has not been defined. Three isoforms of NOS, including neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), are located in the rVLM. We explored the role of NO, derived from different NOS isoforms in the rVLM, in processing cardiac-sympathetic reflexes using whole animal reflex and electrophysiological approaches. We found that, in anesthetized cats, increased mean arterial blood pressure and renal sympathetic nerve activity elicited by epicardial application of bradykinin (BK; 1–10 µg/ml, 50 µl) were significantly attenuated following unilateral rVLM microinjection of the nonselective NOS inhibitor, N-nitro-l-arginine methyl ester (50 nmol/50 nl), or a specific nNOS inhibitor, 7-nitroindazole (7-NI; 5–10 pmol/50 nl; both P < 0.05). In contrast, the responses of mean arterial blood pressure and renal sympathetic nerve activity to cardiac BK stimulation were unchanged by unilateral rVLM microinjection of N-nitro-d-arginine methyl ester (inactive isomer of N-nitro-l-arginine methyl ester, 50 nmol/50 nl), 3–6% methanol (7-NI vehicle), N⁶-(1-iminoethyl)-l-lysine (250 pmol/50 nl; iNOS inhibitor), or N⁵-(1-iminoethyl)-l-ornithine (250 nmol/50 nl; eNOS inhibitor). Furthermore, in separate cats, we noted that iontophoresis of 7-NI (0.1 mM) reduced the increased discharge of cardiovascular sympathoexcitatory rVLM neurons in response to cardiac stimulation with BK (P < 0.05). These neurons were characterized by their responses to inputs from baroreceptors, and their cardiac rhythmicity was determined through frequency and time domain analyses, correlating their discharge to arterial blood pressure and cardiac sympathetic efferent nerve activity. These data suggest that NO, specifically nNOS, mediates sympathetic cardiac-cardiovascular responses through its action in the rVLM.

Melatonin protects against heart ischemia-reperfusion injury by inhibiting mitochondrial permeability transition pore opening

Thu, 24 Sep 2009 16:56:18 PDT

Melatonin, a well-known antioxidant, has been shown to protect against ischemia-reperfusion myocardial damage. Mitochondrial permeability transition pore (MPTP) opening is an important event in cardiomyocyte cell death occurring during ischemia-reperfusion and therefore a possible target for cardioprotection. In the present study, we tested the hypothesis that melatonin could protect heart against ischemia-reperfusion injury by inhibiting MPTP opening. Isolated perfused rat hearts were subjected to global ischemia and reperfusion in the presence or absence of melatonin in a Langerdoff apparatus. Melatonin treatment significantly improves the functional recovery of Langerdoff hearts on reperfusion, reduces the infarct size, and decreases necrotic damage as shown by the reduced release of lactate dehydrogenase. Mitochondria isolated from melatonin-treated hearts are less sensitive than mitochondria from reperfused hearts to MPTP opening as demonstrated by their higher resistance to Ca²⁺. Similar results were obtained following treatment of ischemic-reperfused rat heart with cyclosporine A, a known inhibitor of MPTP opening. In addition, melatonin prevents mitochondrial NAD⁺ release and mitochondrial cytochrome c release and, as previously shown, cardiolipin oxidation associated with ischemia-reperfusion. Together, these results demonstrate that melatonin protects heart from reperfusion injury by inhibiting MPTP opening, probably via prevention of cardiolipin peroxidation.

Nitrite enhances RBC hypoxic ATP synthesis and the release of ATP into the vasculature: a new mechanism for nitrite-induced vasodilation

Cao, Z., Bell, J. B., Mohanty, J. G., Nagababu, E., Rifkind, J. M. — Thu, 24 Sep 2009 16:56:18 PDT

A role for nitric oxide (NO) produced during the reduction of nitrite by deoxygenated red blood cells (RBCs) in regulating vascular dilation has been proposed. It has not, however, been satisfactorily explained how this NO is released from the RBC without first reacting with the large pools of oxyhemoglobin and deoxyhemoglobin in the cell. In this study, we have delineated a mechanism for nitrite-induced RBC vasodilation that does not require that NO be released from the cell. Instead, we show that nitrite enhances the ATP release from RBCs, which is known to produce vasodilation by several different methods including the interaction with purinergic receptors on the endothelium that stimulate the synthesis of NO by endothelial NO synthase. This mechanism was established in vivo by measuring the decrease in blood pressure when injecting nitrite-reacted RBCs into rats. The observed decrease in blood pressure was not observed if endothelial NO synthase was inhibited by N-nitro-l-arginine methyl ester (l-NAME) or when any released ATP was degraded by apyrase. The nitrite-enhanced ATP release was shown to involve an increased binding of nitrite-modified hemoglobin to the RBC membrane that displaces glycolytic enzymes from the membrane, resulting in the formation of a pool of ATP that is released from the RBC. These results thus provide a new mechanism to explain nitrite-induced vasodilation.

Postinfarction gene therapy with adenoviral vector expressing decorin mitigates cardiac remodeling and dysfunction

Thu, 24 Sep 2009 16:56:18 PDT

The small leucine-rich proteoglycan decorin is a natural inhibitor of transforming growth factor-β (TGF-β) and exerts antifibrotic effects in heart and to stimulate skeletal muscle regeneration. We investigated decorin's chronic effects on postinfarction cardiac remodeling and dysfunction. Myocardial infarction (MI) was induced in mice by left coronary artery ligation. An adenoviral vector encoding human decorin (Ad. CAG-decorin) was then injected into the hindlimbs on day 3 post-MI (control, Ad.CAG-LacZ). Four weeks post-MI, the decorin-treated mice showed significant mitigation of the left ventricular dilatation and dysfunction seen in control mice. Although infarct size did not differ between the two groups, the infarcted wall thickness was greater and the segmental length of the infarct was smaller in decorin-treated mice. In addition, cellular components, including myofibroblasts and blood vessels, were more abundant within the infarcted area in decorin-treated mice, and fibrosis was significantly reduced in both the infarcted and noninfarcted areas of the left ventricular wall. Ten days post-MI, there was greater cell proliferation and less apoptosis among granulation tissue cells in the infarcted areas of decorin-treated mice. The treatment, however, did not affect proliferation and apoptosis of salvaged cardiomyocytes. Although decorin gene therapy did not affect TGF-β1 expression in the infarcted heart, it inhibited Smad2/3 activation (downstream mediators of TGF-β signaling). In summary, postinfarction decorin gene therapy mitigated cardiac remodeling and dysfunction by altering infarct tissue noncardiomyocyte dynamics and preventing cardiac fibrosis, accompanying inhibition of Smad2/3 activation.

Hypophosphatemia-mediated hypotension in transgenic mice overexpressing human FGF-23

Liu, P., Bai, X., Wang, H., Karaplis, A., Goltzman, D., Miao, D. — Thu, 24 Sep 2009 16:56:18 PDT

Fibroblast growth factor-23 (FGF-23) is a potent circulating phosphaturic factor associated with renal phosphate wasting. The effects of FGF-23 on skeletal and phosphate homeostasis have been investigated widely; however, the effect of FGF-23 on the cardiovascular system (CVS) is unknown. To assess whether FGF-23 influences the function and structure of the CVS and whether the effect of FGF-23 on the CVS is mediated by FGF receptors directly or indirectly by hypophosphatemia, FGF-23 transgenic mice and their wild-type littermates were fed a normal diet or a high-phosphate diet comprising a normal diet plus 1.25% phosphate in drinking water from weaning for 5 wk, and the phenotypes of the CVS were compared between FGF-23 transgenic mice and their wild-type littermates on the same diet. At the end of this time period, transgenic animals on the normal diet developed hypotension. The left ventricle was appropriately hypertrophic, and plasma catecholamine and renin-angiotensin system components were upregulated, indicating compensatory mechanisms in response to the hypotension. Transgenic mice also exhibited an impaired vascular reactivity and a downregulation of vasoconstrictor receptor gene expression, possibly as pathogenetic factors contributing to the hypotension. The high-phosphate diet improved the hypophosphatemia, resulting in a rescue of the cardiovascular phenotype. This study demonstrates that FGF-23 overexpression can result in abnormalities in the CVS and that the effect of FGF-23 overexpression on the CVS is mediated by the secondary severe hypophosphatemia.

Experimental and theoretical ventricular electrograms and their relation to electrophysiological gradients in the adult rat heart

Weber dos Santos, R., Nygren, A., Otaviano Campos, F., Koch, H., Giles, W. R. — Thu, 24 Sep 2009 16:56:18 PDT

The electrical activity of adult mouse and rat hearts has been analyzed extensively, often as a prerequisite for genetic engineering studies or for the development of rodent models of human diseases. Some aspects of the initiation and conduction of the cardiac action potential in rodents closely resemble those in large mammals. However, rodents have a much higher heart rate and their ventricular action potential is triangular and very short. As a consequence, an interpretation of the electrocardiogram in the mouse and rat remains difficult and controversial. In this study, optical mapping techniques have been applied to an in vitro left ventricular adult rat preparation to obtain patterns of conduction and action potential duration measurements from the epicardial surface. This information has been combined with previously published mathematical models of the rat ventricular myocyte to develop a bidomain model for action potential propagation and electrogram formation in the rat left ventricle. Important insights into the basis for the repolarization waveform in the ventricular electrogram of the adult rat have been obtained. Notably, our model demonstrated that the biphasic shape of the rat ventricular repolarization wave can be explained in terms of the transmural and apex-to-base gradients in action potential duration that exist in the rat left ventricle.

Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis

Thu, 24 Sep 2009 16:56:18 PDT

Atherosclerosis is closely associated with disturbed flow characterized by low and oscillatory shear stress, but studies directly linking disturbed flow to atherogenesis is lacking. The major reason for this has been a lack of an animal model in which disturbed flow can be acutely induced and cause atherosclerosis. Here, we characterize partial carotid ligation as a model of disturbed flow with characteristics of low and oscillatory wall shear stress. We also describe a method of isolating intimal RNA in sufficient quantity from mouse carotid arteries. Using this model and method, we found that partial ligation causes upregulation of proatherogenic genes, downregulation of antiatherogenic genes, endothelial dysfunction, and rapid atherosclerosis in 2 wk in a p47^phox-dependent manner and advanced lesions by 4 wk. We found that partial ligation results in endothelial dysfunction, rapid atherosclerosis, and advanced lesion development in a physiologically relevant model of disturbed flow. It also allows for easy and rapid intimal RNA isolation. This novel model and method could be used for genome-wide studies to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases.

Corrigendum

Thu, 24 Sep 2009 16:56:18 PDT