AJP: Heart and Circulatory Physiology current issue

Limits of isolation and culture: intact vascular endothelium and BKCa

Sandow, S. L., Grayson, T. H. — 2009-06-29

The potential physiological role of plasmalemmal large-conductance calcium-activated potassium channels (BK_Ca) in vascular endothelial cells is controversial. Studies of freshly isolated and cultured vascular endothelial cells provide disparate results, both supporting and refuting a role for BK_Ca in endothelial function. Most studies using freshly isolated, intact, healthy arteries provide little support for a physiological role for BK_Ca in the endothelium, although recent work suggests that this may not be the case in diseased vessels. In isolated and cultured vascular endothelial cells, the autocrine action of growth factors, hormones, and vasoactive substances results in phenotypic drift. Such an induced heterogeneity is likely a primary factor accounting for the apparent differences, and often enhanced BK_Ca expression and function, in isolated and cultured vascular endothelial cells. In a similar manner, heterogeneity in endothelial BK_Ca expression and function in intact arteries may be representative of normal and disease states, BK_Ca being absent in normal intact artery endothelium and upregulated in disease where dysfunction induces signals that alter channel expression and function. Indeed, in some intact vessels, there is evidence for the presence of BK_Ca, such as mRNA and/or specific BK subunits, an observation that is consistent with the potential for rapid upregulation, as may occur in disease. This perspective proposes that the disparity in the results obtained for BK_Ca expression and function from freshly isolated and cultured vascular endothelial cells is largely due to variability in experimental conditions and, furthermore, that the expression of BK_Ca in intact artery endothelium is primarily associated with disease. Although answers to physiologically relevant questions may only be available in atypical physiological conditions, such as those of isolation and culture, the limitations of these methods require open and objective recognition.

A glass of red wine to improve mitochondrial biogenesis? Novel mechanisms of resveratrol

Szabo, G. — 2009-06-29

Extracellular SOD and aged blood vessels

Fukai, T. — 2009-06-29

Resveratrol induces mitochondrial biogenesis in endothelial cells

2009-06-29

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Myocardial TLR4 is a determinant of neutrophil infiltration after global myocardial ischemia: mediating KC and MCP-1 expression induced by extracellular HSC70

Ao, L., Zou, N., Cleveland, J. C., Fullerton, D. A., Meng, X. — 2009-06-29

Cardiac surgery with global myocardial ischemia-reperfusion (I/R) induces a myocardial inflammatory response that impairs cardiac recovery. Chemokines contribute to the overall myocardial inflammatory response through inducing leukocyte infiltration. Although Toll-like receptor 4 (TLR4) has an important role in postischemic myocardial injury, the relative roles of myocardial tissue and leukocyte TLR4 in leukocyte infiltration, as well as the role of TLR4 in myocardial chemokine expression, are unclear. Our recent study, in an isolated mouse heart model of global I/R, found that the 70-kDa heat shock cognate protein (HSC70) is released from cardiac cells and mediates the expression of cardiodepressant cytokines via a TLR4-dependent mechanism. In the present study, we tested the hypotheses that myocardial tissue TLR4 has a major role in mediating neutrophil infiltration and that myocardial TLR4 and extracellular HSC70 contribute to the mechanisms underlying cardiac chemokine response to global I/R. We subjected hearts isolated from TLR4-defective and TLR4-competent mice to global I/R and examined myocardial neutrophil infiltration and expression of keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein-1 (MCP-1). TLR4-defective hearts exhibited reduced neutrophil infiltration regardless of the phenotypes of neutrophils perfused during reperfusion and expressed lower levels of KC and MCP-1. HSC70-specific antibody reduced myocardial expression of KC and MCP-1 after I/R. Furthermore, perfusion of HSC70 increased KC and MCP-1 expression in TLR4-competent hearts but not in TLR4-defective hearts, and HSC70 also induced the chemokine response in macrophages in a TLR4-dependent fashion. A recombinant HSC70 fragment lacking the substrate-binding domain was insufficient to induce chemokine expression in hearts and cells. This study demonstrates that myocardial tissue TLR4, rather than neutrophil TLR4, is the determinant of myocardial neutrophil infiltration after global I/R. TLR4 mediates myocardial chemokine expression, and the mechanisms involve extracellular HSC70. These results imply the HSC70-TLR4 interaction as a novel mechanism underlying the myocardial chemokine response to global I/R.

Relaxin alters cardiac myofilament function through a PKC-dependent pathway

Shaw, E. E., Wood, P., Kulpa, J., Yang, F. H., Summerlee, A. J., Pyle, W. G. — 2009-06-29

The pregnancy hormone relaxin (RLX) is a powerful cardiostimulatory peptide. Despite its well-characterized effects on the heart, the intracellular mechanisms responsible for RLX's positive inotropic effects are unknown. Cardiac myofilaments are the central contractile elements of the heart, and changes in the phosphorylation status of myofilament proteins are known to mediate changes in function. The first objective of this study was to determine whether RLX stimulates myofilament activation and alters the phosphorylation of one or more myofilament proteins. RLX works through a variety of intracellular signaling cascades in different tissue types. Protein kinases A (PKA) and C (PKC) are two common molecules implicated in RLX signaling and are known to affect myofilament function. Thus the second objective of this study was to determine whether RLX mediates its myocardial effects through PKA or PKC activation. Murine myocardium was treated with recombinant H2-RLX, and cardiac myofilaments were isolated. RLX increased cardiac myofilament force development at physiological levels of intracellular Ca²⁺ without altering myofilament ATP consumption. Myosin binding protein C, troponin T, and troponin I phosphorylation levels were increased with RLX treatment. Immunoblot analysis revealed an increase in myofilament-associated PKC-, decreases in PKC- and -β_II, but no effect on PKC-. Inhibition of PKC with chelerythrine chloride or PKC- with rottlerin prevented the RLX-dependent changes in myofilament function and protein phosphorylation. PKA antagonism with H-89 had no effect on the myofilament effects of RLX. This study is the first to show that RLX-dependent changes in myofilament-associated PKC alters myofilament activation in a manner consistent with its cardiostimulatory effects.

Overexpression of CYP2J2 provides protection against doxorubicin-induced cardiotoxicity

2009-06-29

Human cytochrome P-450 (CYP)2J2 is abundant in heart and active in biosynthesis of epoxyeicosatrienoic acids (EETs). Recently, we demonstrated that these eicosanoid products protect myocardium from ischemia-reperfusion injury. The present study utilized transgenic (Tr) mice with cardiomyocyte-specific overexpression of human CYP2J2 to investigate protection toward toxicity resulting from acute (0, 5, or 15 mg/kg daily for 3 days, followed by 24-h recovery) or chronic (0, 1.5, or 3.0 mg/kg biweekly for 5 wk, followed by 2-wk recovery) doxorubicin (Dox) administration. Acute treatment resulted in marked elevations of serum lactate dehydrogenase and creatine kinase levels that were significantly greater in wild-type (WT) than CYP2J2 Tr mice. Acute treatment also resulted in less activation of stress response enzymes in CYP2J2 Tr mice (catalase 750% vs. 300% of baseline, caspase-3 235% vs. 165% of baseline in WT vs. CYP2J2 Tr mice). Moreover, CYP2J2 Tr hearts exhibited less Dox-induced cardiomyocytes apoptosis (measured by TUNEL) compared with WT hearts. After chronic treatment, comparable decreases in body weight were observed in WT and CYP2J2 Tr mice. However, cardiac function, assessed by measurement of fractional shortening with M-mode transthoracic echocardiography, was significantly higher in CYP2J2 Tr than WT hearts after chronic Dox treatment (WT 37 ± 2%, CYP2J2 Tr 47 ± 1%). WT mice also had larger increases in β-myosin heavy chain and cardiac ankryin repeat protein compared with CYP2J2 Tr mice. CYP2J2 Tr hearts had a significantly higher rate of Dox metabolism than WT hearts (2.2 ± 0.25 vs. 1.6 ± 0.50 ng·min^–1·100 µg protein^–1). In vitro data from H9c2 cells demonstrated that EETs attenuated Dox-induced mitochondrial damage. Together, these data suggest that cardiac-specific overexpression of CYP2J2 limited Dox-induced toxicity.

Evidence for role of epoxyeicosatrienoic acids in mediating ischemic preconditioning and postconditioning in dog

2009-06-29

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce marked reductions in infarct size (IS) in canine myocardium either given before an ischemic insult or at reperfusion similar to that produced in ischemic preconditioning (IPC) and postconditioning (POC) protocols. However, no studies have addressed the possibility that EETs serve a beneficial role in IPC or POC. We tested the hypothesis that EETs may play a role in these two phenomena by preconditioning dog hearts with one 5-min period of total coronary occlusion followed by 10 min of reperfusion before 60 min of occlusion and 3 h of reperfusion or by postconditioning with three 30-s periods of reperfusion interspersed with three 30-s periods of occlusion. To test for a role of EETs in IPC and POC, the selective EET antagonists 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) or its derivative, 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxy-propoxy)-ethoxy]-ethyl ester (14,15-EEZE-PEG), were administered 10 min before IPC, 5 min after IPC, or 5 min before POC. In a separate series, the selective EET synthesis inhibitor N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) was administered 10 min before IPC. Infarct size was determined by tetrazolium staining and coronary collateral blood flow at 30 min of occlusion and reperfusion flow at 3 h by radioactive microspheres. Both IPC and POC produced nearly equivalent reductions in IS expressed as a percentage of the area at risk (AAR) [Control 21.2 ± 1.2%, IPC 8.3 ± 2.2%, POC 10.1 ± 1.8% (P < 0.001)]. 14,15-EEZE, 14,15-EEZE-PEG, and MS-PPOH markedly attenuated the cardioprotective effects of IPC and POC (14,15-EEZE and 14,15-EEZE-PEG) at doses that had no effect on IS/AAR when given alone. These results suggest a unique role for endogenous EETs in both IPC and POC.

Slow head-up tilt causes lower activation of muscle sympathetic nerve activity: loading speed dependence of orthostatic sympathetic activation in humans

Kamiya, A., Kawada, T., Shimizu, S., Iwase, S., Sugimachi, M., Mano, T. — 2009-06-29

Many earlier human studies have reported that increasing the tilt angle of head-up tilt (HUT) results in greater muscle sympathetic nerve activity (MSNA) response, indicating the amplitude dependence of sympathetic activation in response to orthostatic stress. However, little is known about whether and how the inclining speed of HUT influences the MSNA response to HUT, independent of the magnitude of HUT. Twelve healthy subjects participated in passive 30° HUT tests at inclining speeds of 1° (control), 0.1° (slow), and 0.0167° (very slow) per second. We recorded MSNA (tibial nerve) by microneurography and assessed nonstationary time-dependent changes of R-R interval variability using a complex demodulation technique. MSNA averaged over every 10° tilt angle increased during inclination from 0° to 30°, with smaller increases in the slow and very slow tests than in the control test. Although a 3-min MSNA overshoot after reaching 30° HUT was observed in the control test, no overshoot was detected in the slow and very slow tests. In contrast with MSNA, increases in heart rate during the inclination and after reaching 30° were similar in these tests, probably because when compared with the control test, greater increases in plasma epinephrine counteracted smaller autonomic responses in the very slow test. These results indicate that slower HUT results in lower activation of MSNA, suggesting that HUT-induced sympathetic activation depends partially on the speed of inclination during HUT in humans.

Abnormal energetics and ATP depletion in pressure-overload mouse hearts: in vivo high-energy phosphate concentration measures by noninvasive magnetic resonance

Gupta, A., Chacko, V. P., Weiss, R. G. — 2009-06-29

³¹P magnetic resonance spectroscopy (MRS) offers a unique means to noninvasively quantify the major cardiac high-energy phosphates, creatine phosphate (PCr) and adenosine 5'-triphosphate (ATP), that are critical for normal myocardial contractile function and viability. Spatially localized ³¹P MRS has been used to quantify the in vivo PCr-to-ATP ratio (PCr/ATP) of murine hearts, including those with pressure-overload hypertrophy induced by thoracic aortic constriction (TAC). To date, there has been no approach for measuring the absolute tissue concentrations of PCr and ATP in the in vivo mouse heart that promise a better understanding of high-energy metabolism. A method to quantify in vivo murine myocardial concentrations of PCr and ATP using an external reference is described, validated, and applied to normal and TAC hearts. This new method does not prolong the scan times in mice beyond those previously required to measure PCr/ATP. The new method renders an [ATP] of 5.0 ± 0.9 (mean ± SD) and [PCr] of 10.4 ± 1.4 µmol/g wet wt in normal mouse hearts (n = 7) and significantly lower values in TAC hearts (n = 10) of 4.0 ± 0.8 and 6.7 ± 2.0 µmol/g wet wt for [ATP] (P < 0.04) and [PCr] (P < 0.001), respectively. The in vivo magnetic resonance [ATP] results are in good agreement with those obtained using an in vitro enzyme luminescent assay of perchloric acid extracts of the same hearts. In conclusion, a validated ³¹P MRS method for quantifying [ATP] and [PCr] in the in vivo mouse heart using spatial localization and an external reference is described and used to demonstrate significant reductions in not only PCr/ATP but [ATP] in hypertrophied TAC hearts.

Reduced EGFR causes abnormal valvular differentiation leading to calcific aortic stenosis and left ventricular hypertrophy in C57BL/6J but not 129S1/SvImJ mice

2009-06-29

Epidermal growth factor receptor (EGFR) signaling contributes to aortic valve development in mice. Because developmental phenotypes in Egfr-null mice are dependent on genetic background, the hypomorphic Egfr^wa2 allele was made congenic on C57BL/6J (B6) and 129S1/SvImJ (129) backgrounds and used to identify the underlying cellular cause of EGFR-related aortic valve abnormalities. Egfr^wa2/wa2 mice on both genetic backgrounds develop aortic valve hyperplasia. Many B6-Egfr^wa2/wa2 mice die before weaning, and those surviving to 3 mo of age or older develop severe left ventricular hypertrophy and heart failure. The cardiac phenotype was accompanied by significantly thicker aortic cusps and larger transvalvular gradients in B6-Egfr^wa2/wa2 mice compared with heterozygous controls and age-matched Egfr^wa2 homozygous mice on either 129 or B6129F1 backgrounds. Histological analysis revealed cellular changes in B6-Egfr^wa2/wa2 aortic valves underlying elevated pressure gradients and progression to heart failure, including increased cellular proliferation, ectopic cartilage formation, extensive calcification, and inflammatory infiltrate, mimicking changes seen in human calcific aortic stenosis. Despite having congenitally enlarged valves, 129 and B6129F1-Egfr^wa2/wa2 mice have normal lifespans, absence of left ventricular hypertrophy, and normal systolic function. These results show the requirement of EGFR activity for normal valvulogenesis and demonstrate that dominantly acting genetic modifiers curtail pathological changes in congenitally deformed valves. These studies provide a novel model of aortic sclerosis and stenosis and suggest that long-term inhibition of EGFR signaling for cancer therapy may have unexpected consequences on aortic valves in susceptible individuals.

IL-18 induction of osteopontin mediates cardiac fibrosis and diastolic dysfunction in mice

2009-06-29

Osteopontin (OPN), a key component of the extracellular matrix, is associated with the fibrotic process during tissue remodeling. OPN and the cytokine interleukin (IL)-18 have been shown to be overexpressed in an array of human cardiac pathologies. In the present study, we determined the role of IL-18 in the regulation of cardiac OPN expression and the subsequent interstitial fibrosis and diastolic dysfunction. We demonstrated parallel increases in IL-18, OPN expression, and interstitial fibrosis in murine models of left ventricular pressure and volume overload. Exogenous recombinant (r)IL-18 administered for 2 wk increased cardiac OPN expression, interstitial fibrosis, and diastolic dysfunction. Stimulation of the T helper (Th)1 lymphocyte phenotype with a selective toll-like receptor (TLR)9 agonist induced cardiac IL-18 and OPN expression, which was associated with increased cardiac fibrillar collagen concentrations and interstitial fibrosis resulting in diastolic dysfunction. rIL-18 induced OPN expression and protein levels in primary of cardiac fibroblast cultures. Conditioned media from TLR9-stimulated T lymphocyte cultures induced IL-18 and OPN expression in cardiac fibroblasts, while blockade of the IL-18 receptor with a neutralizing antibody abolished the increase in OPN expression. Furthermore, a mutation in the transcriptional factor interferon regulatory factor (IRF)1 or IRF1 small interfering RNA (siRNA) resulted in the decreased expression of IL-18 and OPN in cardiac fibroblasts. With pressure overload, IRF1-mutant mice showed downregulation of IL-18 and OPN expression in cardiac tissue, reduced cardiac fibrotic development, and increased left ventricular function compared with wild type. These results provide direct evidence that the induction of IL-18 regulates OPN-mediated cardiac fibrosis and diastolic dysfunction.

Short-term magnesium deficiency results in decreased levels of serum sphingomyelin, lipid peroxidation, and apoptosis in cardiovascular tissues

2009-06-29

The present study tested the hypothesis that short-term dietary deficiency of magnesium (Mg) (21 days) in rats would 1) result in decreased serum(s) [the present study tested the levels of Mg, sphingomyelin (SM), and phosphatidylcholine (PC)]; 2) promote DNA fragmentation, lipid peroxidation (LP), and activation of caspase-3 in cardiac (ventricular and atrial) and vascular(aortic) muscle; and 3) low levels of Mg²⁺ added to drinking water would either prevent or greatly ameliorate these manifestations. The data indicate that short-term Mg deficiency (10% normal dietary intake) resulted in profound reductions in serum-ionized Mg and total Mg with an elevation in serum-ionized calcium (Ca²⁺), significant lowering of serum SM and serum PC, with concomitant LP, DNA fragmentation, and activation of caspase-3 in ventricular (right and left chambers), atrial (right and left chambers) and abdominal aortic smooth muscle. The greater the reduction in serum-ionized Mg, the greater the effects on DNA fragmentation, LP, and caspase-3 activity. The intake of water-borne Mg²⁺ at all levels greatly attenuated or inhibited the reductions in serum SM and serum PC, activation of LP, DNA fragmentation, and the activation of caspase-3; even very low levels of Mg²⁺ in drinking water (i.e., 15 parts·million^–1·day^–1) were cardio- and vascular protective. In addition, we demonstrate that short-term dietary deficiency of Mg probably results in a downregulation of SM synthase and a decreased synthesis of PC.

Reduction of electrical coupling between microvascular endothelial cells by NO depends on connexin37

2009-06-29

We have previously shown that increased nitric oxide (NO) production in sepsis impairs arteriolar-conducted vasoconstriction cGMP independently and that the gap junction protein connexin (Cx) 37 is required for this conducted response. In the present study, we hypothesized that NO impairs interendothelial electrical coupling in sepsis by targeting Cx37. We examined the effect of exogenous NO on coupling in monolayers of cultured microvascular endothelial cells derived from the hindlimb skeletal muscle of wild-type (WT), Cx37 null, Cx40 null, and Cx43^G60S (nonfunctional mutant) mice. To assess coupling, we measured the spread of electrical current injected in the monolayer and calculated the monolayer intercellular resistance (inverse measure of coupling). The NO donor 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (DETA) rapidly and reversibly reduced coupling in cells from WT mice, cGMP independently. NO scavenger HbO₂ did not affect baseline coupling, but it eliminated DETA-induced reduction in coupling. Reduced coupling in response to DETA was also seen in cells from Cx40 null and Cx43^G60S mice, but not in cells from Cx37 null mice. DETA did not alter the expression of Cx37, Cx40, and Cx43 in WT cells analyzed by immunoblotting and immunofluorescence. Furthermore, neither the peroxynitrite scavenger 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III), superoxide scavenger Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, nor preloading of WT cells with the antioxidant ascorbate affected this reduction. We conclude that NO-induced reduction of electrical coupling between microvascular endothelial cells depends on Cx37 and propose that NO in sepsis impairs arteriolar-conducted vasoconstriction by targeting Cx37 within the arteriolar wall.

Catecholaminergic automatic activity in the rat pulmonary vein: electrophysiological differences between cardiac muscle in the left atrium and pulmonary vein

Doisne, N., Maupoil, V., Cosnay, P., Findlay, I. — 2009-06-29

Ectopic activity in cardiac muscle within pulmonary veins (PVs) is associated with the onset and the maintenance of atrial fibrillation in humans. The mechanism underlying this ectopic activity is unknown. Here we investigate automatic activity generated by catecholaminergic stimulation in the rat PV. Intracellular microelectrodes were used to record electrical activity in isolated strips of rat PV and left atrium (LA). The resting cardiac muscle membrane potential was lower in PV [–70 ± 1 (SE) mV, n = 8] than in LA (–85 ± 1 mV, n = 8). No spontaneous activity was recorded in PV or LA under basal conditions. Norepinephrine (10^–5 M) induced first a hyperpolarization (–8 ± 1 mV in PV, –3 ± 1 mV in LA, n = 8 for both) then a slowly developing depolarization (+21 ± 2 mV after 15 min in PV, +1 ± 2 mV in LA) of the resting membrane potential. Automatic activity occurred only in PV; it was triggered at approximately –50 mV, and it occurred as repetitive bursts of slow action potentials. The diastolic membrane potential increased during a burst and slowly depolarized between bursts. Automatic activity in the PV was blocked by either atenolol or prazosine, and it could be generated with a mixture of cirazoline and isoprenaline. In both tissues, cirazoline (10^–6 M) induced a depolarization (+37 ± 2 mV in PV, n = 5; +5 ± 1 mV in LA, n = 5), and isoprenaline (10^–7 M) evoked a hyperpolarization (–11 ± 3 mV in PV, n = 7; –3 ± 1 mV in LA, n = 6). The differences in membrane potential and reaction to adrenergic stimulation lead to automatic electrical activity occurring specifically in cardiac muscle in the PV.

ETA selective receptor antagonism prevents ventricular remodeling in volume-overloaded rats

Murray, D. B., McMillan, R., Brower, G. L., Janicki, J. S. — 2009-06-29

The objective of this study was to investigate the ability of selective endothelin receptor subtype A (ET_A) endothelin receptor antagonism (ETA) to prevent the acute myocardial remodeling process secondary to volume overload. Left ventricular tissue from sham-operated (Sham) and untreated (Fist), and TBC-3214 (Fist + ETA, 25 mg·kg^–1·day^–1)-treated fistula animals was analyzed for mast cell density, matrix metalloproteinase (MMP) activity, and extracellular collagen volume fraction (CVF) 1 and 5 days following the initiation of volume overload. Compared with Fist, ETA treatment prevented the increase in left ventricular mast cell density at 1 day and 5 days. Additionally, at 1 day postfistula, a substantial decrease in MMP-2 activity below Sham levels was observed following endothelin receptor antagonism (1.7 ± 0.7 vs. 0.3 ± 0.3 vs. 0.9 ± 0.2 arbitrary activity units, Fist vs. Fist + ETA vs. Sham, P ≤ 0.05). This same effect was also seen at 5 days postfistula (1.9 ± 0.3 vs. 0.5 ± 0.1 arbitrary activity units, Fist vs. Fist + ETA, P ≤ 0.05). The marked decrease in myocardial CVF seen in Fist hearts (0.7 ± 0.1 vs. 1.6 ± 0.1% myocardial area, Fist vs. Sham, P ≤ 0.05) was prevented by ETA (1.7 ± 0.1% Fist + ETA, P < 0.05 vs. Fist). This preservation of the collagen matrix was also present on day 5 in the TBC-treated group vs. the Fist group (1.0 ± 0.1 vs. 1.4 ± 0.1%, Fist vs. Fist + ETA, P ≤ 0.01). Furthermore, an 8-wk preventative treatment with ETA significantly attenuated the increase in left ventricular end systolic and diastolic volumes compared with untreated fistula hearts. In conclusion, the novel findings of this study indicate that the activation of cardiac mast cells and subsequent MMP activation/collagen degradation during the acute stages of volume overload are prevented by blockade of the ET_A receptor subtype. Furthermore, by preventing these events, ET-1 antagonism was efficacious in attenuating ventricular dilatation and limiting the development of structural and functional deficits.

Targeted disruption of the voltage-dependent calcium channel {alpha}2/{delta}-1-subunit

2009-06-29

Cardiac L-type voltage-dependent Ca²⁺ channels are heteromultimeric polypeptide complexes of ₁-, ₂/-, and β-subunits. The ₂/-1-subunit possesses a stereoselective, high-affinity binding site for gabapentin, widely used to treat epilepsy and postherpetic neuralgic pain as well as sleep disorders. Mutations in ₂/-subunits of voltage-dependent Ca²⁺ channels have been associated with different diseases, including epilepsy. Multiple heterologous coexpression systems have been used to study the effects of the deletion of the ₂/-1-subunit, but attempts at a conventional knockout animal model have been ineffective. We report the development of a viable conventional knockout mouse using a construct targeting exon 2 of ₂/-1. While the deletion of the subunit is not lethal, these animals lack high-affinity gabapentin binding sites and demonstrate a significantly decreased basal myocardial contractility and relaxation and a decreased L-type Ca²⁺ current peak current amplitude. This is a novel model for studying the function of the ₂/-1-subunit and will be of importance in the development of new pharmacological therapies.

Comparative investigation of the left ventricular pressure-volume relationship in rat models of type 1 and type 2 diabetes mellitus

2009-06-29

Diabetes mellitus (DM) is associated with characteristic structural and functional changes of the myocardium, termed diabetic cardiomyopathy. As a distinct entity independent of coronary atherosclerosis, diabetic cardiomyopathy is an increasingly recognized cause of heart failure. A detailed understanding of diabetic cardiac dysfunction, using relevant animal models, is required for the effective prevention and treatment of cardiovascular complications in diabetic patients. We investigated and compared cardiac performance in rat models of type 1 DM (streptozotocin induced) and type 2 DM (Zucker diabetic fatty rats) using a pressure-volume (P-V) conductance catheter system. Left ventricular (LV) systolic and diastolic function was evaluated in vivo at different preloads, including the slope of the end-systolic P-V relation (ESPVR) and end-diastolic P-V relationship (EDPVR), preload recruitable stroke work (PRSW), maximal slope of the systolic pressure increment (dP/dt_max), and its relation to end-diastolic volume (dP/dt_max-EDV) as well as the time constant of LV relaxation and maximal slope of the diastolic pressure decrement. Type 1 DM was associated with decreased LV systolic pressure, dP/dt_max, slope of ESPVR and dP/dt_max-EDV, PRSW, ejection fraction, and cardiac and stroke work indexes, indicating marked systolic dysfunction. In type 2 DM rats, systolic indexes were altered only to a lower extent and the increase of LV stiffness was more pronounced, as indicated by the higher slopes of EDPVR. Our data suggest that DM is characterized by decreased systolic performance and delayed relaxation (mainly in type 1 DM), accompanied by increased diastolic stiffness of the heart (more remarkably in type 2 DM). Based on the sophisticated method of P-V analysis, different characteristics of type 1 and type 2 diabetic cardiac dysfunction can be demonstrated.

NO production and eNOS phosphorylation induced by epinephrine through the activation of {beta}-adrenoceptors

2009-06-29

Epinephrine plays a key role in the control of vasomotor tone; however, the participation of the NO/cGMP pathway in response to β-adrenoceptor activation remains controversial. To evaluate the involvement of the endothelium in the vascular response to epinephrine, we assessed NO production, endothelial NO synthase phosphorylation, and tissue accumulation of cGMP in the perfused arterial mesenteric bed of rat. Epinephrine elicited a concentration-dependent increase in NO (EC₅₀ of 45.7 pM), which was coupled to cGMP tissue accumulation. Both NO and cGMP production were blocked by either endothelium removal (saponin) or NO synthase inhibition (N-nitro-l-arginine). Blockade of β₁- and β₂-adrenoceptors with 1 µM propranolol or β₃-adrenoceptor with 10 nM SR 59230A displaced rightward the concentration-NO production curve evoked by epinephrine. Selective stimulation of β₁-, β₂-, or β₃-adrenoceptors also resulted in NO and cGMP production. Propranolol (1 µM) inhibited the rise in NO induced by isoproterenol or the β₂-adrenoceptor agonists salbutamol, terbutaline, or fenoterol. Likewise, 10 nM SR 59230A reduced the effects of the β₃-adrenoceptor agonists BRL 37344, CGP 12177, SR 595611A, or pindolol. The NO production induced by epinephrine and BRL 37344 was associated with the activation of the phosphatidylinositol 3-kinase/Akt pathway and phosphorylation of eNOS in serine 1177. In addition, in anaesthetized rats, bolus administration of isoproterenol, salbutamol, or BRL 37344 produced NO-dependent reductions in systolic blood pressure. These findings indicate that β₁-, β₂-, and β₃-adrenoceptors are coupled to the NO/cGMP pathway, highlighting the role of the endothelium in the vasomotor action elicited by epinephrine and related β-adrenoceptor agonists.

Exercise training induces a cardioprotective phenotype and alterations in cardiac subsarcolemmal and intermyofibrillar mitochondrial proteins

Kavazis, A. N., Alvarez, S., Talbert, E., Lee, Y., Powers, S. K. — 2009-06-29

Endurance exercise is known to provide cardioprotection against ischemia-reperfusion-induced myocardial injury, and mitochondrial adaptations may play a critical role in this protection. To investigate exercise-induced changes in mitochondrial proteins, we compared the proteome of subsarcolemmal and intermyofibrillar mitochondria isolated from the myocardium of sedentary (control) and exercise-trained Sprague-Dawley rats. To achieve this goal, we utilized isobaric tags for relative and absolute quantitation, which allows simultaneous identification and quantification of proteins between multiple samples. This approach identified a total of 222 cardiac mitochondrial proteins. Importantly, repeated bouts of endurance exercise resulted in significant alterations in 11 proteins within intermyofibrillar mitochondria (seven increased; four decreased) compared with sedentary control animals. Furthermore, exercise training resulted in significant changes in two proteins within subsarcolemmal mitochondria (one increased; one decreased) compared with sedentary control animals. Differentially expressed proteins could be classified into seven functional groups, and several novel and potentially important cardioprotective mediators were identified. We conclude that endurance exercise induces alterations in mitochondrial proteome that may contribute to cardioprotective phenotype. Importantly, based on our findings, pharmacological or other interventions could be used to develop a strategy of protecting the myocardium during an ischemic attack.

Superoxide production by NAD(P)H oxidase and mitochondria is increased in genetically obese and hyperglycemic rat heart and aorta before the development of cardiac dysfunction. The role of glucose-6-phosphate dehydrogenase-derived NADPH

2009-06-29

Increased oxidative stress is a known cause of cardiac dysfunction in animals and patients with diabetes, but the sources of reactive oxygen species [e.g., superoxide anion (O₂^–)] and the mechanisms underlying O₂^– production in diabetic hearts are not clearly understood. Our aim was to determine whether NADPH oxidase (Nox) is a source of O₂^– and whether glucose-6-phosphate dehydrogenase (G6PD)-derived NADPH plays a role in augmenting O₂^– generation in diabetes. We assessed cardiac function, Nox and G6PD activities, NADPH levels, and the activities of antioxidant enzymes in heart homogenates from young (9–11 wk old) Zucker lean and obese (fa/fa) rats. We found that myocardial G6PD activity was significantly higher in fa/fa than in lean rats, whereas superoxide dismutase and glutathione peroxidase activities were decreased (P < 0.05). O₂^– levels were elevated (70–90%; P < 0.05) in the diabetic heart, and this elevation was blocked by the Nox inhibitor gp-91^ds-tat (50 µM) or by the mitochondrial respiratory chain inhibitors antimycin (10 µM) and rotenone (50 µM). Inhibition of G6PD by 6-aminonicotinamide (5 mM) and dihydroepiandrosterone (100 µM) also reduced (P < 0.05) O₂^– production. Notably, the activities of Nox and G6PD in the fa/fa rat heart were inhibited by chelerythrine, a protein kinase C inhibitor. Although we detected no changes in stroke volume, cardiac output, or ejection fraction, left ventricular diameter was slightly increased during diastole and systole, and left ventricular posterior wall thickness was decreased during systole (P < 0.05) in Zucker fa/fa rats. Our findings suggest that in a model of severe hyperlipidema and hyperglycemia Nox-derived O₂^– generation in the myocardium is fueled by elevated levels of G6PD-derived NADPH. Similar mechanisms were found to activate O₂^– production and induce endothelial dysfunction in aorta. Thus G6PD may be a useful therapeutic target for treating the cardiovascular disease associated with type 2 diabetes, if second-generation drugs specifically reducing the activity of G6PD to near normal levels are developed.

A numerical study on the flow of blood and the transport of LDL in the human aorta: the physiological significance of the helical flow in the aortic arch

Liu, X., Pu, F., Fan, Y., Deng, X., Li, D., Li, S. — 2009-06-29

It has been proposed that a mass transfer phenomenon called concentration polarization of low-density lipoproteins (LDLs) may occur in the arterial system and is likely involved in the localization of atherogenesis. To test the hypothesis that concentration polarization of LDL may be suppressed by the helical flow pattern in the human aorta, hence sparing the ascending aorta from atherosclerosis, the effects of aortic torsion, branching, curvature, and taper on blood flow and LDL transport in the lumen were simulated numerically under steady-state flow conditions using four aorta models constructed based on in vivo MRI slices. The results showed that it was the aortic torsion that induced the helical flow in the aortic arch, stabilizing the flow of blood in the aorta, and compensated the adverse effects of the aortic curvature on blood flow and LDL transport. The helical flow reduced the luminal surface LDL concentration in the aortic arch and probably played a role in suppressing severe polarization of LDL at the entrances of the three branches on the arch, hence, protecting them from atherogenesis. The taper of the aorta was another important feature of the aorta that further stabilized the flow of blood and delayed the attenuation of the helical flow, making it move beyond the arch and into the beginning part of the descending aorta. The results therefore may account for why the ascending aorta and the arch are relatively free of atherosclerosis.

Role of coupled gating between cardiac ryanodine receptors in the genesis of triggered arrhythmias

Chen, W., Wasserstrom, J. A., Shiferaw, Y. — 2009-06-29

Mutations in the ryanodine receptor (RyR) have been linked to exercise-induced sudden cardiac death. However, the precise sequence of events linking RyR channel mutations to a whole heart arrhythmia is not completely understood. In this paper, we apply a detailed, mathematical model of subcellular calcium (Ca) release, coupled to membrane voltage, to study how defective RyR channels can induce arrhythmogenic-triggered activity. In particular, we show that subcellular Ca activity, such as spontaneous Ca sparks and Ca waves, is highly sensitive to coupled gating between RyR channels in clusters. We show that small changes in coupled gating can induce aberrant Ca release activity, which, under Ca overload conditions, can induce delayed afterdepolarization (DAD). We systematically investigate the properties of subcellular Ca during DAD induction and show that the voltage time course during a DAD is dependent on the timing and number of spontaneous Ca sparks that transition to Ca waves. These results provide a detailed mechanism for the role of coupled gating in the genesis of triggered arrhythmias.

An internal domain of {beta}-tropomyosin increases myofilament Ca2+ sensitivity

2009-06-29

Tropomyosin (TM) is involved in Ca²⁺-mediated muscle contraction and relaxation in the heart. Striated muscle -TM is the major isoform expressed in the heart. The expression of striated muscle β-TM in the murine myocardium results in a decreased rate of relaxation and increased myofilament Ca²⁺ sensitivity. Replacing the carboxyl terminus (amino acids 258–284) of -TM with β-TM (a troponin T-binding region) results in decreased rates of contraction and relaxation in the heart and decreased myofilament Ca²⁺ sensitivity. We hypothesized that the putative internal troponin T-binding domain (amino acids 175–190) of β-TM may be responsible for the increased myofilament Ca²⁺ sensitivity observed when the entire β-TM is expressed in the heart. To test this hypothesis, we generated transgenic mice that expressed chimeric TM containing β-TM amino acids 175–190 in the backbone of -TM (amino acids 1–174 and 191–284). These mice expressed 16–57% chimeric TM and did not develop cardiac hypertrophy or any other morphological changes. Physiological analysis showed that these hearts exhibited decreased rates of contraction and relaxation and a positive response to isoproterenol. Skinned fiber bundle analyses showed a significant increase in myofilament Ca²⁺ sensitivity. Biophysical experiments demonstrated that the exchanged amino acids did not influence the flexibility of the TM. This is the first study to demonstrate that a specific domain within TM can increase the Ca²⁺ sensitivity of the thin filament and affect sarcomeric performance. Furthermore, these results enhance the understanding of why TM mutations associated with familial hypertrophic cardiomyopathy demonstrate increased myofilament sensitivity to Ca²⁺.

Nonmuscle myosin is regulated during smooth muscle contraction

Yuen, S. L., Ogut, O., Brozovich, F. V. — 2009-06-29

The participation of nonmuscle myosin in force maintenance is controversial. Furthermore, its regulation is difficult to examine in a cellular context, as the light chains of smooth muscle and nonmuscle myosin comigrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-RLC) and nonmuscle myosin (NM-RLC) were purified, and these proteins were resolved by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct RLC isoelectric variants. These spots were identified as phosphorylated NM-RLC (most acidic), nonphosphorylated NM-RLC, phosphorylated SM-RLC, and nonphosphorylated SM-RLC (most basic). During smooth muscle activation, NM-RLC phosphorylation increased. During depolarization, the increase in NM-RLC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II-induced increase in NM-RLC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous nonmuscle myosin IIB knockout mice was significantly less than that of wild-type littermates, suggesting that, in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that, in smooth muscle, the activation of nonmuscle myosin is regulated by Ca²⁺-calmodulin-activated myosin light chain kinase during depolarization and a Rho kinase-dependent pathway during agonist stimulation.

Endothelin receptor blockade combined with phosphodiesterase-5 inhibition increases right ventricular mitochondrial capacity in pulmonary arterial hypertension

2009-06-29

Pulmonary arterial hypertension (PAH) is often treated with endothelin (ET) receptor blockade or phosphodiesterase-5 (PDE5) inhibition. Little is known about the specific effects on right ventricular (RV) function and metabolism. We determined the effects of single and combination treatment with Bosentan [an ET type A (ET_A)/type B (ET_B) receptor blocker] and Sildenafil (a PDE5 inhibitor) on RV function and oxidative metabolism in monocrotaline (MCT)-induced PAH. Fourteen days after MCT injection, male Wistar rats were orally treated for 10 days with Bosentan, Sildenafil, or both. RV catheterization and echocardiography showed that MCT clearly induced PAH. This was evidenced by increased RV systolic pressure, reduced cardiac output, increased pulmonary vascular resistance (PVR), and reduced RV fractional shortening. Quantitative histochemistry showed marked RV hypertrophy and fibrosis. Monotreatment with Bosentan or Sildenafil had no effect on RV systolic pressure or cardiac function, but RV fibrosis was reduced and RV capillarization increased. Combination treatment did not reduce RV systolic pressure, but significantly lowered PVR, and normalized cardiac output, RV fractional shortening, and fibrosis. Only combination treatment increased the mitochondrial capacity of the RV, as reflected by increased succinate dehydrogenase and cytochrome c oxidase activities, associated with an activation of PKG, as indicated by increased VASP phosphorylation. Moreover, significant interactions were found between Bosentan and Sildenafil on PVR, cardiac output, RV contractility, PKG activity, and mitochondrial capacity. These data indicate that the combination of Bosentan and Sildenafil may beneficially contribute to RV adaptation in PAH, not only by reducing PVR but also by acting on the mitochondria in the heart.

Validation of a one-dimensional model of the systemic arterial tree

Reymond, P., Merenda, F., Perren, F., Rufenacht, D., Stergiopulos, N. — 2009-06-29

A distributed model of the human arterial tree including all main systemic arteries coupled to a heart model is developed. The one-dimensional (1-D) form of the momentum and continuity equations is solved numerically to obtain pressures and flows throughout the systemic arterial tree. Intimal shear is modeled using the Witzig-Womersley theory. A nonlinear viscoelastic constitutive law for the arterial wall is considered. The left ventricle is modeled using the varying elastance model. Distal vessels are terminated with three-element windkessels. Coronaries are modeled assuming a systolic flow impediment proportional to ventricular varying elastance. Arterial dimensions were taken from previous 1-D models and were extended to include a detailed description of cerebral vasculature. Elastic properties were taken from the literature. To validate model predictions, noninvasive measurements of pressure and flow were performed in young volunteers. Flow in large arteries was measured with MRI, cerebral flow with ultrasound Doppler, and pressure with tonometry. The resulting 1-D model is the most complete, because it encompasses all major segments of the arterial tree, accounts for ventricular-vascular interaction, and includes an improved description of shear stress and wall viscoelasticity. Model predictions at different arterial locations compared well with measured flow and pressure waves at the same anatomical points, reflecting the agreement in the general characteristics of the "generic 1-D model" and the "average subject" of our volunteer population. The study constitutes a first validation of the complete 1-D model using human pressure and flow data and supports the applicability of the 1-D model in the human circulation.

Reductions in mitochondrial O2 consumption and preservation of high-energy phosphate levels after simulated ischemia in chronic hibernating myocardium

Hu, Q., Suzuki, G., Young, R. F., Page, B. J., Fallavollita, J. A., Canty, J. M. — 2009-06-29

We performed the present study to determine whether hibernating myocardium is chronically protected from ischemia. Myocardial tissue was rapidly excised from hibernating left anterior descending coronary regions (systolic wall thickening = 2.8 ± 0.2 vs. 5.4 ± 0.3 mm in remote myocardium), and high-energy phosphates were quantified by HPLC during simulated ischemia in vitro (37°C). At baseline, ATP (20.1 ± 1.0 vs. 26.7 ± 2.1 µmol/g dry wt, P < 0.05), ADP (8.1 ± 0.4 vs. 10.3 ± 0.8 µmol/g, P < 0.05), and total adenine nucleotides (31.2 ± 1.3 vs. 40.1 ± 2.9 µmol/g, P < 0.05) were depressed compared with normal myocardium, whereas total creatine, creatine phosphate, and ATP-to-ADP ratios were unchanged. During simulated ischemia, there was a marked attenuation of ATP depletion (5.6 ± 0.9 vs. 13.7 ± 1.7 µmol/g at 20 min in control, P < 0.05) and mitochondrial respiration [145 ± 13 vs. 187 ± 11 ng atoms O₂·mg protein^–1·min^–1 in control (state 3), P < 0.05], whereas lactate accumulation was unaffected. These in vitro changes were accompanied by protection of the hibernating heart from acute stunning during demand-induced ischemia. Thus, despite contractile dysfunction at rest, hibernating myocardium is ischemia tolerant, with reduced mitochondrial respiration and slowing of ATP depletion during simulated ischemia, which may maintain myocyte viability.

Biventricular pacing-induced acute response in baroreflex sensitivity has predictive value for midterm response to cardiac resynchronization therapy

2009-06-29

In a previous study we demonstrated that the institution of biventricular pacing in chronic heart failure (CHF) acutely facilitates the arterial baroreflex. The arterial baroreflex has important prognostic value in CHF. We hypothesized that the acute response in baroreflex sensitivity (BRS) after the institution of cardiac resynchronization therapy (CRT) has predictive value for midterm response. One day after implantation of a CRT device in 33 CHF patients (27 male/6 female; age, 66.5 ± 9.5 yr; left ventricular ejection fraction, 28 ± 7%) we measured noninvasive BRS and heart rate variability (HRV) in two conditions: CRT device switched on and switched off (on/off order randomized). Echocardiography was performed before implantation (baseline) and 6 mo after implantation (follow-up). CRT responders were defined as patients in whom left ventricular end-systolic volume at follow-up had decreased by ≥15%. Responders (69.7%) and nonresponders (30.3%) had similar baseline characteristics. In responders, CRT increased BRS by 30% (P = 0.03); this differed significantly (P = 0.02) from the average BRS change (–2%) in the nonresponders. CRT also increased HRV by 30% in responders (P = 0.02), but there was no significant difference found compared with the increase in HRV (8%) in the nonresponders. Receiver-operating characteristic curve analysis revealed that the percent BRS increase had predictive value for the discrimination of responders and nonresponders (area under the curve, 0.69; 95% confidence interval, 0.51–0.87; maximal accuracy, 0.70). Our study demonstrates that a CRT-induced acute BRS increase has predictive value for the echocardiographic response to CRT. This finding suggests that the autonomic nervous system is actively involved in CRT-related reverse remodeling.

ERM protein moesin is phosphorylated by advanced glycation end products and modulates endothelial permeability

2009-06-29

Advanced glycation end products (AGEs) accumulated in different pathological conditions have the potent capacity to alter cellular properties that include endothelial structural and functional regulations. The disruption of endothelial barrier integrity may contribute to AGE-induced microangiopathy and macrovasculopathy. Previous studies have shown that AGEs induced the rearrangement of actin and subsequent hyperpermeability in endothelial cells (ECs). However, the mechanisms involved in this AGE-evoked EC malfunction are not well understood. This study directly evaluated the involvement of moesin phosphorylation in AGE-induced alterations and the effects of the RhoA and p38 MAPK pathways on this process. Using immortalized human dermal microvascular ECs (HMVECs), we first confirmed that the ezrin/radixin/moesin (ERM) protein moesin is required in AGE-induced F-actin rearrangement and hyperpermeability responses in ECs by knockdown of moesin protein expression with small interfering RNA. We then detected AGE-induced moesin phosphorylation by Western blot analysis. The mechanisms involved in moesin phosphorylation were analyzed by blocking AGE receptor binding and inhibiting Rho and MAPK pathways. AGE-treated HMVECs exhibited time- and dose-dependent increases in the Thr⁵⁵⁸ phosphorylation of moesin. The increased moesin phosphorylation was attenuated by preadministrations of AGE receptor antibody, Rho kinase (ROCK), or p38 inhibitor. Suppression of p38 activation via the expression of dominant negative mutants with Ad.MKK6b or Ad.p38 also decreased moesin phosphorylation. The activation of the p38 pathway by transfection of HMVECs with an adenoviral construct of dominant active MKK6b resulted in moesin phosphorylation. These results suggest a critical role of moesin phosphorylation in AGE-induced EC functional and morphological regulations. Activation of the ROCK and p38 pathways is required in moesin phosphorylation.

Determination of rate constants for turnover of myosin isoforms in rat myocardium: implications for in vivo contractile kinetics

2009-06-29

The ventricles of small mammals express mostly -myosin heavy chain (-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express ~10% -MHC on a predominately β-MHC (slow isoform) background. In failing human ventricles, the amount of -MHC is dramatically reduced, leading to the hypothesis that even small amounts of -MHC on a predominately β-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment (f_app) and detachment (g_app) for myosins composed of 100% -MHC or β-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% -MHC or 100% β-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (k_tr) in solutions of varying Ca²⁺ concentrations. The rate of ATP utilization was ~2.5-fold higher in preparations expressing 100% -MHC compared with those expressing only β-MHC, whereas k_tr was 2-fold faster in the -MHC myocardium. From these variables, we calculated f_app to be approximately threefold higher for -MHC than β-MHC and g_app to be twofold higher in -MHC. Mathematical modeling of isometric twitches predicted that small increases in -MHC significantly increased the rate of force development. These results suggest that low-level expression of -MHC has significant effects on contraction kinetics.

Clinical assessment of left ventricular rotation and strain: a novel approach for quantification of function in infarcted myocardium and its border zones

2009-06-29

Left ventricular (LV) circumferential strain and rotation have been introduced as clinical markers of myocardial function. This study investigates how regional LV apical rotation and strain can be used in combination to assess function in the infarcted ventricle. In healthy subjects (n = 15) and patients with myocardial infarction (n = 23), LV apical segmental rotation and strain were measured from apical short-axis recordings by speckle tracking echocardiography (STE) and MRI tagging. Infarct extent was determined by late gadolinium enhancement MRI. To investigate mechanisms of changes in strain and rotation, we used a mathematical finite element simulation model of the LV. Mean apical rotation and strain by STE were lower in patients than in healthy subjects (9.0 ± 4.9 vs. 12.9 ± 3.5° and –13.9 ± 10.7 vs. –23.8 ± 2.3%, respectively, P < 0.05). In patients, regional strain was reduced in proportion to segmental infarct extent (r = 0.80, P < 0.0001). Regional rotation, however, was similar in the center of the infarct and in remote viable myocardium. Minimum and maximum rotations were found at the infarct borders: minimum rotation at the border zone opposite to the direction of apical rotation, and maximum rotation at the border zone in the direction of rotation. The simulation model reproduced the clinical findings and indicated that the dissociation between rotation and strain was caused by mechanical interactions between infarcted and viable myocardium. Systolic strain reflects regional myocardial function and infarct extent, whereas systolic rotation defines infarct borders in the LV apical region. Regional rotation, however, has limited ability to quantify regional myocardial dysfunction.

Effect of a high dose of glucosamine on systemic and tissue inflammation in an experimental model of atherosclerosis aggravated by chronic arthritis

2009-06-29

Glucosamine sulfate (GS) is a glycosaminoglycan with anti-inflammatory and immunoregulatory properties. Here we set out to explore the effect of GS administration on markers of systemic and local inflammation in rabbits with atherosclerosis aggravated by chronic arthritis. Atherosclerosis was induced in rabbits by maintaining them on a hyperlipidemic diet after producing an endothelial lesion in the femoral arteries. Simultaneously, chronic arthritis was induced in these animals by repeated intra-articular injections of ovalbumin in previously immunized rabbits. A group of these rabbits was treated prophylactically with oral GS (500 mg·kg^–1·day^–1), and, when the animals were killed, serum was extracted and peripheral blood mononuclear cells (PBMC) were isolated. Furthermore, the femoral arteries, thoracic aorta, and synovial membranes were examined in gene expression studies and histologically. GS administration reduced circulating levels of the C-reactive protein and of interleukin-6. GS also lowered nuclear factor-B activation in PBMC, and it downregulated the expression of both the CCL2 (monocyte chemoattractant protein) and cyclooxygenase-2 genes in these cells. Lesions at the femoral wall were milder after GS treatment, as reflected by the intimal-to-media thickened ratio and the absence of aortic lesions. Indeed, GS also attenuated the histological lesions in synovial tissue. In a combined rabbit model of chronic arthritis and atherosclerosis, orally administered GS reduced the markers of inflammation in peripheral blood, as well as the femoral and synovial membrane lesions. GS also prevented the development of inflammation-associated aortic lesions. These results suggest an atheroprotective effect of GS.

Recombinant human activated protein C improves endotoxemia-induced endothelial dysfunction: a blood-free model in isolated mouse arteries

2009-06-29

Recombinant human activated protein C (rhAPC) is one of the treatment panels for improving vascular dysfunction in septic patients. In a previous study, we reported that rhAPC treatment in rat endotoxemia improved vascular reactivity, although the mechanisms involved are still under debate. In the present study, we hypothesized that rhAPC may improve arterial dysfunction through its nonanticoagulant properties. Ten hours after injection of LPS in mice (50 mg/kg ip), aortic rings and mesenteric arteries were isolated and incubated with or without rhAPC for 12 h. Aortic rings were mounted in a myograph, after which arterial contractility and endothelium-dependent relaxation were measured in the presence or absence of nitric oxide synthase or cyclooxygenase inhibitors. Flow (shear stress)-mediated dilation with or without the above inhibitors was also measured in mesenteric resistance arteries. Protein expression was assessed by Western blotting. Lipopolysaccharide (LPS) reduced aortic contractility to KCl and phenylephrine as well as dilation to acetylcholine. LPS also reduced flow-mediated dilation in mesenteric arteries. In rhAPC-treated aorta and mesenteric arteries, contractility and endothelial responsiveness to vasodilator drug and shear stress were improved. rhAPC treatment also improved LPS-induced endothelial dysfunction; this effect was associated with an increase in the phosphorylated form of endothelial nitric oxide synthase and protein kinase B as well as cyclooxygenase vasodilatory pathways, thus suggesting that these pathways, together with the decrease in nuclear factor-B activation and inducible nitric oxide synthase expression in the vascular wall, are implicated in the endothelial effect of rhAPC. In conclusion, ex vivo application of rhAPC improves arterial contractility and endothelial dysfunction resulting from endotoxemia in mice. This finding provides important insights into the mechanism underlying rhAPC-induced improvements on arterial dysfunction during septic shock.

Nonmyofilament-associated troponin T fragments induce apoptosis

Jeong, E.-M., Wang, X., Xu, K., Hossain, M. M., Jin, J.-P. — 2009-06-29

Troponin T (TnT) is a striated muscle-specific protein and an abundant component of the myofilaments. Nonmyofilament-associated TnT is rapidly degraded in myocytes, implying an importance in the maintenance of the cellular environment. However, if the level of nonmyofilament-associated TnT or TnT fragments exceeds the degradation capacity, it may cause cytotoxicity. To investigate this hypothesis, we constructed bicistronic vectors to express different portions of TnT polypeptide chain, together with nonfusion green fluorescent protein as a tracer for the transfection. Cytotoxicity of the TnT fragments was studied through forced expression in C₂C₁₂ myoblasts and human embryonic kidney-293 nonmuscle cells and examination of the viability of the transfected cells. The results demonstrated that, in the absence of myofilaments, the conserved COOH-terminal and middle fragments of TnT were highly effective on inducing cell death via apoptosis, whereas the NH₂-terminal variable region was not. As combined effects, nonmyofilament-associated intact cardiac TnT and a COOH-terminal truncated slow TnT fragment found in Amish nemaline myopathy exhibited intermediate cytotoxicity. A particular significance of this finding is that peak releases of TnT or TnT fragments from decomposition of a large number of myofibrils in acute myocardial infarction may breach the cellular protection of proteolytic degradation and result in apoptosis as a potential cause for the loss of cardiomyocytes.

Loss of cerebrovascular Shaker-type K+ channels: a shared vasodilator defect of genetic and renal hypertensive rats

2009-06-29

The cerebral arteries of hypertensive rats are depolarized and highly myogenic, suggesting a loss of K⁺ channels in the vascular smooth muscle cells (VSMCs). The present study evaluated whether the dilator function of the prominent Shaker-type voltage-gated K⁺ (K_V1) channels is attenuated in middle cerebral arteries from two rat models of hypertension. Block of K_V1 channels by correolide (1 µmol/l) or psora-4 (100 nmol/l) reduced the resting diameter of pressurized (80 mmHg) cerebral arteries from normotensive rats by an average of 28 ± 3% or 26 ± 3%, respectively. In contrast, arteries from spontaneously hypertensive rats (SHR) and aortic-banded (Ao-B) rats with chronic hypertension showed enhanced Ca²⁺-dependent tone and failed to significantly constrict to correolide or psora-4, implying a loss of K_V1 channel-mediated vasodilation. Patch-clamp studies in the VSMCs of SHR confirmed that the peak K⁺ current density attributed to K_V1 channels averaged only 5.47 ± 1.03 pA/pF, compared with 9.58 ± 0.82 pA/pF in VSMCs of control Wistar-Kyoto rats. Subsequently, Western blots revealed a 49 ± 7% to 66 ± 7% loss of the pore-forming _1.2- and _1.5-subunits that compose K_V1 channels in cerebral arteries of SHR and Ao-B rats compared with control animals. In each case, the deficiency of K_V1 channels was associated with reduced mRNA levels encoding either or both -subunits. Collectively, these findings demonstrate that a deficit of _1.2- and _1.5-subunits results in a reduced contribution of K_V1 channels to the resting diameters of cerebral arteries from two rat models of hypertension that originate from different etiologies.

Role of cGMP-dependent protein kinase in regulation of pulmonary vascular smooth muscle cell adhesion and migration: effect of hypoxia

2009-06-29

Exposure to prolonged hypoxia can result in pulmonary vascular remodeling and pulmonary hypertension. Hypoxia induces pulmonary vascular smooth muscle cell (PVSMC) proliferation and vascular remodeling by affecting cell adhesion and migration and secretion of extracellular matrix proteins. We previously showed that acute hypoxia decreases cGMP-dependent protein kinase (PKG) activity in PVSMC and that PKG plays a role in maintaining the differentiated contractile phenotype in normoxia. In this study, we investigated the effect of hypoxia on PVSMC adhesion and migration and the role of PKG in these functions. Ovine fetal pulmonary artery SMC were incubated in normoxia (Po₂ ~100 Torr) or hypoxia (Po₂ ~30–40 Torr) or treated with the PKG inhibitor DT-3 for 24 h in normoxia. To further study the role of PKG in the modulation of adhesion and migration, PVSMC were transiently transfected with a full-length PKG1 [PKG-green fluorescent protein (GFP)] or a dominant-negative construct (G1R-GFP). Cell adhesion to extracellular matrix proteins was determined, and integrin-mediated adhesion was assessed by /β-integrin-mediated cell adhesion array. Exposure to hypoxia (24 h) and pharmacological inhibition of PKG1 by DT-3 significantly promoted adhesion mediated by ₄-, β₁-, and ₅β₁-integrins to fibronectin, laminin, and tenacin and also resulted in increased cell migration. Likewise, inhibition of PKG by expression of a dominant-negative PKG1 construct increased cell adhesion and migration, comparable to that induced by hypoxia. Dynamic actin reorganization associated with integrin-mediated cell adhesion is partly regulated by the actin-binding protein cofilin, the (Ser3) phosphorylation of which inhibits its actin-severing activity. We found that increased PKG expression and activity is associated with decreased cofilin (Ser3) phosphorylation, implying a role for PKG in the modulation of cofilin activity and actin dynamics. Together, these findings identify cGMP/PKG1 signaling as central to the functional differences between PVSMC exposed to normoxia versus hypoxia.

Suppression of 5'-AMP-activated protein kinase activity does not impair recovery of contractile function during reperfusion of ischemic hearts

2009-06-29

Activation of 5'-AMP-activated protein kinase (AMPK) may benefit the heart during ischemia-reperfusion by increasing energy production. While AMPK stimulates glycolysis, mitochondrial oxidative metabolism is the major source of ATP production during reperfusion of ischemic hearts. Stimulating AMPK increases mitochondrial fatty acid oxidation, but this is usually accompanied by a decrease in glucose oxidation, which can impair the functional recovery of ischemic hearts. To examine the relationship between AMPK and cardiac energy substrate metabolism, we subjected isolated working mouse hearts expressing a dominant negative (DN) ₂-subunit of AMPK (AMPK-₂ DN) to 20 min of global no-flow ischemia and 40 min of reperfusion with Krebs-Henseleit solution containing 5 mM [U-¹⁴C]glucose, 0.4 mM [9, 10-³H]palmitate, and 100 µU/ml insulin. AMPK-₂ DN hearts had reduced AMPK activity at the end of reperfusion (82 ± 9 vs. 141 ± 7 pmol·mg^–1·min^–1) with no changes in high-energy phosphates. Despite this, AMPK-₂ DN hearts had improved recovery of function during reperfusion (14.9 ± 0.8 vs. 9.4 ± 1.4 beats·min^–1·mmHg·10^–3). During reperfusion, fatty acid oxidation provided 44.0 ± 2.8% of total acetyl-CoA in AMPK-₂ DN hearts compared with 55.0 ± 3.2% in control hearts. Since insulin can inhibit both AMPK activation and fatty acid oxidation, we also examined functional recovery in the absence of insulin. Functional recovery was similar in both groups despite a decrease in AMPK activity and a decreased reliance on fatty acid oxidation during reperfusion (66.4 ± 9.4% vs. 85.3 ± 4.3%). These data demonstrate that the suppression of cardiac AMPK activity does not produce an energetically compromised phenotype and does not impair, but may in fact improve, the recovery of function after ischemia.

Postmyocardial infarction remodeling and coronary reserve: effects of ivabradine and beta blockade therapy

2009-06-29

We compared the effects of heart rate reduction (HRR) by the hyperpolarization-activated pacemaker current (I_f) channel inhibitor ivabradine (MI+Iva) and the β₁-blocker atenolol (MI+Aten) on ventricular remodeling and perfusion after myocardial infarction (MI) in middle-aged (12 mo) Sprague-Dawley rats. Mean HRR was virtually identical in the two treated groups (19%). Four weeks after coronary artery ligation, maximal myocardial perfusion fell in the MI group but was preserved in infarcted rats treated with either Iva or Aten. However, coronary reserve in the remodeled hearts was preserved only with Iva, since Aten treatment elevated baseline perfusion in response to a higher wall stress. The higher maximal perfusion noted in the two treated groups was not due to arteriogenesis or angiogenesis. Plasma levels of angiotensin (ANG) II and myocardial ANG type 1 (AT₁) receptor and transforming growth factor (TGF)-β1 were reduced during the first week of treatment by both Iva and Aten. Moreover, treatment also reduced arteriolar perivascular collagen density. Despite these similar effects of Iva and Aten on vascularity and ANG II, Iva, but not Aten, attenuated the decline in ejection fraction and lowered left ventricular (LV) end-diastolic volume (LVEDV)-to-LV mass ratio, determined by echocardiography. In conclusion, 1) Iva has advantages over Aten in postinfarction therapy that are not due to differential effects of the drugs on heart rate, and 2) age limits growth factor upregulation, angiogenesis, and arteriogenesis in the postinfarcted heart.

Sepiapterin reductase regulation of endothelial tetrahydrobiopterin and nitric oxide bioavailability

2009-06-29

Sepiapterin reductase (SPR) catalyzes the final step of tetrahydrobiopterin (H₄B) biosynthesis and the first step of H₄B regeneration from an exogenous precursor sepiapterin. Despite the potential significance of SPR in regulating H₄B-dependent nitric oxide (NO^•) production, the endothelium-specific sequence and functions of SPR remain elusive. We first cloned endothelial SPR cDNA from bovine aortic endothelial cells (Genebank: DQ978331). In cells transiently transfected with SPR gene, SPR activity (HPLC) was dramatically increased by 19-fold, corresponding to a significant increase in endothelial H₄B content (HPLC) and NO^• production (electron spin resonance). In vivo delivery of SPR gene significantly increased vascular SPR protein expression (mouse vs. bovine antibodies to differentiate endogenous vs. exogenous), activity, H₄B content, and NO^• production, as well as NO^•-dependent vasorelaxation. In endothelial cells transfected with small interfering RNA specific for SPR, ~87% of mRNA were attenuated (real-time quantitative RT-PCR), corresponding to a significant reduction in SPR protein expression and activity, which was associated with decreases in both intracellular H₄B content and NO^• level. Exogenous administration of sepiapterin to endothelial cells significantly upregulated H₄B and NO^• levels, which were attenuated by SPR RNA interference (RNAi). H₄B-stimulated increase in NO^• production, however, was SPR RNAi independent. GTP cyclohydrolase 1 expression and activity, as well as dihydrofolate reductase expression, were not affected by SPR RNAi, whereas dihydrofolate reductase activity was significantly downregulated. These data represent the first to study endothelial SPR functionally and clearly demonstrate an important role of endothelial SPR in modulating H₄B and NO^• bioavailability.

Cardiac MRI of myocardial salvage at the peri-infarct border zones after primary coronary intervention

2009-06-29

The purpose of this study was to use cardiac MRI to define the morphology of the reversibly injured peri-infarct border zone in patients treated with primary percutaneous coronary intervention (PPCI) for acute ST elevation myocardial infarction. In 15 patients, T2-weighted myocardial edema imaging was used to identify the ischemic bed or area at risk (AAR), and late gadolinium enhancement imaging was used to measure infarct size. Images were coregistered, and the boundaries of edema and necrosis were defined using an edge-detection methodology. We observed that infarction always involved the subendocardium but showed variable transmural extension within the AAR. The mean infarct size was 22 ± 19% (range: 8–48%), and the mean AAR was 34 ± 12% (range: 20–57%). The infarcted myocardium was always smaller than the ischemic AAR and involved between 34% and 99% (mean 72 ± 21%) of the ischemic bed primarily due to variation in transmural infarct extension. Although a lateral border zone of potentially viable myocardium was often present, its extent was limited (range: 0–11 mm, mean: 5 ± 4 mm). As a result of this, infarcts occupied the majority (range: 70–100%, mean: 82 ± 13%) of the width of the AAR. The mean fractional wall thickening in the infarcted, peri-infarcted, and remote myocardium was 3.6 ± 16.0%, 40.5 ± 26.4%, and 88.2 ± 39.3%, respectively. These findings demonstrate that myocardial salvage is largely determined by epicardial limitation of the infarct within the ischemic AAR after PPCI. The lateral boundaries of necrosis approximate to the lateral extent of the ischemic bed and systolic wall motion abnormalities extend well beyond the infarct border zone.

Role of store-operated Ca2+ entry in adenosine-induced vasodilatation of rat small mesenteric artery

2009-06-29

Store-operated Ca²⁺ entry (SOCE) has recently been proposed to contribute to Ca²⁺ influx in vascular smooth muscle cells (VSMCs). Adenosine is known for its protective role against hypoxia and ischemia by increasing nutrient and oxygen supply through vasodilation. This study was designed to examine the hypothesis that SOCE have a functional role in adenosine-induced vasodilation. Small mesenteric resistance arteries and mesenteric VSMCs were obtained from rats. Isometric tensions of isolated artery rings were measured by a sensitive myograph system. Laser-scanning confocal microscopy was used to determine the intracellular Ca²⁺ concentration of fluo 3-loaded VSMCs. Adenosine (0.1–100 µM) relaxed artery rings that were precontracted by phenylephrine in a concentration-dependent manner. In cultured mesenteric VSMCs, passive store depletion by thapsigargin and active store depletion by phenylephrine both induced Ca²⁺ influx due to SOCE. Adenosine inhibited SOCE-mediated increases in cytosolic Ca²⁺ levels evoked by the emptying of the stores. In isolated artery rings, adenosine inhibited SOCE-induced contractions due to store depletion. A_2A receptor antagonism with SCH-58261 and adenylate cyclase inhibition with SQ-22536 largely attenuated adenosine responses. The cAMP analog 8-bromo-cAMP mimicked the effects of adenosine on SOCE. Our results indicate a novel mechanism of vasodilatation by adenosine that involves regulation of SOCE through the cAMP signaling pathway due to activation of adenosine A_2A receptors.

Effects of KCNE2 on HCN isoforms: distinct modulation of membrane expression and single channel properties

2009-06-29

Hyperpolarization-activated cation (HCN) channels give rise to an inward current with similar but not identical characteristics compared with the pacemaker current (I_f), suggesting that HCN channel function is modulated by regulatory β-subunits in native tissue. KCNE2 has been proposed to serve as a β-subunit of HCN channels; however, available data remain contradictory. To further clarify this situation, we therefore analyzed the effect of KCNE2 on whole cell currents, single channel properties, and membrane protein expression of all cardiac HCN isoforms in the CHO cell system. On the whole cell level, current densities of all HCN isoforms were significantly increased by KCNE2 without altering voltage dependence or current reversal. While these results correlated well with the KCNE2-mediated 2.2-fold and 1.6-fold increases of membrane protein levels of HCN2 and HCN4, respectively, no effect of KCNE2 on HCN1 expression was obtained. All HCN subtypes displayed faster activation kinetics upon coexpression with KCNE2. Most importantly, for the first time, we demonstrated modulation of single channel function by KCNE2, thus supporting direct functional interaction with HCN subunits. In the presence of KCNE2, the single channel amplitudes and conductance of HCN1, HCN2, and HCN4 were significantly increased versus control recordings. Mean open time was significantly increased in cells coexpressing HCN2 + KCNE2, whereas it was unaffected in HCN1 + KCNE2 cotransfected cells and reduced in HCN4 + KCNE2 cotransfected cells compared with the respective HCN subunits alone. Thus, we demonstrate KCNE2-mediated distinct effects on HCN membrane expression and direct functional modulation of HCN isoforms, further supporting that KCNE2 surves as a regulatory β-subunit of HCN channels.

Region-specific adaptations in determinants of rat skeletal muscle oxygenation to chronic hypoxia

2009-06-29

Chronic exposure to hypoxia is associated with muscle atrophy (i.e., a reduction in muscle fiber cross-sectional area), reduced oxidative capacity, and capillary growth. It is controversial whether these changes are muscle and fiber type specific. We hypothesized that different regions of the same muscle would also respond differently to chronic hypoxia. To investigate this, we compared the deep (oxidative) and superficial (glycolytic) region of the plantaris muscle of eight male rats exposed to 4 wk of hypobaric hypoxia (410 mmHg, Po₂: 11.5 kPa) with those of nine normoxic rats. Hematocrit was higher in chronic hypoxic than control rats (59% vs. 50%, P < 0.001). Using histochemistry, we observed 10% fiber atrophy (P < 0.05) in both regions of the muscle but no shift in the fiber type composition and myoglobin concentration of the fibers. In hypoxic rats, succinate dehydrogenase (SDH) activity was elevated in fibers of each type in the superficial region (25%, P < 0.05) but not in the deep region, whereas in the deep region but not the superficial region the number of capillaries supplying a fiber was elevated (14%, P < 0.05). Model calculations showed that the region-specific alterations in fiber size, SDH activity, and capillary supply to a fiber prevented the occurrence of anoxic areas in the deep region but not in the superficial region. Inclusion of reported acclimatization-induced increases in mean capillary oxygen pressure attenuated the development of anoxic tissue areas in the superficial region of the muscle. We conclude that the determinants of tissue oxygenation show region-specific adaptations, resulting in a marked differential effect on tissue Po₂.

Effect of early versus late AT1 receptor blockade with losartan on postmyocardial infarction ventricular remodeling in rabbits

2009-06-29

To characterize the temporal activation of the renin-angiotensin system after myocardial infarction (MI) in rabbits, we examined cardiac ANG II type 1 receptor (AT₁R) expression and ANG II levels from 3 h to 35 days. The effects of losartan (12.5 mg·kg^–1·day^–1) on functional and histomorphometric parameters when treatment was initiated early (3 h) and late (day 15) post-MI and maintained for different periods of time [short term (4 days), midterm (20 days), and long term (35 days)] were also studied. AT₁R expression increased in the MI zone at 15 and 35 days (P < 0.05). ANG II levels increased (P < 0.05) in the non-MI zone at 24 h and in the MI zone as well as in plasma at 4 days and then progressively decreased until 35 days. The survival rate was significantly lower in untreated MI and early long-term-treated animals. Diastolic pressure-volume curves in MI at 35 and 56 days shifted to the right (P < 0.05). This shift was even more pronounced in long-term-treated groups (P < 0.05). Contractility decreased (P < 0.05 vs. sham) in the untreated and long-term-treated groups and was attenuated in the midterm-treated group. The early administration of losartan reduced RAM 11-positive macrophages from 4.15 ± 0.05 to 3.05 ± 0.02 cells/high-power field (HPF; P < 0.05) and CD45 RO-positive lymphocytes from 2.23 ± 0.05 to 1.48 ± 0.01 cells/HPF (P < 0.05) in the MI zone at 4 days. Long-term treatment reduced the scar collagen (MI: 70.50 ± 2.35% and MI + losartan: 57.50 ± 2.48, P < 0.05), determined the persistency of RAM 11-positive macrophages (3.02 ± 0.13 cells/HPF) and CD45 RO-positive lymphocytes (2.77 ± 0.58 cells/HPF, P < 0.05 vs. MI), and reduced the scar thinning ratio at 35 days (P < 0.05). Consequently, the temporal expressions of cardiac AT₁R and ANG II post-MI in rabbits are different from those described in other species. Long-term treatment unfavorably modified post-MI remodeling, whereas midterm treatment attenuated this harmful effect. The delay in wound healing (early reduction and late persistency of inflammatory infiltrate) and adverse remodeling observed in long-term-treated animals might explain the unfavorable effect observed in rabbits.

Long-acting phosphodiesterase-5 inhibitor, tadalafil, induces sustained cardioprotection against lethal ischemic injury

Ahmad, N., Wang, Y., Ali, A. K., Ashraf, M. — 2009-06-29

The ability of pharmacological preconditioning mimetics to confer long-lasting and sustained cardioprotection may be a logical criterion to develop a drug that can be used clinically for cardioprotection. We propose here that the use of long-acting phosphodiesterase-5 inhibitor, tadalafil, may confer sustained cardioprotection against ischemia. Tadalafil (5 mg/kg) was administered orally to male C57B/6J mice (n = 6 in each treatment subgroup at each time point studied). Hearts were isolated and subjected to 40 min of ischemia and 30 min of reperfusion on Langendorff's apparatus at 1, 12, 24, 36, 48, 60, 72, and 108 h after tadalafil administration. In 1- to 48-h subgroups, tadalafil was given once at 0 h only. In 60- and 72-h subgroups, tadalafil was given twice at 0 and 36 h. Similarly, in the 108-h subgroup, tadalafil was administered at 0, 36, and 72 h. In the same subgroups, wortmannin (15 µg/kg ip), an inhibitor of phosphatidylinositol 3-kinase or 5-hydroxydecanoic acid (5 mg/kg ip), an inhibitor of mitochondrial ATP-sensitive K⁺ channels, was given together with tadalafil, and the hearts were subjected to ischemia-reperfusion at 36 h to determine whether the effect of tadalafil on ischemia-reperfusion injury was abolished. As a result, tadalafil treatment reduced left ventricular end-diastolic pressure and increased left ventricular developed pressure as well as reduced lactate dehydrogenase release. This protection remained till 36–40 h, and thereafter it vanished. The readministration of tadalafil at 36 and 72 h restored the protection till 108 h. Tadalafil treatment accelerated Akt phosphorylation in cardiac tissue and decreased myocyte apoptosis. The administration of wortmannin abolished the beneficial effects of tadalafil on hemodynamic parameters and myocyte apoptosis, together with significantly reduced Akt phosphorylation. 5-Hydroxydecanoic acid also abolished the antiapoptotic effect of tadalafil. It is concluded that tadalafil treatment induces the long-term protection of ischemic myocardium via phosphatidylinositol 3-kinase/Akt signaling pathway.

Carbon monoxide, skeletal muscle oxidative stress, and mitochondrial biogenesis in humans

2009-06-29

Given that the physiology of heme oxygenase-1 (HO-1) encompasses mitochondrial biogenesis, we tested the hypothesis that the HO-1 product, carbon monoxide (CO), activates mitochondrial biogenesis in skeletal muscle and enhances maximal oxygen uptake (Vo_2max) in humans. In 10 healthy subjects, we biopsied the vastus lateralis and performed Vo_2max tests followed by blinded randomization to air or CO breathing (1 h/day at 100 parts/million for 5 days), a contralateral muscle biopsy on day 5, and repeat Vo_2max testing on day 8. Six independent subjects underwent CO breathing and two muscle biopsies without exercise testing. Molecular studies were performed by real-time RT-PCR, Western blot analysis, and immunochemistry. After Vo_2max testing plus CO breathing, significant increases were found in mRNA levels for nuclear respiratory factor-1, peroxisome proliferator-activated receptor- coactivator-1, mitochondrial transcription factor-A (Tfam), and DNA polymerase (Pol) with no change in mitochondrial DNA (mtDNA) copy number or Vo_2max. Levels of myosin heavy chain I and nuclear-encoded HO-1, superoxide dismutase-2, citrate synthase, mitofusin-1 and -2, and mitochondrial-encoded cytochrome oxidase subunit-I (COX-I) and ATPase-6 proteins increased significantly. None of these responses were reproduced by Vo_2max testing alone, whereas CO alone increased Tfam and Pol mRNA, and COX-I, ATPase-6, mitofusin-2, HO-1, and superoxide dismutase protein. These findings provide evidence linking the HO/CO response involved in mitochondrial biogenesis in rodents to skeletal muscle in humans through a set of responses involving regulation of the mtDNA transcriptosome and mitochondrial fusion proteins autonomously of changes in exercise capacity.

Lactosylceramide promotes cell migration and proliferation through activation of ERK1/2 in human aortic smooth muscle cells

Mu, H., Wang, X., Wang, H., Lin, P., Yao, Q., Chen, C. — 2009-06-29

Increased plasma levels of lactosylceramide (LacCer) have been associated with cardiovascular disease. However, it is largely unknown whether LacCer directly contributes to dysfunction of smooth muscle cells (SMCs), a key event in vascular lesion formation. In the present study, we determined the effects and potential mechanisms of LacCer on cell migration and proliferation in human aortic SMCs (AoSMCs). Cell migration and proliferation were determined by a modified Boyden chamber assay and nonradioactive colorimetric (MTS) assay, respectively. We found that LacCer significantly induced AoSMC migration and proliferation in a concentration- and time-dependent manner. In addition, LacCer significantly upregulated the expression of PDGFR-B, integrins (_v and β₃), and matrix metalloproteinases (matrix metalloproteinase-1 and -2) at both mRNA and protein levels, as determined by real-time PCR and Western blot analyses, respectively. Furthermore, LacCer increased superoxide anion production and the transient phosphorylation of ERK1/2 in AoSMCs, as determined by dihydroethidium staining and immunoassay, respectively. Accordingly, LacCer-induced cell migration and proliferation were effectively blocked by antioxidants (seleno-l-methionine and Mn tetrakis porphyrin) and by a specific ERK1/2 inhibitor. Thus, LacCer promotes cell migration and proliferation through oxidative stress and activation of ERK1/2 in AoSMCs. These findings demonstrate the functional role of LacCer in the vascular disease pathogenesis.

Increased myocardial NAD(P)H oxidase-derived superoxide causes the exacerbation of postinfarct heart failure in type 2 diabetes

2009-06-29

Type 2 diabetes adversely affects the outcomes in patients with myocardial infarction (MI), which is associated with the development of left ventricular (LV) failure. NAD(P)H oxidase-derived superoxide (O₂^–) production is increased in type 2 diabetes. However, its pathophysiological significance in advanced post-MI LV failure associated with type 2 diabetes remains unestablished. We thus hypothesized that an inhibitor of NAD(P)H oxidase activation, apocynin, could attenuate the exacerbated LV failure after MI in high-fat diet (HFD)-induced obese mice with type 2 diabetes. Male C57BL/6J mice were fed on either HFD or normal diet (ND) for 8 wk. At 4 wk of feeding, MI was created in mice by ligating the left coronary artery. HFD-fed MI mice were treated with either 10 mmol/l apocynin or vehicle. HFD + MI had significantly greater LV end-diastolic diameter (LVEDD; 5.7 ± 0.1 vs. 5.3 ± 0.2 mm), end-diastolic pressure (12 ± 2 vs. 8 ± 1 mmHg), and lung weight/tibial length (10.1 ± 0.3 vs. 8.7 ± 0.7 mg/mm) than ND + MI, which was accompanied by an increased interstitial fibrosis of noninfarcted LV. Treatment of HFD + MI with apocynin significantly decreased LVEDD (5.4 ± 0.1 mm), LV end-diastolic pressure (9.7 ± 0.8 mmHg), lung weight/tibial length (9.0 ± 0.3 mg/mm), and concomitantly interstitial fibrosis of noninfarcted LV to the ND + MI level without affecting body weight, glucose metabolism, and infarct size. NAD(P)H oxidase activity and O₂^– production were increased in noninfarcted LV tissues from HFD + MI, both of which were attenuated by apocynin to the ND + MI level. Type 2 diabetes was associated with the exacerbation of LV failure after MI via increasing NAD(P)H oxidase-derived O₂^–, which may be a novel important therapeutic target in advanced heart failure with diabetes.

Inhibition of TRPC1/TRPC3 by PKG contributes to NO-mediated vasorelaxation

Chen, J., Crossland, R. F., Noorani, M. M. Z., Marrelli, S. P. — 2009-06-29

Nitric oxide (NO) inhibits transient receptor potential channel 3 (TRPC3) channels via a PKG-dependent mechanism. We sought to determine 1) whether NO inhibition of TRPC3 occurs in freshly isolated smooth muscle cells (SMC); and 2) whether NO inhibition of TRPC3 channels contributes to NO-mediated vasorelaxation. We tested these hypotheses in freshly isolated rat carotid artery (CA) SMC using patch clamp and in intact CA by vessel myograph. We demonstrated TRPC3 expression in whole CA (mRNA and protein) that was localized to the smooth muscle layers. TRPC1 protein was also expressed and coimmunoprecipitated with TRPC3. Whole cell patch clamp demonstrated nonselective cation channel currents that were activated by UTP (60 µM) and completely inhibited by a TRPC channel inhibitor, La³⁺ (100 µM). The UTP-stimulated current (I_UTP) was also inhibited by intracellular application of anti-TRPC3 or anti-TRPC1 antibody, but not by anti-TRPC6 or anti-TRPC4 control antibodies. We next evaluated the NO signaling pathway on I_UTP. Exogenous NO [(Z)-1-{N-methyl-N-[6(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAHMA NONOate)] or a cell-permeable cGMP analog (8-bromo-cGMP) significantly inhibited I_UTP. Preapplication of a PKG inhibitor (KT5823) reversed the inhibition of MAHMA NONOate or 8-bromo-cGMP, demonstrating the critical role of PKG in NO inhibition of TRPC1/TRPC3. Intact CA segments were contracted with UTP (100 µM) in the presence or absence of La³⁺ (100 µM) and then evaluated for relaxation to an NO donor, sodium nitroprusside (1 nM to 1 µM). Relaxation to sodium nitroprusside was significantly reduced in the La³⁺ treatment group. We conclude that freshly isolated SMC express TRPC1/TRPC3 channels and that these channels are inhibited by NO/cGMP/PKG. Furthermore, NO contributes to vasorelaxation by inhibition of La³⁺-sensitive channels consistent with TRPC1/TRPC3.

Vascular endothelial dysfunction with aging: endothelin-1 and endothelial nitric oxide synthase

2009-06-29

To determine whether impaired endothelium-dependent dilation (EDD) in older adults is associated with changes in the expression of major vasoconstrictor or vasodilator proteins in the vascular endothelium, endothelial cells (EC) were obtained from the brachial artery and peripheral veins of 56 healthy men, aged 18–78 yr. Brachial artery EC endothelin-1 (ET-1) [0.99 ± 0.10 vs. 0.57 ± 0.10 ET-1/human umbilical vein EC (HUVEC) intensity, P = 0.01] and serine 1177 phosphorylated endothelial nitric oxide synthase (PeNOS) (0.77 ± 0.09 vs. 0.44 ± 0.07 PeNOS/HUVEC intensity, P < 0.05) (quantitative immunofluorescence) were greater, and EDD (peak forearm blood flow to intrabrachial acetylcholine) was lower (10.2 ± 0.9 vs. 14.7 ± 1.7 ml·100 ml^–1·min^–1, P < 0.05) in older (n = 18, 62 ± 1 yr) vs. young (n = 15, 21 ± 1 yr) healthy men. EDD was inversely related to expression of ET-1 (r = –0.39, P < 0.05). Brachial artery EC eNOS expression did not differ significantly with age, but tended to be greater in the older men (young: 0.23 ± 0.03 vs. older: 0.33 ± 0.07 eNOS/HUVEC intensity, P = 0.08). In the sample with venous EC collections, EDD (brachial artery flow-mediated dilation) was lower (3.50 ± 0.44 vs. 7.68 ± 0.43%, P < 0.001), EC ET-1 and PeNOS were greater (P < 0.05), and EC eNOS was not different in older (n = 23, 62 ± 1 yr) vs. young (n = 27, 22 ± 1 yr) men. EDD was inversely related to venous EC ET-1 (r = –0.37, P < 0.05). ET-1 receptor A inhibition with BQ-123 restored 60% of the age-related impairment in carotid artery dilation to acetylcholine in B6D2F1 mice (5–7 mo, n = 8; 30 mo, n = 11; P < 0.05). ET-1 expression is increased in vascular EC of healthy older men and is related to reduced EDD, whereas ET-1 receptor A signaling tonically suppresses EDD in old mice. Neither eNOS nor PeNOS is reduced with aging. Changes in ET-1 expression and bioactivity, but not eNOS, contribute to vascular endothelial dysfunction with aging.

Skeletal muscle contraction-induced vasodilator complement production is dependent on stimulus and contraction frequency

Dua, A. K., Dua, N., Murrant, C. L. — 2009-06-29

To test the hypothesis that the vasodilator complement that produces arteriolar vasodilation during muscle contraction depends on both stimulus and contraction frequency, we stimulated four to five skeletal muscle fibers in the anesthetized hamster cremaster preparation in situ and measured the change in diameter of arterioles at a site of overlap with the stimulated muscle fibers. Diameter was measured before, during, and after 2 min of skeletal muscle contraction stimulated over a range of stimulus frequencies [4, 20, and 40 Hz; 15 contractions/min (cpm), 250 ms train duration] and a range of contraction frequencies (6, 15, and 60 cpm; 20 Hz stimulus frequency, 250 ms train duration). Muscle fibers were stimulated in the absence and presence of an inhibitor of adenosine receptors [10^–6 M xanthine amine congener (XAC)], an ATP-dependent potassium (K⁺) channel inhibitor (10^–5 M glibenclamide), an inhibitor of a source of K⁺ by inhibition of voltage-dependent K⁺ channels [3 x 10^–4 M 3,4-diaminopyridine (DAP)], and an inhibitor of nitric oxide synthase [10^–6 M N^G-nitro-l-arginine methyl ester (l-NAME) + 10^–7 S-nitroso-N-acetylpenicillamine (a nitric oxide donor)]. l-NAME inhibited the dilations at all stimulus frequencies and contraction frequencies except 60 cpm. XAC inhibited the dilations at all contraction frequencies and stimulus frequencies except 40 Hz. Glibenclamide inhibited all dilations at all stimulus and contraction frequencies, and DAP did not inhibit dilations at any stimulus frequencies while attenuating dilation at a contraction frequency of 60 cpm only. Our data show that the complement of dilators responsible for the vasodilations induced by skeletal muscle contraction differed depending on the stimulus and contraction frequency; therefore, both are important determinants of the dilators involved in the processes of arteriolar vasodilation associated with active hyperemia.

Acid-sensing ion channels contribute to the metaboreceptor component of the exercise pressor reflex

McCord, J. L., Tsuchimochi, H., Kaufman, M. P. — 2009-06-29

The exercise pressor reflex is evoked by both mechanical and metabolic stimuli arising in contracting skeletal muscle. Recently, the blockade of acid-sensing ion channels (ASICs) with amiloride and A-316567 attenuated the reflex. Moreover, amiloride had no effect on the mechanoreceptor component of the reflex, prompting us to determine whether ASICs contributed to the metaboreceptor component of the exercise pressor reflex. The metaboreceptor component can be assessed by measuring mean arterial pressure during postcontraction circulatory occlusion when only the metaboreceptors are stimulated. We examined the effects of amiloride (0.5 µg/kg), A-317567 (10 mM, 0.5 ml), and saline (0.5 ml) on the pressor response to and after static contraction while the circulation was occluded in 30 decerebrated cats. Amiloride (n = 11) and A-317567 (n = 7), injected into the arterial supply of the triceps surae muscles, attenuated the pressor responses both to contraction while the circulation was occluded and to postcontraction circulatory occlusion (all, P < 0.05). Saline (n = 11), however, had no effect on the pressor responses to contraction while the circulation was occluded or to postcontraction circulatory occlusion (both, P > 0.79). Our findings led us to conclude that ASICs contribute to the metaboreceptor component of the exercise pressor reflex.

Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43

Heyman, N. S., Kurjiaka, D. T., Ek Vitorin, J. F., Burt, J. M. — 2009-06-29

Expression of connexin 40 (Cx40) and Cx43 in cardiovascular tissues varies as a function of age, injury, and development with unknown consequences on the selectivity of junctional communication and its acute regulation. We investigated the PKC-dependent regulation of charge selectivity in junctions composed of Cx43, Cx40, or both by simultaneous assessment of junctional permeance rate constants (B_dye) for dyes of similar size but opposite charge, N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; +1) and Alexa 350 (–1). The ratio of dye rate constants (B_NBD-M-TMA/B_{Alexa 350}) indicated that Cx40 junctions are cation selective (10.7 ± 0.5), whereas Cx43 junction are nonselective (1.22 ± 0.14). In coexpressing cells, a broad range of junctional selectivities was observed with mean cation selectivity increasing as the Cx40 to Cx43 expression ratio increased. PKC activation reduced or eliminated dye permeability of Cx43 junctions without altering their charge selectivity, had no effect on either permeability or charge selectivity of Cx40 junctions, and significantly increased the cation selectivity of junctions formed by coexpressing cells (approaching charge selectivity of Cx40 junctions). Junctions composed of Cx43 truncated at residue 257 (Cx43tr) were also not charge selective, but when Cx43tr was coexpressed with Cx40, a broad range of junctional selectivities that was unaffected by PKC activation was observed. Thus, whereas the charge selectivities of homomeric/homotypic Cx43 and Cx40 junctions appear invariant, the selectivities of junctions formed by cells coexpressing Cx40 and Cx43 vary considerably, reflecting both their relative expression levels and phosphorylation-dependent regulation. Such regulation could represent a mechanism by which coexpressing cells such as vascular endothelium and atrial cells regulate acutely the selective intercellular communication mediated by their gap junctions.

Periadventitial adipose tissue impairs coronary endothelial function via PKC-{beta}-dependent phosphorylation of nitric oxide synthase

Payne, G. A., Bohlen, H. G., Dincer, U. D., Borbouse, L., Tune, J. D. — 2009-06-29

Endogenous periadventitial adipose-derived factors have been shown to contribute to coronary vascular regulation by impairing endothelial function through a direct inhibition of endothelial nitric oxide synthase (eNOS). However, our understanding of the underlying mechanisms remains uncertain. Accordingly, this study was designed to test the hypothesis that periadventitial adipose tissue releases agents that attenuate coronary endothelial nitric oxide production via a protein kinase C (PKC)-β-dependent mechanism. Isometric tension studies were conducted on isolated canine circumflex coronary arteries with and without natural amounts of periadventitial adipose tissue. Adipose tissue significantly diminished coronary endothelial-dependent vasodilation and nitric oxide production in response to bradykinin and acetylcholine. The selective inhibition of endothelial PKC-β with ruboxistaurin (1 µM) abolished the adipose-induced impairment of bradykinin-mediated coronary vasodilation and the endothelial production of nitric oxide. Western blot analysis revealed a significant increase in eNOS phosphorylation at the inhibitory residue Thr⁴⁹⁵ in arteries exposed to periadventitial adipose tissue. This site-specific phosphorylation of eNOS was prevented by the inhibition of PKC-β. These data demonstrate that periadventitial adipose-derived factors impair coronary endothelial nitric oxide production via a PKC-β-dependent, site-specific phosphorylation of eNOS at Thr⁴⁹⁵.

The effects of propofol on vascular function in mesenteric arteries of the aging rat

Gragasin, F. S., Davidge, S. T. — 2009-06-29

Hypotension following administration of propofol, an anesthetic agent, is strongly predicted by advanced age and is partly due to direct vasodilation. We hypothesized that propofol increases nitric oxide (NO)-mediated vasodilation by enhancing its bioavailability in the aged adult vasculature, leading to greater vasodilation than in the young adult. Small mesenteric arteries from rats aged 13–15 versus 3 to 4 mo were compared in this study. Reactivity to propofol (1–100 µM) alone and with the addition of acetylcholine (ACh; 0.1–10 µM) in endothelial-intact and dunuded arteries following phenylephrine constriction was assessed using myography. N^G-nitro-l-arginine methyl ester (l-NAME) and meclofenamate (Meclo) were used to inhibit NO and prostaglandin synthesis, respectively. Superoxide dismutase (SOD) and catalase were used as antioxidants during ACh relaxation and were compared with propofol in aging arteries. Propofol alone induced greater relaxation in 1) endothelial-intact compared with denuded arteries and 2) aged compared with young arteries, which were inhibited by l-NAME. ACh-induced relaxation was greater in young compared with aged control arteries; however, propofol pretreatment increased this relaxation in aged but not in young arteries. Additionally, propofol inhibited ACh-induced relaxation in arteries treated with l-NAME + Meclo [relaxation attributed to endothelium-derived hyperpolarizing factor (EDHF)]. Pretreatment with SOD and catalase increased relaxation to ACh in aged arteries similar to propofol. In conclusion, propofol causes relaxation in small mesenteric arteries in an endothelial-dependent and independent manner and increases ACh-induced relaxation in aged arteries. Interestingly, propofol inhibits EDHF-mediated relaxation but increases availability of NO, which leads to overall vascular relaxation.

Transmural pressure and axial loading interactively regulate arterial remodeling ex vivo

Lawrence, A. R., Gooch, K. J. — 2009-06-29

Physiological axial strains range between 40 and 60% in arteries, resulting in stresses comparable to those due to normal blood pressure or flow. To investigate the contribution of axial strain to arterial remodeling and function, porcine carotid arteries were cultured for 9 days at physiological and reduced axial stretch ratios in the presence of normotensive and hypertensive transmural pressures by ex vivo perfusion techniques. Consistent with previous in vivo studies, vessels cultured with physiological levels of axial strain and exposed to hypertensive pressure had greater mass, wall area, and outer diameter relative to those cultured at the same axial stretch ratio and normotensive pressure. Reducing the amount of axial strain resulted in mass loss and decreased cell proliferation. Culture in a hypertensive pressure environment at reduced axial strain produced arteries with greater contractility in response to norepinephrine. Arteries cultured at reduced axial strain with the matrix metalloproteinase inhibitor GM6001 maintained their masses over culture, indicating a possible mechanism for this model of axial stretch-dependent remodeling. Although not historically considered one of the primary stimuli for remodeling, multiple linear regression analysis revealed that axial strain had an impact similar to or greater than transmural pressure on various remodeling indexes (i.e., outer diameter, wall area, and wet mass), suggesting that axial strain is a primary mediator of vascular remodeling.

A novel system for the reconstruction of a coronary artery lumen profile in real time: a preclinical validation

2009-06-29

Accurate sizing of vessel diameter is important for understanding the physiology of blood vessels as well as the treatment of coronary and peripheral artery disease. The objective of this study was to validate a novel catheter-based system [the LumenRECON (LR) system] for the real-time reconstruction of lumen cross-sectional area (CSA) along the length of a vessel segment. A total of 22 swine (20 Yorkshire and 2 atherosclerotic Ossabaw swine) were used to evaluate the accuracy, reproducibility, and safety of the system compared with intravascular ultrasound (IVUS). The CSA of the right coronary artery, left anterior descending coronary artery, and left circumflex artery were determined by IVUS and the LR system over a 3- to 4-cm segment in 12 Yorkshire and 2 atherosclerotic Ossabaw swine and 2 postmortem atherosclerotic human hearts. In eight chronic animals, the effect of the LR catheter on the vessel wall was evaluated at 1 day and 2 wk (4 animals each) after the intervention. A Bland-Altman plot of the LR and IVUS data showed a mean difference between the two measurements of 0.055 mm in diameter, which was not statistically significant from zero, indicating a lack of bias in the comparison of the LR system with IVUS. The root mean square error of the two measurements was 10.2% of the mean IVUS diameter. The repeatability of the LR system was assessed using duplicate measurements. The mean of the difference between the two measurements was nearly zero, and the repeatability coefficient was within 4.5% of the mean of the two measurements. No injury or intimal hyperplasia was found acutely or chronically after the use of the LR system. This study establishes the accuracy, reproducibility, and safety of a nonimaging 2.7-Fr catheter for lumen sizing of coronary arteries. The system provides a continuous quantitative axial profile of the mean vessel lumen in real time and may have significant utility in vascular research and clinically in the catheterization laboratory.