AJP: Heart and Circulatory Physiology recent issues

Ryanodine receptor Ca2+ sensitivity and excitation-contraction coupling in muscular dystrophy and heart failure: similar and yet different

Shannon, T. R. — Mon, 23 Nov 2009 12:52:55 PST

The challenge of traditional Chinese medicines for allopathic practitioners

Karalliedde, L. D., Kappagoda, C. T. — Mon, 23 Nov 2009 12:52:55 PST

Differential phosphoinositide 3-kinase signaling: implications for PTCA?

Rocic, P. — Mon, 23 Nov 2009 12:52:55 PST

Is endogenous ouabain a physiological regulator of cardiovascular and renal function?

Buckalew, V. — Mon, 23 Nov 2009 12:52:55 PST

Neuregulin-1 attenuated doxorubicin-induced decrease in cardiac troponins

Mon, 23 Nov 2009 12:52:55 PST

Neuregulin-1 (NRG1) is a potential therapeutic agent for the treatment of doxorubicin (Dox)-induced heart failure. NRG1, however, activates the erbB2 receptor, which is frequently overexpressed in breast cancers. It is, therefore, important to understand how NRG1, via erbB2, protects the heart against Dox cardiotoxicity. Here, we studied NRG1-erbB2 signaling in Dox-treated mice hearts and in isolated neonatal rat ventricular myocytes (NRVM). Male C57BL/6 mice were treated with recombinant NRG1 before and daily after a single dose of Dox. Cardiac function was determined by catheterization. Two-week survival was analyzed by the Kaplan-Meier method. Cardiac troponins [cardiac troponin I (cTnI) and cardiac troponin T (cTnT)] and phosphorylated Akt protein levels were determined in mice hearts and in NRVM by Western blot analysis. Activation of caspases and ubiquitinylation of troponins were determined in NRVM by caspase assay and immunoprecipitation. NRG1 significantly improved survival and cardiac function in Dox-treated mice. NRG1 reduced the decrease in cTnI, cTnT, and cardiac troponin C (cTnC) and maintained Akt phosphorylation in Dox-treated mice hearts. NRG1 reduced the decrease in cTnI and cTnT mRNA and proteins in Dox-treated NRVM. Inhibition of erbB2, phosphoinositide 3-kinase (PI3K), Akt, and mTOR blocked the protective effects of NRG1 on cTnI and cTnT in NRVM. NRG1 significantly reduced Dox-induced caspase activation, which degraded troponins, in NRVM. NRG1 reduced Dox-induced proteasome degradation of cTnI. NRG1 attenuates Dox-induced decrease in cardiac troponins by increasing transcription and translation and by inhibiting caspase activation and proteasome degradation of troponin proteins. NRG1 maintains cardiac troponins by the erbB2-PI3K pathway, which may lessen Dox-induced cardiac dysfunction.

Effect of global ischemia and reperfusion during ventricular fibrillation in myopathic human hearts

Mon, 23 Nov 2009 12:52:55 PST

The effect of lack of global coronary perfusion on myocardial activation rate, wavebreak, and its temporal progression during human ventricular fibrillation (VF) is not known. We tested the hypothesis that global myocardial ischemia decreases activation rate and spatiotemporal organization during VF in myopathic human hearts, while increasing wavebreak, and that a short duration of reperfusion can restore these spatiotemporal changes to baseline levels. The electrograms were acquired during VF in a human Langendorff model using global mapping consisting of two 112-electrode arrays placed on the epicardium and endocardium simultaneously. We found that global myocardial ischemia results in slowing of the global activation rate (combined endo and epi), from 4.89 ± 0.04 Hz. to 3.60 ± 0.04 Hz. during the 200 s of global ischemia (no coronary flow) (P < 0.01) in eight myopathic hearts. Two minutes of reperfusion contributed to reversal of the slowing with activation rate value increasing close to VF onset (4.72 ± 0.04 Hz). In addition, during the period of ischemia, an activation rate gradient between the endocardium (3.76 ± 0.06 Hz) and epicardium (3.45 ± 0.06 Hz) was observed (P < 0.01). There was a concomitant difference in wavebreak index (that provides a normalized parameterization of phase singularities) between the epicardium (11.29 ± 2.7) and endocardium (3.25 ± 2.7) during the 200 s of ischemia (P = 0.02). The activation rate, gradient, and wavebreak changes were reversed by short duration (2 min) of reperfusion. Global myocardial ischemia of 3 min leads to complex spatiotemporal changes during VF in myopathic human hearts; these changes can be reversed by a short duration of reperfusion.

Hypersensitivity of excitation-contraction coupling in dystrophic cardiomyocytes

Ullrich, N. D., Fanchaouy, M., Gusev, K., Shirokova, N., Niggli, E. — Mon, 23 Nov 2009 12:52:56 PST

Duchenne muscular dystrophy represents a severe inherited disease of striated muscle. It is caused by a mutation of the dystrophin gene and characterized by a progressive loss of skeletal muscle function. Most patients also develop a dystrophic cardiomyopathy, resulting in dilated hypertrophy and heart failure, but the cellular mechanisms leading to the deterioration of cardiac function remain elusive. In the present study, we tested whether defective excitation-contraction (E-C) coupling contributes to impaired cardiac performance. "E-C coupling gain" was determined in cardiomyocytes from control and dystrophin-deficient mdx mice. To this end, L-type Ca²⁺ currents (I_CaL) were measured with the whole cell patch-clamp technique, whereas Ca²⁺ transients were simultaneously recorded with confocal imaging of fluo-3. Initial findings indicated subtle changes of E-C coupling in mdx cells despite matched Ca²⁺ loading of the sarcoplasmic reticulum (SR). However, lowering the extracellular Ca²⁺ concentration, a maneuver used to unmask latent E-C coupling problems, was surprisingly much better tolerated by mdx myocytes, suggesting a hypersensitive E-C coupling mechanism. Challenging the SR Ca²⁺ release by slow elevations of the intracellular Ca²⁺ concentration resulted in Ca²⁺ oscillations after a much shorter delay in mdx cells. This is consistent with an enhanced Ca²⁺ sensitivity of the SR Ca²⁺-release channels [ryanodine receptors (RyRs)]. The hypersensitivity could be normalized by the introduction of reducing agents, indicating that the elevated cellular ROS generation in dystrophy underlies the abnormal RyR sensitivity and hypersensitive E-C coupling. Our data suggest that in dystrophin-deficient cardiomyocytes, E-C coupling is altered due to potentially arrhythmogenic changes in the Ca²⁺ sensitivity of redox-modified RyRs.

Traditional Chinese medication Tongxinluo dose-dependently enhances stability of vulnerable plaques: a comparison with a high-dose simvastatin therapy

Mon, 23 Nov 2009 12:52:56 PST

This study was carried out to test the hypothesis that Tongxinluo (TXL) as a Chinese herbal medicine enhances stability of vulnerable plaque dose dependently via lipid-lowering and anti-inflammation effects, similar to a high-dose simvastatin therapy. After abdominal aortic balloon injury, 75 rabbits were fed a 1% cholesterol diet for 10 wk and were then divided into five groups for 8-wk treatment: control group, low-dose TXL group, moderate-dose TXL group, high-dose TXL group, and high-dose simvastatin group. At the end of week 16, an adenovirus containing p53 was injected into the abdominal aortic plaques. Two weeks later, plaque rupture was induced by pharmacological triggering. The incidence of plaque rupture in all treatment groups (14.3%, 7.1%, 7.7%, and 7.1%) was significantly lower than that in control group (73.3%; P > 0.01). TXL dose-dependently lowered serum lipid levels and inhibited systemic inflammation. Corrected acoustic intensity and fibrous cap thickness of the aortic plaques were significantly increased, whereas plaque area, plaque burden, vulnerable index, and expression of oxidized low-density lipoprotein (ox-LDL) receptor 1, matrix metalloproteinase 1 (MMP-1), MMP-3, tissue inhibitor of MMP 1, and NF-B in plaques were markedly reduced in all treatment groups when compared with the control group. Similar to high-dose simvastatin group, high-dose TXL group exhibited a low serum level of low-density lipoprotein cholesterol and ox-LDL, a low expression level of systemic and local inflammatory factors and a low plaque vulnerability index, with no differences in the incidence of plaque rupture among all treatment groups. TXL dose-dependently enhances the stability of vulnerable plaques and prevents plaques from rupture. Simvastatin and TXL offer similar protection in terms of lipid-lowering, anti-inflammation, and antioxidation effects.

Differential regulation of vascular smooth muscle and endothelial cell proliferation in vitro and in vivo by cAMP/PKA-activated p85{alpha}PI3K

Mon, 23 Nov 2009 12:52:56 PST

cAMP inhibits proliferation in most cell types, triggering different and sometimes opposing molecular pathways. p85 (phosphatidylinositol 3-kinase regulatory subunit) is phosphorylated by cAMP/PKA in certain cell lineages, but its effects on vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are unknown. In the present study, we evaluated 1) the role of p85 in the integration of cAMP/PKA-dependent signaling on the regulation of VSMC and EC growth in vitro; and 2) the effects of PKA-modified p85 on neointimal hyperplasia and endothelial healing after balloon injury in vivo. Plasmid constructs carrying wild-type and PKA-modified p85 were employed in VSMCs and ECs in vitro and after balloon injury in rat carotid arteries in vivo. cAMP/PKA reduced VSMC proliferation through p85 phosphorylation. Transfected PKA-activated p85 binds p21^ras, reducing ERK1/2 activation and VSMC proliferation in vitro. In contrast, EC proliferation inhibition by cAMP is independent from PKA modification of p85 and ERK1/2 inhibition; indeed, PKA-activated p85 did not inhibit per se ERK1/2 activation and proliferation in ECs in vitro. Interestingly, cAMP reduced both VSMC and EC apoptotic death through p85 phosphorylation. Accordingly, PKA-activated p85 triggered Akt activation, reducing both VSMC and EC apoptosis in vitro. Finally, compared with controls, vascular gene transfer of PKA-activated p85 significantly reduced neointimal formation after balloon injury in rats, without inhibiting endothelial regeneration of the injured arterial segment. In conclusions, PKA-activated p85 integrates cAMP/PKA signaling differently in VSMCs and ECs. By reducing neointimal hyperplasia without inhibiting endothelial regeneration, it exerts a protective effect against restenosis after balloon injury.

Physiological roles of endogenous ouabain in normal rats

Nesher, M., Dvela, M., Igbokwe, V. U., Rosen, H., Lichtstein, D. — Mon, 23 Nov 2009 12:52:56 PST

Endogenous ouabain (EO)-like compounds are synthesized in and released from the adrenal gland. Although EO has been implicated in several pathological states such as hypertension and heart and kidney failure, its physiological roles in normal animal have not been elucidated. To address this issue, we studied the effects of reduction in plasma EO resulting from antiouabain antibody administration. Normal rats were treated for 28 days with antiouabain antibodies or rabbit IgG as control. Infusions were delivered through a jugular vein cannula by osmotic pumps, and blood pressure was monitored by tail-cuff plethysmography. The animals were housed in metabolic cages to measure water and food consumption and urine excretion. After 28 days, the thoracic aorta was isolated and used to study phenylephrine-induced contraction and atrial natriuretic peptide (ANP)-induced vasorelaxation. The adrenal gland cortex was enlarged in the antiouabain antibody-treated rats. Moreover, on the second day of treatment, there was a significant transient reduction in natriuresis in the antiouabain antibody-treated rats, suggesting that EO is a natriuretic hormone. Reduction in natriuresis was also observed when EO levels were reduced by active immunization resulting from sequential injection of ouabain-albumin. Furthermore, following 28 days of treatment, the response to phenylephrine was significantly lowered and that to ANP was significantly increased in aortic rings from antiouabain antibody-treated rats. These findings show for the first time that circulatory ouabain plausibly originating in the adrenal has physiological roles controlling vasculature tone and sodium homeostasis in normal rats.

Myocardial reperfusion injury management: erythropoietin compared with postconditioning

Mon, 23 Nov 2009 12:52:56 PST

Ischemic postconditioning (IPost) and erythropoietin (EPO) have been shown to attenuate myocardial reperfusion injury using similar signaling pathways. The aim of this study was to examine whether EPO is as effective as IPost in decreasing postischemic myocardial injury in both Langendorff-isolated-heart and in vivo ischemia-reperfusion rat models. Rat hearts were subjected to 25 min ischemia, followed by 30 min or 2 h of reperfusion in the isolated-heart study. Rats underwent 45 min ischemia, followed by 24 h of reperfusion in the in vivo study. In both studies, the control group (n = 12; ischemia-reperfusion only) was compared with IPost (n = 16; 3 cycles of 10 s reperfusion/10 s ischemia) and EPO (n = 12; 1,000 IU/kg) at the onset of reperfusion. The following resulted. First, in the isolated hearts, IPost or EPO significantly improved postischemic recovery of left ventricular developed pressure. EPO induced better left ventricular developed pressure than IPost at 30 min of reperfusion (73.18 ± 10.23 vs. 48.11 ± 7.92 mmHg, P < 0.05). After 2 h of reperfusion, the infarct size was significantly lower in EPO-treated hearts compared with IPost and control hearts (14.36 ± 0.60%, 19.11 ± 0.84%, and 36.21 ± 4.20% of the left ventricle, respectively; P < 0.05). GSK-3β phosphorylation, at 30 min of reperfusion, was significantly higher with EPO compared with IPost hearts. Phosphatidylinositol 3-kinase and ERK1/2 inhibitors abolished both EPO- and IPost-mediated cardioprotection. Second, in vivo, IPost and EPO induced an infarct size reduction compared with control (40.5 ± 3.6% and 28.9 ± 3.1%, respectively, vs. 53.7 ± 4.3% of the area at risk; P < 0.05). Again, EPO decreased significantly more infarct size and transmurality than IPost (P < 0.05). In conclusion, with the use of our protocols, EPO showed better protective effects than IPost against reperfusion injury through higher phosphorylation of GSK-3β.

Transforming growth factor-{beta} signaling in hypertensive remodeling of porcine aorta

Mon, 23 Nov 2009 12:52:56 PST

A porcine aortic coarctation model was used to examine regulation of gene expression in early hypertensive vascular remodeling. Aortic segments were collected proximal (high pressure) and distal (low pressure) to the coarctation after 2 wk of sustained hypertension (mean arterial pressure > 150 mmHg). Porcine 10K oligoarrays used for gene expression profiling of the two regions of aorta revealed downregulation of cytoskeletal and upregulation of extracellular region genes relative to the whole genome. A genomic database search for transforming growth factor-β (TGF-β) control elements showed that 19% of the genes that changed expression due to hypertension contained putative TGF-β control elements. Real-time RT-PCR and microarray analysis showed no change in expression of TGF-β₁, TGF-β₂, TGF-β₃, or bone morphogenetic proteins-2 and -4, yet immunohistochemical staining for phosphorylated SMAD2, an indicator of TGF-β signaling, and for phosphorylated SMAD1/5/8, an indicator of signaling through the bone morphogenetic proteins, showed the highest percentage of positively stained cells in the proximal aortic segments of occluded animals. For TGF-β signaling, this increase was significantly different than for sham-operated controls. Western blot analysis showed no difference in total TGF-β₁ protein levels with respect to treatment or aortic segment. Immunohistochemistry showed that the protein levels of latency-associated peptide was decreased in proximal segments of occluded animals. Collectively, these results suggest that activation of TGF-β, but not altered expression, may be a major mechanism regulating early hypertensive vascular remodeling.

Refined approach for quantification of in vivo ischemia-reperfusion injury in the mouse heart

Mon, 23 Nov 2009 12:52:56 PST

Cardiac ischemia-reperfusion experiments in the mouse are important in vivo models of human disease. Infarct size is a particularly important scientific readout as virtually all cardiocirculatory pathways are affected by it. Therefore, such measurements must be exact and valid. The histological analysis, however, remains technically challenging, and the resulting quality is often unsatisfactory. For this report we have scrutinized each step involved in standard double-staining histology. We have tested published approaches and challenged their practicality. As a result, we propose an improved and streamlined protocol, which consistently yields high-quality histology, thereby minimizing experimental noise and group sizes.

Estrogen replacement therapy in diabetic ovariectomized female rats potentiates postischemic leukocyte adhesion in cerebral venules via a RAGE-related process

Mon, 23 Nov 2009 12:52:56 PST

In this study, we tested the hypothesis that the documented transformation of 17β-estradiol (E₂) from a counterinflammatory hormone in nondiabetic (ND) rats to a proinflammatory agent in rats with diabetes mellitus (DM) is due to an enhanced contribution from the receptor for advanced glycation end products (RAGE). Rhodamine 6G-labeled leukocytes were observed through a closed cranial window in rats. In vivo pial venular leukocyte adherence and infiltration were measured over 10 h reperfusion after transient forebrain ischemia in DM (streptozotocin) versus ND intact, ovariectomized (OVX), and E₂-replaced (for 7–10 days) OVX (OVE) females. The role of RAGE was examined in two ways: 1) RAGE knockdown via topical application of RAGE antisense versus missense oligodeoxynucleotide or 2) intracerebroventricular injection of the RAGE decoy inhibitor, soluble RAGE. Among diabetic rats, the lowest levels of cortical RAGE mRNA and immunoreactivity of the RAGE ligand, AGE, were seen in OVX females, with significantly higher levels exhibited in intact and OVE females. However, results from the analysis of cortical RAGE protein only partially tracked those findings. When comparing ND to DM rats, cortical AGE immunoreactivity was significantly lower in OVE and intact females but similar in OVX rats. In DM rats, the level of postischemic leukocyte adhesion and infiltration (highest to lowest) was OVE > intact >> untreated OVX. In NDs, adhesion was highest in the untreated OVX group. Leukocyte extravasation was observed at >6 h postischemia but only in diabetic OVE and intact females and in ND OVX (untreated) rats. Pretreatment with RAGE antisense-oligodeoxynucleotide or soluble RAGE attenuated postischemic leukocyte adhesion and prevented infiltration but only in the diabetic OVE and intact groups. These results indicate that the exacerbation of postischemic leukocyte adhesion by chronic E₂ replacement therapy in diabetic OVX females involves a RAGE-related mechanism. Targeting RAGE may restore the neuroprotective effect of E₂ replacement therapy in diabetic females.

Generation of nitric oxide from nitrite by carbonic anhydrase: a possible link between metabolic activity and vasodilation

Aamand, R., Dalsgaard, T., Jensen, F. B., Simonsen, U., Roepstorff, A., Fago, A. — Mon, 23 Nov 2009 12:52:56 PST

In catalyzing the reversible hydration of CO₂ to bicarbonate and protons, the ubiquitous enzyme carbonic anhydrase (CA) plays a crucial role in CO₂ transport, in acid-base balance, and in linking local acidosis to O₂ unloading from hemoglobin. Considering the structural similarity between bicarbonate and nitrite, we hypothesized that CA uses nitrite as a substrate to produce the potent vasodilator nitric oxide (NO) to increase local blood flow to metabolically active tissues. Here we show that CA readily reacts with nitrite to generate NO, particularly at low pH, and that the NO produced in the reaction induces vasodilation in aortic rings. This reaction occurs under normoxic and hypoxic conditions and in various tissues at physiological levels of CA and nitrite. Furthermore, two specific inhibitors of the CO₂ hydration, dorzolamide and acetazolamide, increase the CA-catalyzed production of vasoactive NO from nitrite. This enhancing effect may explain the known vasodilating effects of these drugs and indicates that CO₂ and nitrite bind differently to the enzyme active site. Kinetic analyses show a higher reaction rate at high pH, suggesting that anionic nitrite participates more effectively in catalysis. Taken together, our results reveal a novel nitrous anhydrase enzymatic activity of CA that would function to link the in vivo main end products of energy metabolism (CO₂/H⁺) to the generation of vasoactive NO. The CA-mediated NO production may be important to the correlation between blood flow and metabolic activity in tissues, as occurring for instance in active areas of the brain.

Expression of constitutively active cGMP-dependent protein kinase inhibits glucose-induced vascular smooth muscle cell proliferation

Wang, S., Li, Y. — Mon, 23 Nov 2009 12:52:56 PST

Previously, we have demonstrated that cGMP-dependent protein kinase (PKG) activity is downregulated in vessels from diabetic animals or in vascular smooth muscle cells (VSMCs) exposed to high-glucose conditions, contributing to diabetes-associated vessel dysfunction. However, whether decreased PKG activity plays a role in hyperglycemia-induced proliferation of VSMCs is unknown. In this report, high-glucose-mediated decreased PKG activity in VSMCs was restored by transfection of cells with expression vector for the catalytic domain of PKG-I (PKG-CD, constitutive active PKG). The effect of glucose on cell proliferation was determined. Our data demonstrated that high glucose exposure stimulated VSMC proliferation and G₁ to S phase progression of the cell cycle, which was inhibited by restoration of PKG activity. Expression of constitutively active PKG inhibited G₁ phase exit in VSMCs under high glucose conditions, which was accompanied by an inhibition of retinoblastoma protein (Rb) phosphorylation (a key switch for G₁ to S phase cell cycle progression). Glucose-induced cyclin E expression and cyclin E-cyclin-dependent kinase 2 activity was also reduced by expression of PKG-CD in VSMCs. Moreover, expression of PKG-CD suppressed glucose-induced p27 degradation. These data demonstrate that restoring the high-glucose-mediated decrease in PKG activity in VSMCs inhibits glucose-induced abnormal VSMC proliferation occurring upstream of Rb phosphorylation. Our work provides the first direct evidence linking decreased PKG activity to high glucose-induced proliferation and cell cycle progression in VSMCs, suggesting that strategies to increase PKG activity might be useful in preventing abnormal VSMC proliferation in diabetic patients and might provide treatments for diabetes-associated proliferative vascular diseases.

Increased phase synchronization and decreased cerebral autoregulation during fainting in the young

Ocon, A. J., Kulesa, J., Clarke, D., Taneja, I., Medow, M. S., Stewart, J. M. — Mon, 23 Nov 2009 12:52:56 PST

Vasovagal syncope may be due to a transient cerebral hypoperfusion that accompanies frequency entrainment between arterial pressure (AP) and cerebral blood flow velocity (CBFV). We hypothesized that cerebral autoregulation fails during fainting; a phase synchronization index (PhSI) between AP and CBFV was used as a nonlinear, nonstationary, time-dependent measurement of cerebral autoregulation. Twelve healthy control subjects and twelve subjects with a history of vasovagal syncope underwent 10-min tilt table testing with the continuous measurement of AP, CBFV, heart rate (HR), end-tidal CO₂ (ETco₂), and respiratory frequency. Time intervals were defined to compare physiologically equivalent periods in fainters and control subjects. A PhSI value of 0 corresponds to an absence of phase synchronization and efficient cerebral autoregulation, whereas a PhSI value of 1 corresponds to complete phase synchronization and inefficient cerebral autoregulation. During supine baseline conditions, both control and syncope groups demonstrated similar oscillatory changes in phase, with mean PhSI values of 0.58 ± 0.04 and 0.54 ± 0.02, respectively. Throughout tilt, control subjects demonstrated similar PhSI values compared with supine conditions. Approximately 2 min before fainting, syncopal subjects demonstrated a sharp decrease in PhSI (0.23 ± 0.06), representing efficient cerebral autoregulation. Immediately after this period, PhSI increased sharply, suggesting inefficient cerebral autoregulation, and remained elevated at the time of faint (0.92 ± 0.02) and during the early recovery period (0.79 ± 0.04) immediately after the return to the supine position. Our data demonstrate rapid, biphasic changes in cerebral autoregulation, which are temporally related to vasovagal syncope. Thus, a sudden period of highly efficient cerebral autoregulation precedes the virtual loss of autoregulation, which continued during and after the faint.

Type 1 diabetic cardiomyopathy in the Akita (Ins2WT/C96Y) mouse model is characterized by lipotoxicity and diastolic dysfunction with preserved systolic function

Mon, 23 Nov 2009 12:52:56 PST

Diabetic cardiomyopathy is an important contributor to diastolic and systolic heart failure. We examined the nature and mechanism of the cardiomyopathy in Akita (Ins2^WT/C96Y) mice, a model of genetic nonobese type 1 diabetes that recapitulates human type 1 diabetes. Cardiac function was evaluated in male Ins2^WT/C96Y and their littermate control (Ins2^WT/WT) mice using echocardiography and tissue Doppler imaging, in vivo hemodynamic measurements, as well as ex vivo working heart preparation. At 3 and 6 mo of age, Ins2^WT/C96Y mice exhibited preserved cardiac systolic function compared with Ins2^WT/WT mice, as evaluated by ejection fraction, fractional shortening, left ventricular (LV) end-systolic pressure and maximum rate of increase in LV pressure in vivo, cardiac work, cardiac power, and rate-pressure product ex vivo. Despite the unaltered systolic function, Ins2^WT/C96Y mice exhibited significant and progressive diastolic dysfunction at 3 and 6 mo of age compared with Ins2^WT/WT mice as assessed by tissue and pulse Doppler imaging (E-wave velocity, isovolumetric relaxation time) and by in vivo hemodynamic measurements (LV end-diastolic pressure, time constant of LV relaxation, and maximum rate of decrease in LV pressure). We found no evidence of myocardial hypertrophy or fibrosis in the Ins2^WT/C96Y myocardium. Consistent with the lack of fibrosis, expression of procollagen- type I, procollagen- type III, and fibronectin were not increased in these hearts. Ins2^WT/C96Y hearts showed significantly reduced sarcoplasmic reticulum Ca²⁺-ATPase 2a (cardiac sarcoplasmic reticulum Ca²⁺ pump) levels, elevated β-myosin heavy chain isoform, increased long-chain fatty acids, and triacylglycerol with evidence of lipotoxicity, as indicated by a significant rise in ceramide, diacylglycerol, and lipid deposits in the myocardium. Consistent with metabolic perturbation, and a switch to fatty acid oxidation from glucose oxidation in Ins2^WT/C96Y hearts, expression of mitochondrial long-chain acyl-CoA dehydrogenase and pyruvate dehydrogenase kinase isoform 4 were increased. Insulin treatment reversed the diastolic dysfunction, the elevated B-type natriuretic peptide and β-myosin heavy chain, and the reduced sarcoplasmic reticulum Ca²⁺-ATPase 2a levels with abolition of cardiac lipotoxicity. We conclude that early type 1 diabetic cardiomyopathy is characterized by diastolic dysfunction associated with lipotoxic cardiomyopathy with preserved systolic function in the absence of interstitial fibrosis and hypertrophy.

Myocardial fibrosis and apoptosis, but not inflammation, are present in long-term experimental diabetes

Mon, 23 Nov 2009 12:52:56 PST

The aim of this paper is to study the myocardial damage secondary to long-term streptozotocin-induced type 1 diabetes mellitus (DM1). Normotensive and spontaneously hypertensive rats (SHR) received either streptozotocin injections or vehicle. After 22 or 6 wk, DM1, SHR, DM1/SHR, and control rats were killed, and the left ventricles studied by histology, quantitative PCR, Western blot, ELISA, and electromobility shift assay. Cardiomyocyte cultures were also performed. The expression of profibrotic factors, transforming growth factor-β (TGF-β₁), connective tissue growth factor, and matrix proteins was increased, and the TGF-β₁-linked transcription factors phospho-Smad3/4 and activator protein-1 were activated in the DM1 myocardium. Proapoptotic molecules FasL, Fas, Bax, and cleaved caspase-3 were also augmented. Myocardial injury in long-term hypertension shared these features. In addition, hypertension was associated with activation of NF-B, increased inflammatory cell infiltrate, and expression of the mediators [interleukin-1β (IL-1β), tumor necrosis factor-, monocyte chemoattractant protein 1, vascular cell adhesion molecule 1, angiotensinogen, and oxidants], which were absent in long-term DM1. At this stage, the combination of DM1 and hypertension resulted in nonsignificant additive effects. Moreover, the coexistence of DM1 blunted the inflammatory response to hypertension. Anti-inflammatory IL-10 and antioxidants were induced in long-term DM1 and DM1/SHR hearts. Myocardial inflammation was, however, observed in the short-term model. In cultured cardiomyocytes, IL-10, TGF-β₁, and catalase blocked the glucose-stimulated expression of proinflammatory genes. Fibrosis and apoptosis are features of long-term myocardial damage in experimental DM1. Associated hypertension does not induce additional changes. Myocardial inflammation is present in hypertension and short-term DM1, but is not a key feature in long-term DM1. Local reduction of proinflammatory factors and expression of anti-inflammatory and antioxidant molecules may underlie this effect.

Status epilepticus induces cardiac myofilament damage and increased susceptibility to arrhythmias in rats

Metcalf, C. S., Poelzing, S., Little, J. G., Bealer, S. L. — Mon, 23 Nov 2009 12:52:57 PST

Status epilepticus (SE) is a seizure or series of seizures that persist for >30 min and often results in mortality. Death rarely occurs during or immediately following seizure activity, but usually within 30 days. Although ventricular arrhythmias have been implicated in SE-related mortality, the effects of this prolonged seizure activity on the cardiac function and susceptibility to arrhythmias have not been directly investigated. We evaluated myocardial damage, alterations in cardiac electrical activity, and susceptibility to experimentally induced arrhythmias produced by SE in rats. SE resulted in seizure-related increases in blood pressure, heart rate, and the first derivative of pressure, as well as modest, diffuse myocyte damage assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining. Ten to twelve days following seizures, electrocardiographic recordings showed arrhythmogenic alterations in cardiac electrical activity, denoted by prolonged QT interval corrected for heart rate and QT dispersion. Finally, SE increased susceptibility to experimentally induced (intravenous aconitine) cardiac arrhythmias. These data suggest that SE produces tachycardic ischemia following the activation of the sympathetic nervous system, resulting in cardiac myofilament damage, arrhythmogenic alterations in cardiac electrical activity, and increased susceptibility to ventricular arrhythmias.

Shear-induced interaction of platelets with von Willebrand factor results in glycoprotein Ib{alpha} shedding

Cheng, H., Yan, R., Li, S., Yuan, Y., Liu, J., Ruan, C., Dai, K. — Mon, 23 Nov 2009 12:52:57 PST

Shear-induced platelet adhesion through the interaction of glycoprotein (GP) Ib with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture is a prerequisite for the physiological hemostatic process or pathological thrombus formation in stenosed arteries. Here we show that shear-induced interaction of platelets with immobilized VWF results in GPIb ectodomain shedding. Washed platelets were exposed to VWF-coated glass capillary or cone-and-plate viscometer at different shear rates, and GPIb ectodomain was shed from platelets, while a small mass of GPIb COOH-terminal peptide, ~17 kDa, was increased correspondingly. The extent of GPIb shedding was enhanced with the concentration of immobilized VWF and the time duration of constant shear stress, whereas it was obviously reduced with the decreased number of adherent platelets. Pretreatment of platelets with membrane-permeable calpain inhibitors and metalloproteinase inhibitor abolished shear-induced GPIb shedding. Furthermore, GPIb shedding was obviously diminished by anti-integrin-_IIbβ₃ monoclonal antibody SZ21, phosphatidylinositol 3-kinase inhibitor wortmannin, and cell-permeable calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. These results indicate that shear-induced platelet-VWF interaction results in calpain and metalloproteinase-dependent GPIb ectodomain shedding. These findings not only have a physiological implication in understanding the presence of glycocalicin in normal circulation, but also suggest a novel mechanism for the negative regulation of platelet function and the limitation of platelet thrombus infinite formation under pathophysiological flow conditions.

Rate-dependent AV nodal refractoriness: a new functional framework based on concurrent effects of basic and pretest cycle length

Tadros, R., Billette, J. — Mon, 23 Nov 2009 12:52:57 PST

The atrioventricular (AV) node filters atrial impulses. Underlying rate-dependent refractory properties are assessed with the effective (ERPN; longest nonconducted atrial cycle length) and functional (FRPN; shortest His bundle cycle) refractory period determined with premature protocols at different basic rates. Fast rates prolong ERPN and shorten FRPN, but these effects vary with subjects, age, and species. We propose that these opposite and variable effects reflect the net sum of concurrent cumulative and noncumulative effects associated with basic (BCL) and pretest cycle length (PTCL), respectively. To test this hypothesis, we assessed selective and combined effects of five BCL (S₁S₁) and six PTCL (S₁S₂) on ERPN, FRPN, and their subintervals (ERPN = A₂H₂ + H₂A₃ and FRPN = H₂A₃ + A₃H₃, where A is atrium and H is His bundle) with S₁S₂S₃ protocols in six rabbit heart preparations. At control BCL, PTCL shortenings prolonged ERPN (113 ± 12 vs. 101 ± 14 ms, P < 0.01) as a net result of prolonged A₂H₂ and curtailed H₂A₃. At control PTCL, BCL shortenings increased ERPN (127 ± 20 vs. 101 ± 14 ms, P < 0.01) by prolonging A₂H₂. FRPN did not vary with BCL but decreased (163 ± 6 vs. 175 ± 10 ms, P < 0.01) with PTCL that curtailed H₂A₃. Equal BCL and PTCL shortenings as in standard protocols prolonged ERPN but left FRPN unchanged. Notably, ERPN and FRPN significantly correlated through their H₂A₃ subinterval. In conclusion, BCL and PTCL are both important determinants of AV nodal refractoriness and together account for rate-induced changes in ERPN and FRPN observed during standard premature protocols. ERPN and FRPN are related variables. Similar functional rules may govern nodal refractory behavior during supraventricular tachyarrhythmias.

Glutathione peroxidase deficiency exacerbates ischemia-reperfusion injury in male but not female myocardium: insights into antioxidant compensatory mechanisms

Mon, 23 Nov 2009 12:52:57 PST

The female sex has been associated with increased resistance to acute myocardial ischemia-reperfusion (I/R) injury. While enhanced antioxidant capacity has been implicated in female cardioprotection, there is little evidence to support this assumption. Previous studies have shown an important role of cellular glutathione peroxidase (GPx1) in protection of the myocardium from I/R injury. Whether GPx1 is mechanistic in the protection of female myocardium, post-I/R, has not been examined. We utilized a murine model with homozygous deletion of GPx1 and examined its impact on postischemic myocardial recovery in male and female mice. Following I/R, male GPx1(–/–) hearts were more susceptible to contractile and diastolic dysfunction, and this was associated with increased protein carbonyls, a marker of oxidative stress. In contrast, GPx1 deficiency in female hearts did not exacerbate dysfunction or oxidative stress post-I/R. Both wild-type and GPx1(–/–) female hearts exhibited reduced creatine kinase leakage and a more favorable ascorbate redox status compared with males. Following I/R, female GPx1(–/–) hearts showed a comparable decrease in glutathione redox status as their male counterparts; however, they exhibited a greater decrease in nitrate-to-nitrite ratio, suggesting a higher consumption of nitrate in female GPx1(–/–) hearts. Our findings demonstrate that GPx1 is critical for cardioprotection during I/R in male, but not female, mice. The maintenance of cardioprotection in female mice lacking GPx1 post-I/R may be due to an improved ascorbate redox homeostasis and enhanced nitrate-to-nitrite conversion, which would predictably be accompanied by enhanced production of cardioprotective nitric oxide.

RC time constant of single lung equals that of both lungs together: a study in chronic thromboembolic pulmonary hypertension

Mon, 23 Nov 2009 12:52:57 PST

The product of resistance, R, and compliance, C (RC time), of the entire pulmonary circulation is constant. It is unknown if this constancy holds for individual lungs. We determined R and C in individual lungs in chronic thromboembolic pulmonary hypertension (CTEPH) patients where resistances differ between both lungs. Also, the contribution of the proximal pulmonary arteries (PA) to total lung compliance was assessed. Patients (n = 23) were referred for the evaluation of CTEPH. Pressure was measured by right heart catheterization and flows in the main, left, and right PA by magnetic resonance imaging. Total, left, and right lung resistances were calculated as mean pressure divided by mean flow. Total, left, and right lung compliances were assessed by the pulse pressure method. Proximal compliances were derived from cross-sectional area change A and systolic-diastolic pressure difference P (A/P) in main, left, and right PA, multiplied by vessel length. The lung with the lowest blood flow was defined "low flow" (LF), the contralateral lung "high flow" (HF). Total resistance was 0.57 ± 0.28 mmHg·s^–1·ml^–1, and resistances of LF and HF lungs were 1.57 ± 0.2 vs. 1.00 ± 0.1 mmHg·s^–1·ml^–1, respectively, P < 0.0001. Total compliance was 1.22 ± 1.1 ml/mmHg, and compliances of LF and HF lung were 0.47 ± 0.11 and 0.62 ± 0.12 ml/mmHg, respectively, P = 0.01. Total RC time was 0.49 ± 0.2 s, and RC times for the LF and HF lung were 0.45 ± 0.2 and 0.45 ± 0.1 s, respectively, not different. Proximal arterial compliance, given by the sum of main, right, and left PA compliances, was only 19% of total lung compliance. The RC time of a single lung equals that of both lungs together, and pulmonary arterial compliance comes largely from the distal vasculature.

EDHF function in the ductus arteriosus: evidence against involvement of epoxyeicosatrienoic acids and 12S-hydroxyeicosatetraenoic acid

Mon, 23 Nov 2009 12:52:57 PST

We have previously shown (Ref. 2) that endothelium-derived hyperpolarizing factor (EDHF) becomes functional in the fetal ductus arteriosus on removal of nitric oxide and carbon monoxide. From this, it was proposed that EDHF originates from a cytochrome P-450 (CYP450)-catalyzed reaction being inhibited by the two agents. Here, we have examined in the mouse ductus whether EDHF can be identified as an arachidonic acid product of a CYP450 epoxygenase and allied pathways. We did not detect transcripts of the mouse CYP2C subfamily in vessel, while CYP2J subfamily transcripts were expressed with CYP2J6 and CYP2J9. These CYP2J hemoproteins were also detected in the ductus by immunofluorescence microscopy, being colocalized with the endoplasmic reticulum in both endothelial and muscle cells. Distinct CYP450 transcripts were also detected and were responsible for -hydroxylation (CYP4A31) and 12R-hydroxylation (CYP4B1). Mass spectrometric analysis showed formation of epoxyeicosatrienoic acids (EETs) in the intact ductus, with 11,12- and 14,15-EETs being more prominent than 5,6- and 8,9-EETs. However, their yield did not increase with nitric oxide/carbon monoxide suppression, nor did it abate with endothelium removal. No evidence was obtained for formation of 12R-hydroxyeicosatrienoic acid and -hydroxylation products. 2S-hydroxyeicosatetraenoic acid was instead detected, and, contrary to data implicating this compound as an alternative EDHF, its suppression with baicalein did not modify the EDHF-mediated relaxation to bradykinin. We conclude that none of the more common CYP450-linked arachidonic acid metabolites appears to qualify as EDHF in mouse ductus. We speculate that some novel eicosanoid or a totally unrelated compound requiring CYP450 for its synthesis accounts for EDHF in this vessel.

Doxorubicin induces senescence or apoptosis in rat neonatal cardiomyocytes by regulating the expression levels of the telomere binding factors 1 and 2

Mon, 23 Nov 2009 12:52:57 PST

Low or high doses of doxorubicin induce either senescence or apoptosis, respectively, in cardiomyocytes. The mechanism by which different doses of doxorubicin may induce different stress-response cellular programs is not well understood. A recent study showed that the level of telomere dysfunction may induce senescence or apoptosis. We investigated the pathways to both apoptosis and senescence in neonatal rat cardiomyocytes and in H9c2 cells exposed to a single pulsed incubation with low or high doses of doxorubicin. High-dose doxorubicin strongly reduces TRF2 expression while enhancing TRF1 expression, and it determines early apoptosis. Low-dose doxorubicin induces downregulation of both TRF2 and TRF1, and it also increases the senescence-associated-β-galactosidase activity, downregulates the checkpoint kinase Chk2, induces chromosomal abnormalities, and alters the cell cycle. The involvement of TRF1 and TRF2 with apoptosis and senescence was assessed by short interfering RNA interference. The cells maintain telomere dysfunction and a senescent phenotype over time and undergo late death. The increase in the phase >4N and the presence of micronuclei and anaphase bridges indicate that cells die by mitotic catastrophe. p38 modulates TRF2 expression, whereas JNK and cytoplasmic p53 regulate TRF1. Pretreatment with specific inhibitors of MAPKs and p53 may either attenuate the damage induced by doxorubicin or shift the cellular response to stress from senescence to apoptosis. In conclusion, various doses of doxorubicin induce differential regulation of TRF1 and TRF2 through p53 and MAPK, which is responsible for inducing either early apoptosis or senescence and late death due to mitotic catastrophe.

Does conduit artery diameter vary according to the anthropometric characteristics of children or men?

Mon, 23 Nov 2009 12:52:57 PST

Arterial measurements are commonly undertaken to assess acute and chronic adaptations to exercise. Despite the widespread adoption of scaling practices in cardiac research, the relevance of scaling for body size and/or composition has not been addressed for arterial measures. We therefore investigated the relationships between brachial artery diameter and body composition in 129 children aged 9 to 10 yr (75 girls and 54 boys), and 50 men aged 16–49 yr. Body composition variables (total, lean, and fat mass in the whole body, arm, and forearm) were assessed by dual-energy X-ray absorptiometry, and brachial artery diameter was measured using high-resolution ultrasound. Bivariate correlations were performed, and arterial diameter was then scaled using simple ratios (y/x) and allometric approaches after log-log least squares linear regression and production of allometric exponents (b) and construction of power function ratios (y/x^b). Size independence was checked via bivariate correlations (x:y/x; x:y/x^b). As a result, significant correlations existed between brachial artery diameter and measures of body mass and lean mass in both cohorts (r = 0.21–0.48, P < 0.05). There were no significant relationships between diameter and fat mass. All b exponents were significantly different from 1 (0.08–0.50), suggesting that simple ratio scaling approaches were likely to be flawed. This was confirmed when ratio scaling produced negative residual size correlations, whereas allometric scaling produced size-independent indexes (r = 0.00 to 0.03, P > 0.05). In conclusion, when between- or within-group comparisons are performed under circumstances where it is important to control for differences in body size or composition, allometric scaling of artery diameter should be adopted rather than ratio scaling. Our data also suggest that scaling for lean or total mass may be more appropriate than scaling for indexes of fat mass.

Improving insulin sensitivity via activation of PPAR-{gamma} increases telomerase activity in the heart of OLETF rats

Makino, N., Maeda, T., Oyama, J.-i., Higuchi, Y., Mimori, K. — Mon, 23 Nov 2009 12:52:58 PST

This study was conducted to examine telomere biology in terms of improving insulin sensitivity in a type 2 diabetic animal model: Otsuka Long-Evans Tokushima fatty (OLETF) rats. To improve insulin sensitivity, pioglitazone (PG; 10 mg·kg^–1·day^–1) was administrated to OLETF rats from 20 to 40 wk of age, and the effects of treatment were compared with those in untreated OLETF or control Long-Evans Tokushima Otsuka fatty rats. At the end of the study, the homeostasis model assessment of insulin resistance significantly increased in OLETF rats but decreased in OLETF rats treated with PG. No shortening of telomere length was observed in the heart tissue of OLETF rats, whereas telomerase activity was decreased in OLETF heart tissue. The mRNA expression of both telomerase reverse transcriptase and telomere repeat binding factor 2 was downregulated in the hearts of OLETF rats. The protein expression of phospho-Akt, insulin-like growth factor, and endothelial nitric oxide synthase was reduced in OLETF rats. On the other hand, myocardial matrix metalloproteinase-9 expression was elevated in OLETF rats. The changes observed in OLETF rats were inhibited by PG treatment. However, protein and mRNA expression of Sirt1, a lifespan modulator, were attenuated in OLETF rat hearts, although they were enhanced in OLETF rats with PG treatement. Myocardial fibrosis was less extensive and diastolic dysfunction more greatly ameliorated in PG-treated OLETF rats than in OLETF rats. These findings suggest that improving insulin sensitivity via the activation of peroxisom proliferator-activated receptor- may exert regulatory effects on cardiac telomere biology and may have desirable morphological and functional effects on the diabetic heart.

Right ventricular free wall pacing improves cardiac pump function in severe pulmonary arterial hypertension: a computer simulation analysis

Mon, 23 Nov 2009 12:52:58 PST

In pulmonary arterial hypertension (PAH), duration of myofiber shortening is prolonged in the right ventricular (RV) free wall (RVfw) compared with that in the interventricular septum and left ventricular free wall. This interventricular mechanical asynchrony eventually leads to right heart failure. We investigated by computer simulation whether, in PAH, early RVfw pacing may improve interventricular mechanical synchrony and, hence, cardiac pump function. A mathematical model of the human heart and circulation was used to simulate left ventricular and RV pump mechanics and myofiber mechanics. First, we simulated cardiovascular mechanics of a healthy adult at rest. Size and mass of heart and blood vessels were adapted so that mechanical tissue load was normalized. Second, compensated PAH was simulated by increasing mean pulmonary artery pressure to 32 mmHg while applying load adaptation. Third, decompensated PAH was simulated by increasing mean pulmonary artery pressure further to 79 mmHg without further adaptation. Finally, early RVfw pacing was simulated in severely decompensated PAH. Time courses of circumferential strain in the ventricular walls as simulated were similar to the ones measured in healthy subjects (uniform strain patterns) and in PAH patients (prolonged RVfw shortening). When simulating pacing in decompensated PAH, RV pump function was best upon 40-ms RVfw preexcitation, as evidenced by maximal decrease of RV end-diastolic volume, reduced RVfw myofiber work, and most homogeneous distribution of workload over the ventricular walls. Thus our simulations indicate that, in decompensated PAH, RVfw pacing may improve RV pump function and may homogenize workload over the ventricular walls.

Structural adaptation of microvessel diameters in response to metabolic stimuli: where are the oxygen sensors?

Reglin, B., Secomb, T. W., Pries, A. R. — Mon, 23 Nov 2009 12:52:58 PST

Maintenance of functional vascular networks requires structural adaptation of vessel diameters in response to hemodynamic and metabolic conditions. The mechanisms by which diameters respond to the metabolic state are not known, but may involve the release of vasoactive substances in response to low oxygen by tissue ("tissue signaling", e.g., CO₂, adenosine), by vessel walls ("wall signaling", e.g., prostaglandins, adenosine), and/or by red blood cells (RBCs) ("RBC signaling", e.g., ATP and nitric oxide). Here, the goal was to test the potential of each of these locations of oxygen-dependent signaling to control steady-state vascular diameters and tissue oxygenation. A previously developed theoretical model of structural diameter adaptation based on experimental data on microvascular network morphology and hemodynamics was used. Resulting network characteristics were analyzed with regard to tissue oxygenation (Oxdef; percentage of tissue volume with Po₂ < 1 Torr) and the difference between estimated blood flow velocities and corresponding experimental data [velocity error (V_err); root mean square deviation of estimated vs. measured velocity]. Wall signaling led to Oxdef < 1% and to the closest hemodynamic similarity (V_err: 0.60). Tissue signaling also resulted in a low oxygen deficit, but a higher V_err (0.73) and systematic diameter deviations. RBC signaling led to widespread hypoxia (Oxdef: 4.7%), unrealistic velocity distributions (V_err: 0.81), and shrinkage of small vessels. The results suggest that wall signaling plays a central role in structural control of vessel diameters in microvascular networks of given angioarchitecture. Tissue-derived and RBC-derived signaling of oxygen levels may be more relevant for the regulation of angiogenesis and/or smooth muscle tone.

Simultaneous variation of ventricular pacing site and timing with biventricular pacing in acute ventricular failure improves function by interventricular assist

Mon, 23 Nov 2009 12:52:58 PST

The goal of this work was to investigate the hemodynamic effects of simultaneous left ventricular (LV) pacing site (LVPS) and interventricular pacing delay (VVD) variation with biventricular pacing (BiVP) during acute LV failure. Simultaneously varying LVPS and VVD with BiVP has been shown to improve hemodynamics during acute right ventricular (RV) failure. However, effects during acute LV failure have not been reported. In six open-chest pigs, acute LV volume overload was induced by regurgitant flow via an aortic-LV conduit. Epicardial BiVP was implemented with right atrial and ventricular leads and a custom LV pacing array. Fifty-four LVPS-VVD combinations were tested in random order. Cardiac output was evaluated by aortic flow probe, ventricular systolic function by maximum rate of ventricular pressure change, and mechanical interventricular synchrony by normalized RV-LV pressure diagram area. Simultaneous LVPS-VVD variation improved all measures of cardiac function. The observed effect was different for each functional index, with evidence of LVPS-VVD interaction. Compared with effects of LVPS-VVD variation in a model of acute RV failure, hemodynamic changes were markedly different. However, in both models, maximum rate of ventricular pressure change of the failing ventricle was improved with synchronous interventricular contraction, suggesting that, in acute ventricular failure, BiVP can recruit the unstressed ventricle to support systolic function of the failing one. Thus simultaneously varying LVPS and VVD with BiVP during acute ventricular failure can improve cardiac function by "interventricular assist", with hemodynamic effects dependent on the type of failure. This supports the potential utility of temporary BiVP for the treatment of acute ventricular failure commonly seen after cardiac surgery.

Abnormal nitric oxide production in aged rat mesenteric arteries is mediated by NAD(P)H oxidase-derived peroxide

Zhou, X., Bohlen, H. G., Unthank, J. L., Miller, S. J. — Mon, 23 Nov 2009 12:52:58 PST

Previous work in our laboratory showed increased basal periarterial nitric oxide (NO) and H₂O₂ concentrations in the spontaneously hypertensive rat, characterized by oxidant stress, as well as impaired flow-mediated NO production that was corrected by a reduction of periarterial H₂O₂. Aging is also associated with an increase in vascular reactive oxygen species and results in abnormal vascular function. The current study was designed to assess the role of H₂O₂ in regulating NO production during vascular aging. In vivo, real-time NO and H₂O₂ concentrations were measured by microelectrodes in mesenteric arteries of retired breeder (aged; 8–12 mo) and young (2 to 3 mo) Wistar-Kyoto rats under conditions of altered flow. The results in aged rats revealed elevated basal NO (1,611 ± 286 vs. 793 ± 112 nM, P < 0.05) and H₂O₂ concentrations (16 ± 2 vs. 9 ± 1 µM, P < 0.05) and a flow-mediated increase in H₂O₂ but not NO production. Pretreatment of aged rats with the antioxidant apocynin lowered both basal H₂O₂ (8 ± 1 µM) and NO (760 ± 102 nM) to young levels and restored flow-mediated NO production. Similar results were obtained with the NAD(P)H oxidase inhibitor gp91ds-tat. In addition, acute incubation with topical polyethylene-glycolated catalase lowered the baseline NO concentration and restored flow-mediated NO production. Taken together, the data indicate that elevated baseline and suppressed flow-mediated NO production in aged Wistar-Kyoto rats are mediated by NAD(P)H oxidase-derived H₂O₂.

Developmental regulation of cardiovascular function is dependent on both genotype and environment

Knight, B. S., Sunn, N., Pennell, C. E., Adamson, S. L., Lye, S. J. — Mon, 23 Nov 2009 12:52:58 PST

Adverse developmental environments can increase the risk of adult cardiovascular disease, but not all individuals are affected, suggesting the importance of genotype. Genetically distinct mouse strains allow the genetic dissection of complex traits; however, they have not been used to evaluate the developmental origins of adult cardiovascular disease. Our objective was to determine the effect of prenatal nutrient restriction (R) on adult cardiovascular function in A/J (AJ) and C57BL/6J (B6) mice and whether a postnatal high-fat (HF) diet exacerbates these effects. Pregnant AJ and B6 mice underwent a 30% R or ad libitum diet, and their offspring underwent a HF or control diet. Hypertension (+17 mmHg; P < 0.001) was observed in B6R mice at 9 wk, and their arterial pressure tended to remain high at 25 wk (+13 mmHg; not significant). In AJR mice, the normal decrement in arterial pressure over this age range in this strain was abolished. Heart rate prematurely increased in B6R and decreased in AJR (all; P < 0.05) mice from 9 to 25 wk. There was no effect of postnatal HF diet on these relationships. The Tei index (from a 26-wk microultrasound) was increased in both AJR and B6R mice (all; P < 0.05), suggesting an improved global myocardial performance. Neither R nor HF alone changed diastolic (ratio of E wave to A wave) or systolic (%fractional shortening) function in either strain; however, R and HE combined improved diastolic function in B6 (P < 0.05) but not in AJ mice. Therefore, there are strain-dependent alterations in adult cardiovascular function in response to prenatal nutrient restriction. Unexpectedly, a postnatal HF diet did not exacerbate the effects of prenatal nutrient restriction on postnatal cardiovascular outcomes.

Maturation and the role of PKC-mediated contractility in ovine cerebral arteries

Goyal, R., Mittal, A., Chu, N., Shi, L., Zhang, L., Longo, L. D. — Mon, 23 Nov 2009 12:52:58 PST

Ca²⁺-independent pathways such as protein kinase C (PKC), extracellular-regulated kinases 1 and 2 (ERK1/2), and Rho kinase 1 and 2 (ROCK1/2) play important roles in modulating cerebral vascular tone. Because the roles of these kinases vary with maturational age, we tested the hypothesis that PKC differentially regulates the Ca²⁺-independent pathways and their effects on cerebral arterial contractility with development. We simultaneously examined the responses of arterial tension and intracellular Ca²⁺ concentration and used Western immunoblot analysis to measure ERK1/2, RhoA, 20 kDa regulatory myosin light chain (MLC₂₀), PKC-potentiated inhibitory protein of 17 kDa (CPI-17), and caldesmon. Phorbol 12,13-dibutyrate (PDBu)-mediated PKC activation produced a robust contractile response, which was increased a further 20 to 30% by U-0126 (MEK inhibitor) in cerebral arteries of both age groups. Of interest, in the fetal cerebral arteries, PDBu leads to an increased phosphorylation of ERK2 compared with ERK1, whereas in adult arteries, we observed an increased phosphorylation of ERK1 compared with ERK2. Also, in the present study, RhoA/ROCK played a significant role in the PDBu-mediated contractility of fetal cerebral arteries, whereas in adult cerebral arteries, CPI-17 and caldesmon had a significantly greater role compared with the fetus. PDBu also led to an increased MLC₂₀ phosphorylation, a response blunted by the inhibition of myosin light chain kinase only in the fetus. Overall, the present study demonstrates an important maturational shift from RhoA/ROCK-mediated to CPI-17/caldesmon-mediated PKC-induced contractile response in ovine cerebral arteries.

Caspase-mediated protein kinase C-{delta} cleavage is necessary for apoptosis of vascular smooth muscle cells

Mon, 23 Nov 2009 12:52:58 PST

Apoptotic death of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling and various vascular diseases. We have previously shown that protein kinase C- (PKC-) plays a critical role in SMC apoptosis. In this study, we tested the importance of PKC- proteolytic cleavage and tyrosine phosphorylation within the apoptosis pathway. Using hydrogen peroxide as a paradigm for oxidative stress, we showed that proteolytic cleavage of PKC- occurred in SMCs that underwent apoptosis, while tyrosine phosphorylation was detected only in necrotic cells. Furthermore, using a peptide (z-DIPD-fmk) that mimics the caspase-3 binding motif within the linker region of PKC-, we were able to prevent the cleavage of PKC-, as well as apoptosis. Inhibition of PKC- with rottlerin or small-interfering RNA diminished caspase-3 cleavage, caspase-3 activity, cleavage of poly (ADP-ribose) polymerase, cleavage of PKC-, and DNA fragmentation, confirming the previously reported role of PKC- in initiation of apoptosis. In contrast, z-DIPD-fmk markedly diminished caspase-3 activity, cleavage of PKC-, and DNA fragmentation without affecting cleavage of caspase-3 and poly (ADP-ribose) polymerase. Taken together, our data suggest that caspase-3-mediated PKC- cleavage underlies SMC apoptosis induced by oxidative stress, and that PKC- acts both upstream and downstream of caspase-3.

Mitochondrial-derived hydrogen peroxide inhibits relaxation of bovine coronary arterial smooth muscle to hypoxia through stimulation of ERK MAP kinase

Gao, Q., Zhao, X., Ahmad, M., Wolin, M. S. — Mon, 23 Nov 2009 12:52:58 PST

Mitochondrial reactive oxygen species (ROS) are potentially important in vascular oxygen-sensing mechanisms because hypoxia appears to be a stimulus for mitochondrial ROS generation; however, scavenging of endogenous ROS does not alter relaxation of endothelium-denuded bovine coronary arteries (BCA) to hypoxia. The purpose of this study was to investigate the influence of increasing mitochondrial ROS on the relaxation of BCA to hypoxia. Increasing mitochondrial superoxide with inhibitors of electron transport (10 µM rotenone and antimycin) and by opening mitochondrial ATP-dependent K⁺ channels with 100 µM diazoxide were observed in this study to attenuate relaxation of BCA precontracted with 30 mM KCl to hypoxia by 68–76% and 38%, respectively. This effect of rotenone is not prevented by inhibiting NADPH oxidase (Nox) activation or scavenging superoxide with Peg-SOD; however, it is reversed 85% and 26% by increasing the consumption of intracellular peroxide by 0.1 mM ebselen and 32.5 U/ml Peg-catalase. Because inhibition of extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase (10 µM PD-98059), but not src kinase or rho kinase, also reverses the effects of rotenone by 69%, the peroxide-elicited force-enhancing effects of ERK appear to be attenuating the response to hypoxia. Rotenone increased the phosphorylation of ERK (by 163%). Activation of ERK in BCA with 0.1 mM peroxide or endogenous peroxide generated by stimulating Nox2 with a stretch treatment or contraction with 100 nM U-46619 also attenuated relaxation to hypoxia. Thus coronary arterial relaxation to hypoxia may be attenuated by pathophysiological conditions associated with increased peroxide generation by mitochondria or other sources that stimulate ERK.

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

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

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

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

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

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

Angiotensin II, mechanotransduction, and pulsatile arterial hemodynamics in hypertension

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

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

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

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

Pathways to embryonic heart failure

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

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

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

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

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

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

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

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

Increased susceptibility of aged hearts to ventricular fibrillation during oxidative stress

Mon, 26 Oct 2009 13:43:28 PDT

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

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

Mon, 26 Oct 2009 13:43:28 PDT

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

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

Mon, 26 Oct 2009 13:43:28 PDT

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

Impaired function of coronary BKCa channels in metabolic syndrome

Mon, 26 Oct 2009 13:43:28 PDT

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

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

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

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

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

Mon, 26 Oct 2009 13:43:28 PDT

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

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

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

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

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

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

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

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

Mon, 26 Oct 2009 13:43:29 PDT

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

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

Mon, 26 Oct 2009 13:43:29 PDT

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

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

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

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

O-GlcNAc signaling attenuates ER stress-induced cardiomyocyte death

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

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

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

Mon, 26 Oct 2009 13:43:29 PDT

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

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

Mon, 26 Oct 2009 13:43:29 PDT

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

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

Mon, 26 Oct 2009 13:43:29 PDT

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

Calpain inhibition preserves myocardial structure and function following myocardial infarction

Mon, 26 Oct 2009 13:43:29 PDT

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

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

Mon, 26 Oct 2009 13:43:29 PDT

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

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

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

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

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

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

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

Adenylyl cyclase type 5 protein expression during cardiac development and stress

Mon, 26 Oct 2009 13:43:29 PDT

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Mon, 26 Oct 2009 13:43:29 PDT

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

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

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

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

eNOS uncoupling and endothelial dysfunction in aged vessels

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

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

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

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

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

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

Mon, 26 Oct 2009 13:43:30 PDT

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

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

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

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

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

Mon, 26 Oct 2009 13:43:30 PDT

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

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

Mon, 26 Oct 2009 13:43:30 PDT

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

Resveratrol attenuates mitochondrial oxidative stress in coronary arterial endothelial cells

Mon, 26 Oct 2009 13:43:30 PDT

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

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

Mon, 26 Oct 2009 13:43:30 PDT

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

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

Mon, 26 Oct 2009 13:43:30 PDT

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

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

Mon, 26 Oct 2009 13:43:30 PDT

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

Endogenous regulation of cardiovascular function by apelin-APJ

Mon, 26 Oct 2009 13:43:30 PDT

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

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

Mon, 26 Oct 2009 13:43:30 PDT

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

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

Mon, 26 Oct 2009 13:43:30 PDT

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

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

Mon, 26 Oct 2009 13:43:30 PDT

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

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

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

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

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

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

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

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

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

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