AJP: Heart and Circulatory Physiology current issue

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.