摘要:

Abstract—Spontaneous sarcoplasmic reticulum (SR) Ca2+release causes delayed afterdepolarizations (DADs) via Ca2+-induced transient inward currents (Iti). However, no quantitative data exists regarding (1) Ca2+dependence of DADs, (2) Ca2+required to depolarize the cell to threshold and trigger an action potential (AP), or (3) relative contributions of Ca2+-activated currents to DADs. To address these points, we evoked SR Ca2+release by rapid application of caffeine in indo 1-AM–loaded rabbit ventricular myocytes and measured caffeine-induced DADs (cDADs) with whole-cell current clamp. The SR Ca2+load of the myocyte was varied by different AP frequencies. The cDAD amplitude doubled for every 88±8 nmol/L of &Dgr;[Ca2+]i(simple exponential), and the &Dgr;[Ca2+]ithreshold of 424±58 nmol/L was sufficient to trigger an AP. Blocking Na+-Ca2+exchange current (INa/Ca) by removal of [Na]oand [Ca2+]o(or with 5 mmol/L Ni2+) reduced cDADs by >90%, for the same &Dgr;[Ca2+]i. In contrast, blockade of Ca2+-activated Cl–current (ICl(Ca)) with 50 &mgr;mol/L niflumate did not significantly alter cDADs. We conclude that DADs are almost entirely due toINa/Ca, notICl(Ca)or Ca2+-activated nonselective cation current. To trigger an AP requires 30 to 40 &mgr;mol/L cytosolic Ca2+or a [Ca2+]itransient of 424 nmol/L. Current injection, simulatingItis with different time courses, revealed that fasterItis require less charge for AP triggering. Given that spontaneous SR Ca2+release occurs in waves, which are slower than cDADs or fastItis, the true &Dgr;[Ca2+]ithreshold for AP activation may be ≈3-fold higher in normal myocytes. This provides a safety margin against arrhythmia in normal ventricular myocytes.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Abstract—Cultured neonatal rat cardiac myocytes have been used extensively to study cellular and molecular mechanisms of cardiac hypertrophy. However, there are only a few studies in cultured mouse myocytes despite the increasing use of genetically engineered mouse models of cardiac hypertrophy. Therefore, we characterized hypertrophic responses in low-density, serum-free cultures of neonatal mouse cardiac myocytes and compared them with rat myocytes. In mouse myocyte cultures, triiodothyronine (T3), norepinephrine (NE) through a &bgr;-adrenergic receptor, and leukemia inhibitory factor induced hypertrophy by a 20% to 30% increase in [3H]phenylalanine-labeled protein content. T3 and NE also increased &agr;-myosin heavy chain (MyHC) mRNA and reduced &bgr;-MyHC. In contrast, hypertrophic stimuli in rat myocytes, including &agr;1-adrenergic agonists, endothelin-1, prostaglandin F2&agr;, interleukin 1&bgr;, and phorbol 12-myristate 13-acetate (PMA), had no effect on mouse myocyte protein content. In further contrast with the rat, none of these agents increased atrial natriuretic factor or &bgr;-MyHC mRNAs. Acute PMA signaling was intact by extracellular signal–regulated kinase (ERK1/2) and immediate-early gene (fos/jun) activation. Remarkably, mouse but not rat myocytes had hypertrophy in the absence of added growth factors, with increases in cell area, protein content, and the mRNAs for atrial natriuretic factor and &bgr;-MyHC. We conclude that mouse myocytes have a unique autonomous hypertrophy. On this background, T3, NE, and leukemia inhibitory factor activate hypertrophy with different mRNA phenotypes, but certain Gq- and protein kinase C–coupled agonists do not.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Abstract—Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis, hypertension, and restenosis, in part by promoting vascular smooth muscle cell (VSMC) growth. Many VSMC growth factors are secreted by VSMC and act in an autocrine manner. Here we demonstrate that cyclophilin A (CyPA), a member of the immunophilin family, is secreted by VSMCs in response to oxidative stress and mediates extracellular signal–regulated kinase (ERK1/2) activation and VSMC growth by reactive oxygen species. Human recombinant CyPA can mimic the effects of secreted CyPA to stimulate ERK1/2 and cell growth. The peptidyl-prolyl isomerase activity is required for ERK1/2 activation by CyPA. In vivo, CyPA expression and secretion are increased by oxidative stress and vascular injury. These findings are the first to identify CyPA as a secreted redox-sensitive mediator, establish CyPA as a VSMC growth factor, and suggest an important role for CyPA and enzymes with peptidyl-prolyl isomerase activity in the pathogenesis of vascular diseases.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Abstract—Electric shock is the only effective therapy against ventricular fibrillation. However, shocks are also known to cause electroporation of cell membranes. We sought to determine the impact of electroporation on ventricular conduction and defibrillation. We optically mapped electrical activity in coronary-perfused rabbit hearts during electric shocks (50 to 500 V). Electroporation was evident from transient depolarization, reduction of action potential amplitude, and upstroke dV/dt. Electroporation was voltage dependent and significantly more pronounced at the endocardium versus the epicardium, with thresholds of 229±81 versus 318±84 V, respectively (P=0.01, n=10), both being above the defibrillation threshold of 181.3±45.8 V. Epicardial electroporation was localized to a small area near the electrode, whereas endocardial electroporation was observed at the bundles and trabeculas throughout the entire endocardium. Higher-resolution imaging revealed that papillary muscles (n=10) were most affected. Electroporation and conduction block thresholds in papillary muscles were 281±64 V and 380±79 V, respectively. We observed no arrhythmia in association with electroporation. Further, preconditioning with high-energy shocks prevented reinduction of fibrillation by 50-V shocks, which were otherwise proarrhythmic. Endocardial bundles are the most susceptible to electroporation and the resulting conduction impairment. Electroporation is not associated with proarrhythmic effects and is associated with a reduction of vulnerability.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Abstract—Multiple mutations in cardiac troponin I (cTnI) have been associated with familial hypertrophic cardiomyopathy. Two mutations are located in the cTnI inhibitory domain, a highly negatively charged region that alternately binds to either actin or troponin C, depending on the intracellular concentration of calcium. This region is critical to the inhibition of actin-myosin crossbridge formation when intracellular calcium is low. We modeled one of the inhibitory domain mutations, arginine145→glycine (TnI146Glyin the mouse sequence), by cardiac-specific expression of the mutated protein in transgenic mice. Multiple lines were generated with varying degrees of expression to establish a dose relationship; the severity of phenotype could be correlated directly with transgene expression levels. Transgenic mice overexpressing wild-type cTnI were generated as controls and analyzed in parallel with the TnI146Glyanimals. The control mice showed no abnormalities, indicating that the phenotype of TnI146Glywas not simply an artifact of transgenesis. In contrast, TnI146Glymice showed cardiomyocyte disarray and interstitial fibrosis and suffered premature death. The functional alterations that seem to be responsible for the development of cardiac disease include increased skinned fiber sensitivity to calcium and, at the whole organ level, hypercontractility with diastolic dysfunction. Severely affected lines develop a pathology similar to human familial hypertrophic cardiomyopathy but within a dramatically shortened time frame. These data establish the causality of this mutation for cardiac disease, provide an animal model for understanding the resultant pathogenic structure-function relationships, and highlight the differences in phenotype severity of the troponin mutations between human and mouse hearts.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Abstract—Previous studies have suggested that oxygen-derived free radicals are involved in the pathophysiology of myocardial ischemia/reperfusion (MI/R) injury. Specifically, neutrophils have been shown to mediate postischemic ventricular arrhythmias and myocardial necrosis. We hypothesized that MI/R injury would be reduced in the absence (−/−) of NADPH oxidase. Heterozygous control mice (n=23) and NADPH oxidase–/–mice (n=24) were subjected to 30 minutes of coronary artery occlusion and 24 hours of reperfusion. Myocardial area at risk per left ventricle was similar in heterozygous control hearts (55±3%) and NADPH oxidase–/–hearts (61±4%). Contrary to our hypothesis, the size of infarct area at risk was similar in the heterozygous control mice (42±4%) and NADPH oxidase–/–mice (34±5%) (P=not significant). In addition, echocardiographic examination of both groups revealed that left ventricle fractional shortening was similar in NADPH oxidase–/–mice (n=8; 27±2.5%) and heterozygous control mice (n=10; 23.3±3.3%) after MI/R. Superoxide production, as detected by cytochrome c reduction, was significantly impaired (P<0.01) in NADPH oxidase–/–mice (n=6) compared with heterozygous mice (n=7) (0.04±0.03 versus 2.2±0.08 nmol O2·min–1·106cells–1). Intravital microscopy of the inflamed mesenteric microcirculation demonstrated that leukocyte rolling and adhesion were unaffected by the absence of NADPH oxidase. Oyster glycogen-stimulated neutrophil transmigration into the peritoneum was also similar in both the heterozygous control mice and NADPH oxidase–/–mice (P=not significant). These findings suggest that NADPH oxidase does not contribute to the development of myocardial injury and dysfunction after MI/R.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Abstract—We sought to determine whether adenovirus-mediated gene transfer in vivo of calcitonin gene–related peptide (CGRP), a potent vasodilator, ameliorates cerebral vasoconstriction after experimental subarachnoid hemorrhage (SAH). Arterial blood was injected into the cisterna magna of rabbits to mimic SAH 5 days after injection of AdRSVCGRP (8×108pfu), AdRSV&bgr;gal (control virus), or vehicle. After injection of AdRSVCGRP, there was a 400-fold increase in CGRP in cerebrospinal fluid. Contraction of the basilar artery to serotonin in vitro was greater in rabbits after SAH than after injection of artificial cerebrospinal fluid (P<0.001). Contraction to serotonin was less in rabbits with SAH after AdRSVCGRP than after AdRSV&bgr;gal or vehicle (P<0.02). Basal diameter of the basilar artery before SAH (measured with digital subtraction angiogram) was 13% greater in rabbits treated with AdRSVCGRP than in rabbits treated with vehicle or AdRSV&bgr;gal (P<0.005). In rabbits treated with vehicle or AdRSV&bgr;gal, arterial diameter after SAH was 25±3% smaller than before SAH (P<0.0005). In rabbits treated with AdRSVCGRP, arterial diameter was similar before and after SAH and was reduced by 19±3% (P<0.01) after intracisternal injection of CGRP-(8-37) (0.5 nmol/kg), a CGRP1receptor antagonist. To determine whether gene transfer of CGRP after SAH may prevent cerebral vasoconstriction, we constructed a virus with a cytomegalovirus (CMV) promoter, which results in rapid expression of the transgene product. Treatment of rabbits with AdCMVCGRP after experimental SAH prevented constriction of the basilar artery 2 days after SAH. Thus, gene transfer of CGRP prevents cerebral vasoconstriction in vivo after experimental SAH.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Abstract—Both cGMP-dependent and -independent mechanisms have been implicated in the regulation of vascular tone by NO. We analyzed acetylcholine (ACh)- and NO-induced relaxation in pressurized small arteries and aortic rings from wild-type (wt) and cGMP kinase I–deficient (cGKI–/–) mice. Low concentrations of NO and ACh decreased the spontaneous myogenic tone in wt but not in cGKI–/–arteries. However, contractions of cGKI–/–arteries and aortic rings were reduced by high concentrations (10 &mgr;mol/L) of 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). Iberiotoxin, a specific blocker of Ca2+-activated K+(BKCa) channels, only partially prevented the relaxation induced by DEA-NO or ACh in pressurized vessels and aortic rings. DEA-NO increased the activity of BKCachannels only in vascular smooth muscle cells isolated from wt cGKI+/+mice. These results suggest that low physiological concentrations of NO decrease vascular tone through activation of cGKI, whereas high concentrations of DEA-NO relax vascular smooth muscle independent of cGKI and BKCa. NO-stimulated, cGKI-independent relaxation was antagonized by the inhibition of soluble guanylyl cyclase or cAMP kinase (cAK). DEA-NO increased cGMP to levels that are sufficient to activate cAK. cAMP-dependent relaxation was unperturbed in cGKI–/–vessels. In conclusion, low concentrations of NO relax vessels by activation of cGKI, whereas in the absence of cGKI, NO can relax small and large vessels by cGMP-dependent activation of cAK.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID