实验性心肌梗塞心肌肌浆网钙释放通道变化

目的 :探讨急性心肌梗塞心肌肌浆网钙释放通道的变化。　　方法 :将兔分为急性心肌梗塞组和对照组 ,急性心肌梗塞组结扎兔冠状动脉左前降支 6小时后制成急性心肌梗塞模型 ,用氚标记的兰尼定 (3H- ryanodine)对心肌肌浆网钙释放通道进行放射配基分析。　　结果 :兔实验性急性心肌梗塞梗塞区心肌肌浆网钙释放通道总结合位点数 (Bmax值 )较对照组显著降低 (93.91± 9.6 8fmol/ mg.pro比 15 5 .74± 2 3.5 0 fmol/ mg.pro,P<0 .0 1) ;两组心肌肌浆网钙释放通道平衡解离常数 (Kd值 )无显著变化(P>0 .0 5 )。　　结论 :急性心肌梗塞时心肌肌浆网钙释放通道密度降低 ,而其亲和力则无改变。     

大鼠实验性糖尿病对异丙肾上腺素所致心肌坏死的影响

异丙肾上腺素(ISP)是一种强有力的心脏毒性物质。给动物注射一定剂量可引起明显心肌坏死。糖尿病时可发生心肌病改变,为探讨糖尿病心肌对致损伤因素的反应特点,本实验研究了糖尿病状态对ISP所致心肌坏死的影响。由于甲状腺功能状态可影响儿茶酚胺的反应性,故同时也探讨了糖尿病大鼠血清甲状腺激素水平对这种心肌坏死的影响。     

异丙肾上腺素对犬心房肌细胞动作电位及L型钙电流的影响

目的研究两种浓度的异丙肾上腺素（Isoproterenol，ISO）对犬心房肌细胞（AP）及L型钙电流（ICa，L）的作用。方法采用离体灌注和消化的方法获取心房肌细胞，用全细胞记录技术记录单个心房肌细胞AP以及ICa，L。结果低浓度ISO（10nmol／L）可延长APD，可使90％AP时程（APD90）延长34．4％，并降低AP平台期水平。高浓度ISO（1μmol／L）可减少APD，APD90减少32．1％。两种浓度的ISO均可诱发AP后除极及触发活动。10nmol／L和1μmol／L ISO分别增加ICa，L 36．7％和49．3％。结论两种浓度的ISO对心肌细胞ICa，L均有促进作用，Ca^2＋内流引起的肌浆网Ca^2＋释放可能是房性心律失常的发生机制之一。     

左旋卡尼汀对异丙肾上腺素致心肌损害的影响

目的:观察左旋卡尼汀对异丙肾上腺素致心肌损害的保护作用并探讨其机制。方法:采用大鼠皮下注射异丙肾上腺素造成心肌损害模型。心肌切片,HE染色,光镜下观察心肌损害程度;分离心肌线粒体,测定膜脂流动性（LFU）。结果:注射左旋卡尼汀可减轻心肌损害程度;改善线粒体的损伤,改善LFU。结论:左旋卡尼汀能减轻异丙肾对心肌的损害,对线粒体膜脂流动性损伤具有明显的保护作用。     

心肌细胞内钙释放对心肌细胞PTEN表达的影响

目的探讨雷尼丁（Ryanodine,RY）介导的心肌细胞内钙释放对PTEN mRNA和蛋白表达影响。方法新生Wistar乳鼠心肌细胞原代培养。用不同浓度的RY（0.0110μmol/L）促进心肌细胞内钙释放,特异的钙敏感指示剂Fura-2/AM测定心肌细胞内钙浓度（[Ca2+]i）。逆转录聚合酶链式反应（RT-PCR）、Western blot分别检测PTEN mRNA和蛋白表达。结果RY在0.011μmol/L浓度范围内,能浓度依赖性地促进心肌细胞内钙释放,RY在10μmol/L时,心肌细胞内钙浓度低于RY在1μmol/L时的效应,但仍高于对照组;RY在0.011μmol/L浓度范围内能浓度依赖性促进心肌细胞PTEN mRNA和蛋白表达,RY在10μmol/时,心肌细胞PTEN mRNA和蛋白表达低于1μmol/L的效应,但仍高于对照组。结论RY介导的心肌细胞内钙释放可影响心肌细胞PTEN基因表达,其表达在RY处于0.011μmol/L范围内具有浓度依赖性。     

异丙肾上腺素对大鼠内皮素基因表达的影响

目的　探讨异丙肾上腺素（ＩＳＰ）对内皮素基因表达的影响。方法　通过ＩＳＰ心肌损伤模型,用放免及分子生物学的方法测定血浆内皮素－１（ＥＴ－１）放免活性及心肌和主动脉 ＥＴ－１ｍ ＲＮＡ 的表达。结果　ＩＳＰ致血浆中ＥＴ－１ 放免活性明显升高,使心肌及主动脉ＥＴ－１ ｍ ＲＮＡ 高表达。结论　表明ＩＳＰ使血管内皮细胞ＥＴ－１ ｍ ＲＮＡ 高表达,内皮素分泌增多可能是其致心肌损伤的一个重要原因。     

白细胞介素8对异丙肾上腺素所致大鼠心肌细胞坏死的影响

近年发现白细胞介素8(IL-8)具有抑制中性粒细胞与血管内皮细胞粘附的作用,本工作用重组IL-8治疗异丙肾上腺素(ISO)所致大鼠心肌坏死。与单纯ISO组比较,重组IL-8治疗明显减轻ISO引起的心肌坏死,使心肌丙二醛含量减少、降低心肌髓过氧化物酶活性,使血浆内皮素放免活性明显降低。实验结果提示,IL-8可以通过抑制白细胞粘附和浸润,抑制内皮素释放,防治缺血心肌坏死。     

异丙肾上腺素对犬心房肌细胞动作电位及L型钙电流的影响

目的研究两种浓度的异丙肾上腺素(Isoproterenol,ISO)对犬心房肌细胞(AP)及L型钙电流(ICa,L)的作用。方法采用离体灌注和消化的方法获取心房肌细胞,用全细胞记录技术记录单个心房肌细胞AP以及ICa,L。结果低浓度ISO(10nmol/L)可延长APD,可使90%AP时程(APD90)延长34.4%,并降低AP平台期水平。高浓度ISO(1μmol/L)可减少APD,APD90减少32.1%。两种浓度的ISO均可诱发AP后除极及触发活动。10nmol/L和1μmol/LISO分别增加ICa,L36.7%和49.3%。结论两种浓度的ISO对心肌细胞ICa,L均有促进作用,Ca2+内流引起的肌浆网Ca2+释放可能是房性心律失常的发生机制之一。     

尼莫地平对缺血心肌细胞内钙的影响及心肌保护作用

应用Fura-2荧光技术和原子吸收分光光度法测定大鼠心肌内钙，结果发现；对照组、缺血组和治疗组大鼠心肌细胞内游离钙（MyoCai）分别为139．5±7.4、525．9±106．7和202．2±53.5nmol／L；对照组和缺血组大鼠心肌细胞总钙（MyoCat）分别为119.0±17．5和135．8±26.5mmol／kg·干重；MyoCai和MyoCat间无相关性。研究表明：缺血早期MyoCai明显增高，但MyoCat变化不明显；尼莫地平能阻止缺血心肌细胞钙超载的发展和加重。     

内皮素和降钙素基因相关肽对异丙肾上腺素致心肌损伤的影响

本实验在大鼠异丙肾上腺素（ＩＳＯ）心肌损伤模型上，观察了内皮素（ＥＴ）及降钙素基因相关肽（ＣＧＲＰ）对ＩＳＯ损伤心肌作用的影响．结果表明：ＥＴ明显加重ＩＳＯ对心肌的损伤，ＣＧＲＰ则能减轻ＩＳＯ对心肌的损伤及ＩＳＯ与ＥＴ对心肌的协同损伤．提示心肌病理情况下，ＥＴ是一种促损伤因素，而ＣＧＲＰ是抗损伤因素．     

Ca2+ blinks: rapid nanoscopic store calcium signaling

Luminal Ca(2+) in the endoplasmic and sarcoplasmic reticulum (ER/SR) plays an important role in regulating vital biological processes, including store-operated capacitative Ca(2+) entry, Ca(2+)-induced Ca(2+) release, and ER/SR stress-mediated cell death. We report rapid and substantial decreases in luminal [Ca(2+)], called "Ca(2+) blinks," within nanometer-sized stores (the junctional cisternae of the SR) during elementary Ca(2+) release events in heart cells. Blinks mirror small local increases in cytoplasmic Ca(2+),orCa(2+) sparks, but changes of [Ca(2+)] in the connected free SR network were below detection. Store microanatomy suggests that diffusional strictures may account for this paradox. Surprisingly, the nadir of the store depletion trails the peak of the spark by about 10 ms, and the refilling of local store occurs with a rate constant of 35 s(-1), which is approximately 6-fold faster than the recovery of local Ca(2+) release after a spark. These data suggest that both local store depletion and some time-dependent inhibitory mechanism contribute to spark termination and refractoriness. Visualization of local store Ca(2+) signaling thus broadens our understanding of cardiac store Ca(2+) regulation and function and opens the possibility for local regulation of diverse store-dependent functions.     

缬沙坦对心力衰竭家兔心肌肌浆网钙调控相关蛋白基因的影响

目的探讨家兔慢性心力衰竭(心衰)时心肌肌浆网钙泵(SERCA2)、受磷蛋白(PLB)基因表达的改变及血管紧张素Ⅱ受体拮抗剂缬沙坦长期干预的意义。方法18只家兔随机分为3组,假手术组、心衰组和缬沙坦组,每组6只。通过超容量负荷联合压力负荷建立家兔心衰模型,术后7周观察左心室结构、血流动力学的变化及SERCA2、PLB基因的表达。结果与假手术组比较,心衰组左心室重量指数、心率、左心室舒张末压显著升高(P<0.05),左心室缩短率及LVEF明显降低(P<0.05);与心衰组比较,缬沙坦组左心室重量指数、心率、左心室舒张末压显著降低(P<0.05),左心室缩短率及左心室射血分数明显升高(P<0.05);心衰组SERCA2、PLBmRNA显著低于假手术组(P<0.05),缬沙坦组SERCA2、PLBmRNA显著高于心衰组(P<0.05)。结论缬沙坦长期干预心衰,能够改善心脏舒缩功能,可能与其上调肌浆网的钙调控蛋白SERCA2、PLB基因表达有关。     

Transport and release of calcium by sarcoplasmic reticulum in chemically skinned ventricular muscle of the rat

Summary Strips of rat ventricle were treated with EGTA (5 mM) for 24 h at 4°C and used to perform isotopical measurements of transport and release of Ca²⁺ in the SR in situ.⁴⁵Ca accumulated by these preparations showed dependence on time of incubation until it reached saturation after 30 min. Rate and capacity of Ca²⁺ accumulation were calculated in 0.075 nmol mg ww^–1 min^–1 and 0.402 nmol mg ww^–1, respectively. These values were increased by a factor of 2.6 and 8.6 when K oxalate was present in the incubation media as could be expected for Ca²⁺ transported by SR.Ca²⁺ release was assayed on⁴⁵Ca desaturation curves at three free Ca²⁺ concentrations: 0.3, 1 and 10 M. Significant increases in the velocity of Ca²⁺ efflux and net release of Ca²⁺ were induced only by 1 M free Ca²⁺, and the Ca²⁺ release could be inhibited by 75% when 50 M of ruthenium red was included in the washout solution.These results are in agreement with those obtained in assessing the SR function by mechanical measurements in skinned cardiac cells or by biochemical determinations in isolated cardiac SR vesicles. In spite of the fact that the resolution time is not as high as that required for the physiological handling of Ca²⁺ by SR, this methodology looks promising for approaching the SR function in cardiac pathologies as well as the effects of drugs on transport and release of Ca²⁺ by cardiac SR.This work was supported by Grant PID 306220088 from the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina (CONICET).     

Sarcolipin and phospholamban as regulators of cardiac sarcoplasmic reticulum Ca2+ ATPase

The cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a) plays a critical role in maintaining the intracellular calcium homeostasis during cardiac contraction and relaxation. It has been well documented over the years that altered expression and activity of SERCA2a can lead to systolic and diastolic dysfunction. The activity of SERCA2a is regulated by two structurally similar proteins, phospholamban (PLB) and sarcolipin (SLN). Although, the relevance of PLB has been extensively studied over the years, the role SLN in cardiac physiology is an emerging field of study. This review focuses on the advances in the understanding of the regulation of SERCA2a by SLN and PLB. In particular, it highlights the similarities and differences between the two proteins and their roles in cardiac patho-physiology.     

心房颤动病人心房肌肌浆网Ca2+泵和Ca2+释放通道的变化

目的 探讨心房颤动病人心房肌肌浆网Ca2 泵(sarcoplasmic reticulum Ca2 ATPase,SERCA2a)及钙释放通道2型雷尼丁受体(type 2 ryanodine receptor,RYR2)mRNA表达的变化.方法 39例风湿心脏病二尖瓣关闭不全接受外科手术者,分为3组,窦性心律组13例,心房颤动持续小于6个月组11例,心房颤动持续超过6个月组15例.手术时取右心房肌约100 mg,用逆转录-聚合酶链反应检测心房肌SERCA2a和RYR2的mRNA表达.结果 心房颤动者肌浆网SERCA2a、RYR2的mRNA较窦性心律者下调,而且下调随心房颤动持续时间延长而明显.结论 心房颤动病人SERCA2a、RYR2的mRNA下调,提示肌浆网SERCA2a、RYR2与心房颤动的发生和维持有关.     

缬沙坦对心力衰竭家兔肌质网钙ATP酶表达及功能的影响

目的 探讨家兔慢性心力衰竭时心肌肌质网钙ATP酶（SERCA2）表达和功能的改变及血管紧张素Ⅱ受体拮抗剂缬沙坦长期干预的意义。方法27只家兔随机分为3组,假手术组、心力衰竭组和缬沙坦组各9只。通过超容量负荷联合压力负荷建立家兔心力衰竭模型,于术后7周观察左心室结构、血流动力学的变化及SERCA2的表达和功能的改变。结果与假手术组比较,心力衰竭组左心室质量指数【（1．32±0．06）vS（3．61±0．09）g／kg】、左心室舒张末压【（-0．50±1．05）vs（23．00±2．37）mmHg）】显著升高（P〈0．05）,左心室短轴缩短率【（37．83±3．58）％vs（17．38±3．13）％】及左室射血分数【（72±5）％vS（38±6）％】明显降低（P〈0．05）;与心力衰竭组比较,缬沙坦组左心室质量指数和左心室舒张末压显著降低【（2．07±0．14）g/kg;（2．17±0．72）mmHg;P〈0．05】;左心室短轴缩短率及左室射血分数明显升高【（33．8±2．9）％;（65±4）％;P〈0．05]。心力衰竭组SERCA2的表达【（0．69±0．04）vS（1．02±0．02）】和功能【（54．4±7．9）％w（95．5±2．1）％】显著低于假手术组（P〈0．05）。缬沙坦组SERCA2的表达和功能显著高于心力衰竭组[（0．91±0．02）;（81．7±4．9）％;P〈0．05]。结论缬沙坦长期干预,能够改善心力衰竭心脏舒缩功能,可能与增加SERCA2的表达和提高其功能有关。     

Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during beta-adrenergic stimulation

We aimed to define the relative contribution of both PKA and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during beta-adrenergic receptor (betaAR) stimulation. Rat hearts were perfused with increasing concentrations of the beta-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca(2+) influx to the cell by low [Ca](o) plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [(3)H]-ryanodine binding and fast Ca(2+) release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca(2+). Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under betaAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the beta-induced increase in the channel activity as indicated by the enhancement of the [(3)H]-ryanodine binding and the velocity of fast SR Ca(2+) release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca(2+) release during betaAR stimulation.     

培哚普利对心衰大鼠心肌SR Ca2+释放通道密度和mRNA表达的影响

目的 :探讨培哚普利对慢性心衰心肌肌浆网 （SR） Ca2 +释放通道 （Ry R2 ）密度和 m RNA表达的影响及意义。方法 :通过结扎大鼠左冠脉建立慢性心衰模型 ,以培哚普利进行干预 ,对照观察血流动力学、[3 H ]- ryanodine与左室心肌 Ry R2 最大结合量 （Bmax）和 Kd 值、Ry R2 m RNA表达水平。结果 :心衰组 （F组 ）与对照组 （C组 ）相比 ,L VEDP显著升高 （P<0 .0 1） ,+dp/dtmax,- dp/dtmax显著低于 C组 （P<0 .0 1）。F组 [3 H]- ryanodine与 Ry R2 最大结合量 Bmax显著低于 C组 （P<0 .0 1） ,P组显著高于 F组 （P<0 .0 1） ,3组 Kd 值无显著差异 （P>0 .0 5 ） ;F组 Ry R2 m RNA表达水平显著低于 C组 （P<0 .0 1） ,P组显著高于 F组 （P<0 .0 1）。结论 :培哚普利长期干预慢性心衰 ,能够增加 Ry R2基因表达 ,增加 Ry R2 密度 ,可能与其改善心肌收缩功能有关     

微电极阵列技术研究肌质网钙ATP酶过表达对大鼠衰竭心肌电活动的改善作用

目的 探索重组腺病毒(rAd)介导的肌质网Ca2+-ATP酶(SERCA2a)过表达对大鼠心肌梗死后心力衰竭心肌电活动节律和传导的改善作用,并探讨可能的电活动机制.方法 将26只成年雄性SD大鼠随机分为3组:假手术组(n=l0),空病毒对照组(rAd.β-gal组,n=8)和肌质网Ca2+-ATP酶(SERCA2a)转染组(rAd.SERCA2a组,n=8).假手术组仅开胸不结扎动脉,rAd.β-gal组和rAd.SERCA2a组分别进行左冠状动脉前降支结扎建立大鼠心肌梗死后心力衰竭动物模型,同时分别将携带β-gal和SERCA2a基因的重组腺病毒(rAd)导入衰竭心脏,术后2周超声心电图检测心脏舒张功能和收缩功能,心电图监测体表心电活动以及微电极阵列(MEA)技术监测离体心脏组织电活动情况.结果 rAd携带SERCA2a与β-gal基因均成功转入大鼠衰竭心脏.rAd.SERCA2a组可改善心功能,与假手术组相比心室舒张末期容积与心室收缩末期容积轻微增加[(0.41±0.13)cm2对(0.39±0.02)cm2,(0.08±0.02)cm2对(0.06±0.01)cm2,P＞0.05],左心室射血分数[(0.82±0.05)对(0.86±0.01),P＞0.05]和短轴缩短率[(46.6±2.32)％对(49.58±1.71)％,P＞0.05]无明显改变.与假手术组相比,rAd.β-gal组体表心电图QT间期延长[(111.02±7.42) ms对(94.7±1.55) ms,n=6,P＜0.05],室性早搏发生率达71.5％ (5/7),而rAd.SERCA2a组QT间期缩短[(81.45±4.97)ms对(94.7±1.55)ms,n=6,P＜0.05],室性早搏发生率达14.3％(1/7).MEA记录可发现rAd.SERCA2a组心率与假手术组相比差异无统计学意义[(435±31)次/min对(442 ±22)次/min,n=6,P＞0.05],与rAd.β-gal组相比,rAd.SERCA2a组最大场电位[(0.82±0.39)mV对(0.64±0.13) mV,n=6,P＜0.05]、最小场电位[(1.88±0.57) mV对(1.35±0.12) mV n=6,P＜0.05]、场电位时限[(124.17±21.08)ms对(113.23±12.02) ms n=6,P＜0.05]均延长;rAd.β-gal组梗死区与梗死对侧区心肌组织场电位时限差异有统计学意义[(60.36±2.08)ms对(103.24±7.35) ms,n=5,P＜0.05],并且60通道记录梗死区心肌组织场电位时限离散度大于rAd.SERCA2a组[(38.5ms±4.62)ms对(26.88±5.09) ms,n=5];rAd.SERCA2a组传导基本一致,使心肌梗死面心室肌组织电活动呈均一性传导.结论 SERCA2a转基因治疗可以显著改善心力衰竭大鼠的左心室收缩功能、舒张功能,同时可以降低心肌梗死后心力衰竭伴发心律失常的发生,改善心脏电活动的均一传导.MEA技术是一项检测心血管疾病动物模型心脏组织电生理节律和频率以及传导活动的理想技术.