MECHANICAL LOAD EFFECT ON CARDIAC EXCITATION-CONTRACTION COUPLING

摘要 心脏必须调整其收缩力量以抵消血压的变化(由于体力活动， 身体位置、情绪压力等)以维持心输出量。心肌细胞具有固有的 能够感知机械载荷，并相应微调收缩压力。我们和其他人之前的工作 研究发现，增加肌条或单个肌细胞的机械负荷会导致肌条或单个肌细胞 胞浆钙瞬变(CAT)，增加收缩力量以部分补偿所增加的 机械载荷。CAT的持续增加需要增加肌浆中的钙含量 网状结构(SR)，它必须来自整个肌膜上钙离子的净增加。然而， 机械负荷与控制钙离子进入的机制尚不清楚。看似合理的机制 这种净增加包括机械负荷引起的通过L型钙通道的钙内流增加 (ICAL)或Na+-Ca~(2+)交换器(INCX)外流减少。然而，研究机械载荷如何影响 ICAL或INCX一直受到使心肌细胞承受机械负荷的技术困难的阻碍 同时进行膜片钳实验。现在我们已经克服了这个障碍。创新：最近 我们发明了膜片钳凝胶技术，使我们能够同时测量膜电压 或电流，SR或胞浆钙离子，以及嵌入在粘弹性水凝胶中的单个肌细胞的收缩 控制电池上的机械载荷。初步数据显示，机械载荷增加了周期性，延长了 动作电位(AP)，并增加心肌细胞肌浆网钙离子含量。因此我们假设 这种机械负荷增加了AP期间的钙离子内流，从而提高了SR的钙含量和钙离子的释放 增加CAT，可以部分补偿增加的机械负荷。虽然这是一种补偿 CAT的增加可能有利于抵消机械载荷的适度增加，我们进一步假设 过度负荷导致肌质网钙超载，导致肌浆网钙离子过度升高或INCx降低，导致心肌梗死 致心律失常的自发性钙离子释放。衰竭的心脏不能产生足够的力量来维持足够的 心输出量。我们假设，CAT的代偿性增加随着负荷的增加在HF时被钝化。 我们的跨学科团队将开发新的膜片钳凝胶技术并测试假说 利用健康兔和心力衰竭兔的心肌细胞，以及转基因小鼠来实现三个特定的目的。 (1)系统研究机械负荷对心动周期心肌细胞E-C偶联的调节作用。(2) 破译AP时ICAL和INCX及钙离子内流的机械电转导效应。(3)了解如何 病理性高负荷可导致心衰时钙离子调节失调和致心律失常。这样做的结果是 项目将阐明机械负荷如何影响代偿性心脏兴奋-收缩偶联。 心力衰竭患者对生理负荷的反应以及机械转导如何受损导致 心律失常和收缩功能障碍。

ABSTRACT The heart must adjust its contractile force to counteract the blood pressure changes (due to physical activity, body position, emotional stress, etc.) to maintain cardiac output. Cardiac muscle cells possess the intrinsic ability to sense mechanical load and fine-tune contractile force accordingly. Previous work by us and others found that increasing mechanical loading on muscle strips or single myocytes results in an increase of the cytosolic Ca2+ transient (CaT) that increases the contractile force to partially compensate for the added mechanical load. A sustained increase in CaT requires augmenting the Ca2+ content of the sarcoplasmic reticulum (SR), which must come from a net increase of Ca2+ across the sarcolemma. However, the mechanism that links mechanical loading to the control of Ca2+ entry remains unknown. Plausible mechanisms for this net increase include a mechanical load-induced increase of Ca2+ influx through L-type Ca2+ channels (ICaL) or a decreased efflux from Na+-Ca2+ exchanger (INCX). However, studying how mechanical loading affects ICaL or INCX has been hampered by the technical difficulty of subjecting myocytes to mechanical loads while simultaneously doing patch-clamp experiments. Now we have overcome this obstacle. Innovation: Recently we invented the Patch-Clamp-in-Gel technique that enables us to simultaneously measure membrane voltage or current, SR or cytosolic Ca2+, and contraction in single myocytes embedded in a viscoelastic hydrogel that controls mechanical load on the cell. Preliminary data reveal that mechanical load increases ICaL, prolongs the action potential (AP), and increases SR Ca2+ content in rabbit ventricular myocytes. Therefore we hypothesize that mechanical load increases Ca2+ entry during AP to elevate the SR Ca2+ content and Ca2+ release that increases CaT, which can partially compensate for increased mechanical load. While a compensatory increase in CaT might be beneficial to offset moderate increases in mechanical loading, we further posit that excessive loading causes SR Ca2+ overload by excessive ICaL increase or INCX decrease, leading to arrhythmogenic spontaneous Ca2+ release. Failing hearts cannot generate enough force to maintain adequate cardiac output. We hypothesize that the compensatory increase of CaT with increased load is blunted in HF. Our interdisciplinary team will develop the new Patch-Clamp-in-Gel technique and test the hypotheses using ventricular myocytes from healthy and HF rabbit, and transgenic mouse to achieving three specific aims. (1) Systematically investigate how mechanical load regulates myocyte E-C- coupling during cardiac cycle. (2) Decipher mechano-electro-transduction effects on ICaL and INCX and Ca2+ entry during AP. (3) Understand how pathological high load may cause Ca2+ dysregulation and arrhythmogenic activities in HF. The outcome of this project will elucidate how mechanical load affects cardiac excitation-contraction coupling in compensatory response to physiological loading, and how mechanotransduction is impaired in heart failure patients to cause arrhythmias and contractile dysfunction.