MECHANICAL LOAD EFFECT ON CARDIAC EXCITATION-CONTRACTION COUPLING

摘要 心脏必须调整其收缩力以抵消血压变化（由于体力活动， 身体姿势、情绪压力等）维持心输出量心肌细胞具有内在的 感知机械负荷并相应地微调收缩力的能力。我们和其他人以前的工作 发现增加肌肉条或单个肌细胞上的机械负荷会导致 胞质Ca 2+瞬变（CaT），增加收缩力以部分补偿增加的收缩力。 机械负荷CaT的持续增加需要增加肌浆内Ca 2+含量， 网状层（SR），这必须来自肌膜上Ca 2+的净增加。但 将机械负荷与Ca 2+进入控制联系起来的机制仍然未知。可能机制 这种净增加包括机械负荷诱导的通过L型Ca 2+通道的Ca 2+内流增加 （ICaL）或Na+-Ca 2+交换器（INCX）的流出减少。然而，研究机械负荷如何影响 ICaL或INCX受到使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或胞质Ca 2+，以及包埋在粘弹性水凝胶中的单个肌细胞的收缩， 控制电池上的机械负载。初步数据显示，机械负荷增加ICaL， 动作电位（AP），并增加SR Ca ~（2+）含量。因此我们假设 机械负荷增加了AP期间的Ca 2+进入，从而提高了SR Ca 2+含量和Ca 2+释放， 增加CaT，这可以部分补偿增加的机械负荷。虽然补偿性 CaT的增加可能有利于抵消机械负荷的适度增加，我们进一步证实， 过度负荷通过过度增加ICaL或降低INCX导致SR Ca 2+超载，导致 致炎性自发性Ca 2+释放。衰竭的心脏无法产生足够的力量来维持足够的 心输出量我们推测，随着负荷的增加，CaT的代偿性增加在HF中是钝化的。 我们的跨学科团队将开发新的膜片钳凝胶技术，并测试假设 利用健康兔、心衰兔和转基因小鼠的心室肌细胞，达到三个特定的目的。 (1)系统研究机械负荷如何调节心动周期中的心肌细胞E-C偶联。（二） 解读AP期间机械电转导对ICaL和INCX以及Ca 2+内流的影响。(3)了解如何 病理性高负荷可引起HF中Ca ~（2+）失调和促凋亡活性。这场 该项目将阐明机械负荷如何影响心脏兴奋-收缩耦合在代偿性 对生理负荷的反应，以及心力衰竭患者的机械传导如何受损， 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.