LPS引起心肌细胞肌浆网钙漏流的机制及在内毒素心功能损伤中的作用

Myocardial depression is a well-recognized manifestation of organ dysfunction in sepsis. The development of myocardial depression is related to high motality of sepsis. Septic cardiac dysfunction is characterized by impairment of systolic and diastolic function and reduced inotropic response to catecholamine stimulation. It is well known that the major source of Ca2+ for myocyte contraction is from sarcoplasmic reticulum (SR). The SR Ca2+ content determines the cardiac contractile function. Reduced SR Ca2+ content results in decrease of systolic Ca2+ transient and impairment of myocyte contractility. Under physiological conditions, the cardiac ryanodine receptors (RyR2), the SR Ca2+ release channels, are closed during diastole to prevent Ca2+ leak from SR and maintain SR Ca2+ content. If RyR2 cannot be normally closed during diastole, it will enhance SR Ca2+ leak and result in partial depletion of SR Ca2+ content. Our preliminary data show that lipopolysacchride (LPS) stimulation decreased myocyte contractility in cultured rat ventricular myocytes. Meanwhile, we found that the frequency of spontaneous Ca2+ sparks in cardiomyocytes during diastole was significantly increased, indicating that RyR2 was hyperactive and SR Ca2+ leak was enhanced. Importantly, the result suggested that LPS reduced SR Ca2+ content, which may be an important reason for myocardial depression in sepsis. Therefore, we proposed an mechanism underlying the pathogenesis of cardiac dysfunction in sepsis: LPS enhanced Ca2+ spark-mediated SR Ca2+ leak, which caused partial depletion of SR Ca2+ content, leading to reduced systolic Ca2+ transient and impairment of myocyte contractility. This hypothesis will be tested in LPS-stimulated cardiomyocytes and septic mouse model. Furthermore, we will investigate the cellular and molecular mechanisms underlying the abnormal hyperactive RyR2 upon LPS stimulation. It has been suggested that the mode of the interdomain interaction between the N terminal and central domains in RyR2 plays a key role in RyR2 channel regulation and the pathogenesis of heart failure. The interdomain interaction can be modulated by the oxidative status of RyR2. It has been suggested that oxidative modification of RyR2 results in abnomal interdomain interaction (unzipped) of RyR2. LPS induces intracellular reactive oxygen species (ROS) production through multiple mechanisms. Therefore, we propose that LPS-induced hyperactive RyR and subsequent SR Ca2+ leak is related to defective interdomain interaction within RyR2 due to oxidative modification of the channel. Through this project, we hope to elucidate the mechanisms underlying myocardial depression in sepsis and develope new therapeutic strategies against the disease.

心功能损害是脓毒症和感染性休克发展过程中常见的并发症，也是造成患者死亡的重要原因。肌浆网(SR)是心脏收缩的主要钙离子来源，钙库容量决定心肌收缩功能。我们前期工作在LPS刺激造成收缩力减弱的心肌细胞，发现舒张期钙火花频率显著增加，表明SR钙释放通道-ryanodine receptor (RyR2)异常开放，SR钙漏流增强，提示SR钙库容量降低。本项目拟在LPS刺激的心肌细胞模型和内毒素心功能障碍动物模型，查明SR钙漏流引起内毒素心功能损害的机制。RyR2的N-末端和中心片段的正常相互作用是保证舒张期RyR2通道正常关闭的关键，受RyR2氧化应激的调控。LPS增强细胞氧化应激反应，因此，我们提出RyR2异常开放的可能分子机制：LPS通过氧化修饰引起RyR2分子变构，使上述片段间作用异常，降低RyR2稳定性使其在舒张期异常开放。本研究旨在阐明内毒素心功能障碍的机制，为临床治疗提供新思路。

有研究表明，肌浆网 (SR)Ca2 +漏流在脓毒症心肌收缩功能障碍中起到了重要的作用。然而，肌浆网Ca2 +漏流的机制并不清楚，且缺乏直接证据。在这里，我们分别在LPS处理的小鼠心肌细胞和利用盲肠结扎穿孔法（CLP）制作的多种微生物感染的脓毒症小鼠模型上，观察心肌细胞收缩和Ca2 +变化。发现LPS降低了收缩期钙瞬变和心肌细胞收缩力，减少了肌浆网钙容量。同时，LPS增加了钙火花介导的肌浆网钙漏流。为探讨LPS刺激引起的钙火花介导的钙漏流和肌浆网钙容量减少之间的因果关系，我们应用了RyR阻断剂tetracaine,抑制钙火花介导的钙漏流，同时观察对肌浆网钙负荷的影响。结果发现tetracaine显著降低钙火花频率，并且很大程度上恢复了经LPS处理而减少的肌浆网负荷，心肌收缩力增强，提示SR钙漏流是导致肌浆网钙负荷减少的一个重要的原因，也提示内毒素引起心肌细胞SR钙漏流的机制与RyR有关。JTV-519是一个新的抗肌浆网钙漏流的药物，我们应用JTV-519治疗后，发现可以恢复SR负荷，改善心脏功能。并且没有显著影响促炎细胞因子的产生，证实脓毒症时心脏收缩功能受损的重要原因是肌浆网钙漏流，从而强调了JTV-519在脓毒症心肌病时潜在的治疗作用。在LPS处理的心肌细胞，RyR2的线粒体活性氧（mitoROS）和氧化应激均增加，而RyR2相关的 FK506绑定蛋白1B（FKBP12.6）却下降。Toll样受体4（TLR4）是LPS的受体，在脓毒症心脏功能障碍中起着至关重要的作用。TLR4激活的信号通路可以诱导线粒体功能障碍，及细胞内ROS的过度积累。为探讨TLR4激活对细胞内氧化应激的贡献，以及随之带来的肌浆网钙漏流，我们用TLR4特殊抑制剂TAK-242预处理。发现TAK- 242降低了氧化应激，减少了SR漏流，并使得钙调控和心肌细胞收缩正常。与此一致的，在脓毒症小鼠，TLR4基因敲除能显著改善心脏功能，并纠正异常钙调控。这项研究证明了肌浆网钙漏流在脓毒症心肌病的发展中起到了重要的作用，并强调了JTV-519可以通过阻止肌浆网钙漏流起到潜在的治疗作用。此外，本研究还发现TLR4激活引发了mitoROS产生， RyR2氧化应激增强，从而导致肌浆网钙漏流增加。