Regulation of Contractility During Ischemia-Reperfusion

描述（由申请人提供）：心肌梗死很普遍，每年诊断出约100万患者。为了更好地理解导致心肌细胞损伤和死亡的细胞条件，实验模型模拟了心肌经历的缺血-再灌注（I-R）。ATP随I-R的减少通常被认为是收缩力下降背后的驱动力。然而，最近的研究表明，收缩丝的内在变化，如蛋白质水解或氧化还原依赖性蛋白质修饰，也影响I-R期间的收缩性。本申请中的初步数据表明，心肌收缩力的下降发生在缺血30分钟时，并在再灌注60分钟时基本上逆转。收缩力的可逆性下降是独立的ATP的可用性，这表明收缩丝的内在变化最好地描述了下降。然而，这一时间范围不足以使缺血期间的蛋白质水解通过再灌注期间的蛋白质合成和重新组装来挽救。因此，这些收缩性的变化可能反映了可逆的，共价修饰的蛋白质的收缩丝，而不是他们的蛋白水解。与这一假设相一致，初步数据表明，在I-R过程中发生了肌动蛋白的可逆修饰，影响了它与原肌球蛋白的相互作用。因此，该资助申请旨在研究I-R期间的纤维收缩性，并表征收缩性变化背后的收缩丝蛋白质的可逆修饰。该申请将测试缺血-再灌注导致心肌细丝蛋白质可逆共价修饰的假设，从而通过细丝调节蛋白质结合的变化限制收缩力。该假设将通过以下方式进行检验：i）确定I-R对心肌纤维收缩性的影响; ii）表征肌动蛋白的I-R依赖性修饰，并确定I-R是否导致对其他细丝蛋白的共价修饰; iii）确定肌动蛋白修饰对细丝组装以及肌动蛋白激活的肌球蛋白ATP酶的影响。这些发现将提供新的洞察I-R期间的收缩缺陷的性质，强调心肌细丝蛋白的状态及其对收缩性的影响。

DESCRIPTION (provided by applicant): Myocardial infarction is prevalent with -1 million patients diagnosed each year. To better understand the cellular conditions leading to cardiomyocyte damage and death, experimental models have mimicked the ischemia - reperfusion (I-R) experienced by the myocardium. The decrease in ATP with I-R is often considered the driving force behind the contractility decline. However, recent research suggests that changes intrinsic to the contractile filaments, such as protein proteolysis or redox-dependent protein modifications, also influence contractility during I-R. The preliminary data in this application indicate that a decline in cardiac muscle contractility occurs with 30' of ischemia and is largely reversed by 60' of reperfusion. The reversible decline in contractility was independent of ATP availability, suggesting that intrinsic changes to the contractile filaments best described the decline. However, this timeframe is insufficient for protein proteolysis during ischemia to be rescued by protein synthesis and re-assembly during reperfusion. Therefore, these changes in contractility may reflect reversible, covalent modifications to proteins of the contractile filaments rather than their proteolysis. Consistent with this hypothesis, preliminary data demonstrate that a reversible modification of actin occurs during I-R, affecting it's interaction with tropomyosin. Therefore, this grant application aims to investigate fibre contractility during I-R, and characterize the reversible modifications to proteins of the contractile filaments that underlie the changes in contractility. The application will test the hypothesis that ischemia-reperfusion results in reversible, covalent modifications to proteins of the cardiac muscle thin filament, consequently limiting contractility through changes in the association of thin filament regulatory proteins. This hypothesis will be examined by: i) determining the effect of I-R on the contractility of cardiac muscle fibres; ii) characterizing the I-R dependent modification of actin, and determining if I-R results in covalent modifications to other thin filament proteins; iii) determining the effect of modification of actin on thin filament assembly as well as the actin activated myosin ATPase. These findings will provide novel insight into the nature of the contractile deficit during I-R, with emphasis on the state of the cardiac muscle thin filament proteins and their effect on contractility.