Regulation of Contractility During Ischemia-Reperfusion

心肌梗死很普遍，每年有100万名患者被诊断出来。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 心肌所经历的缺血-再灌注(I-R)。随着I-R，ATP的降低通常是 被认为是收缩能力下降背后的驱动力。然而，最近的研究表明， 收缩细丝固有的变化，如蛋白质分解或氧化还原依赖的蛋白质 修饰性也会影响I-R期间的收缩。本申请中的初步数据表明， 心肌收缩能力的下降在缺血30‘时发生，并在60’时基本逆转 再灌流。收缩能力的可逆性下降与三磷酸腺苷的可获得性无关，表明 收缩细丝的内在变化最好地描述了这种下降。然而，这一时间框架是 缺血时蛋白质分解不足，可通过蛋白质合成和重组来挽救 再灌流。因此，这些收缩性能的变化可能反映了可逆的共价修饰 收缩细丝的蛋白质，而不是它们的蛋白分解。与这一假设一致，初步 数据表明，肌动蛋白在I-R过程中发生可逆的修饰，影响其与 原肌球蛋白。因此，这项拨款申请旨在研究I-R期间的纤维收缩性能，以及 表征对收缩细丝蛋白质的可逆修饰，这些修饰是导致 伸缩性。这项应用将检验缺血-再灌注导致可逆、共价的假设 修饰心肌细丝的蛋白质，从而限制收缩能力 细丝调节蛋白关联性的变化。这一假设将通过以下方式进行检验：i) 确定I-R对心肌纤维收缩能力的影响；ii)表征I-R依赖性 肌动蛋白的修饰，以及确定I-R是否导致对其他细丝蛋白的共价修饰；iii) 肌动蛋白修饰对细丝组装以及肌动蛋白激活肌球蛋白的影响 ATPase。这些发现将为了解I-R期间收缩缺陷的性质提供新的见解， 强调心肌细丝蛋白的状态及其对收缩性能的影响。

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.