Fast myosin binding protein-C and cardiac contractility in heart failure

项目摘要：心力衰竭(HF)是一种复杂的临床综合征，也是 成人死亡率。这次更新的总体目标是继续阐明行动机制 肌球蛋白结合蛋白-C(MyBP-C)家族蛋白对心脏功能的影响。MyBP-C蛋白调节 心肌和骨骼肌的收缩结构和功能。本提案的重点是 将MyBP-C蛋白之一，快速肌球蛋白结合蛋白-C(fMyBP-C)的作用定义为阴性 心脏收缩能力的调节剂。FMyBP-C在心脏中的表达水平最低，但显著高于正常水平 心衰时心肌细胞内的含量增加。在初步研究中，一种新型的转基因小鼠表达了 FMyBP-C(fMyBP-CTG)特异性地存在于心肌细胞中，其水平与在HF中观察到的水平相似 心肌肥厚，最大肌力降低，肌丝对钙的敏感性降低。相比之下， 纯合子fMyBP-C基因敲除(fMyBP-CKO)小鼠，当受到横断性主动脉缩窄的攻击时 (TAC)诱导的压力超负荷，与对照组相比，显示出显著的心功能改善。 此外，体外研究表明，fMyBP-C通过以下方式负向调节心肌细胞的收缩能力 增加肌球蛋白的超松弛状态，肌球蛋白的相互作用和细丝的激活。然而， 调节fMyBP-C及其影响的分子机制 肌节的功能，是完全未知的。 根据初步发现，我们的中心假设是fMyBP-C上调 减少肌动球蛋白的相互作用，防止细丝和粗丝，从而降低心力衰竭期间的心功能 相互作用，减少力量产生的速度，导致收缩功能障碍和心衰。 特异性目标1将使用一种新的fMyBP-CTG小鼠模型来确定fMyBP-C的充分性 肌球蛋白在心肌细胞中的表达与心肌收缩能力、肌动球蛋白相互作用和交叉性的关系 与对照组相比，包括cMyBP-C转基因小鼠和空白小鼠。使用 FMyBP-CKO小鼠，特异性AIM 2将确定fMyBP-C加重(TAC)诱导的必要性 与对照组相比，使用fMyBP-CKO小鼠的压力超载HF。出于同样的目的，一种新的Mybpc2- MScarlet(荧光)敲入小鼠模型将用于定位TAC-C过程中fMyBP-C的表达 诱导压力过载。利用这些小鼠模型，特定的AIM 3将阐明分子 利用fMyBP-CTG和FMBP-CTG的心肌细胞降低心肌收缩能力的机制(S) FMyBP-CKO小鼠，以及cMyBP-C/fMyBP-C的重组蛋白和嵌合体在不同的 体外生物物理、生化和功能研究，并与野生型对照进行比较。加在一起，这些 研究将确定fMyBP-C负性调节肌力的必要性和充分性及其原因 高频。这一结果将有助于确定治疗心力衰竭的潜在靶点。

PROJECT SUMMARY: Heart failure (HF) is a complex clinical syndrome and a predominant cause of mortality in adults. The overall objective of this renewal is to continue to elucidate the mechanism of action of the myosin binding protein-C (MyBP-C) family of proteins on cardiac function. MyBP-C proteins regulate contractile structure and function in both cardiac and skeletal muscles. The present proposal is focused on defining the role of one of the MyBP-C proteins, fast myosin binding protein-C (fMyBP-C), as a negative regulator of cardiac contractility. fMyBP-C is minimally expressed at baseline in the heart but is significantly increased in the cardiomyocytes during HF. In the pilot studies, a novel transgenic mouse that expresses fMyBP-C (fMyBP-CTg) specifically in cardiomyocytes and at levels similar to those observed in HF develops cardiac hypertrophy, decreased maximal force and reduced myofilament Ca2+ sensitivity. In contrast, homozygous fMyBP-C knockout (fMyBP-CKO) mice, when challenged with transverse aortic coarctation (TAC)-induced pressure overload, shows significantly improved cardiac function, compared to controls. Furthermore, in vitro studies revealed that fMyBP-C negatively modulates cardiomyocyte contractility by increasing super-relaxed state of myosin, actomyosin interactions and thin filament activation. However, the molecular mechanisms underlying the regulation of fMyBP-C and, in turn, its impact on sarcomere function, are completely unknown. On the basis of the preliminary findings, our central hypothesis is that fMyBP-C upregulation reduces cardiac function during HF by reducing actomyosin interactions, preventing thin and thick filament interactions and decreasing the rate of force generation, resulting in contractile dysfunction and HF. SPECIFIC AIM 1 will use a novel fMyBP-CTg mouse model to determine the sufficiency of fMyBP-C expression in the cardiomyocytes in reducing cardiac contractility, actomyosin interactions and cross- bridges and developing HF, compared to controls, including cMyBP-C transgenic and null mice. Using the fMyBP-CKO mice, SPECIFIC AIM 2 will define the necessity of fMyBP-C to exacerbate (TAC)-induced pressure overload HF using fMyBP-CKO mice, compared to controls. In the same aim, a novel Mybpc2- mScarlet (fluorescence) knock-in mouse model will be used to fate map fMyBP-C expression during TAC- induced pressure overload. Using these mouse models, SPECIFIC AIM 3 will elucidate the molecular mechanism(s) underlying reduced cardiac contractility using cardiomyocytes from the fMyBP-CTg and fMyBP-CKO mice, as well as recombinant proteins and chimeras of cMyBP-C/fMyBP-C, on various biophysical, biochemical and functional studies in vitro, compared to wild-type controls. Together, these studies will determine the necessity and sufficiency of fMyBP-C to negatively regulate inotropy and cause HF. The results will lead to the identification of potential therapeutic targets to treat HF.