Mechanical Stress-Dependent Remodeling of the Cardiac Microtubule Network

心力衰竭通常以心脏组织硬化为标志，这损害了心脏的放松能力。微管网络-心肌细胞骨架的一部分-提供了一种内部刚度，可以阻止心肌细胞的收缩和舒张。我们最近发现，心肌细胞微管网络的刚度是由翻译后脱酪氨酸和微管，脱酪氨酸，和细胞骨架交联剂在心力衰竭中一直升高，伴随着心肌细胞刚度的增加。我们的研究结果表明，减少脱酪氨酸减少微管网络密度和收缩缺陷的心肌细胞从晚期心力衰竭患者支持脱酪氨酸作为一个治疗目标。与此同时，这些发现提出了关于心力衰竭中微管网络重塑的驱动过程以及微管网络随时间持续增加的后果的重要问题。因此，拟议的研究将测试心脏微管网络的重塑是对改变的机械应力的可逆适应的假设，当持续时，会导致病理性肥大和收缩功能障碍。三个目标下的研究将解决这一假设的多个组成部分。目的1实验将确定机械应力是否足以驱动微管网络的细胞自主重塑，使用机械生物学工具包来分离三个关键机械应力源（预加载，后加载和基质刚度）对微管网络重塑的贡献。目的2实验将我们的机械操作扩展到组织和器官水平，以表征相关体内环境下的微管网络重塑。目的3研究将采用体外和体内遗传操作，以确定在存在和不存在慢性压力超负荷的情况下，脱酪氨酸的持续增加是否有助于病理性心脏肥大。我们的整体研究设计使用新颖和互补的实验方法，既利用模型系统的优势，又减轻其缺点。这包括来自人类心肌的原代心肌细胞，以补充来自动物模型和工程组织构建体的发现。这种跨物种、多尺度的方法平衡了简化论的严谨性和综合相关性的双重目标，从而促进了最终的临床翻译。这项工作将为心力衰竭中微管网络变化的原因提供深入了解，并帮助确定预防或逆转这些变化是否具有治疗益处。

Heart failure is often marked by stiffening of cardiac tissue that impairs the heart’s ability to relax. The microtubule network – a part of the cardiomyocyte cytoskeleton – provides an internal stiffness that can impede cardiomyocyte contraction and relaxation. We have recently found that cardiomyocyte microtubule network stiffness is tightly regulated by post-translational detyrosination and that microtubules, detyrosination, and cytoskeletal cross-linkers are consistently elevated in heart failure, with concomitant increases in cardiomyocyte stiffness. Our findings that reducing detyrosination lessens microtubule network density and contractile defects in cardiomyocytes from patients with advanced heart failure supports detyrosination as a therapeutic target. At the same time, these findings raise important questions about the processes driving remodeling of the microtubule network in heart failure and the consequences of sustained increases in the microtubule network over time. Accordingly, the proposed research will test the hypothesis that remodeling of the cardiac microtubule network is a reversible adaptation to altered mechanical stress, which when sustained, contributes to pathological hypertrophy and contractile dysfunction. Studies under three aims will address the multiple components of this hypothesis. Aim 1 experiments will determine if mechanical stress is sufficient to drive cell-autonomous remodeling of the microtubule network using a mechanobiology toolkit to isolate the contribution of three key mechanical stressors (pre-load, after-load, and matrix stiffness) on microtubule network remodeling. Aim 2 experiments will extend our mechanical manipulations to the tissue and organ level to characterize microtubule network remodeling under relevant in vivo contexts. Aim 3 studies will employ in vitro and in vivo genetic manipulations to determine whether sustained increases in detyrosination contribute to pathologic cardiac hypertrophy in the presence and absence of chronic pressure overload. Our overall study design uses novel and complementary experimental approaches to both exploit strengths of model systems and mitigate their shortcomings. This includes primary cardiomyocytes from human myocardium to complement findings from animal models and engineered tissue constructs. This cross-species, multi-scale approach balances the dual goals of reductionist rigor and integrative relevance that furthers ultimate clinical translation. Together this work will provide insight into the causes of microtubule network changes in heart failure and help determine if preventing or reversing these changes is therapeutically beneficial.