Mechanical regulation of maturation and pathology of engineered human heart tissues

摘要 人类诱导多能干细胞衍生的心肌细胞（hiPSC-CM）的出现提供了令人兴奋的 研究人类心脏病和体外发育的机会。然而，hiPSC-CM在结构上是 功能上不成熟，更像胎儿而不是成人CM，这从它们对 糖酵解而不是氧化代谢，弱收缩和兴奋性，以及表达不成熟的 包括肌球蛋白和肌钙蛋白的结构蛋白的同种型。这使得hiPSC-CM不足以用于以下研究： 成人获得性心脏病，如心力衰竭（HF），这仍然是死亡的主要原因， 美国和全世界。CM的机械环境被广泛认为是一个关键的调节器， 心脏组织发育、生理学和疾病。特别是机械载荷的动态变化 CM在出生后发育过程中所经历的变化可能在它们获得成熟的成年体中起关键作用 表型。我假设，动态的，随时间变化的拉伸（预负荷）和阻力的介绍， 对由hiPSC-CM制成的人类工程化心脏组织（hEHT）的收缩（后负荷）将增加它们的 结构和功能成熟度。此外，我预测hEHT的机械过载将诱导 HF进展的病理特征。为了验证这些假设，我写了一本小说 生物反应器，其中施加在hEHT上的机械预载荷和后载荷可以随时间独立地变化， 通过施加拉伸和电刺激进行培养。利用这个平台，我将系统地研究如何 逐渐增加的前负荷和后负荷的不同状态影响结构，收缩力的产生， 动作电位的传播以及hEHT的转录组学和代谢特性。另外，我将聘请 实时成像，以识别和表征所观察到的机械转导机制 hEHT的功能变化。对于机械过载制度，诱导分子和功能 HF表型的特征，我将研究不同的辅助治疗是否与应用的机械 类似于使用左心室辅助装置（LVAD）的卸载可以逆转结构和功能 hEHTs的缺陷。完成后，这些研究将确定体外CM的新机械生物学驱动因素 成熟和进一步的HF疾病和治疗的分子理解。

ABSTRACT The advent of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offers exciting opportunities to study human cardiac disease and development in vitro. However, hiPSC-CMs are structurally and functionally immature and more closely resemble fetal than adult CMs as evident from their reliance on glycolytic instead of oxidative metabolism, weak contractions and excitability, and expression of immature isoforms of structural proteins including myosin and troponin. This makes hiPSC-CMs inadequate for studies of adult-acquired cardiac diseases, such as heart failure (HF), which remains the leading cause of death in the United States and worldwide. The mechanical environment of CMs is widely recognized as a key regulator of cardiac tissue development, physiology, and disease. In particular, dynamic changes in mechanical load experienced by CMs during postnatal development may play a key role in their acquisition of a mature adult phenotype. I hypothesize that presentation of dynamic, time-varying stretch (preload) and resistance to contraction (afterload) to human engineered heart tissues (hEHTs) made of hiPSC-CMs will increase their structural and functional maturity. Furthermore, I predict that mechanical overload of hEHTs will induce pathological features characteristic of HF progression. To test these hypotheses, I have developed a novel bioreactor where mechanical preload and afterload imposed on hEHTs can be independently varied with time of culture via application of stretch and electrical stimulation. Using this platform, I will systematically study how different regimes of progressively increased preload and afterload affect structure, contractile force generation, propagation of action potentials, and transcriptomic and metabolic properties of hEHTs. Additionally, I will employ real-time imaging to identify and characterize the mechanotransduction mechanisms underlying the observed functional changes in the hEHTs. For mechanical overload regimes that induce molecular and functional signatures of an HF phenotype, I will study if different adjuvant therapies combined with applied mechanical unloading akin to use of left ventricular assist devices (LVADs) can reverse-remodel structural and functional deficits in hEHTs. When completed, these studies will identify new mechanobiological drivers of in vitro CM maturation and further the molecular understanding of HF disease and therapy.