Mechanical regulation of maturation and pathology of engineered human heart tissues

摘要 人类诱导多能干细胞来源的心肌细胞(hiPSC-CMS)的出现提供了令人兴奋的 研究人类心脏疾病和体外发育的机会。然而，HiPSC-CMS在结构上是 在功能上不成熟，比成年CMS更像胎儿，这从他们对 糖酵解代替氧化代谢，弱收缩和兴奋性，表达不成熟 结构蛋白的异构体，包括肌球蛋白和肌钙蛋白。这使得HiPSC-CMS不适合于研究 成人获得性心脏病，如心力衰竭(HF)，它仍然是导致 美国和全世界。CMS的机械环境被广泛认为是一个关键的调节因素 心脏组织发育、生理学和疾病。尤其是机械载荷的动态变化 CMS在出生后发育过程中的经历可能在他们获得成熟成年人的过程中起到关键作用 表型。我假设动态、时变拉伸(预加载)和阻力的呈现 由hiPSC-CMS制成的人工程心脏组织的收缩(后负荷)将增加其 结构和功能的成熟度。此外，我预测hEHTs的机械过载将导致 心力衰竭进展的病理特征。为了验证这些假设，我开发了一本小说 生物反应器，施加在hEHTs上的机械预负荷和后负荷可以独立地随时间变化 通过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 不同的递增预载荷和后载荷影响结构、收缩压力的产生， 动作电位的传播，以及hEHTs的转录和代谢特性。此外，我还将聘用 实时成像以识别和表征所观察到的机械转导机制 HEHTs的功能变化。对于诱导分子和功能的机械过载状态 一个HF表型的特征，我将研究不同的辅助治疗是否结合应用机械治疗 类似于使用左心室辅助装置(LVAD)的卸载可以反向重塑结构和功能 HEHTs的赤字。这些研究完成后，将确定体外CM的新的机械生物学驱动因素 成熟度和加深对心力衰竭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.