Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA.
Stem Cells. 2020 Oct 1;38(10):1254-1266. doi: 10.1002/stem.3235. Epub 2020 Jun 16.
Advancing maturation of stem cell-derived cardiac muscle represents a major barrier to progress in cardiac regenerative medicine. Cardiac muscle maturation involves a myriad of gene, protein, and cell-based transitions, spanning across all aspects of cardiac muscle form and function. We focused here on a key developmentally controlled transition in the cardiac sarcomere, the functional unit of the heart. Using a gene-editing platform, human induced pluripotent stem cell (hiPSCs) were engineered with a drug-inducible expression cassette driving the adult cardiac troponin I (cTnI) regulatory isoform, a transition shown to be a rate-limiting step in advancing sarcomeric maturation of hiPSC cardiac muscle (hiPSC-CM) toward the adult state. Findings show that induction of the adult cTnI isoform resulted in the physiological acquisition of adult-like cardiac contractile function in hiPSC-CMs in vitro. Specifically, cTnI induction accelerated relaxation kinetics at baseline conditions, a result independent of alterations in the kinetics of the intracellular Ca transient. In comparison, isogenic unedited hiPSC-CMs had no cTnI induction and no change in relaxation function. Temporal control of adult cTnI isoform induction did not alter other developmentally regulated sarcomere transitions, including myosin heavy chain isoform expression, nor did it affect expression of SERCA2a or phospholamban. Taken together, precision genetic targeting of sarcomere maturation via inducible TnI isoform switching enables physiologically relevant adult myocardium-like contractile adaptations that are essential for beat-to-beat modulation of adult human heart performance. These findings have relevance to hiPSC-CM structure-function and drug-discovery studies in vitro, as well as for potential future clinical applications of physiologically optimized hiPSC-CM in cardiac regeneration/repair.
推进干细胞衍生心肌的成熟是心脏再生医学进展的主要障碍。心肌成熟涉及无数基因、蛋白质和基于细胞的转变，涵盖了心肌形态和功能的各个方面。我们在这里关注的是心肌收缩单位（心肌的功能单位）中一个关键的发育控制转变。使用基因编辑平台，通过药物诱导表达盒对人诱导多能干细胞（hiPSC）进行工程改造，该表达盒驱动成人肌钙蛋白 I（cTnI）调节同工型的表达，该转变被证明是推进 hiPSC 心肌（hiPSC-CM）向成人状态的肌节成熟的限速步骤。研究结果表明，诱导成人 cTnI 同工型可使 hiPSC-CM 在体外获得类似于成人的心肌收缩功能。具体来说，cTnI 诱导可加速基础条件下的弛豫动力学，这一结果与细胞内 Ca 瞬变动力学的改变无关。相比之下，同基因未编辑的 hiPSC-CM 没有 cTnI 诱导，也没有弛豫功能的改变。成人 cTnI 同工型诱导的时间控制不会改变其他发育调节的肌节转变，包括肌球蛋白重链同工型的表达，也不会影响 SERCA2a 或肌浆球蛋白的表达。总之，通过诱导型 TnI 同工型转换对肌节成熟进行精确的遗传靶向，可以实现与生理相关的成人心肌样收缩适应性，这对于调节成人人心功能的跳动至关重要。这些发现与 hiPSC-CM 的结构-功能以及体外药物发现研究有关，也与未来生理优化的 hiPSC-CM 在心脏再生/修复中的潜在临床应用有关。
