Mitochondria play key roles in the differentiation and maturation of human cardiomyocytes (CMs). As human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold potential in the treatment of heart diseases, we sought to identify key mitochondrial pathways and regulators, which may provide targets for improving cardiac differentiation and maturation. Proteomic analysis was performed on enriched mitochondrial protein extracts isolated from hiPSC-CMs differentiated from dermal fibroblasts (dFCM) and cardiac fibroblasts (cFCM) at time points between 12 and 115 days of differentiation, and from adult and neonatal mouse hearts. Mitochondrial proteins with a 2-fold change at time points up to 120 days relative to 12 days were subjected to Ingenuity Pathway Analysis (IPA). The highest upregulation was in metabolic pathways for fatty acid oxidation (FAO), the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS) and branched chain amino acid (BCAA) degradation. The top upstream regulators predicted to be activated were peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1-α), the insulin receptor (IR) and the retinoblastoma protein (Rb1) transcriptional repressor. IPA and immunoblotting showed upregulation of the mitochondrial LonP1 protease - a regulator of mitochondrial proteostasis, energetics and metabolism. LonP1 knockdown increased FAO in neonatal rat ventricular cardiomyocytes (nRVMs). Our results support the notion that LonP1 upregulation negatively regulates FAO in cardiomyocytes to calibrate the flux between glucose and fatty acid oxidation. We discuss potential mechanisms by which IR, Rb1 and LonP1 regulate the metabolic shift from glycolysis to OXPHOS and FAO. These newly identified factors and pathways may help in optimizing the maturation of iPSC-CMs.

中文翻译：

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人诱导多能干细胞分化心肌细胞线粒体生物发生的蛋白质组学分析

线粒体在人心肌细胞 (CM) 的分化和成熟中起关键作用。由于人类诱导多能干细胞衍生的心肌细胞 (hiPSC-CM) 在治疗心脏病方面具有潜力，我们试图确定关键的线粒体途径和调节因子，它们可能为改善心脏分化和成熟提供靶点。在分化 12 至 115 天之间的时间点，对从从真皮成纤维细胞 (dFCM) 和心脏成纤维细胞 (cFCM) 分化的 hiPSC-CM 以及从成年和新生小鼠心脏中分离出来的富集线粒体蛋白提取物进行蛋白质组学分析。对在长达 120 天（相对于 12 天）的时间点发生 2 倍变化的线粒体蛋白进行 Ingenuity Pathway Analysis (IPA)。最高的上调是在脂肪酸氧化 (FAO)、三羧酸 (TCA) 循环、氧化磷酸化 (OXPHOS) 和支链氨基酸 (BCAA) 降解的代谢途径中。预计将被激活的上游调节因子是过氧化物酶体增殖物激活受体 γ 共激活因子 1 α (PGC1-α)、胰岛素受体 (IR) 和视网膜母细胞瘤蛋白 (Rb1) 转录抑制因子。IPA 和免疫印迹显示线粒体 LonP1 蛋白酶的上调——线粒体蛋白质稳态、能量学和新陈代谢的调节剂。LonP1 敲低增加了新生大鼠心室心肌细胞 (nRVM) 中的FAO。我们的结果支持LonP1上调负调节心肌细胞中的FAO以校准葡萄糖和脂肪酸氧化之间的通量的观点。我们讨论了 IR、Rb1 和 LonP1 调节从糖酵解到 OXPHOS 和 FAO 的代谢转变的潜在机制。这些新发现的因素和途径可能有助于优化 iPSC-CM 的成熟。