Abstract
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent an infinite cell source for cardiovascular disease modeling, drug screening and cell therapy. Despite extensive efforts, current approaches have failed to generate hPSC-CMs with fully adult-like phenotypes in vitro, and the immature properties of hPSC-CMs in structure, metabolism and electrophysiology have long been impeding their basic and clinical applications. The prenatal-to-postnatal transition, accompanied by severe nutrient starvation and autophagosome formation in the heart, is believed to be a critical window for cardiomyocyte maturation. In this study, we developed a new strategy, mimicking the in vivo starvation event by Earle’s balanced salt solution (EBSS) treatment, to promote hPSC-CM maturation in vitro. We found that EBSS-induced starvation obviously activated autophagy and mitophagy in human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Intermittent starvation, via 2-h EBSS treatment per day for 10 days, significantly promoted the structural, metabolic and electrophysiological maturation of hESC-CMs. Structurally, the EBSS-treated hESC-CMs showed a larger cell size, more organized contractile cytoskeleton, higher ratio of multinucleation, and significantly increased expression of structure makers of cardiomyocytes. Metabolically, EBSS-induced starvation increased the mitochondrial content in hESC-CMs and promoted their capability of oxidative phosphorylation. Functionally, EBSS-induced starvation strengthened electrophysiological maturation, as indicated by the increased action potential duration at 90% and 50% repolarization and the calcium handling capacity. In conclusion, our data indicate that EBSS intermittent starvation is a simple and efficient approach to promote hESC-CM maturation in structure, metabolism and electrophysiology at an affordable time and cost.
Highlights
Cardiovascular disease has become the number one killer of human health (Tan and Ye, 2018)
Our data indicated that Earle’s balanced salt solution (EBSS) treatments for 1 to 5 h efficiently induced autophagy in H1 into spontaneously beating cardiomyocytes (H1-CMs), and the 2-h treatment of EBSS was chosen for further studies
We observed a significant increase in the oxygen consumption rate (OCR) of EBSS-treated H1-CMs (Figure 3A)
Summary
Cardiovascular disease has become the number one killer of human health (Tan and Ye, 2018). Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), show enormous potential for the development of cardiac disease models, drug discovery and cell therapy (Ye et al, 2015; Gintant et al, 2017; Hu et al, 2018) These applications are hampered by the immature phenotypes of hPSC-derived cardiomyocytes (hPSC-CMs) that resemble fetal CMs. The major hallmarks of cardiomyocyte maturation include maturational hypertrophy, well-formed and organized myofibrils, increased DNA content and mitochondrial density, as well as the ability to perform β-oxidation of fatty acids, functional electrophysiology and calcium handling (Matsui et al, 2007). Our results indicate that intermittent EBSS starvation provides a new direction for promoting the maturation of cardiomyocytes for better application in disease modeling, drug screening and cell therapy
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