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Autophagy, a critical catabolic process for cell survival against different types of stress, has a role in the differentiation of various cells, such as cardiomyocytes. Adenosine 5ʹ‐monophosphate (AMP)‐activated protein kinase (AMPK) is an energy‐sensing protein kinase involved in the regulation of autophagy. In addition to its direct role in regulating autophagy, AMPK can also influence other cellular processes by regulating mitochondrial function, posttranslational acetylation, cardiomyocyte metabolism, mitochondrial autophagy, endoplasmic reticulum stress, and apoptosis. As AMPK is involved in the control of various cellular processes, it can influence the health and survival of cardiomyocytes. This study investigated the effects of an AMPK inducer (Metformin) and an autophagy inhibitor (Hydroxychloroquine) on the differentiation of human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs). The results showed that autophagy was upregulated during cardiac differentiation. Furthermore, AMPK activation increased the expression of CM‐specific markers in hPSC‐CMs. Additionally, autophagy inhibition impaired cardiomyocyte differentiation by targeting autophagosome‐lysosome fusion. These results indicate the significance of autophagy in cardiomyocyte differentiation. In conclusion, AMPK might be a promising target for the regulation of cardiomyocyte generation by in vitro differentiation of pluripotent stem cells.

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