Investigation into the difference in mitochondrial-cytosolic calcium coupling between adult cardiomyocyte and hiPSC-CM using a novel multifunctional genetic probe.

Ca cycling plays a critical role in regulating cardiomyocyte (CM) function under both physiological and pathological conditions. Mitochondria have been implicated in Ca handling in adult cardiomyocytes (ACMs). However, little is known about their role in the regulation of Ca dynamics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In the present study, we developed a multifunctional genetically encoded Ca probe capable of simultaneously measuring cytosolic and mitochondrial Ca in real time. Using this novel probe, we determined and compared mitochondrial Ca activity and the coupling with cytosolic Ca dynamics in hiPSC-CMs and ACMs. Our data showed that while ACMs displayed a highly coordinated beat-by-beat response in mitochondrial Ca in sync with cytosolic Ca, hiPSC-CMs showed high cell-wide variability in mitochondrial Ca activity that is poorly coordinated with cytosolic Ca. We then revealed that mitochondrial-sarcoplasmic reticulum (SR) tethering, as well as the inter-mitochondrial network connection, is underdeveloped in hiPSC-CM compared to ACM, which may underlie the observed spatiotemporal decoupling between cytosolic and mitochondrial Ca dynamics. Finally, we showed that knockdown of mitofusin-2 (Mfn2), a protein tethering mitochondria and SR, led to reduced cytosolic-mitochondrial Ca coupling in ACMs, albeit to a lesser degree compared to hiPSC-CMs, suggesting that Mfn2 is a potential engineering target for improving mitochondrial-cytosolic Ca coupling in hiPSC-CMs. Physiological relevance: The present study will advance our understanding of the role of mitochondria in Ca handling and cycling in CMs, and guide the development of hiPSC-CMs for healing injured hearts.