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

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