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

Abstract

Ca²⁺ cycling plays a critical role in regulating cardiomyocyte (CM) function under both physiological and pathological conditions. Mitochondria have been implicated in Ca²⁺ handling in adult cardiomyocytes (ACMs). However, little is known about their role in the regulation of Ca²⁺ dynamics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In the present study, we developed a multifunctional genetically encoded Ca²⁺ probe capable of simultaneously measuring cytosolic and mitochondrial Ca²⁺ in real time. Using this novel probe, we determined and compared mitochondrial Ca²⁺ activity and the coupling with cytosolic Ca²⁺ dynamics in hiPSC-CMs and ACMs. Our data showed that while ACMs displayed a highly coordinated beat-by-beat response in mitochondrial Ca²⁺ in sync with cytosolic Ca²⁺, hiPSC-CMs showed high cell-wide variability in mitochondrial Ca²⁺ activity that is poorly coordinated with cytosolic Ca²⁺. 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 Ca²⁺ dynamics. Finally, we showed that knockdown of mitofusin-2 (Mfn2), a protein tethering mitochondria and SR, led to reduced cytosolic-mitochondrial Ca²⁺ coupling in ACMs, albeit to a lesser degree compared to hiPSC-CMs, suggesting that Mfn2 is a potential engineering target for improving mitochondrial-cytosolic Ca²⁺ coupling in hiPSC-CMs. Physiological relevance: The present study will advance our understanding of the role of mitochondria in Ca²⁺ handling and cycling in CMs, and guide the development of hiPSC-CMs for healing injured hearts.

Keywords: Ca2+ cycling; Genetically encoded Ca2+ probe; Mitochondrial network; hiPSC-CM.

Figure 1.

Representative confocal images showing mitochondrial-specific expression of RCaMP (yellow) in HEK293 cells, evidenced by the overlap with Mitotracker Deep Red (red). Scalebar: 10 μm. A total of 5 cell cultures were examined.

Figure 2.

A) Representative confocal images showing subcellular expression of GCaMP (green) and RCaMP (yellow) in NRVMs stained with Mitotracker Deep Red (red). B) Pearson Correlation analysis of colocalization between GCaMP, RCaMP, and Mitotracker. C) Cytosolic & mitochondrial Ca²⁺ recordings in paced NRVMs, along with ΔΨ_m. D) Ru360 abolished fluctuations in mitochondrial Ca²⁺ and ΔΨ_m in paced NRVMs. A total of 4 NRVM cultures were examined. *: P < 0.05 vs GCaMP-RCaMP. #: P < 0.05 vs RCaMP-MitoTracker.

Figure 3.

A-B) Representative confocal images showing expression and localization of GCaMP (green) and RCaMP (yellow) in adult mouse cardiomyocytes (ACMs) (A) and hiPSC-derived cardiomyocytes (hiPSC-CMs) (B) stained with Mitotracker Deep Red (red). C-D) Pearson Correlation analysis of colocalization between GCaMP, RCaMP, and Mitotracker in ACMs (C) and hiPSC-CM (D). E-F) Cell-wide simultaneous recordings of cytosolic Ca²⁺ and mitochondrial Ca²⁺ in ACMs (E) and hiPSC-CMs (F) paced at 1 Hz. A total of 10 ACMs from 4 mouse hearts and 8 hiPSC-CMs from 4 cultures were analyzed. *: P < 0.05 vs GCaMP-RCaMP. #: P < 0.05 vs RCaMP-MitoTracker.

Figure 4.

A-B) Immunostaining of MFN1 (green) and RyR2 (red) proteins in adult mouse cardiomyocytes (ACMs) (A) and hiPSC-derived cardiomyocytes (hiPSC-CMs) (B). C-D) Line-scan profiles of MFN1 & RyR2 protein colocalization in ACMs (C) and hiPSC-CMs (D). E) Distance between mitochondria and SR in ACMs and hiPSC-CMs. F) Latency between cytosolic and mitochondrial Ca²⁺ peaks in ACMs and hiPSC-CMs. G) Coupling coefficient between cytosolic and mitochondrial Ca²⁺ in ACMs and hiPSC-CMs. A total of 8 ACMs from 4 mouse hearts and 8 hiPSC-CMs from 4 cultures were analyzed. *: P < 0.05 vs ACM.

Figure 5.

A-B) Representative traces of cytosolic and mitochondrial Ca²⁺ recordings in adult cardiomyocytes (ACMs) (A) and hiPSC-derived cardiomyocytes (hiPSC-CMs) (B). C) Distribution of inter-mitochondrial distances in ACMs and hiPSC-CMs. D) Distribution of mitochondria-SR distances in ACMs and hiPSC-CMs. A total of 12 ACMs from 4 mouse hearts and 10 hiPSC-CMs from 4 cultures were analyzed.

Figure 6.

A) Representative western blot and quantification of MFN2 protein expression in wildtype (WT) and Mfn2 KO cardiac tissue. B-C) Representative traces of Ca²⁺ in the cytosol and individual mitochondria of WT (B) and Mfn2 KO (C) CMs. D) Latency between cytosolic and individual mitochondrial Ca²⁺ peaks in WT and Mfn2 KO CMs. E) Coupling coefficient of cytosolic and mitochondrial Ca²⁺ activity in WT and Mfn2 KO CMs. For the western blotting, samples from 4 different wildtype and 4 different Mfn2 KO hearts were examined. For Ca²⁺ measurement, a total of 13 cardiomyocytes from 4 wildtype hearts and 10 cardiomyocytes from 4 Mfn2 KO hearts were analyzed. *: P < 0.05 vs. WT.