Regulation of SR and mitochondrial Ca2+ signaling by L-type Ca2+ channels and Na/Ca exchanger in hiPSC-CMs

Abstract

Rationale & methods: While signaling of cardiac SR by surface membrane proteins (I_Ca & I_NCX) is well studied, the regulation of mitochondrial Ca²⁺ by plasmalemmal proteins remains less explored. Here we have examined the signaling of mitochondria and SR by surface-membrane calcium-transporting proteins, using genetically engineered targeted fluorescent probes, mito-GCamP6 and R-CEPIA1er.

Results: In voltage-clamped and TIRF-imaged cardiomyocytes, low Na⁺ induced SR Ca²⁺ release was suppressed by short pre-exposures to ∼100 nM FCCP, suggesting mitochondrial Ca²⁺ contribution to low Na⁺ triggered SR Ca²⁺release. Even though low Na⁺- or caffeine-triggered SR Ca²⁺ release activated global mitochondrial Ca²⁺ uptake, focal mitochondrial Ca²⁺ signals varied in kinetics and magnitude, showing uptake or release of calcium, depending on cellular location of mitochondria. In spontaneously pacing cells, sustained caffeine exposures depleted the SR Ca²⁺ content activating mitochondrial Ca²⁺ uptake followed by sustained mitochondrial pacing. Spontaneous hiPSCCMs pacing was strongly suppressed by L-type calcium channels blockers, but not by inhibiting SERCA2a by CPA.

Conclusion: Spontaneous hiPSCCMs pacing is triggered by influx of calcium through L-type Ca²⁺ channel that gates the release of SR pools supplemented by NCX-mediated mitochondrial calcium contribution.

Keywords: Genetically targeted fluorescence probes; I(NCX) and I(Ca); Mitochondria Ca(2+) signaling; Sarcoplasmic reticulum; hiPSC—CMs.