Regulation of SR and mitochondrial Ca 2+ signaling by L-type Ca 2+ channels and Na/Ca exchanger in hiPSC-CMs

While signaling of cardiac SR by surface membrane proteins (I & I ) is well studied, the regulation of mitochondrial Ca 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. In voltage-clamped and TIRF-imaged cardiomyocytes, low Na induced SR Ca release was suppressed by short pre-exposures to ∼100 nM FCCP, suggesting mitochondrial Ca contribution to low Na triggered SR Ca release. Even though low Na - or caffeine-triggered SR Ca release activated global mitochondrial Ca uptake, focal mitochondrial Ca 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 Ca content activating mitochondrial Ca 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. Spontaneous hiPSCCMs pacing is triggered by influx of calcium through L-type Ca channel that gates the release of SR pools supplemented by NCX-mediated mitochondrial calcium contribution.