Intracellular Ca²⁺ modulates PKA compartmentalization and dynamics in human iPSC-derived cardiomyocytes

Introduction
The automaticity of human induced Pluripotent Stem Cell-derived cardiomyocytes (hiPSC-CMs, CMs) is regulated by a coupled clock system of Ca²⁺ and membrane clocks. These clocks are coupled by local Ca²⁺ releases (LCRs) and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent signaling.
Material and Methods
In this study we investigate the PKA dynamics in the cytosol and mitochondria, and the crosstalk with Ca²⁺ signaling. Here we tested three hypotheses: (i) PKA dynamics are different in the cytosol than in the mitochondrial compartments; (ii) The activity of the Ca²⁺-activated adenylyl cyclases (ACs) correlates with the spontaneously beating rate of CMs; (iii) PKA compartmentalization is regulated by intracellular Ca²⁺concentration. In the process of investigating these hypotheses, we measured in CMs the spatiotemporal PKA dynamics and their regulation by intracellular Ca²⁺ and compared PKA dynamics in CMs and rabbit sinoatrial node cells (SANCs).
Results and Discussion
The major finding were: (i) Reduction in PKA activity or mitochondrial Ca²⁺ leads to ATP supply to demand imbalance, measured by flavoprotein Florence activity. (ii) In CMs, the PKA inhibitor H-89 caused distinct compartmentalization of PKA activity in the cytosol, mitochondrial matrix (MM) and the outer mitochondrial membrane (OMM), an effect abolished by the Ca²⁺ chelator BAPTA. (iii) ACs 1 and 8 were detected for the first time in CMs. Inhibition of their activity with BAPTA reduced PKA activity in all compartments. (iv) PKA dynamics and its Ca²⁺-dependent activity were similar in CMs and rabbit SANCs.
Conclusion
In conclusion, intracellular Ca²⁺-mediated PKA compartmentalization is present in CMs and rabbit SANCs.