Intracellular Ca²⁺ Modulates PKA Compartmentalization and Dynamics in Human iPSC-Derived Cardiomyocytes

The automaticity of human-induced Pluripotent Stem Cell-derived cardiomyocytes (hiPSC-CMs) is governed by coupled Ca²⁺ and membrane clocks, coordinated through local Ca²⁺ releases (LCRs) and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling. We investigated the role of PKA in hiPSC-CM energetics by measuring its dynamics in the cytosol and mitochondria, and its crosstalk with Ca²⁺. We tested three hypotheses: (i) Ca²⁺-activated PKA signaling regulates energy balance; (ii) adenylyl cyclase activity correlates with spontaneous beating; and (iii) PKA compartmentalization is Ca²⁺-dependent. We also compared hiPSC-CMs with rabbit sinoatrial node cells (SANCs). The key findings are: (i) PKA inhibition (H-89), Ca²⁺ chelation (BAPTA), or mitochondrial Ca²⁺ blockade (Ru360) led to energy imbalance; (ii) H-89 induced compartmentalized PKA activity in the cytosol, mitochondrial matrix, and outer mitochondrial membrane; (iii) Ca²⁺ chelation with BAPTA reduced PKA activity globally; and (iv) PKA dynamics and Ca²⁺-dependent regulation were similar in hiPSC-CMs and rabbit SANCs. In conclusion, intracellular Ca²⁺-mediated PKA compartmentalization is present in hiPSC-CMs and rabbit SANCs.