Spatiotemporal profiling of protein kinase A activity in spontaneously beating hiPSC-derived cardiac organoids.

Protein kinase A (PKA) phosphorylates proteins crucial for rhythm modulation, with its dysregulation linked to arrhythmias. This study investigated PKA activity's spatiotemporal dynamics in spontaneously beating cardiac organoids. We hypothesized that PKA activity would respond to autonomic stimulation and exhibit spatial heterogeneity upon drug-induced modulation. Forskolin (activator) and H-89 (inhibitor) altered PKA activity ratios from 1 to 1.52 ± 0.03 and 0.89 ± 0.03, respectively. Hill equation-based regression showed a high fit of PKA activity behavior for all four tested drugs (forskolin, H-89, isoproterenol, or carbachol) at concentrations. Responses to forskolin or isoproterenol, which increase PKA activity, showed higher heterogeneity (10.1 ± 0.8% or 8.7 ± 1%) compared to responses to H-89 or carbachol (4.3 ± 0.8% or 4.4 ± 1.2%), which decrease PKA activity. These results reveal the intricate spatial dynamics of PKA activity in cardiac organoids and its dependence on PKA activation levels.