SERCA2a gain of function in patient-derived R14Del hiPSC-CMs: Calcium Signaling and Excitation–Contraction in Cardiac, Skeletal and Smooth Muscle

Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA2a). Heterozygous PLN-R14del mutation is associated with an arrhythmogenic dilated cardiomyopathy (DCM), whose pathogenesis has been attributed to SERCA2a “superinhibition.” The aim of the project is to test in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CM) harvested from a PLN-R14del carrier whether (1) Ca²⁺ dynamics and protein localization were compatible with SERCA2a superinhibition and (2) functional abnormalities could be reverted by pharmacological SERCA2a activation with PST3093. Ca²⁺ transients (CaT) were recorded at 36°C in hiPSC-CMs clusters during field stimulation. SERCA2a and PLN were immunolabeled in single hiPSC-CMs. Mutant (MUT) preparations were compared with isogenic WT ones obtained by mutation reversal. WT and MUT differed for the following properties: (1) CaT time to peak (t_peak) and half-time of CaT decay were shorter in MUT, (2) several CaT profiles were identified in WT, whereas “hyperdynamic” ones largely prevailed in MUT, (3) whereas t_peak rate-dependently declined in WT, it was shorter and rate independent in MUT, and (4) diastolic Ca²⁺ rate-dependently accumulated in WT, but not in MUT. When applied to WT, PST3093 changed all of the above properties to resemble those of MUT; when applied to MUT, PST3093 had no effect. Preferential perinuclear SERCA2a-PLN localization was lost in MUT hiPSC-CMs. In conclusion, functional data converge to argue for PLN-R14del incompetence in inhibiting SERCA2a in the tested case, thus weakening the rationale for therapeutic SERCA2a activation. Mechanisms alternative to SERCA2a superinhibition should be considered in the pathogenesis of DCM, including dysregulation of Ca²⁺-dependent transcription.