Aims: The heterozygous phospholamban (PLN) mutation R14del (PLN R14del+/-) is associated with a severe arrhythmogenic cardiomyopathy (ACM) developing in the adult. "Superinhibition" of SERCA2a by PLN R14del is widely assumed to underlie the pathogenesis, but alternative mechanisms such abnormal energy metabolism have also been reported. This work aims to 1) to evaluate Ca2+ dynamics and energy metabolism in a transgenic (TG) mouse model of the mutation prior to cardiomyopathy development; 2) to test whether they are causally connected. Methods and Results: Ca2+ dynamics, energy metabolism parameters, reporters of mitochondrial integrity, energy and redox homeostasis were measured in ventricular myocytes of 8-12 weeks-old, phenotypically silent, TG mice. Mutation effects were compared to pharmacological PLN antagonism and analysed during modulation of sarcoplasmic reticulum (SR) and cytosolic Ca2+ compartments. Transcripts and proteins of relevant signalling pathways were evaluated. The mutation was characterized by hyperdynamic Ca2+ handling, similar to that induced by PLN antagonism. Albeit all components of energy metabolism were depressed at rest, functional signs of mitochondrial damage or energy starvation were absent and cell energy charge was preserved. The response of mitochondrial O2 consumption to SERCA2a blockade was lost in mutant myocytes (SR-mitochondrial uncoupling) and ER-stress signalling was activated. Conclusions: 1) PLN R14del+/- loses its ability to inhibit SERCA2a, which argues against SERCA2a superinhibition as a mechanism of ACM; 2) depression of resting energy metabolism may at least partly reflect impairment of SR-mitochondrial coupling; 3) ER-stress may be an early factor in the pathogenesis.

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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 (tpeak) 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 tpeak 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.

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