Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs.

There is increasing momentum toward the development of gene therapy for heart failure (HF) that is defined by impaired calcium (Ca) transport and reduced contractility. We have used FRET (fluorescence resonance energy transfer) between fluorescently-tagged SERCA2a (the cardiac Ca pump) and PLB (phospholamban, ventricular peptide inhibitor of SERCA) to test directly the effectiveness of loss-of-inhibition/gain-of-binding (LOI/GOB) PLB mutants (PLB) that were engineered to compete with the binding of inhibitory wild-type PLB (PLB). Our therapeutic strategy is to relieve PLB inhibition of SERCA2a by using the reserve adrenergic capacity mediated by PLB to enhance cardiac contractility. Using a FRET assay, we determined that the combination of a LOI PLB mutation (L31A) and a GOB PLB mutation (I40A) results in a novel engineered LOI/GOB PLB (L31A/I40A) that effectively competes with PLB binding to cardiac SERCA2a in HEK293-6E cells. We demonstrated that co-expression of PLB enhances SERCA Ca-ATPase activity by increasing enzyme Ca affinity (1/K) in PLB-inhibited HEK293 cell homogenates. For an initial assessment of PLB physiological effectiveness, we used human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from a healthy individual. In this system, we observed that adeno-associated virus 2 (rAAV2)-driven expression of PLB enhances the amplitude of SR Ca release and the rate of SR Ca re-uptake. To assess therapeutic potential, we used a hiPSC-CM model of dilated cardiomyopathy (DCM) containing PLB mutation R14del, where we observed that rAAV2-driven expression of PLB rescues arrhythmic Ca transients and alleviates decreased Ca transport. Thus, we propose that PLB transgene expression is a promising gene therapy strategy that directly targets the underlying pathophysiology of abnormal Ca transport and thus contractility in underlying systolic heart failure.