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 ²⁺ ) transport and reduced contractility. We have used FRET (fluorescence resonance energy transfer) between fluorescently-tagged SERCA2a (the cardiac Ca ²⁺ 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 _M ) that were engineered to compete with the binding of inhibitory wild-type PLB (PLB _WT ). Our therapeutic strategy is to relieve PLB _WT 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 _M (L31A/I40A) that effectively competes with PLB _WT binding to cardiac SERCA2a in HEK293-6E cells. We demonstrated that co-expression of PLB _M enhances SERCA Ca-ATPase activity by increasing enzyme Ca ²⁺ affinity (1/K _Ca ) in PLB _WT -inhibited HEK293 cell homogenates. For an initial assessment of PLB _M 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 _M enhances the amplitude of SR Ca ²⁺ release and the rate of SR Ca ²⁺ 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 _M rescues arrhythmic Ca ²⁺ transients and alleviates decreased Ca ²⁺ transport. Thus, we propose that PLB _M transgene expression is a promising gene therapy strategy that directly targets the underlying pathophysiology of abnormal Ca ²⁺ transport and thus contractility in underlying systolic heart failure.
Competing Interests: Declaration of Competing Interest None.
(Published by Elsevier Ltd.)