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

Abstract

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 HEK 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 *

Linking Biochemical and Structural States of SERCA: Achievements, Challenges, and New Opportunities

“…Except for the stoichiometry of the complexes between these micropeptides and SERCA [138], the structure-function relationships of these new regulators remain unknown largely because of the absence of experimental structures of these micropeptides or their complexes with SERCA. Addressing these gaps and challenges will yield a complete picture of the mechanisms for SERCA regulation and will also open new venues for the development and discovery of regulator-based therapies targeting SERCA [137,139].…”

Circular RNA Expression for Dilated Cardiomyopathy in Hearts and Pluripotent Stem Cell–Derived Cardiomyocytes

“…However, it is worthy of noting that using hiPSC as a disease model faces some challenges as follows: In a hiPSC-CM model, cLQTS2 and Kv11.1 activators could restore normal heart signaling, but at the same time there may be a hazard of overcorrection that reduces itself being pro-arrhythmic (Perry et al, 2020). HiPSC-CM do not express other components that make up the protein of cardiomyocytes, including key Ca2+ processing components and contractile elements, despite it could remedy arrhythmic Ca2+ transients and alleviates declined Ca2+ transport in a DCM model; this leads to limited observations (Stroik et al, 2020). But the advantage is always obvious too.…”

Genetic Cardiomyopathies: The Lesson Learned from hiPSCs

“…An in-frame R14 deletion (R14Del) mutation in another protein of the desmosome, phospholamban ( PLB ), was also associated with calcium-handling abnormalities and myofibrillar disarray that were reversed after correction by genome editing [ 66 ]; more recently, using in the same iPSC lines, rAAV2-driven expression of a PLB mutant that activates the cardiac Ca 2+ pump SERCA2a has proved to rescue arrhythmic Ca 2+ transients and to alleviate decreased Ca 2+ transport. Thus, the authors proposed SERCA2a-activating PLB mutant transgene expression as a promising gene therapy strategy to directly target the underlying pathophysiology of abnormal Ca 2+ transport and the consequent contractility defects leading to systolic heart failure [ 67 ].…”