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

Daniel R. Stroik, Delaine K. Ceholski, Philip A. Bidwell, Justyna Mleczko, Paul F. Thanel, Forum Kamdar, Joseph M. Autry, Razvan L. Cornea, David D. Thomas

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

There is increasing momentum toward the development of gene therapy for heart failure (HF) that is defined by impaired calcium (Ca2+) transport and reduced contractility. We have used FRET (fluorescence resonance energy transfer) between fluorescently-tagged SERCA2a (the cardiac Ca2+ 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 (PLBM) that were engineered to compete with the binding of inhibitory wild-type PLB (PLBWT). Our therapeutic strategy is to relieve PLBWT 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 PLBM (L31A/I40A) that effectively competes with PLBWT binding to cardiac SERCA2a in HEK293-6E cells. We demonstrated that co-expression of PLBM enhances SERCA Ca-ATPase activity by increasing enzyme Ca2+ affinity (1/KCa) in PLBWT-inhibited HEK293 cell homogenates. For an initial assessment of PLBM 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 PLBM enhances the amplitude of SR Ca2+ release and the rate of SR Ca2+ 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 PLBM rescues arrhythmic Ca2+ transients and alleviates decreased Ca2+ transport. Thus, we propose that PLBM transgene expression is a promising gene therapy strategy that directly targets the underlying pathophysiology of abnormal Ca2+ transport and thus contractility in underlying systolic heart failure.

Original languageEnglish (US)
Pages (from-to)59-65
Number of pages7
JournalJournal of Molecular and Cellular Cardiology
Volume138
DOIs
StatePublished - Jan 2020

Keywords

  • Calcium transport
  • Cardiomyocyte
  • Dilated cardiomyopathy
  • Gene therapy
  • Phospholamban
  • SERCA

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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