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

Research output: Contribution to journalArticle

Authors

External Institution(s)

  • University of Minnesota Twin Cities
  • Icahn School of Medicine at Mount Sinai

Details

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

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.

    Research areas

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

Citation formats

APA

Stroik, D. R., Ceholski, D. K., Bidwell, P. A., Mleczko, J., Thanel, P. F., Kamdar, F., ... Thomas, D. D. (2020). Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs. Journal of Molecular and Cellular Cardiology, 138, 59-65. https://doi.org/10.1016/j.yjmcc.2019.11.147

Harvard

Stroik, DR, Ceholski, DK, Bidwell, PA, Mleczko, J, Thanel, PF, Kamdar, F, Autry, JM, Cornea, RL & Thomas, DD 2020, 'Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs', Journal of Molecular and Cellular Cardiology, vol. 138, pp. 59-65. https://doi.org/10.1016/j.yjmcc.2019.11.147