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

Autor: Stroik DR; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States of America., Ceholski DK; Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York City, New York 10029, United States of America., Bidwell PA; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States of America; Division of Cardiology, Department of Medicine, University of Minnesota, Minneapolis, MN 55455, United States of America., Mleczko J; Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York City, New York 10029, United States of America., Thanel PF; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States of America., Kamdar F; Division of Cardiology, Department of Medicine, University of Minnesota, Minneapolis, MN 55455, United States of America., Autry JM; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States of America., Cornea RL; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States of America., Thomas DD; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States of America. Electronic address: ddt@umn.edu.
Jazyk: angličtina
Zdroj: Journal of molecular and cellular cardiology [J Mol Cell Cardiol] 2020 Jan; Vol. 138, pp. 59-65. Date of Electronic Publication: 2019 Nov 18.
DOI: 10.1016/j.yjmcc.2019.11.147
Abstrakt: 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.)
Databáze: MEDLINE
Externí odkaz: