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

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

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Induced Pluripotent Stem Cells
Cardiac Myocytes
Fluorescence Resonance Energy Transfer
HEK293 Cells
Calcium
Genetic Therapy
Mutation
Systolic Heart Failure
Dependovirus
Dilated Cardiomyopathy
Transgenes
Adrenergic Agents
Adenosine Triphosphatases
Heart Failure
Peptides
Enzymes
Therapeutics

Keywords

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

PubMed: MeSH publication types

  • Journal Article

Cite this

Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs. / Stroik, Daniel R.; Ceholski, Delaine K.; Bidwell, Philip A.; Mleczko, Justyna; Thanel, Paul F.; Kamdar, Forum; Autry, Joseph M.; Cornea, Razvan L.; Thomas, David D.

In: Journal of Molecular and Cellular Cardiology, Vol. 138, 01.2020, p. 59-65.

Research output: Contribution to journalArticle

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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.",
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AU - Ceholski, Delaine K.

AU - Bidwell, Philip A.

AU - Mleczko, Justyna

AU - Thanel, Paul F.

AU - Kamdar, Forum

AU - Autry, Joseph M.

AU - Cornea, Razvan L.

AU - Thomas, David D.

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AB - 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.

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