TY - JOUR T1 - Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs JF - bioRxiv DO - 10.1101/699975 SP - 699975 AU - Daniel R. Stroik AU - Delaine K. Ceholski AU - Justyna Mleczko AU - Paul F. Thanel AU - Philip A. Bidwell AU - Joseph M. Autry AU - Razvan L. Cornea AU - David D. Thomas Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/08/08/699975.abstract N2 - There is increasing momentum toward the development of gene therapy for heart failure (HF), cardiomyopathy, and other progressive cardiac diseases that correlate with impaired calcium (Ca2+) transport and reduced contractility. We have used FRET between fluorescently-tagged SERCA2a (the cardiac Ca2+ pump) and PLB (its ventricular peptide regulator) to directly test the effectiveness of loss-of-function/gain-of-binding 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 utilizing the reserve adrenergic capacity of PLB to enhance non-stimulated cardiac contractility. We determined that the combination of a loss- of-inhibitory function PLB mutation (L31A) and a gain-of-binding PLB mutation (I40A) results in a novel engineered PLBM (L31A/I40A) that effectively displaces PLBWT binding to cardiac SERCA2a in HEK293-6E cells, as assessed by a structural assay based on FRET spectroscopy. Functional assays demonstrated that co-expression of L31A/I40A-PLBM enhances SERCA Ca-ATPase activity by increasing enzyme Ca2+ affinity (1/KCa) in PLBWT-inhibited HEK cell homogenates. For an initial assessment of physiological relevance, human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from a healthy individual were used to determine the effect of PLBM in a native SERCA2a/WT-PLB cardiac system, finding that recombinant adeno-associated virus 2 (rAAV2)-driven expression of L31A/I40A-PLBM enhances the amplitude of SR Ca2+ release and the rate of SR Ca2+ re-uptake in cardiomyocytes. To assess therapeutic potential, a hiPSC-CM dilated cardiomyopathy model (DCM) containing PLB mutation R14del was used to characterize Ca2+ transients and the effect of viral-driven expression of L31A/I40A-PLBM, demonstrating that PLBM rescues arrhythmic Ca2+ transients and alleviates decreased Ca2+ transport in DCM cultured cells. We propose that PLBM transgene expression is an effective gene therapy strategy for cardiomyopathies associated with impaired Ca2+ transport and decreased cardiac contractility.HighlightsFRET-based assays demonstrate that the combination of two PLB mutations: L31A-PLBM (loss of inhibitory function) and I40A-PLBM (associated with increased SERCA binding), result in a PLBM (L31A/I40A) capable of displacing PLBWT.Ectopic expression of the L31A/I40A-PLBM construct in HEK293-6E cells expressing PLBWT increased Ca-ATPase activity.In hiPSC-CMs, direct measurement of the effect(s) of virally-delivered L31A/I40A-PLBM on SR Ca2+ transport showed significantly enhancement in Ca-release amplitude and removal compared to controls.rAAV2.L31A/I40A-PLBM corrected irregular Ca2+ transients and improved Ca2+ transport in a hereditary dilated cardiomyopathy model in hiPSC-CMs (R14del-PLB).These results suggest that gene transfer of PLBM is viable therapy for cardiac diseases associated with impaired Ca2+ transport and muscle contractility.Ca2+(calcium)DCM(dilated cardiomyopathy)ER(endoplasmic reticulum)FLT(fluorescence lifetime)FRET(fluorescence resonance energy transfer)GOI(gain of inhibitory functionbinding)GFP(green fluorescent protein)hiPSC-CM(human induced pluripotent stem cell-derived cardiomyocyte)HEK(human embryonic kidney)HF(heart failure)LOI(loss of inhibitory function) mAb (monoclonal antibody) miRNA (microRNA)pAb(polyclonal antibody) PLB (phospholamban)PLBM(phospholamban mutant)PLBWT(wild type phospholamban)rAAV(recombinant adeno-associated virus)RFP(red fluorescent protein)SERCA2a(sarcoendoplasmic reticulum calcium ATPase 2a)SR(sarcoplasmic reticulum) ER -