Heart Failure-Inducible Gene Therapy Targeting Protein Phosphatase 1 Prevents Progressive Left Ventricular Remodeling

Yosuke Miyazaki,Koichi Yoshimura,Masunori Matsuzaki,Hidekazu Aoyama,Shizuka N Fujimoto,Masahiko Hoshijima,Hideko Kasahara,Yasuhiro Ikeda,Kozo Shiraishi,Hiroki Aoki,Makoto Inui

doi:10.1371/journal.pone.0035875

Abstract

BackgroundThe targeting of Ca2+ cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR) Ca2+ ATPase, or ablation of phospholamban (PLN) and associated protein phosphatase 1 (PP1) protein complexes. We previously reported that PP1β, one of the PP1 catalytic subunits, predominantly suppresses Ca2+ uptake in the SR among the three PP1 isoforms, thereby contributing to Ca2+ downregulation in failing hearts. In the present study, we investigated whether heart-failure-inducible PP1β-inhibition by adeno-associated viral-9 (AAV9) vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice.MethodsWe created an adeno-associated virus 9 (AAV9) vector encoding PP1β short-hairpin RNA (shRNA) or negative control (NC) shRNA. A heart failure inducible gene expression system was employed using the B-type natriuretic protein (BNP) promoter conjugated to emerald-green fluorescence protein (EmGFP) and the shRNA sequence. AAV9 vectors (AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA) were injected into the tail vein (2×1011 GC/mouse) of muscle LIM protein deficient mice (MLPKO), followed by serial analysis of echocardiography, hemodynamic measurement, biochemical and histological analysis at 3 months.ResultsIn the MLPKO mice, BNP promoter activity was shown to be increased by detecting both EmGFP expression and the induced reduction of PP1β by 25% in the myocardium. Inducible PP1βshRNA delivery preferentially ameliorated left ventricular diastolic function and mitigated adverse ventricular remodeling. PLN phosphorylation was significantly augmented in the AAV9-BNP-EmGFP-PP1βshRNA injected hearts compared with the AAV9-BNP-EmGFP-NCshRNA group. Furthermore, BNP production was reduced, and cardiac interstitial fibrosis was abrogated at 3 months.ConclusionHeart failure-inducible molecular targeting of PP1β has potential as a novel therapeutic strategy for heart failure.

Highlights

Heart failure is a leading cause of morbidity and mortality in developed countries and afflicts more than 55 million people in the United States [1]
We investigated whether heart-failure-inducible inhibition of PP1b by associated viral-9 (AAV9)-mediated short hairpin RNA (shRNA) gene transfer is beneficial for preventing heart failure progression in muscle LIM proteindeficient (MLPKO) mice cardiomyopathy
The B-type natriuretic protein (BNP) promoter sequence [20] was generously provided by Dr La Pointes of the Henry Ford Hospital, and pZAC2.1 and AAV9 construction plasmids were kindly provided by Dr James Wilson, the University of Pennsylvania (UPenn)

Summary

Introduction

Heart failure is a leading cause of morbidity and mortality in developed countries and afflicts more than 55 million people in the United States [1]. Patients with chronic heart failure manifest a progressive form of cardiac dysfunction that is characterized by either reduced left systolic and diastolic ventricular function, or both sides, with ventricular remodeling, arrhythmia, and intracardiac conduction disturbances [2]. The targeting of Ca2+ cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR) Ca2+ ATPase, or ablation of phospholamban (PLN) and associated protein phosphatase 1 (PP1) protein complexes. We investigated whether heart-failure-inducible PP1b-inhibition by adeno-associated viral-9 (AAV9) vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice

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Discussion

Conclusion