Proteomic analysis identifies key differences in the cardiac interactomes of dystrophin and micro-dystrophin.

Hong Wang,Stan C Froehner,Marvin E Adams,Nalinda B Wasala,Liwen Zhang,Eric K Johnson,Kwan-Leong Hau,Federica Montanaro,Charlotte S Scott,Dongsheng Duan,Yongping Yue,Elena Marrosu,Aaron J Trask,Daniel Brayson

doi:10.1093/hmg/ddab133

Abstract

ΔR4-R23/ΔCT micro-dystrophin (μDys) is a miniaturized version of dystrophin currently evaluated in a Duchenne muscular dystrophy (DMD) gene therapy trial to treat skeletal and cardiac muscle disease. In pre-clinical studies, μDys efficiently rescues cardiac histopathology, but only partially normalizes cardiac function. To gain insights into factors that may impact the cardiac therapeutic efficacy of μDys, we compared by mass spectrometry the composition of purified dystrophin and μDys protein complexes in the mouse heart. We report that compared to dystrophin, μDys has altered associations with α1- and β2-syntrophins, as well as cavins, a group of caveolae-associated signaling proteins. In particular, we found that membrane localization of cavin-1 and cavin-4 in cardiomyocytes requires dystrophin and is profoundly disrupted in the heart of mdx5cv mice, a model of DMD. Following cardiac stress/damage, membrane-associated cavin-4 recruits the signaling molecule ERK to caveolae, which activates key cardio-protective responses. Evaluation of ERK signaling revealed a profound inhibition, below physiological baseline, in the mdx5cv mouse heart. Expression of μDys in mdx5cv mice prevented the development of cardiac histopathology but did not rescue membrane localization of cavins nor did it normalize ERK signaling. Our study provides the first comparative analysis of purified protein complexes assembled in vivo by full-length dystrophin and a therapeutic micro-dystrophin construct. This has revealed disruptions in cavins and ERK signaling that may contribute to DMD cardiomyopathy. This new knowledge is important for ongoing efforts to prevent and treat heart disease in DMD patients.

Highlights

Patients with Duchenne muscular dystrophy (DMD) lack sufficient expression of a functional dystrophin protein in all striated muscles, leading to loss of ambulation by 13 years of age, progressive respiratory insufficiency and dilated cardiomyopathy (1)
Our findings reveal a R previously unsuspected disruption of cavins and ERK signaling in DMD cardiomyopathy that is C not corrected by μDys
Two R proteins we previously reported as part of the cardiac dystrophin-associated protein complex (DAPC), cypher and αB-crystallin (20), were C identified in control IPs (Table S1) and were excluded from further analyses

Summary

INTRODUCTION

Patients with Duchenne muscular dystrophy (DMD) lack sufficient expression of a functional dystrophin protein in all striated muscles, leading to loss of ambulation by 13 years of age, progressive respiratory insufficiency and dilated cardiomyopathy (1). While we could not confirm a previously reported interaction of dystrophin with IP caveolin-3 (47), the major caveolar protein in cardiac cells, a functional link between dystrophin R and caveolae is supported by our finding that ERK signaling, a pathway known to be in part C regulated via caveolae and cavin-4(26), is impaired in mdx5cv mice. This disruption of ERK US signaling is not secondary to the presence of cardiac pathology for three reasons. A two-way repeated O measure ANOVA was used for superplots involving nested measurements of membrane UNC fluorescence intensity for n=3 mice per group

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IPT Parameter Unit

Findings

MW P value FC