Allele-specific silencing of mutant Myh6 transcripts in mice suppresses hypertrophic cardiomyopathy

Abstract

Dominant mutations in sarcomere proteins such as the myosin heavy chains (MHC) are the leading genetic causes of human hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy. We found that expression of the HCM-causing cardiac MHC gene (Myh6) R403Q mutation in mice can be selectively silenced by an RNA interference (RNAi) cassette delivered by an adeno-associated virus vector. RNAi-transduced MHC(403/+) mice developed neither hypertrophy nor myocardial fibrosis, the pathologic manifestations of HCM, for at least 6 months. Because inhibition of HCM was achieved by only a 25% reduction in the levels of the mutant transcripts, we suggest that the variable clinical phenotype in HCM patients reflects allele-specific expression and that partial silencing of mutant transcripts may have therapeutic benefit.

Fig. 1. Selective silencing of Myh6 R403Q expression by AAV-9-mediated RNAi

(A) Schematic representation of FVB, 129SvEv, 129SvEv mutant (R403Q) transcript and RNAi sequences. (B) Quantitative real-time PCR analysis of wild-type Myh6 (white bar) and mutant Myh6 R403Q (black bar) expression after transduction of the 403m and 403i constructs (n=4). Levels of the transcripts were normalized to control LacZ RNAi. (C) Quantitative real-time PCR analysis of FVB Myh6 (white bar) and 129SvEv Myh6 R403Q (black bar) expression after transduction of the 129i construct (n=4). Levels of the transcripts were normalized to control LacZ RNAi. Data are presented as the mean ± s.d.

Fig. 2. In vivo effect of Myh6 R403Q silencing

(A) Cardiac histopathology from MHC^403/+ mice transduced with control RNAi (left) and 403i RNAi (right). Masson trichrome staining reveals marked fibrosis (blue) in MHC^403/+ mice transduced with control RNAi. Bar =1 mm. (B) Hematoxylin and eosin staining shows myocyte disarray in MHC^403/+ mice transduced with control RNAi (left) and normal myocyte architecture in mice transduced with 403i RNAi (right). Bar = 100 µm. (C) Quantification of myocardial fibrosis in MHC^403/+ mice transduced with control RNAi (black bar, n=4) and 403i RNAi (white bar, n=4). (D) An electrocardiogram of MHC^403/+ mice transduced with control RNAi (left top panel) and 403i RNAi (right top panel). Mice transduced with control RNAi have prolonged QRS (ventricular conduction) interval and high voltage P waves consistent with LV hypertrophy and atrial enlargement. The bottom panel presents QRS intervals from mice transduce with control RNAi (black bar, n=5) and 403i RNAi (white bar, n=6). (E) Quantitative real-time PCR analysis of Nppa (left panel) and Nppb (right panel) expression after transduction of control RNAi (black bar) and two different doses of 403i constructs (white bar) (n=5). Levels of the transcripts were normalized to transcript levels from age matched wild-type hearts. Data are presented as the mean ± s.d.