Molecular genetic and functional characterization implicate muscle-restricted coiled-coil gene (MURC) as a causal gene for familial dilated cardiomyopathy

Abstract

Background: Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are classic forms of systolic and diastolic heart failure, respectively. Mutations in genes encoding sarcomere and cytoskeletal proteins are major causes of HCM and DCM. MURC, encoding muscle-restricted coiled-coil, a Z-line protein, regulates cardiac function in mice. We investigated potential causal role of MURC in human cardiomyopathies.

Methods and results: We sequenced MURC in 1199 individuals, including 383 probands with DCM, 307 with HCM, and 509 healthy control subjects. We found 6 heterozygous DCM-specific missense variants (p.N128K, p.R140W, p.L153P, p.S307T, p.P324L, and p.S364L) in 8 unrelated probands. Variants p.N128K and p.S307T segregated with inheritance of DCM in small families (χ(2)=8.5, P=0.003). Variants p.N128K, p.R140W, p.L153P, and p.S364L were considered probably or possibly damaging. Variant p.P324L recurred in 3 independent probands, including 1 proband with a TPM1 mutation (p.M245T). A deletion variant (p.L232-R238del) was present in 3 unrelated HCM probands, but it did not segregate with HCM in a family who also had a MYH7 mutation (p.L907V). The phenotype in mutation carriers was notable for progressive heart failure leading to heart transplantation in 4 patients, conduction defects, and atrial arrhythmias. Expression of mutant MURC proteins in neonatal rat cardiac myocytes transduced with recombinant adenoviruses was associated with reduced RhoA activity, lower mRNA levels of hypertrophic markers and smaller myocyte size as compared with wild-type MURC.

Conclusions: MURC mutations impart loss-of-function effects on MURC functions and probably are causal variants in human DCM. The causal role of a deletion mutation in HCM is uncertain.

Figure 1

Pedigree of families with MURC mutations: Squares and circles represent male and female members. Full and open circles and squares indicated affected and unaffected (normal) individuals, respectively. Gray squares and circles indicate individuals that were not studied. The mutation and wild type codons are listed under those members that were studied. Individual III-3, who is 31 years old is a mutation carrier but is phenotypically normal (non-penetrance). Individual IV-1 in Pedigree F018 is only 5 years old and non-contributory to co-segregation analysis.

Figure 2

Multiple sequence alignment (MSA) of MURC variants across species: MSA is performed using software package PRRN ((http://align.genome.jp/prrn/) to infer evolutionary conservation of the amino acids affected by the MURC non-synonymous variants identified in probands with DCM. All available sequences from different species are included.

Figure 3

Effects of MURC mutations on RhoA activation and expression of molecular markers of cardiac hypertrophy: Panel A. shows a bar graph representing RhoA activity in neonatal rat cardiac myocytes (NRCM) infected with recombinant adenoviruses at a multiplicity of infection (MOI) of 10. The groups included Ad-LacZ (expressing β-galactosidase, as a control), wild type human MURC (Ad-hMURC-WT) and each of the specific mutant MURCs (Ad-N128K, Ad-R140W, Ad-L153P, Ad-S307T, Ad-P324L, or Ad-S364L) that were identified in patients with DCM. (N=3 independent experiments per group. Panels B, C and D represent mRNA expression levels of atrial natriuretic peptide (ANP, gene Nppa), B-type or Brain natriuretic peptide (BNP, gene name Nppb) and skeletal α-actin (SkA, gene name Acta1) in cardiac myocytes transduced at an MOI of 10 with control recombinant adenoviruses or viruses expressing WT or each of the six mutant MURC proteins. Cells were harvested 48 hr after the infection for extraction of RNA and qPCR. Panel E. shows an immunoblot of myocytes proteins extracted 48 h after transduction with the recombinant viruses and probed with an anti-FLAG antibody to detect expression of the WT or mutant MURC proteins (upper blot). Immunoblotting was performed to assess equal expression level of the WT and mutant MURC proteins among the groups. The lower blot shows expression of Gapdh, which was used as a control for loading conditions. * indicates p<0.05 and ** indicates p<0.01 as compared to WT MURC.

Figure 4

Induction of cardiac myocyte hypertrophy: Panel A shows representative immunofluorescence images of NRCM transduced with a control virus or recombinant viruses, at an MOI of 10, expressing WT mutant MURC (Ad-LacZ, Ad-hMURC-WT, Ad-N128K, Ad-R140W, Ad-L153P, Ad-S307T, Ad-P324L, or Ad-S364L). The yellow bar indicates 20 μm scale. Panel B shows a bar graph depicting the mean and standard error of myocyte surface area in each experimental group. NRCM were infected with the recombinant viruses and stained with fluorescein isothiocyanate-conjugated phalloidin 48 hours after the transduction. Myocyte surface area was measured in at least 100 cells per each group. * indicates p<0.05 and ** indicates p<0.01 as compared to WT MURC).