Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. HCM is caused by mutations in sarcomeric genes, but in >40% of patients, the mutation is not yet identified. We hypothesized that FHL1, encoding four-and-a-half-LIM domains 1, could be another disease gene since it has been shown to cause distinct myopathies, sometimes associated with cardiomyopathy. We evaluated 121 HCM patients, devoid of a mutation in known disease genes. We identified three novel variants in FHL1 (c.134delA/K45Sfs, c.459C>A/C153X and c.827G>C/C276S). Whereas the c.459C>A variant was associated with muscle weakness in some patients, the c.134delA and c.827G>C variants were associated with isolated HCM. Gene transfer of the latter variants in C2C12 myoblasts and cardiac myocytes revealed reduced levels of FHL1 mutant proteins, which could be rescued by proteasome inhibition. Contractility measurements after adeno-associated virus transduction in rat-engineered heart tissue (EHT) showed: (i) higher and lower forces of contraction with K45Sfs and C276S, respectively, and (ii) prolonged contraction and relaxation with both mutants. All mutants except one activated the fetal hypertrophic gene program in EHT. In conclusion, this study provides evidence for FHL1 to be a novel gene for isolated HCM. These data, together with previous findings of proteasome impairment in HCM, suggest that FHL1 mutant proteins may act as poison peptides, leading to hypertrophy, diastolic dysfunction and/or altered contractility, all features of HCM.

Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5–6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5+6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5+6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM.

Ranolazine improved tolerance to high workload in mouse HCM cardiomyocytes, not by blocking late Na(+) current, but by antagonizing β-adrenergic stimulation and slightly desensitizing myofilaments to Ca(2+). This effect did not translate in therapeutic efficacy in vivo.

Based on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomyopathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding density were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes markedly down-regulated α-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal conditions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine- or endothelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-014-0451-8) contains supplementary material, which is available to authorized users.

No abstract

No abstract

Hypertrophic cardiomyopathy (HCM) is a myocardial disease associated with mutations in sarcomeric genes. Three mutations were found in ANKRD1, encoding ankyrin repeat domain 1 (ANKRD1), a transcriptional co-factor located in the sarcomere. In the present study, we investigated whether expression of HCM-associated ANKRD1 mutations affects contraction parameters after gene transfer in engineered heart tissues (EHTs). EHTs were generated from neonatal rat heart cells and were transduced with adeno-associated virus encoding GFP or myc-tagged wild-type (WT) or mutant (P52A, T123M, or I280V) ANKRD1. Contraction parameters were analyzed from day 8 to day 16 of culture, and evaluated in the absence or presence of the proteasome inhibitor epoxomicin for 24 h. Under standard conditions, only WT- and T123M-ANKRD1 were correctly incorporated in the sarcomere. T123M-ANKRD1-transduced EHTs exhibited higher force and velocities of contraction and relaxation than WT- P52A- and I280V-ANKRD1 were highly unstable, not incorporated into the sarcomere, and did not induce contractile alterations. After epoxomicin treatment, P52A and I280V were both stabilized and incorporated into the sarcomere. I280V-transduced EHTs showed prolonged relaxation. These data suggest different impacts of ANKRD1 mutations on cardiomyocyte function: gain-of-function for T123M mutation under all conditions and dominant-negative effect for the I280V mutation which may come into play only when the proteasome is impaired.

A growing line of evidence indicates a dysfunctional ubiquitin-proteasome system (UPS) in cardiac diseases. Anti-hypertrophic effects and improved cardiac function have been reported after treatment with proteasome inhibitors in experimental models of cardiac hypertrophy. Here we tested whether proteasome inhibition could also reverse the disease phenotype in a genetically-modified mouse model of hypertrophic cardiomyopathy (HCM), which carries a mutation in Mybpc3, encoding the myofilament protein cardiac myosin-binding protein C. At 7 weeks of age, homozygous mutant mice (KI) have 39% higher left ventricular mass-to-body-weight ratio and 29% lower fractional area shortening (FAS) than wild-type (WT) mice. Both groups were treated with epoxomicin (0.5 mg/kg/day) or vehicle for 1 week via osmotic minipumps. Epoxomicin inhibited the chymotrypsin-like activity by ~50% in both groups. All parameters of cardiac hypertrophy (including the fetal gene program) were not affected by epoxomicin treatment in both groups. In contrast, FAS was 12% and 35% higher in epoxomicin-treated than vehicle-treated WT and KI mice, respectively. To identify which genes or pathways could be involved in this positive effect, we performed a transcriptome analysis in KI and WT neonatal cardiac myocytes, treated or not with the proteasome inhibitor MG132 (1 μM, 24 h). This revealed 103 genes (four-fold difference; 5% FDR) which are commonly regulated in both KI and WT cardiac myocytes. Thus, even in genetically-modified mice with manifest HCM, proteasome inhibition showed beneficial effects, at least with regard to cardiac function. Targeting the UPS in cardiac diseases remains therefore a therapeutic option.