SH3-Binding Glutamic Acid Rich-Deficiency Augments Apoptosis in Neonatal Rat Cardiomyocytes.

Congenital heart disease (CHD) is one of the most common birth defects in humans, present in around 40% of newborns with Down's syndrome (DS). The SH3 domain-binding glutamic acid-rich (SH3BGR) gene, which maps to the DS region, belongs to a gene family encoding a cluster of small thioredoxin-like proteins sharing SH3 domains. Although its expression is confined to the cardiac and skeletal muscle, the physiological role of SH3BGR in the heart is poorly understood. Interestingly, we observed a significant upregulation of SH3BGR in failing hearts of mice and human patients with hypertrophic cardiomyopathy. Along these lines, the overexpression of SH3BGR exhibited a significant increase in the expression of hypertrophic markers (Nppa and Nppb) and increased cell surface area in neonatal rat ventricular cardiomyocytes (NRVCMs), whereas its knockdown attenuated cellular hypertrophy. Mechanistically, using serum response factor (SRF) response element-driven luciferase assays in the presence or the absence of RhoA or its inhibitor, we found that the pro-hypertrophic effects of SH3BGR are mediated via the RhoA-SRF axis. Furthermore, SH3BGR knockdown resulted in the induction of apoptosis and reduced cell viability in NRVCMs via apoptotic Hippo-YAP signaling. Taking these results together, we here show that SH3BGR is vital for maintaining cytoskeletal integrity and cellular viability in NRVCMs through its modulation of the SRF/YAP signaling pathways.

Dysbindin deficiency Alters Cardiac BLOC-1 Complex and Myozap Levels in Mice.

Dysbindin, a schizophrenia susceptibility marker and an essential constituent of BLOC-1 (biogenesis of lysosome-related organelles complex-1), has recently been associated with cardiomyocyte hypertrophy through the activation of Myozap-RhoA-mediated SRF signaling. We employed sandy mice (Dtnbp1_KO), which completely lack Dysbindin protein because of a spontaneous deletion of introns 5-7 of the Dtnbp1 gene, for pathophysiological characterization of the heart. Unlike in vitro, the loss-of-function of Dysbindin did not attenuate cardiac hypertrophy, either in response to transverse aortic constriction stress or upon phenylephrine treatment. Interestingly, however, the levels of hypertrophy-inducing interaction partner Myozap as well as the BLOC-1 partners of Dysbindin like Muted and Pallidin were dramatically reduced in Dtnbp1_KO mouse hearts. Taken together, our data suggest that Dysbindin's role in cardiomyocyte hypertrophy is redundant in vivo, yet essential to maintain the stability of its direct interaction partners like Myozap, Pallidin and Muted.

The E3 ubiquitin ligase HectD3 attenuates cardiac hypertrophy and inflammation in mice.

Myocardial inflammation has recently been recognized as a distinct feature of cardiac hypertrophy and heart failure. HectD3, a HECT domain containing E3 ubiquitin ligase has previously been investigated in the host defense against infections as well as neuroinflammation; its cardiac function however is still unknown. Here we show that HectD3 simultaneously attenuates Calcineurin-NFAT driven cardiomyocyte hypertrophy and the pro-inflammatory actions of LPS/interferon-γ via its cardiac substrates SUMO2 and Stat1, respectively. AAV9-mediated overexpression of HectD3 in mice in vivo not only reduced cardiac SUMO2/Stat1 levels and pathological hypertrophy but also largely abolished macrophage infiltration and fibrosis induced by pressure overload. Taken together, we describe a novel cardioprotective mechanism involving the ubiquitin ligase HectD3, which links anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and Stat1. In a broader perspective, these findings support the notion that cardiomyocyte growth and inflammation are more intertwined than previously anticipated.

Cardiac α-Actin (ACTC1) Gene Mutation Causes Atrial-Septal Defects Associated With Late-Onset Dilated Cardiomyopathy.

BACKGROUND: Familial atrial septal defect (ASD) has previously been attributed primarily to mutations in cardiac transcription factors. Here, we report a large, multi-generational family (78 members) with ASD combined with a late-onset dilated cardiomyopathy and further characterize the consequences of mutant α-actin. METHODS: We combined a genome-wide linkage analysis with cell biology, microscopy, and molecular biology tools to characterize a novel ACTC1 (cardiac α-actin) mutation identified in association with ASD and late-onset dilated cardiomyopathy in a large, multi-generational family. RESULTS: Using a genome-wide linkage analysis, the ASD disease locus was mapped to chromosome 15q14 harboring the ACTC1 gene. In 15 affected family members, a heterozygous, nonsynonymous, and fully penetrant mutation (p. Gly247Asp) was identified in exon 5 of ACTC1 that was absent in all healthy family members (n=63). In silico tools predicted deleterious consequences of this variant that was found absent in control databases. Ultrastructural analysis of myocardial tissue of one of the mutation carriers showed sarcomeric disarray, myofibrillar degeneration, and increased apoptosis, while cardiac proteomics revealed a significant increase in extracellular matrix proteins. Consistently, structural defects and increased apoptosis were also observed in neonatal rat ventricular cardiomyocytes overexpressing the mutant, but not native human ACTC1. Molecular dynamics studies and additional mechanistic analyses in cardiomyocytes confirmed actin polymerization/turnover defects, thereby affecting contractility. CONCLUSIONS: A combined phenotype of ASD and late-onset heart failure was caused by a heterozygous, nonsynonymous ACTC1 mutation. Mechanistically, we found a shared molecular mechanism of defective actin signaling and polymerization in both cardiac development and contractile function. Detection of ACTC1 mutations in patients with ASD may thus have further clinical implications with regard to monitoring for (late-onset) dilated cardiomyopathy.

A systematic view on E3 ligase Ring TRIMmers with a focus on cardiac function and disease.

Ubiquitination, a post-translational modification via ubiquitin-proteasome-system, is one of the vital cellular processes involved in intracellular signaling, cell death, transcriptional control, etc. Importantly, it prevents the aggregation of non-functional, misfolded or unfolded, potentially toxic proteins to maintain cellular protein homeostasis. Ubiquitination is accomplished by the concerted action of three enzymatic steps involving E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. Tripartite motif-containing (TRIM) proteins are one of the integral members of E3 ubiquitin ligases in metazoans modulating essential cellular pathways. For long, MuRFs (Muscle ring finger proteins) were the most extensively studied TRIMs for their cardiac function. Recent research advances in the field and our analysis presented here, however, demonstrated broader and ever increasing involvement of additional TRIM E3 ligases in the pathophysiology of heart. In this review, we summarize the known cardiac E3 ligases and their targets, and discuss their role and importance in cardiac proteostasis, pathophysiology and potential therapeutic implications with specific focus on TRIM E3 ligases.

TRIM24 protein promotes and TRIM32 protein inhibits cardiomyocyte hypertrophy via regulation of dysbindin protein levels.

We have previously shown that dysbindin is a potent inducer of cardiomyocyte hypertrophy via activation of Rho-dependent serum-response factor (SRF) signaling. We have now performed a yeast two-hybrid screen using dysbindin as bait against a cardiac cDNA library to identify the cardiac dysbindin interactome. Among several putative binding proteins, we identified tripartite motif-containing protein 24 (TRIM24) and confirmed this interaction by co-immunoprecipitation and co-immunostaining. Another tripartite motif (TRIM) family protein, TRIM32, has been reported earlier as an E3 ubiquitin ligase for dysbindin in skeletal muscle. Consistently, we found that TRIM32 also degraded dysbindin in neonatal rat ventricular cardiomyocytes as well. Surprisingly, however, TRIM24 did not promote dysbindin decay but rather protected dysbindin against degradation by TRIM32. Correspondingly, TRIM32 attenuated the activation of SRF signaling and hypertrophy due to dysbindin, whereas TRIM24 promoted these effects in neonatal rat ventricular cardiomyocytes. This study also implies that TRIM32 is a key regulator of cell viability and apoptosis in cardiomyocytes via simultaneous activation of p53 and caspase-3/-7 and inhibition of X-linked inhibitor of apoptosis. In conclusion, we provide here a novel mechanism of post-translational regulation of dysbindin and hypertrophy via TRIM24 and TRIM32 and show the importance of TRIM32 in cardiomyocyte apoptosis in vitro.