Proteome modulation in H9c2 cardiac cells by microRNAs miR-378 and miR-378.

MicroRNAs are a novel class of powerful endogenous regulators of gene expression. MiR-378 and miR-378* are localized in the first intron of the Ppargc1b gene that codes the transcriptional co-activator PGC-1ß. The latter regulates energy expenditure as well as mitochondrial biogenesis. The miR-378:miR-378* hairpin is highly expressed in cardiac cells. To better assess their role in cardiomyocytes, we identified miR-378 and miR-378* targets via a proteomic screen. We established H9c2 cellular models of overexpression of miR-378 and miR-378* and identified a total of 86 down-regulated proteins in the presence of either one of these miRs. Functional annotation clustering showed that miR-378 and miR-378* regulate related pathways in cardiomyocytes, including energy metabolism, notably glycolysis, cytoskeleton, notably actin filaments and muscle contraction. Using bioinformatics algorithms we found that 20 proteins were predicted as direct targets of the miRs. We validated eight of these targets by quantitative RT-PCR and luciferase reporter assay. We found that miR-378 targets lactate dehydrogenase A and impacts on cell proliferation and survival whereas miR-378* targets cytoskeleton proteins actin and vimentin. Proteins involved in endoplasmic reticulum stress response such as chaperone and/or calcium buffering proteins GRP78, PPIA (cyclophilin A), calumenin, and GMMPA involved in glycosylation are repressed by these miRs. Our results show that the miR-378/378* hairpin establishes a connection among energy metabolism, cytoskeleton remodeling, and endoplasmic reticulum function through post-transcriptional regulation of key proteins involved in theses pathways.

An SRF/miR-1 axis regulates NCX1 and annexin A5 protein levels in the normal and failing heart.

AIMS: The expression of the sodium/calcium exchanger NCX1 increases during cardiac hypertrophy and heart failure, playing an important role in Ca(2+) extrusion. This increase is presumed to result from stress signalling induced changes in the interplay between transcriptional and post-transcriptional regulations. We aimed to determine the impact of the SRF transcription factor known to regulate the NCX1 promoter and microRNA genes, on the expression of NCX1 mRNA and protein and annexin A5 (AnxA5), a Ca(2+)-binding protein interacting with NCX1 and increased during HF. METHODS AND RESULTS: NCX1 mRNA was decreased while the protein was increased in the failing heart of the cardiomyocyte-restricted SRF knock-out mice (SRF(HKO)). The induction of NCX1 mRNA by the pro-hypertrophic drug phenylephrine observed in control mice was abolished in the SRF(HKO) though the protein was strongly increased. AnxA5 protein expression profile paralleled the expression of NCX1 protein in the SRF(HKO). MiR-1, a microRNA regulated by SRF, was decreased in the SRF(HKO) and repressed by phenylephrine. In vitro and in vivo manipulation of miR-1 levels and site-directed mutagenesis showed that NCX1 and AnxA5 mRNAs are targets of miR-1. AnxA5 overexpression slowed down Ca(2+) extrusion during caffeine application in adult rat cardiomyocytes. CONCLUSION: Our study reveals the existence of a complex regulatory loop where SRF regulates the transcription of NCX1 and miR-1, which in turn functions as a rheostat limiting the translation of NCX1 and AnxA5 proteins. The decrease of miR-1 and increase of AnxA5 appear as important modulators of NCX1 expression and activity during heart failure.

Muscle creatine kinase deficiency triggers both actin depolymerization and desmin disorganization by advanced glycation end products in dilated cardiomyopathy.

Alterations in the balance of cytoskeleton as well as energetic proteins are involved in the cardiac remodeling occurring in dilated cardiomyopathy (DCM). We used two-dimensional DIGE proteomics as a discovery approach to identify key molecular changes taking place in a temporally controlled model of DCM triggered by cardiomyocyte-specific serum response factor (SRF) knock-out in mice. We identified muscle creatine kinase (MCK) as the primary down-regulated protein followed by α-actin and α-tropomyosin down-regulation leading to a decrease of polymerized F-actin. The early response to these defects was an increase in the amount of desmin intermediate filaments and phosphorylation of the αB-crystallin chaperone. We found that αB-crystallin and desmin progressively lose their striated pattern and accumulate at the intercalated disk and the sarcolemma, respectively. We further show that desmin is a preferential target of advanced glycation end products (AGE) in mouse and human DCM. Inhibition of CK in cultured cardiomyocytes is sufficient to recapitulate both the actin depolymerization defect and the modification of desmin by AGE. Treatment with either cytochalasin D or glyoxal, a cellular AGE, indicated that both actin depolymerization and AGE contribute to desmin disorganization. Heat shock-induced phosphorylation of αB-crystallin provides a transient protection of desmin against glyoxal in a p38 MAPK-dependent manner. Our results show that the strong down-regulation of MCK activity contributes to F-actin instability and induces post-translational modification of αB-crystallin and desmin. Our results suggest that AGE may play an important role in DCM because they alter the organization of desmin filaments that normally support stress response and mitochondrial functions in cardiomyocytes.

Inducible mouse model of chronic intestinal pseudo-obstruction by smooth muscle-specific inactivation of the SRF gene.

BACKGROUND & AIMS: Serum response factor (SRF) regulates the expression of muscle genes and immediate early genes. We investigated the consequences of inactivating this transcription factor SRF in adult gastrointestinal smooth muscle cells. METHODS: SRF-floxed mice were crossed with SM-CreER(T2)(ki) mice expressing a tamoxifen-inducible recombinase in smooth muscle cells. Tamoxifen was injected into 12-week-old animals to activate the CreER(T2) and excise the SRF gene. RESULTS: SRF was down-regulated in the smooth muscle cells of the gastrointestinal tract, urinary bladder, and aorta. The mutant mice developed severe dilation of the intestinal tract associated with food stasis and air-fluid levels in the lumen 13 days after tamoxifen treatment. Mutant mice displayed cachexia and died between days 13 and 22. The dilation was associated with a thinning of the muscularis propria and was also observed in the urinary bladder. Ex vivo colonic contraction induced by electric field stimulation and carbachol was impaired in the mutant mice before the occurrence of the dilation phenotype. The expression of several genes, including those encoding smooth muscle actin, the heavy chain of smooth muscle myosin, and smoothelin, was 60% to 70% lower in mutants than in controls, and mutants also had a lower F/G actin ratio. CONCLUSIONS: SRF plays a central role in maintaining visceral smooth muscle contractile function in adults. Mice with a smooth muscle cell-specific SRF mutation develop a severe motility disorder resembling chronic intestinal pseudo-obstruction in humans and may be used as an inducible model of this disorder.