Dataset Information

Titin splicing

ABSTRACT: Titin is a striated muscle-specific giant elastic protein and largely responsible for the generation of the diastolic force in the cardiac myocyte. Cardiac titin undergoes developmental changes in isoform expression during the course of cardiac development. At present, at least five size classes of titin isoforms (N2B and N2BA-A1, A2, N1, N2) have been identified using SDS agarose gel electrophoresis. The larger titin isoform N2BAs gradually decreased with ages, in contrast, the smaller titin isoform N2B increased in normal cardiomyocyte, and cardiac myocytes containing a higher proportion of the smaller titin isoform N2B have stronger passive tension than that with a lower proportion of the larger titin isoform N2BAs. Recently we found an autosomal dominant mutation which caused totally opposite cardiac titin isoform expression as compared to developmental stages. The larger total titin isoform N2BA increased in mutant rat cardiac myocytes with ages instead of the smaller cardiac titin isoform N2B. For the moment, mechanism of titin isoform switch is still unknown, therefore, the mutant rats will give us nevol sight to elucidate the titin splicing mechanism. Keywords: Titin isoforms, autosomal dominant mutation

ORGANISM(S): Mus musculus Rattus norvegicus

PROVIDER: GSE11137 | GEO | 2008/06/10

SECONDARY ACCESSION(S): PRJNA106927

REPOSITORIES: GEO

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Project description:Titin is a striated muscle-specific giant elastic protein and largely responsible for the generation of the diastolic force in the cardiac myocyte. Cardiac titin undergoes developmental changes in isoform expression during the course of cardiac development. At present, at least five size classes of titin isoforms (N2B and N2BA-A1, A2, N1, N2) have been identified using SDS agarose gel electrophoresis. The larger titin isoform N2BAs gradually decreased with ages, in contrast, the smaller titin isoform N2B increased in normal cardiomyocyte, and cardiac myocytes containing a higher proportion of the smaller titin isoform N2B have stronger passive tension than that with a lower proportion of the larger titin isoform N2BAs. Recently we found an autosomal dominant mutation which caused totally opposite cardiac titin isoform expression as compared to developmental stages. The larger total titin isoform N2BA increased in mutant rat cardiac myocytes with ages instead of the smaller cardiac titin isoform N2B. For the moment, mechanism of titin isoform switch is still unknown, therefore, the mutant rats will give us nevol sight to elucidate the titin splicing mechanism. Experiment Overall Design: Total RNA was extracted using TRIzol according to manufacturer instruction and further purified by the RNeasy mini kit (Qiagen, Valencia, CA). Double stranded cDNA was synthesized from total RNA (SuperScript II system; Invitrogen). An in vitro transcription reaction was then performed to obtain biotin-labeled cRNA from the double-stranded cDNA (Enzo BioArray High Yield RNA Transcript Labeling kit; Enzo Diagnostics, Farmingdale, NY). The cRNA was fragmented before hybridization, and then mixed in a hybridization mixture containing probe array controls, BSA, and herring sperm DNA. A cleanup procedure was performed on the hybridization cocktail using an RNeasy spin column (Qiagen), after which it was applied to the Affymetrix Rat 230 2.0 probe array. Total eighteen hybridization experiments were performed in which each stage (1-day, 20-day and 49-day) was represented by three normal and three mutant individual ventricular RNA extracts. Hybridization was allowed to continue for 16 h at 45Â°C in a GeneChip 640 hybridization oven, after which the arrays were washed and stained with phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, OR). Images were scanned using a GeneArray scanner (Agilent Technologies, Palo Alto, CA) and GeneChip cel files were subsequently processed by the log scale robust multi-array analysis (RMA). The log scale robust multi-array analysis (RMA) estimates are based upon a robust average of log2 (B (PM)), where B (PM) are background corrected perfect match intensities. three replicates were available for these conditions: three wild types and three homozygotes for 1-day, three wild types and three homozygotes for 20-day, and three wild types and three homozygotes for 49-day.

Project description:Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ~1:200-500 and predisposes individuals to sudden death and heart failure. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and Results: Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured primary cardiac non-myocytes and analyzed the conditioned media by proteomics, we found that several ECM proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially expressed. We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac non-myocytes drives upregulation of ECM genes including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac non-myocytes. Conclusions: Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac non-myocytes. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes.

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Titin splicing

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