| BackgroundHomologous myofibrillogenesis regulator 1(hMR-1) is a novel characterized human functional gene cloned from human skeletal muscle cDNA library by our research group. It was found that hMR-1 gene locates on the human chromosome 2q35, with a full length of 755 bps and encodes a 142 amino acid-protein. Previous studies suggested that MR-1 found in human myofibrils by immunohistochemistry was highly expressed in myocardium and skeletal muscle, MR-1 is significantly up-regulated in hypertrophied cardiomyocytes induced by angiotensinⅡ(AngⅡ) whereas its silencing using RNAi would abolish the AngⅡ-induced hypertrophy, indicating that MR-1 is involved in hypertrophy. However, whether MR-1 induces hypertrophy directly or not and the mechanism is still unclear. The results from yeast two-hybrid screen and in vitro GST pull-down assay indicated that MR-1 could interact with the contractile apparatus associated proteins, i.e. myosin light chain-2 (MRLC), myomesin-1 andβ-enolase etc. These findings suggested that MR-1 induces cardiac hypertrophy by regulating the myofibrillogenesis.ObjectiveIn order to validate the hypothesis that MR-1 induces cardiac hypertrophy by promoting myofibrillogenesis, we aimed to verify the direct contribution of MR-1 to hypertrophy in vitro and influence of MR-1 on myofibrillogenesis and myofibrillar orgnization, as well as the regulatory mechnisms. Thus, we were to prepare the anti-MR-1 antibody, to construct the MR-1 eucaryotic expressing vector and to establish the MR-1 silencing method at first.Methods1. Antibody preparation; superior epitopes were selected based on the bioinformatics analysis, synthesized and mixed for immunization; anti-serum was purified by immunoaffinity chromatography and the specificity and application were measured as well. Plasmid construction; the full length of hMR-1 mRNA from NCBI GenBank database was cloned to the MCS of pcDNA3.1-HisB(-)-Myc vector. RNA interference (RNAi) assay; The rat original MR-1 (rMR-1) stably-modified stealth siRNA was designed, synthesized and then selected according to the silencing effect.2. Neonatal rat cardiomyocytes culture and hypertrophy model in vitro; cardiomyocytes were cultured and incubated with angiotensin II (Ang II) 1x10-7 mol/L for 48h to develop hypertrophy, to validate the effect, hallmarks i.e. mean area of cell surface analyzed by professional software, protein synthesis velocity assessed by [3H]-Leucine incorporation and mRNA levels of atrial natriuretic factor and brain natriuretic peptide measured by RT-PCR were all employed.3. Investigation on myofibrillogenesis and myofibril-alignment; striated-like F-actin pattern which indicated the myofibrillogenesis was characterized by specific staining with FITC-phalloidin.4. Stuadies on interesting proteins; the sublocation, collocation and translocation of thosed molecules were detected using immunocytofluorescent assay, whereas their expression at mRNA and protein levels were measured using RT-PCR and Western Blot, respectively.5. Measurement of [Ca2+]i and activity of calcineurin (CaN); cytoplasmic free Ca2+ was probed by fluo-3AM whereafter its fluorescent density was semi-quantitatively analyzed; enzymatic activity of CaN was measured by chromatometry.6. Image analysis, data management and statistics analysis; semi-quantitative analysis on mean cell surface area, integrated optical density of bands and fluorescent density of [Ca2+]i were performed with software Image Pro-Plus (version 4.11), experimental data were represented by mean±standard deviation ((?)±s). SPSS 13.0 statistics software was introduced. Bonferroni's test was employed to analyze statistical differences between every two-sample among several groups. P<0.05 was considered statistically significant.Results1. The rabbit anti-hMR-1 polyclonal antibody detects either human original or rat original epitopes, available for both Western Blot and immunocytofluorescent assays were successfully prepared; the hMR-1 eukaryotic expression plasmid pcDNA3.1-Myc/HisB(-)-hMRl for exogenous hMR-1 overexpression in neonatal rat cardiomyocytes was constructed; the stealth siRNA selected according to its silencing effect was transfected to cardiomyocytes and successfully inhibited the expression of endogenous rMR-1.2. The mean surface area, incorporation of [3H]-Leucine and transcriptional levels of ANF and BNP were all significantly increased with MR-1-overexpression (P<0.05); MR-1-RNAi decreased the transcription of ANF and BNP (P<0.05) instead of the mean surface area and protein synthesis. (P>0.05) In particular, it would reverse the Ang II-induced increases on those three hypertrophic hallmarks.3. Cardiomyocytes in the normal culture exhibited a nonstriated and nonmuscle-like F-actin, which mainly distributed along the internal surface of the cellular membrane, whereas a quite different organization that periodic and characteristic striated-like F-actin was observed in MR-1-overexpressed myocytes,25.0±6.7% of which, however, exhibited an irregularly organized F-actin, suggesting a promoted but derangement myofibrillogenesis. MR-1-silenced myocytes showed a stress fiber-like F-actin, which is similar to the random-RNAi control, otherwise, MR-1-RNAi would abolish the striated-like F-actin induced by AngⅡ.4. MR-1-involved regulation in myofibrillogenesis has been recognized detailed as follows; 1) Whereas MRLC collocates with MR-1 extensively in cytoplasm, especially the peri-nucleus area where they both enriched, the nucleus-located myomesin-1 does not, unless its translocation is initiated that myomesin-1 shifts to cytoplasm where its partial collocation with MR-1 was found.2) Myomesin-1 and MRLC were significantly increased at mRNA and protein levels in MR-1-overexpressed culture; MR-1-RNAi does not affect their expression instead of the AngⅡ-induced upregulation.3) MR-1-overexpression promotes myomesin-1 shuttling from nucleus to cytoplasm, this phenomenon would not be affected by MR-1-RNAi, but RNAi may reverse the AngⅡ-induced translocation of myomesin-1.4) Overexpression of SUMO-1 promotes myomesin-1 translocation and the translocation involved myofibrillogenesis, which provide evidence that MR-1 may serve a crucial role in the SUMOylated modification of myomesin-1 and SUMOylated myomesin-1 mediated myofibrillogenesis.5) [Ca2+]i, CaN-activity, myocytes enhancer factor 2C (MEF-2C) and phospho-MEF-2C levels were found significantly increased in MR-1-overexpressed myocytes (P<0.05) but not any significant disparity in MR-1-RNAi culture (P>0.05); nevertheless, RNAi applied myocytes would reverse the AngⅡ-induced upregulation of those molecules.Conclusion1. Overexpression of MR-1 directly induces hypertrophy in neonatal rat cardiomyocytes.2. MR-1 induces hypertrophy in neonatal rat cardiomyocytes by promoting myofibrillogenesis and the myofibril-alignment.3. MR-1 induces hypertrophy in cardiomyocytes through regulating myomesin-1 and MRLC mediated myofibrillogenesis. (major conclusion)4. MR-1 may play an important role in the classic cardiac hypertrophy "Ca2+→CaN→MEF-2C" signaling pathway. |
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