| BackgroundMyocardial ischemia-reperfusion (I/R) injury is a major problem to besolved in cardiovascular field. To investigate the mechanism of inhibition ofcardiomyocyte injury will provide a new target for myocardial I/R prevention andtherapy.Myofibrillogenesis regulator1(MR-1), a human gene (AF417001) clonedfrom a human skeleton muscle cDNA library and found by Li in2004, is highlyexpressed in myocardium, skeletal muscle, kidney and liver. Previous studydemonstrated that MR-1was located in cardiac myofibrils. Moreover, MR-1overexpression attenuated renal tubular epithelial cell injury induced by H/R,which suggested that MR-1attenuated cell injury.Microfilament (MF) system, mainly consisting of actin and actin-bindingproteins such as α-actinin, maintains cell survival and the structure and function ofcardiac myofibrils, which mainly consist of fibrous actin (F-actin). MR-1islocated in cardiac myofibrils and interacts with myosin regulatory light chain-2(MLC-2), which is an unique physiological substrate of myosin light chainkinase (MLCK). MLC-2plays a key role in the regulation of F-actindepolymerization/polymerization. MR-1may attenuate H/R injury incardiomyocytes through MLCK/MLC-2/F-actin pathway.In addition, endoplasmic reticulum stress (ERS) is the initial reaction whencellular stress occurs. When ERS-associated apoptosis induced by H/R occurs,protein kinase R-like ER kinase (PERK) activation phosphorylates eukaryotictranslation initiator factor2α (eIF2α), which upregulates the expression ofactivating transcription factor4(ATF4), and then consequently increases theexpression of C/EBP homologous protein (CHOP), down-regulating Bcl-2protein and up-regulating Bax protein. Nuclear factor erythroid2-related factor2(Nrf2) is located in the cytoskeleton in unstressed conditions. According to thelatest reports, Nrf2is the downstream factor of PERK. Nrf2translocates to nucleus induced by I/R, forms amounts of clusters in nucleus, and combines withthe promoter of ATF4to upregulate ATF4expression. It demostrates thatNrf2/ATF4pathway mediated by PERK may be a significant mechanism inH/R-induced apoptosis. MR-1may attenuate H/R-induced cardiomyocyteapoptosis through PERK/Nrf2pathway.ObjectiveThe effects of MR-1on H/R-induced cardiomyocytes and microfilamentinjury were studied to verified that MR-1protected cardiomyocytes against H/Rinjury through attenuating microfilament damage. The effects of MR-1onH/R-induced ERS-associated apoptosis were investigated to demonstrate thatMR-1protected cardiomyocytes against H/R injury by attenuating ERS-associatedapoptosis.Methods1. Establishment of cardiomyocytes model of H/R injury: Under sterile conditions,cultured neonatal rat cardiomyocytes were placed in three gas incubator treatedwith hypoxia for8hours/reoxygenation for16hours.2. Analysis of cardiomyocyte apoptosis: Cells were labelled with Annexin V andPropidium Iodide, and apoptosis rate was analysed by flow cytometry.3. Analysis of lactate dehydrogenase (LDH) activity: LDH activity incardiomyocyte-culturing medium was measured spectrophotometrically by the useof the LDH assay kit to estimate LDH leakage under different treatmentconditions.4. Cell viability assessment: It was determined by using the CCK-8assay.5. Actin cytoskeleton observation: F-actin fluorescence staining withphalloidin-FITC and α-actinin labeled by immunofluorescence staining wereemployed to view actin cytoskeleton in cardiomyocytes. The protein expression ofF-actin and G-actin was detected by Western Blot.6. Detection of F-actin assembly-regulated factors: The reverse transcriptionreaction and polymerase chain reaction (RT-PCR) were employed to detected theexpression of MLC-2mRNA. The protein expression and phosphorylation ofMLC-2and MLCK were detected by Western Blot.7. Analysis of ERS-associated apoptotic pathway: Western Blot was employed todetect ERS-associated protein expression as follows: GRP78, Nrf2, ATF4, CHOP, Bcl-2and Bax protein, PERK protein expression and phosphorylation. In addition,the expression of Nrf2protein in cytoplasm and nucleus was respectively detectedby the assay of cytoplasmic and nuclear protein separation. Nrf2localization wasobserved with confocal laser scanning microscopy by the staining of Nrf2.8. Data processing and statistical analysis: The results were presented as themean±SD. SPSS13.0statistics software was introduced. Significant differencesamong groups were assessed by one-way analysis of variance (ANOVA)followed by Student-Newman-Keuls tests. P-values less than0.05wereconsidered stastically significant. Pearson bivariate correlation analysis wasapplied to determine the correlation between variables.Results1. MR-1attenuates H/R-induced cardiomyocyte injury1.1MR-1attenuates H/R-induced cardiomyocyte apoptosis: In vitro culturedcardiomyocytes model of H/R injury, we found that the apoptosis rate wasincreased with H/R compared with the control treatment. While overexpressingMR-1before H/R treatment significantly reduced H/R-induced cardiomyocyteapoptosis. However, knocking down MR-1before H/R treatment increasedH/R-induced cardiomyocyte apoptosis.1.2MR-1attenuates the increase of LDH activity induced by H/R: LDH activityin cardiomyocyte-culturing medium in the H/R group was significantly higherthan that in the control group, MR-1overexpression reduced the increase of LDHactivity induced by H/R. There was no effect on H/R-induced increase of LDHactivity by knocking down MR-1.1.3MR-1attenuates the decrease of cell viability induced by H/R: Cell viability inthe H/R group was significantly decreased as compared with the control group.MR-1overexpression significantly attenuated the decrease of cell viabilityinduced by H/R. MR-1knockdown aggravated the decrease of cell viabilityinduced by H/R.2. MR-1attenuates H/R-induced injury in cardiomyocytes by promoting therestoration of microfilament2.1MR-1attenuates H/R-induced F-actin and α-actinin injury in cardiomyocytes:H/R induced disorderly distribution in F-actin and α-actinin as compared withcontrols. MR-1overexpression significantly attenuated H/R-induced injury in F-actin and α-actinin in cardiomyocytes. MR-1knockdown aggravatedH/R-induced F-actin and α-actinin injury in cardiomyocytes.2.2MR-1promotes the restoration of microfilament by regulating MLCK/MLC-2pathway, thereby reducing the H/R-induced cardiomyocyte injury: In the H/Rgroup, the expression and phosphorylation of MLCK and MLC-2were increasedas compared with the control group. MR-1overexpression increased H/R-inducedexpression and phosphorylation of MLCK and MLC-2. MR-1knockdowndecreased H/R-induced expression and phosphorylation of MLCK and MLC-2.3. MR-1inhibits of H/R-induced ERS-associated apoptosis in cardiomyocytes3.1MR-1inhibits ERS-associated apoptotic pathway to reduce H/R-inducedapoptosis in cardiomyocytes: In the H/R group, GRP78and CHOP proteinexpression was increased and Bcl-2/Bax ratio was decreased as compared with thecontrol group. MR-1overexpression reduced the increase of protein expression ofGRP78and CHOP and the decrease of Bcl-2/Bax ratio induced by H/R. MR-1knockdown enhanced H/R-induced expression of GRP78and CHOP, andH/R-induced decrease of Bcl-2/Bax ratio.3.2MR-1attenuates H/R-induced apoptosis in cardiomyocytes by inhibitingPERK/Nrf2pathway in cardiomyocytes: Compared with the control group, PERKphosphorylation, and ATF4protein expression was increased and Nrf2translocated to nucleus in the H/R group. MR-1overexpression reducedH/R-induced PERK phosphorylation, Nrf2nuclear translocation and ATF4proteinexpression. MR-1knockdown increased H/R-induced PERK phosphorylation,Nrf2nuclear translocation and ATF4protein expression.Conclusion1. MR-1protected cardiomyocytes from H/R injury.2. MR-1attenuated H/R-induced injury by promoting the restoration ofmicrofilament in cardiomyocytes.3. MR-1attenuated H/R-induced apoptosis by inhibiting ERS-associatedapoptosis in cardiomyocytes. |
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