| Abnormalities in the myocardial translatome of mice induced by pressure overload and its mechanismBackgroundHeart failure is a common and complex cardiac disease with high morbidity,mortality and disability rates,which seriously endangers people's life and health.Under chronic pressure overload or other pathological factors,the heart develops compensatory myocardial hypertrophy,which can maintain normal or even enhanced systolic function;however,if the pathological factors persist,the myocardium undergoes a decompensated phase and cardiac function gradually deteriorates,eventually leading to heart failure.Therefore,studying the molecular mechanism of myocardial transition from compensated to decompensated is important for understanding the pathogenesis of heart failure and identifying potential therapy targets for heart failure.For a long time,the research on gene expression regulation has mainly focused on transcriptional regulation,and relatively little research has been done on the role of translational regulation in gene expression.It has been shown that translational regulation plays a key role in protein expression,cellular homeostasis,and cell proliferation,growth and differentiation,and that abnormal translation is closely associated with the development of many diseases.Some studies have found that translation regulation plays an important role in compensatory myocardial hypertrophy and heart failure induced by pressure overload;however,existing studies have only revealed the changes in the translation of certain specific genes caused by pressure overload and their significance,and lack of studies on the changes in the translation of all of the myocardial genes and their overall characteristics(i.e.,translatome).Filling this research gap will help reveal the panoramic view of the effects of pressure overload on myocardial protein translation and provide a comprehensive understanding of the mechanisms underlying the onset of myocardial decompensation.Ribosome profiling based on RNA high-throughput sequencing is one of the most commonly used translatome research techniques which can accurately detect the translation levels of genes on a genome-wide scale.This technique has been widely used in studies on tumor research and other fields,and it has been found that there are obvious differences in the translation and translational regulation of different gene clusters in tumor tissues,and these differences are closely related to the occurrence and development of tumors.This technology also provides a powerful tool for elucidating the panoramic view of the effects of pressure overload on myocardial protein translation,but there are no reports of related studies so far.In addition,phosphorylation of eukaryotic initiation factor 2?(eukaryotic Initiation Factor 2?,eIF2?)and the mammalian target of rapamycin(mammalian Target Of Rapamycin,mTOR)signaling pathway are the key regulatory mechanisms of eukaryotic translation,which function mainly in the translation initiation phase and affect the translation efficiency of genes,that is,the rate of protein production per mRNA.It has been shown that eIF2? phosphorylation levels are significantly increased and the mTOR signaling pathway is significantly activated in pressure overloaded hearts.Both play important and complex roles in pressure overload-induced ventricular remodeling and heart failure.Based on the above understanding,we propose the following scientific hypothesis:that pressure overload significantly alters the myocardial translatome,especially the translation levels of those genes important for maintaining cardiac function,and that these changes are closely related to myocardial decompensation and heart failure induced by sustained pressure overload,and that eIF2? phosphorylation may be an important mechanism for these changes.To test this hypothesis,we investigated the abnormal changes and mechanisms of myocardial translatome during pressure overload-induced myocardial decompensation and heart failure using ribosome profiling.Objectives1.To clarify the global changes and characteristics of myocardial translatome of mice induced by pressure overload.2.To reveal the relationship between abnormal myocardial translatome and pressure overload-induced myocardial decompensation and heart failure in mice and its mechanism.Methods and materials1.Animal groupingC57BL/6 mice were randomly divided into sham-operated(Sham)group and transverse aortic constriction(TAC)group,where the TAC group was further divided into 2-week post-TAC group(TAC-2W),5-week post-TAC group(TAC-5W),TAC5W+green fluorescent protein(Green Fluorescent Protein,GFP)-expressing AdenoAssociated Viruses(Adeno-Associated Viruses,AAV)group(AAV-GFP),and TAC5W+eIF2? phosphorylation-deficient mutant-expressing Adeno-Associated Viruses group(AAV-eIF2?-S51A).2.Establishment of animal modelA mouse model of cardiac pressure overload was established according to the TAC using a thoracotomy-free technique as reported in the literature.3.Echocardiography and carotid Doppler ultrasoundEchocardiography and Doppler ultrasound were performed under isoflurane general anesthesia using a high-resolution small animal ultrasound system to assess cardiac function.4.Virus transfectionOne week before TAC,eIF2?-S51A virus and GFP control virus were injected into tail vein of mice and transfection efficiency was checked one week later.5.Sample collection and processingTissue samples of the left ventricle of mice in the Sham group and the 2nd and 5th week after TAC were collected,and part of the samples were stored at-80? and the other part were stored in 4%paraformaldehyde solution for subsequent experiments.6.Ribosome profilingThe left ventricular tissue was lysed,the supernatant was retained after centrifugation,enzymatically treated with ribonuclease ?(Ribonuclease ?,RNase ?),followed by sucrose density gradient ultracentrifugation,isolation of ribosomes,and extraction of RNA bound to ribosomes(i.e.,ribosome footprints),then perform highthroughput RNA sequencing and data analysis to compare the changes in myocardial translatome of each group of mice.7.Isolation of mouse left ventricular cardiomyocytesThe Langendorff perfusion system and collagenase ? were used to isolate left ventricular cardiomyocytes of each group of mice.8.Polysome isolationPolysomes were isolated from left ventricular tissue according to the methods reported in the literature and RNA bound to them was extracted.9.Western blottingThe total protein was extracted with lysate buffer and quantified,then the protein samples were separated by electrophoresis,and the separated proteins were transferred to polyvinylidene fluoride(Polyvinylidene Fluoride,PVDF)membranes by electrotransferring,blocked by non-fat milk and incubated with primary antibody overnight at 4?,followed by incubation of secondary antibody at room temperature,and finally the protein bands were visualized with chemiluminescent solution and imaging system.10.ImmunohistochemistryAfter inactivation of endogenous peroxidase with 3%hydrogen peroxide,the antigen was repaired by microwave heating,blocked by goat serum and incubated with primary antibody at 4? overnight,followed by incubation of secondary antibody at room temperature,and the target protein was visualized and photographed by microscopy after coloring.11.Quantitative polymerase chain reactionReverse transcription and quantitative polymerase chain reaction(quantitative PCR,qPCR)were performed according to the method provided in the PCR kit which use TaqMan method.12.Deoxyribonucleotide end-transferase-mediated nick labeling stainingApoptosis in left ventricular tissue was detected by deoxyribonucleotide endtransferase-mediated nick labeling(TdT-mediated dUTP nick labeling,TUNEL)staining using the method provided in the kit.13.Data processing and statistical analysisGene function annotation and function enrichment analysis were performed using various bioinformatics tools such as DAVID.Shapiro-Wilk test was used to test the normality of the data.Two-tailed t-test and one-way analysis of variance were used to compare two and multiple groups of normally distributed data,respectively.P<0.05 was considered statistically significant.Results1.A mouse model of cardiac pressure overload was successfully establishedThe results of echocardiography and Doppler ultrasonography showed that the aortic arch of the mice in the TAC group was significantly narrowed and the flow rate of the right carotid artery was significantly increased,while the flow rate of the left carotid artery was significantly decreased compared with that of the Sham group.These results indicated that TAC induced cardiac pressure overload and the animal model was successfully established.2.TAC induced myocardium from compensatory hypertrophy to decompensation in miceThe results of echocardiography showed that compared with the Sham group,the interventricular septal end diastole(Interventricular Septal end diastole,IVSd)and left ventricular posterior wall end diastole(Left Ventricular Posterior Wall end diastole,LVPWd)were significantly increased in the TAC-2W group,whereas left ventricular internal diameter end diastole(Left Ventricular Internal Diameter end diastole,LVIDd),left ventricular fractional shortening(Fractional Shortening,FS)and left ventricular ejection fraction(Ejection Fraction,EF)were not significantly changed;at 5 weeks after TAC,EF and FS were significantly decreased by approximately 50%,whereas the left ventricular internal diameter was significantly enlarged.These results suggest that at 2 weeks after TAC,pressure overload mainly induced compensatory hypertrophy in mice,whereas myocardial decompensation occurred in mice at 5 weeks after TAC under the sustained pressure overload.3.Chronic pressure overload induced significant changes in the myocardial translatome,and the translation efficiency of myocardial genes was significantly decreased during myocardial decompensationThe results of the integrated analysis of the data from each group showed a total of 2537 transcripts were detected in the myocardium of both the Sham,TAC-2W and TAC-5W groups.The translation efficiency of these 2537 genes was significantly altered in both the TAC-2W and TAC-5W groups of mice compared to the Sham group:the average translation efficiency of these genes was significantly increased at 2 weeks after TAC.However,this change was reversed at 5 weeks after TAC.Compared to the TAC-2 W group,the translation efficiency of a total of 4346 transcripts was significantly downregulated in the TAC-5 W group,and the frequency of the distribution of ribosome density on these 4346 transcripts was also significantly decreased.4.Genes with significantly down-regulated translation efficiency during myocardial decompensation have important roles in the regulation of cardiac functionThe functional enrichment analysis of the above 4346 genes with significantly down-regulated translation efficiency during myocardial decompensation showed that they were mainly involved in biological processes or signaling pathways that are important in maintaining normal cardiac function,including energy metabolism,myocardial structure and function,vascular development,and angiogenesis.Validation of key node molecules in these genes further showed that,compared with the TAC-2 W group,eight pro-angiogenic genes including vascular endothelial growth factor receptor 1(Vascular Endothelial Growth Factor Receptor 1,FLT1)and vascular endothelial growth factor A(Vascular Endothelial Growth Factor A,VEGFA),and the energy metabolism gene malate dehydrogenase 2(Malate Dehydrogenase 2,MDH2)and the cytochrome c oxidase assembly homolog COX 15(Cytochrome C Oxidase Assembly Homolog COX15,COX15),the muscle-specific gene GATA binding protein 4(GATA Binding Protein 4,GATA4)and desmin(Desmin)were all showed significantly reduced translation efficiency in the TAC-5W group.5.No evidence of press overload causing termination of the translation elongation processTo verify the effect of press overload on the translation elongation process,we analyzed ribosome footprints data from the Sham,TAC-2W,and TAC-5W groups and compared the positional distribution and average abundance of ribosome footprints on the open reading frame end regions of all transcripts,as well as on the five angiogenesisrelated genes.The results showed that,there was no significant aggregation or preference in the positional distribution of ribosome footprints on transcripts,suggesting that pressure overload may not cause termination of the translation elongation process.6.Significant abnormalities in the regulatory mechanisms of protein translation during myocardial decompensationWestern blotting results showed that the phosphorylation levels of mTOR and its downstream target molecules,4E binding protein(4E Binding Protein,4EBP)and ribosomal S6 protein kinase(p70S6 Kinase,p70S6K),were significantly increased in the TAC-2W group compared with the Sham group.Phosphorylation levels of mTOR,4EBP,and p70S6K were significantly decreased in the TAC-5W group compared with the TAC-2W group.The phosphorylation level of eIF2? was only slightly increased in the TAC-2W group,but significantly increased in the TAC-5W group.7.Inhibition of eIF2? phosphorylation significantly improved cardiac functions in TAC miceInhibition of eIF2? phosphorylation by transfection with eIF2a-S51A virus significantly improved cardiac function,increased the translation efficiency of important myocardial genes(FLT1,MDH2,COX15,GATA4,Desmin and VEGFA),increased the density of cardiac microvessels and reduced cardiomyocyte apoptosis in mice 5 weeks after TAC.Conclusions1.Pressure overload can significantly alter the myocardial translatome in mice:the translation efficiency is significantly increased during myocardial compensation,but significantly decreased during myocardial decompensation,including many functionally important myocardial genes.2.Pressure overload can induce sustained activation of eIF2?.Inhibition of eIF2?phosphorylation can significantly improve cardiac function,improve the translation efficiency of important myocardial genes,increase angiogenesis and reduce myocardial apoptosis in TAC mice.Dynamic changes in mitochondrial distribution of endothelial miRNAs induced by ROS and its mechanismBackgroundIn recent years,the morbidity and mortality of cardiovascular diseases have remained high,causing huge social and economic losses,and have become a major global public health problem.A large number of studies have demonstrated that vascular endothelial injury is the key initiating factor and one of the core mechanisms in the development of various cardiovascular diseases,and reactive oxygen species(Reactive Oxygen Species,ROS)is an important factor that cause vascular endothelial injury.However,the mechanism of ROS-induced endothelial injury is very complex and has not been fully elucidated so far.Therefore,uncovering the pathological mechanisms of ROS-induced endothelial injury is essential to understand the pathogenesis of cardiovascular diseases and to develop effective prevention and treatment methods.MicroRNAs(miRNAs)are a class of non-coding small RNAs with regulatory functions,which play an important role in both endothelial homeostasis and ROSinduced endothelial injury.MiRNAs are widely distributed in the cytosol and various subcellular organelles,such as nucleus,nucleolus,endoplasmic reticulum,and mitochondria.This regionalized distribution of miRNAs plays an important role in maintaining cellular homeostasis.Especially those miRNAs distributed in mitochondria,the changes in their species and abundance are closely related to the regulation of normal physiological functions and the development of many diseases,especially cardiovascular diseases.For example,the redistribution of miR-378 in the mitochondria of cardiomyocytes can promote the development of diabetic cardiomyopathy.However,the dynamic changes in mitochondrial distribution of miRNA during ROS-induced endothelial injury and its functional significance are still lacking in systematic research.Numerous studies have shown that the vast majority of miRNAs in mitochondria are encoded by the cellular nuclear genome,matured in the cytosol and then translocated to the mitochondria.Elucidating the mitochondrial transport mechanism of miRNA is a key point to fully understand the changing characteristics of mitochondrial distribution of miRNA and its pathophysiological significance.However,studies on the mitochondrial transport mechanism of miRNA are still scarce and there are still many questions that need to be elucidated.Recently,Shepherd et al.found that polynucleotide phosphorylase(Polynucleotide Phosphorylase,PNPASE)could be involved in the mitochondrial transport of miR-378 in cardiomyocytes.Considering the complexity of mitochondrial miRNA and their transport systems,it is obvious that there are many unknown proteins other than PNPASE that participate in and regulate the mitochondrial transport of miRNA.Therefore,the main content of our work in this part is to investigate the dynamic changes of miRNA mitochondrial distribution and its functional significance during ROS-induced endothelial injury,and to preliminarily reveal the mechanism of miRNA mitochondrial transport.Objectives1.To reveal the dynamic changes in mitochondrial distribution of endothelial miRNAs and its functional significance during ROS-induced endothelial injury.2.To preliminarily identify the mechanism of miRNA mitochondrial transport and the effect of ROS on this mechanism,that is,the mechanism of ROS-induced changes in mitochondrial distribution of endothelial miRNAs.Methods and Materials1.Cell culture?treatment and transfectionHuman umbilical vein endothelial cells(Human Umbilical Vein Endothelial Cells,HUVECs)were cultured in complete endothelial cell medium and stimulated with H2O2 or glucose oxidase(Glucose Oxidase,GO)to establish the model of ROS-induced endothelial injury.Cells were transfected with Lipofectamine RNAiMAX or Lipofectamine 3000.2.Subcellular isolationThe cytosol and mitochondria of HUVECs were isolated and purified according to the methods reported in the literature.3.qPCRqPCR was performed according to the method provided in the kit.The synthetic single-stranded oligonucleotide was used to generate a standard curve for absolute quantitative qPCR.4.miRNA sequencing and data analysisHiseq SE50 was used for miRNA sequencing of whole endothelial cells and endothelial mitochondria,followed by data analysis to obtain miRNA expression matrix,and then bioinformatics analysis for miRNA target gene prediction,functional enrichment and regulatory networks.5.Western blottingAfter the total protein was extracted with lysate buffer and quantified,the protein samples were separated by gel electrophoresis,and the separated proteins were transferred to PVDF membrane by electro-transfer,blocked by 5%non-fat milk and incubated with primary antibody overnight at 4?,followed by incubation of secondary antibody at room temperature,and finally the protein bands were visualized with chemiluminescent solution and imaging system.6.ImmunoprecipitationThe lysates of endothelial mitochondria were successively incubated with antibodies and Protein A/G agarose beads,the agarose beads were retained after centrifugation,resuspended in protein loading buffer and boiled,and the supernatant was collected after centrifugation again,followed by protein immunoblotting to detect the expression of the corresponding protein.7.MitoTracker and immunofluorescence stainingMitochondria were labeled with MitoTracker,then the cells were fixed and permeabilized,followed by successive incubation with primary antibody and fluorescently labeled secondary antibody,and then observed and photographed under a laser confocal microscope after staining the nucleus.8.RNA immunoprecipitation(RIP)RIP was performed using the corresponding antibodies and Millipore's RNA binding protein immunoprecipitation kit.9.Pull down assayBiotinylated miR-301a-3p pull-down experiment was performed according to the methods reported in literature.10.Label-free quantitative proteomic analysisLabel-free quantitative proteomic analysis was performed according to the methods reported in literature.11.Bio-Layer interferometry analysis(BLI)Biotinylated RNA oligonucleotides were bound to the streptavidin-modified biosensor,and then incubated with protein samples of different concentrations to detect the binding kinetics of the two.12.Annexin V/Propidium Iodide and EdU stainingEndothelial cell apoptosis and proliferation were detected according to the methods provided by the Annexin V/Propidium iodide apoptosis kit and the EdU staining kit,respectively.13.MTT assayThe effect of different concentrations of GO on endothelial cell viability was examined according to the method provided in the MTT kit.14.Luciferase assayDNA fragments containing wild or mutated miRNA binding site sequences in the target genes were cloned into dual luciferase expression vectors,and then the constructed vectors and miRNA mimics or control oligonucleotides were cotransfected into HEK293T cells,and the luciferase activity of each group was detected after 72 hours.15.Statistical analysisThe Shapiro-Wilk test was used to evaluate the normality of the data.The Wilcoxon rank sum test was used to compare two groups of non-normally distributed data.Two-tailed t-tests and one-way analysis of variance were used for comparison of differences between two and multiple groups of normally distributed data,respectively.P<0.05 was considered statistically significant.Results1.ROS could induce endothelial injuryH2O2 significantly induced endothelial cell apoptosis and proliferation inhibition.Meanwhile,GO could also induce endothelial injury in a concentration-dependent manner.2.ROS could significantly reduce the amounts of several miRNAs in endothelial mitochondria,but did not affect their expression in the whole cellsThe results of miRNA sequencing showed that H2O2 significantly reduced the amounts of dozens of miRNAs in the mitochondria of HUVECs,but did not affect their expression in the whole cells.Among them,miR-301a-3p and miR-381-3p were two of the most significantly changed high-abundance miRNAs.The results of qPCR and absolute quantitative PCR further confirmed the results of miRNA sequencing.3.MiRNAs exhibiting significantly reduced mitochondrial distribution were closely associated with ROS-induced endothelial injuryFunctional enrichment analysis showed that the above miRNAs with significantly reduced mitochondrial distribution but unchanged total cellular expression were significantly enriched in multiple biological processes and signaling pathways related to ROS-induced endothelial injury.Among them,miR-381-3p was one of the key node molecules in the potential apoptosis and cell proliferation regulatory network of these miRNAs.4.MiR-381-3p promotes H2O2-induced apoptosis and proliferation inhibition in endothelial cells through nuclear factor I/A and low-density lipoprotein receptorrelated protein 6 and their downstream moleculesTo unravel the functional significance of ROS-induced changes in mitochondrial distribution of endothelial miRNAs,we investigated in depth the role and mechanism of miR-381-3p in H2O2-induced endothelial injury.Western blotting,qPCR and luciferase assays all showed that miR-381-3p could directly target nuclear factor I/A(Nuclear Factor IA,NFIA)and low-density lipoprotein receptor related protein 6(LDL Receptor Related Protein 6,LRP6),and inhibited their expression in endothelial cells.The results of further functional study showed that miR-381-3p could promote H2O2induced endothelial injury by targeting NFIA and LRP6 and their downstream molecules(P53,P21,MYC,and cyclin D1).5.Musashi RNA binding protein 2,which is abundant in mitochondria,specifically binds to miR-301a-3p and miR-381-3p and promotes their distribution in mitochondriaTo reveal the mechanism of miRNA mitochondrial distribution and identify the proteins involved in miRNA mitochondrial transport,we performed biotinylated miRNA pull-down assay and label-free quantitative proteomics analysis using miR301a-3p,which is highly abundant in endothelial mitochondria.The results showed that Musashi RNA binding protein 2(Musashi RNA Binding Protein 2,MSI2)was the most abundant in the pull-down product of biotinylated miR-301a-3p,and MSI2 also specifically bound to endogenous miR-301a-3p and miR-381-3p.In addition,MSI2 was abundant in the mitochondria of a variety of cells.Inhibition of MSI2 expression in endothelial cells significantly reduced the abundance of miR-301a-3p and miR-3813p in the mitochondria,while increasing their abundance in the cytosol.We also found that MSI2 did not interact with Argonaute2 protein(Argonaute2,Ago2)in HUVEC mitochondria.These results suggested that MSI2 could promote mitochondrial transport of miR-301a-3p and miR-381-3p in endothelial cells,and that this effect may not be dependent on Ago2 protein.6.Oxidative stress could significantly decrease the expression of MSI2Both H2O2 and GO significantly decreased the expression of MSI2 in HUVECs,while promoting redistribution of MSI2 between cytosol and mitochondria,that is,decreasing the amount of MSI2 in the mitochondria but increasing it in the cytosol.It has been shown that Kruppel-like factor 4(Krüppel-Like Factor 4,KLF4)could regulate MSI2 expression,but we found that KLF4 was not involved in the regulation of MSI2 by H2O2.Conclusions1.During reactive oxygen species-induced endothelial injury,the amounts of many miRNAs,including miR-301a-3p and miR-381-3p,in mitochondria was significantly reduced,but the expression of these miRNAs in endothelial cells was not significantly changed.Moreover,these miRNAs were closely related to ROS-induced endothelial injury.2.MiR-381-3p with significantly reduced mitochondrial distribution could promote H2O2-induced apoptosis and proliferation inhibition in endothelial cells through the target genes NFIA and LRP6 and their downstream molecules(P53,P21,MYC,and cyclin D1).3.MSI2 could specifically bind to miR-301a-3p and miR-381-3p and promote their distribution in endothelial mitochondria,which may be an important component of the miRNA mitochondrial transport system. |
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