| Background:Myocardial infarction is one of cardiovascular diseases that severely threaten human health.Dozens of clinical trials and basic experiments have proved that Bone marrow mesenchymal stem cells(BMSCs)allograft is a promising method to improve heart function after myocardial infarction in addition to operations.However,BMSCs face an array of challenges within the hypoxic and ischemic micro-environment that especially curtail their survival after transplantation.Actually,it is believed that BMSCs therapy works mainly through its paracrine pathway,and the microenvironment of BMSCs in the organism makes it exhibit completely different biological effects.Therefore,survival of BMSCs may be of great importance to long term effects and improving their maintanence in hypoxic and ischemic environment will play a key role in transplantation therapy.Objective:Due to the lack of oxygen under the hypoxic and ischemic environment,MSCs display functional and metabolic changes in proteomics to counter external stimuli.Understanding such proteomic alterations is necessary and requires cell-selective and real-time measurement of the extent to which proteins levels are adjusted as external environmental change.Apoptosis-inducing factor(AIF)may be one of the most critical factors.Subsequently,the AIF-knockdown BMSCs were cultured in an ischemic and hypoxic environment or transplanted into the infarcted myocardium,further confirming the importance of AIF in the survival of BMSCs after transplantation and further recovery of cardiac function after infarction.Methods:To solve this problem,we apply a metabolic labeling method for cell-type-specific proteome with noncanonical amino acids(ncAAs),as an important tool to interrogate proteome dynamics of MSCs.In this method,mutant methionyl-tRNA synthetase(MetRS)L274G is targeted to specific cells of interest,enabling incorporation of methionine surrogate azidonorleucine(ANL),an ncAA,into newly-synthesized polypeptides site-selectively at N-terminal positions.Then MetRSL274G-BMSCs cell line was constructed.The sensitivity and specifity of this labeling method were identified by BONCAT and FUNCAT.Nascent BMSC proteomes were then quantitatively analyzed by liquid chromatography/mass spectrometry(LC/MS)and bioinformatics analysis revealing BMSCs’ adaptive responses and functional alteration in the harsh environment excluding interference of preexisting proteins.We found that apoptosis inducing factor(AIF)were considered one of the most important factor that influence survival of BMSCs.BMSCs with down-regulated AIF were transplanted into mouse infarcted hearts to observe their therapeutic effect on infarcted hearts.Results:1.Murine MSCs were transduced with lentivirus MetRSL274G and supplemented with ANL;the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging,spanning all molecular weights and by fluorescent non-canonical amino-acid tagging,displaying strong fluorescent signal.2.The LC/MS analysis results showed that totally 1326 and 1323 of ANL-labeled proteins were identified respectively in Sham and OGD group after BMSCs were cultured in hypoxic and serum-free condition.Notably,there were 1219 proteins in the two groups,representing 92±0.2%of all identified proteins(Figure 3C).As expected,proteomes were largely overlapped.79 of these proteins were significantly regulated after OGD(1.5 fold change,p<0.05),among which nucleoproteins is the most abundant.3.Proteins related to the regulation of apoptosis and actin cytoskeleton were closely asscociated to the death of BMSCs under the condition of ischemia and hypoxia.The expression of AIF-m in mitochondria was 6.17 times lower than that in normal culture group(P<0.05),while soluble AIF-c in nucleus with apoptotic effect was significantly increased(P<0.05).4.MetRSL274G-transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment.The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1,3,and 7 after cell administration,identified by LC/MS.Among all the 713-863 identified proteins(in Sham and MI hearts,three time-points each),648 were shared by all 6 groups,accounting for 82±5%of total proteins in each group,and enriched under mitochondrion,extracellular exosomes,oxidation-reduction process and poly(A)RNA binding.Notably,26,110 and 65 proteins were significantly up-regulated and 11,28 and 19 proteins were down-regulated in the infarcted vs.Sham heart at the three time-points,respectively(1.5 fold change,p<0.05).5.BMSCs respond to the ischemic/hypoxic environment by altering proteins in the complements and coagulation cascades and regulators of actin cytoskeleton and apoptosis.6.Cathepsin B,prelamin-A/C and apoptosis inducing factor(AIF)are the most important factor causing the death of BMSCs after transplantation.Among them,AIF was the most important.After BMSCs were transplanted to infarcted border zone in mice,the expression of AIF-m in mitochondria was significantly decreased by 1.57 and 3.52 times on day 1 and 7(P<0.05),respectively.7.BMSCs with down-regulated AIF expression were successfully constructed by lentivirus infection(infectious effect was 97.5%).Compared with normal BMSCs,AIF expression in infected BMSCs decreased by 59.93%(P<0.01).Compared with normal BMSCs,the expression of AIF in LV-AIF-shRNA BMSCs decreased by 23.83%(P<0.01),the apoptosis rate decreased by 52.20%(P<0.05),and the cell viability increased by 11.30%(P<0.05)after 12h culture in hypoxic and serum-free media.8.AIF down-regulation may significantly attenuate apoptosis and.increase 2.84-fold survival rate of BMSCs in infarcted hearts(P<0.01).Moreover,better recovery of cardiac function and lesson fibrosis were dectected after AIF was down-regulated in transplanted BMSCs.BNP levels in myocardial infarction mice decreased by 17.8%(P<0.01).EF increased by 3.13%in absolute value after 4 weeks of mice.AIF protein expression(immunofluorescence),myocardial fibrosis degree(Masson staining)and infarct extent(TTC staining)were significantly reduced in infarct area.Conclusion:1.MetRSL274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts,which reflect the functional states,adaptive response,and reparative effects of BMSCs that may be leveraged to improve cardiac repair.2.Regulation of actin cytoskeleton,extracellular exosome,apoptosis and extracellular matrix organization occupied an important position in cellular activities of BMSCs which were exposed to infarcted myocardium or hypoxic and glucose/serum-free condition.3.After BMSCs were transplanted into the infarcted heart,the regulation of extracellular matrix,extracellular exosomes,response to stress and apoptosis were more obvious.BMSCs could regulate complement and coagulation cascade,actin cytoskeleton and apoptosis in respons to the ischemic and hypoxic environment in the recipient.4.AIF is one of the most important factors that determine BMSCs’ survival after cultured in hypoxic and serum-free enviorment of transplanted into infarcted border zone.AIF down-regulation in transplanted BMSCs may significantly increase survival rate of BMSCs in infarcted hearts and result in better recovery of cardiac function and lesson fibrosis. |
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