Investigation Of Cardiac Telocytes Synergizing Cardiac Stem Cell Therapy For Myocardial Infarction And Their Included Mechanism

Objectives:（1）To investigate the different therapeutic effects of Cardiac Telocytes（CTs）,Bone Marrow Mesenchymal Stem Cells（MSCs）and Cardiac Stem Cells（CSCs）transplantations for myocardial infarction（MI）;（2）To compare the therapeutic effects of different synergetic methods of CTs and CSCs for MI;（3）To explore the therapeutic effects of CTs exosomes transplantation for MI and the underlying mechanism;（4）To explore the therapeutic effects and the mechanism of CTs-derived miR-21-5p included in CTs exosomes for MI;（5）To compare the therapeutic effects of different cell therapies,CTs exosomes therapy and miR-21-5p therapy for MI;（6）To predict the possible mechanism of CTs exosome proteome in regeneration of infarcted myocardial by bioinformatic methods;（7）To investigate related signaling pathways for the differential expression genes between the young and old CTs.Methods:（1）Adherent method was applied to isolate MSCs,and Magnetic activated cell sorting was applied to isolate CTs and CSCs.Rat myocardial infarction model was established by left anterior descending coronary artery（LAD）ligation.The intramyocardial injections were conducted with 5×10⁵ CTs,CSCs,MSCs respectively as well as 4 different synergetic methods between CTs and CSCs[（1）5×10⁵CSCs which were co-cultured with CTs for 48 h in a transwell system（CSCs-co）;（2）mix 2.5×10⁵CTs and 2.5×10⁵CSCs,which CSCs were precultured in a hypoxic incubator（5%O₂）for 48 h before mixing（CTs+CSCs-hyp）;（3）mix normal-cultured2.5×10⁵CTs and 2.5×10⁵CSCs which were co-cultured with CTs for 48 h in a transwell system)（CTs-nor+CSCs-co）;（4）5×10⁵ CTs（2.5×10⁵）and CSCs（2.5×10⁵）mix-cultured for 48 h（CTs+CSCs-co）]or PBS for MI.Echocardiograms were applied to evaluate the cardiac function after 4 weeks of cell therapy.The masson’s trichrome staining was applied to analyze infarction size,fibrosis of heart and wall thickness.The immunohistochemistry strainings for v WF was applied to investigate the vessel density in the infarct zone and the border zone.The immunohistochemistry strainings for PH3+α-SA and Ki67+c Tn I were applied to investigate the number of proliferative of cardiomyocytes in the infarct zone and border zone.The immunohistochemistry stainings for c-Kit+CD34 and c-Kit+Sca-1 were applied to investigate the survival of CTs and CSCs in infarct zone respectively.（2）Transmission electron microscopy was used for observe CTs and CTs-secreted exosomes.The intramyocardial injections were conducted with CTs exsomes or PBS for MI.Echocardiograms were applied to evaluate the cardiac function after 4 weeks of CTs exsomes therapy.The masson’s trichrome staining was used to analyze infarction size,fibrosis of heart and wall thickness.The immunohistochemistry strainings forα-SMA and MMP2 were applied to investigate the myofibroblast cells density and the expression of MMP2 in the infarct zone.The immunohistochemistry strainings for v WF and Ki67+c Tn I were applied to investigate the vessel density and the number of proliferations of cardiomyocyte in the infarct zone and border zone respectively.The immunohistochemistry straining for c-Kit+Sca-1 was applied to investigate the survival of CSCs in infarct zone.Di I-stained CTs exosomes were co-cultured with CSCs for 6 h to observe whether CTs exosomes can be taken up by CSCs.The flow cytometry,live/dead cell and CCK8 assay were applied to investigate the possible protective effect of CTs exosomes for CSCs in ischemic and hypoxic environment.（3）Expression level of miR-21-5p in CSCs,which was co-cultured with CTs for48 h,was detected by q-PCR.The intramyocardial injections of miR-21-5p agomir and the scramble mimics control were conducted respectively in rat MI model.In 7 days afrer injection,echocardiogram was applied to evaluate the cardiac function.The masson’s trichrome staining was utilized to analyze infarction size.The immunohistochemistry straining for Ki67+c Tn I was applied to investigate the number of proliferations of cardiomyocyte in the infarct zone and border zone.The immunohistochemistry straining for c-Kit+Sca-1 was used to investigate the survival of CSCs in infarct zone.The potential target genes of miR-21-5p were predicted by bioinformatics and verified by q-PCR and dual-luciferase reporter system.The siRNA of verified target genes was used for confirm the function of miR-21-5p.（4）One-way analysis of variance was used to compare the therapeutic effects among the different cells therapies,CTs exosomes therapy and miR-21-5p therapy for MI.（5）Normoxia and hypoxia CTs exosomes proteome were analyzed by label-free protein mass spectrum quantification,and the possible network and pathway for cardiac repair were analyzed by Ingenuity Systems Pathway Analysis（IPA）.（6）The microarray assay was applied to compare the differential gene expression profile between young and old CTs,and analyze the possible regulated biological process,pathway for cardiac functions by IPA.Results:（1）In 4 weeks after injection of CTs,MSCs or CSCs for MI,it was found that the difference of CTs treated group compared with MSCs or CSCs group in ejection fraction（EF）,fractional shortening（FS）,left ventricular end systolic diameter（LVESD）and left ventricular end diastolic diameter（LVEDD）was not statistically significant（P>0.05）.The infarction size of CTs treated group was decreased singificantly compared with MSCs group（P<0.05）but not CSCs group（P>0.05）.Both the collagen area of the infarct zone（CAIZ）and the perivascular collagen volume area（PVCA）of CTs treated group decreased significantly compared with MSCs group or CSCs group respectively（P<0.05）.The difference of CTs treated group compared with MSCs or CSCs group in the wall thickness in the border zone of the LV（WTBZ）and the thickness of the LV of the infarcted myocardium（TIM）was not statistically significant（P>0.05）.In the infarct zone,the vessel density of CTs group increased significantly compared with MSCs or CSCs group respectively（P<0.05）;in the border zone,the vessel density of CTs group was also significantly larger than the MSCs group（P<0.05）but not CSCs group（P>0.05）.The difference of the number of proliferative cardiomyocytes which were PH3⁺or Ki67⁺in the infarct zone and the border zone between CTs-treated group and MSCs-treated or CSCs-treated group was not statistically significant（P>0.05）.The difference of the survival CTs and the survival CSCs in infarcted zone between CTs-treated group and MSCs-or CSCs-treated group was also not statistically significant（P>0.05）.（2）It was found that EF and FS of the CTs+CSCs-co treated group was significantly higher than CTs,MSCs or CSCs treated group respectively（P<0.01）.The LVESD of CTs+CSCs-co treated group was significantly smaller than the CTs,MSCs and CSCs treated group respectively（P<0.05）.The LVEDD of CTs+CSCs-co treated group was significantly smaller than the CTs and MSCs treated group（P<0.05）.The infarction size of CTs+CSCs-co treated group was significantly smaller than the CTs-,MSC-s or CSCs-treated group respectively（P<0.05）.The CAIZ of CTs+CSCs-co treated group was significantly smaller than the CTs-,MSCs-or CSCs-treated group respectively（P<0.05）,while the PVCA of CTs+CSCs-co treated group was significantly smaller than the CSCs-or MSCs-treated group respectively（P<0.01）but not the CTs-treated group（P>0.05）.The WTBZ of CTs+CSCs-co treated group was similar to the CTs-,MSCs-or CSCs-treated group respectively（P>0.05）,and the TIM of CTs+CSCs-co treated group was significantly larger than the CSCs or MSCs group respectively（P<0.05）and similar to CTs-treated group（P>0.05）.The vessel density in both the infarct zone and the border zone of CTs+CSCs-co treated group were significantly higher than the CTs-,MSCs-or CSCs-treated group respectively（P<0.01）.The number of proliferative cardiomyocytes of CTs+CSCs-co treated group,which were PH3⁺/a-SA⁺or Ki67⁺/c Tn I⁺in the infarct zone and the border zone,was significantly higher than the CTs-,MSCs-or CSCs-treated group respectively（P<0.05）.The density of survival CTs and the density of survival CSCs in the infarct zone of the CTs+CSCs-co treated group was also signficantly higher than the CTs-,MSCs-or CSCs-treated group respectively（P<0.05）.（3）Using transmission electron microscopy,unique morphology of CTs and CTs-secreting exosome in rat myocardium were observed.In addition,it was found that CTs exosomes was able to increase the EF（P<0.05）and FS（P<0.05）and decrease LVESD（P<0.01）significantly compared with the PBS control group in MI.The infarction size of CTs exosomes treated group was significantly smaller the PBS group（P<0.05）.The PVCA,CAIZ and myofibroblast density in infarct zone ware significantly decreased compared with PBS group（P<0.01,respectively）.The WTBZ of CTs exosomes treated group was significantly larger than the PBS group（P<0.05）,and the TIM was similar to the PBS group（P>0.05）,and the expression of MMP2 in infarct zone of CTs exosomes treated group was significantly decreased（P<0.05）.The vessel density in the infarct zone of CTs exosomes treated group was significantly larger than the PBS group（P<0.05）,and the vessel density in border zone of CTs exosomes treated group was similar to the PBS group（P>0.05）.The number of proliferative cardiomyocytes in infarct zone and border zone of CTs exosomes treated group,which were Ki67⁺/c Tn I⁺,was significantly larger than the PBS group（P<0.01）.The density of survival CSCs in infarct zone of the CTs exosomes treated group was also significantly larger than the PBS group（P<0.01）.Furthermore,it was found that CTs exosome was able to be taken up by CSCs and significantly decreased apoptosis and increased cell viability of CSCs in ischemic and hypoxic environment compared with PBS control group（P<0.05）.（4）It was found that the expression of miR-21-5p in CSCs was significantly up-regulated after co-cultured with CTs for 48h.In 7-day after miR-21-5p agomir injection for MI,the EF and FS was significantly increased respectively（P<0.05）,the LVESD and LVEED was significantly decreased respectively（P<0.05）;and the infarction size was decreased significantly（P<0.05）,the density of proliferative of cardiomyocytes in the infarct and the border zone and survival CSCs in the infarct zone were significantly increased compared with scramble control group（P<0.05）.Overexpression of miR-21-5p in CSCs significantly decreased the percentage of cells undergoing apoptosis in ischemic and hypoxic environment（P<0.05）.Cdip1 was identified as a target gene of miR-21-5p in CSCs by dual-luciferase reporter system.Down-regulation of Cdip1in CSCs by siRNA significantly decreased apoptosis in ischemic and hypoxic environment（P<0.05）.（5）It was found that the EF and FS of the different cell treated groups observed in present study,CTs exosome treated group and miR-21-5p treated group significantly were increased and the infarction size was significantly decreased compared with PBS control group（P<0.05）.The EF and FS of CTs+CSCs-co treated group was the largest and the infarction size was the smallest.（6）The 450 and 454 proteins were identified in normoxia and hypoxia inducing CTs exosomes respectively.IPA analysis revealed the proteins were involved in MI via regulating angiogenesis,heart fibrosis,cell proliferation,cell death and survival.（7）The 1948and 1407 genes of old CTs were found to be up-regulated and down-regulated respectively compared with young CTs via microarray analysis.IPA analysis revealed that cardiovascular system development and cardiovascular disease was highly correlated with both the differential genes and the differential genes whose fold changes were greater than five.Conclusions:（1）CTs therapy exerted greater protective effects on the infarct size,myocardial angiogenic and anti-fibrotic effects for MI than BMMSCs and CSCs.（2）The therapeutic effect of CTs+CSCs-co treated group for MI was superior to the CT,CSC and BMMSC treatments alone.（3）CT exosomes transplanted therapy for MI was able to decrease the infarction size and heart fibrosis,improve the cardiac function and reconstruction of LV,increased angiogenesis,myocyte proliferation and survival of CSCs.（4）miR-21-5p transplanted therapy for MI was able to decrease the infarction size,improve the cardiac function,increased myocyte proliferation and survival of CSCs.One of the molecular mechanisms of miR-21-5p mediated protection for CSCs under ischemic and hypoxic environment was down-regulation of the expression of pro-apoptotic gene Cdip1.（5）The proteins from CT exosomes might play an important role in regeneration of MI.（6）Some of the genes which are expressed in difference profile between young and old CTs might play an important role for heart aging,cardiac phathology and regeneration.