GATA-4Enhances MSCs Mediated Cardioprotection In Acute Myocardial Infarction And Mechanism Investigation

Objective:The purpose of this study was to observe the influence of zinc finger transcriptionfactor GATA-4over-expressed mesenchymal stem cells(MSCs) on ischemic myocardiumpost myocardial infarction and to illustrate the relationship between GATA-4, miR-195andBcl-w and apoptosis as well as the possible mechanism.Methods:1. MSCs were isolated and cultured from bone marrow of the Spraque-Dawley(SD)rat.2. the GATA-4gene CDS sequence was copied and insert into the pLVX-IRES-ZsGreen1vector and the recombinant plasmid of pLVX-IRES-ZsGreen1-GATA-4wastransduced into the HEK293T cells using Lenti-X Lentiviral Expression Systems.After24h, supernatant contain the virus was filtered and incubated with MSCs for12h in the presence of10μg/mL polybrene. Stable transduced clones were obtained bypuromycin selection (3μg/mL for5days).3. GATA-4over-expression was verified by immunostaining, RT-PCR and western blot.Negative control MSCs were transduced with pLVX-IRES-ZsGreen1(MSC^Null).4. Cardiomyocytes(CM) which was isolated and cultured from neonatal Spraque-Dawley(SD)rat(aged2-5d),was co-cultured with MSCs on a transwell collagen-coatedmembrane inserts。5. Neonatal CMs were divided into five groups: normoxial control, hypoxia control,co-culture with MSCs group, co-culture with MSC^Nullgroup and co-culture withMSC^GATA-4group。 Apoptotic cells were labeled with annexin V-PE and examined under fluorescence microscope and flow cytometer followinghypoxia(4%CO2+95%N2) for24hrs. Cell injury estimated by LDH release from CMswas measured after hypoxia for36hrs.6. The myocardial infarction SD rats(aged2-3months) modeled by LAD permanentligation were divided into five groups:sham group, MSC^basgroup,MSC^Nullgroup andMSC^GATA-4group.50ul medium,106cells of MSCs, MSC^Nulland MSC^GATA-4in50ulIMDM medium were injected into ischemic border of the infarction area respectively.7. The hearts were harvested and sliced into0.15cm sections from the short axis whenMSCs were transplanted for1week. Myocardial infarction area(MIA) was measuredafter slices were stained by TTC. The hearts were stopped at the diastole stage andfixed in4%paraformaldhyde after MSCs transplantation for4weeks. Hearts wereembedded in paraffin and sectioned (5μm thick) for Masson's TrichromeStaining.the MIA estimated by fibrous area was measured by Image pro plus6.0software.8. Microarray analysis of miRNA isolated from all kinds of MSCs found expression ofmiR-195were depressed in MSC^GATA-4cells.9. over-express miR-195by transduce the miR-195pre-microRNA recombinated lentivirusvector into MSCs. GATA-4expression were silenced by transducing GATA-4-siRNAinto MSCs.10. GATA-4,Bcl-w protein and miRNA levers in MSCs,MSC^Nullgroup,MSC^GATA-4group,GATA-4-siRNA group and miR-195group were measure by western blot and halfquantitative RT-PCR as well as miR-195lever in MSC group,MSC^GATA-4group andmiR-195group.11. A luciferase reporter plasmids containing Bcl-w3′UTR were constructed andco-transfected HEK293T cells with miR-195pre-microRNA recombinated lentivirusvector. The ration of fire luciferase/renilla luciferase activity was measured andanalyzed to determine whether miR-195was combined with3′UTR sites of Bcl-wmRNA and regulated the expression of Bcl-w. Results:1. The recombinated GATA-4lentivirus vector was constructed successfully, Stable MSCsclones with GATA-4over-expressed were obtained by puromycin selection.2. Treating hypoxia for24hrs,the percentage of CMs apoptosis reached33.7±1.92%,ismuch higher than that of normoxia(8.4±1.1%)。The percentage of apoptosis wasdecreased when co-culture with MSCs (25.40±2.1%)and MSC^Null(26.6±1.5%)(p<0.05vs hypoxia group), Whereas there was no significant difference between thetwo groups。When co-culture with MSC^GATA-4cells,the apoptosis percentage wasdramatically decreased to19.7±1.4%(p<0.05vs MSC^Nullgroup)。3. After hypoxia for36hrs,Cell injury estimated by LDH release from CMs wassignificantly increased. In hypoxia group, it was420.82±20.55%folds of that innormoxia. The ratio changed to320.00±22.55%and330.62±26.30%(p<0.05vsnormoxial group) when co-culture with MSCs or MSC^Null, and250.87±20.39%whenco-cultured with MSC^GATA-4(p<0.05vs MSC^Nullgroup).4. when MSCs transplanted for1week, the none-ischemic areas of all sections of themyocardial infarction hearts were stained into red with TTC staining while theischemic area into pale. The MIA was calculated according to the measurement of allthe ischemic area. As a result, the MIA of MSC^basgroup(16.38±2.51%) and MSC^Nullgroup(15.45±1.81%) were reduced(n=4, p<0.05vs Medium group22.06±3.06%). TheMIA of MSC^GATA-4group was reduced to9.52±1.98%进一步的(p<0.05vsMSC^Basand MSC^Null).5. when MSCs transplanted for4week, the infarction area has been replaced by fibrousscar tissue. The collagen fibers was stained blue by Masson's Trichrome staining. Thefibrous area of MSC^basgroup and MSC^Nullgroup were reduced(n=4, p<0.05vsMedium group). The fibrous area of MSC^GATA-4group was significantlyreduced(p<0.05vs MSC^Basand MSC^Null).6. miR-195expression in MSC^GATA-4was0.58±0.14times of that in MSC^Null(p<0.05)inmicroarray scanning, and0.52±0.09times in RT-PCR (p<0.05vs MSC^Null).whenGATA-4expression was silenced by GATA-4-siRNA, the expression of miR-195 increased to2.36±0.24times of that in MSC^Null(p<0.05).7. Bcl-w expression in GATA-4over-expressioned MSCs(MSC^GATA-4) increased, and wasreversed when GATA-4gene was silenced by GATA-4-siRNA. When hypoxia for24hrs, Bcl-w protein lever was equal in MSC and MSC^Nullgroup(p>0.05), butincreased dramatically in MSC^GATA-4group(p<0.05vs MSC and MSC^Nullgroup).When GATA-4gene was silenced, both expression of Bcl-w protein and mRNA weresignificantly decreased(p<0.05vs MSC^GATA-4).8. when MSC^GATA-4was transduced with miR-195pre-microRNA recombinated lentivirusvector, miR-195was over-expressed in MSC^GATA-4cells, followed by Bcl-w proteinlever decreasing(p<0.05vs MSC^GATA-4).9. A luciferase reporter plasmids containing Bcl-w3′UTR were constructed successfully.When co-transfected HEK293T cells with miR-195pre-microRNA recombinatedlentivirus vector, the luciferase activity of Bcl-w3′UTR reporter was depressed, whilethe empty vector as control failed. It was proofed that miR-195was combined with3′UTR sites of Bcl-w mRNA and regulated the expression of Bcl-w.Conclusions:1. The recombinated GATA-4lentivirus vector was constructed successfully, Stable MSCsclones with GATA-4over-expressed were obtained by puromycin selection.2. GATA-4enhanced MSCs mediated cardioprotection in hypoxia condition in vitro,decreased cardiomyocytes apoptosis after hypoxia for24hrs and ischemic injury afterhypoxia for36hrs.3. GATA-4enhanced MSCs mediated cardioprotection in Acute Myocardial Infarctionheart in vivo, decreased ischemic area and fibrous area.4. miR-195combines with3′UTR sites of Bcl-w mRNA and depresses the expression ofBcl-w. GATA-4elevates Bcl-w expression and enhances anti-apoptosis function ofMSCs in ischemic heart post MI by decreasing miR-195expression...