The Experimental Study Of S100A1 Gene Combining Mesenchymal Stem Cells Transplantation For Chronic Heart Failure In Rats

Background and Objectives Chronic heart failure is an important cause of coronary heart disease death.Myocardiocyte reduction and molecular defect are crucial in chronic heart failure(CHF).Mesenchymal stem cells(MSCs) has the potential to regenerate and mutidifferentiate into myocardiocyte like cells.Evidence suggests that transplanting MSCs into the failing heart can prevent deleterious remodeling and improve recovery.But the stem cells transplantation does not solve the molecular defect.Ca²⁺ carrying defect is an important aspect of molecular defect.Increasing sarcoplasmic reticulum Ca²⁺-ATPase(SERCA2a) expression can improve the systolic function but impaired survival in response to ischemic cardiac injury under clinical conditions.S100A1 protin is a Ca²⁺-depending inotropic factor in heart,interacts both with the SR(sarcoplasmic reticulum,SR) Ca²⁺ release channel(RyR2) and the SERCA2a in a Ca²⁺ dependent manner,resulting in enhanced Ca²⁺ induced SR Ca²⁺ release and SR Ca²⁺ reuptake,respectively.In addition,S100A1 can decrease the diastolic SR Ca²⁺ leak through attenuated Ca²⁺ spark frequency,suggesting a biphasic Ca²⁺ dependent modulation of the RyR2 through interaction with S100A1.Evidence suggest that S100A1 protin expression decreased in failuring heart and increasing S100A1 protin expression in the failing heart can improve cardic function and inhibite left ventricular remodeling and does not increase arrhythymal rate.Wether S100A1 gene combing MSCs transplantation for CHF can improve the cardic function more,there is no report.In order to inquire this question,we study the cardic fucion in rat CHF model by echocardiography in the control group,MSCs group,rAAV1/2-S100A1 group,MSCs and rAAV1/2-S100A1 group.Methods Rat bone marrow-derived MSCs were harvested from 6-to 8-week-old(150-80g) male Sprague-Dawley rats.After the bone marrow plugs were flushed out from tibial and femoral bones,the whole marrow samples were collected and cultured with Dulbecco's modified Eagle's medium-low glucose(L-DMEM).After identification the specific surface antigens CD34 and CD44 by immunohistochemistry,the third passage MSCs were incubated with superparamagnetic iron oxide(SPIO) for 24hours.The labeling efficiency was tested through Prussian blue staining,the cells viability was tested through Trypan blue rejection method.After that,fifty SD rats were randomized into experiment and sham-operated groups.After being anesthetized and artificially ventilated,a thoracotomy was performed through the fourth left intercostal space.Cryoinfarction was produced by applying steal cryoprobes of 6mm in diameter which were immersed in liquid nitrogen for 10 min to the anterior left ventricular free wall for 3 times.The during time was 15s,15s,10s respectively.By electrocardiogram(ECG),echocardiography, hematoxylin-eosin(H & E) staining and triphenyltetrazolium chloride(TTC) staining to evaluation the infarct size and heart function.The survival rate during operation and in 4 weeks after operation was observed.Then the CHF rats were devided four groups:control group,MSCs group, rAAV1/2-S100A1 group,MSCs and rAAV1/2-S100A1 group.MSCs were labeled with superparamagnetic iron oxide(SPIO).After performed thoracotomy,0.9%sodium chloride solution, mesenchymal stem cells,recombinant adeno-associated viral 1/2 vector carrying S100A1 gene,the mixtures of mesenchymal stem cells and recombinant adeno-associated viral 1/2 vector carrying S100A1 gene were respectively injected into intramyocardial of heart failure rats in different groups.Then at 4 weeks after treament,we evaluate the cardiac function by echocardiography, identify the transplanted mesenchymal stem cells through Prussian blue staining.Results Most cells were spindle-like in shape and adhered to the plastic tissue culture dishes. Immunohistochemistry showed that 96%cells express CD44 but no cells express CD34. After incubated with SPIO(superparamagnetic iron oxide),the labeling efficiency and the cells viability is 90%,98%,respectively.Infarct evidences were found in 35 of 40 experiment rats which survive over 4 weeks after operation.The infarct size was 35.62±3.74%in all experiment rats.The left ventricular ejection fraction(LVEF) was 46.55±6.80%,the fractional shortening(FS) was 20.61±3.61%.Transmural cryoinfarcted and distinct infarct boundary were observed by the H & E staining and triphenyltetrazolium chloride staining.No infarction was observed in sham-operation group.The heart function decreased significantly in the experiment rats than the sham-operated group(P＜0.01). The survival rate after 4 weeks was 87.5%.4 weeks afert treatment,the MSCs group,MSCs and rAAV1/2-S100A1 group were found Prussian blue staining positive cells;no positive cells were found in the control group.In the MSCs and rAAV1/2-S100A1 group,rAAV1/2-S100A1 group,MSCs group,the left ventricular ejection fraction increased 15.41±6.75%(P＜0.01),7.85±2.96%(P＜0.01),5.63±2.60%(P＜0.01) than before treatmeat respectively.And the fractional shortening in the MSCs and rAAV1/2-S100A1 group,rAAV1/2-S100A1 group,MSCs group increasrd 6.17±3.46%(P＜0.01),3.02±1.25%(P＜0.01),1.58±0.59%than before treatment respectively.The left ventricular end diastolic diameter(LVEDD) in the MSCs and rAAV1/2-S100A1 group,rAAV1/2-S100A1 group decreased 1.87±0.64mm(P＜0.01),1.00±0.62mm(P＜0.05) than before treatment respectively.In contrast to rAAV1/2-S100A1 group or MSCs group,the cardic function improved remarkably in MSCs and rAAV1/2-S100A1 group(P＜0.05).In the control group, left ventricular ejection fraction and fractional shortening decreased 8.44±3.74%(P＜0.01),4.64±1.37%(P＜0.01) respectively.The left ventricular end diastolic diameter increased 0.9±0.14mm(P＜0.01) than before treatment too.Conclusions By adherence the whole marrow samples can culture MSCs,and SPIO can lable MSCs efficiently and safely.Using ultralow temperature cryoinjury may be an ideal method to establish the rat model of acute myocardial infarction.This method can decrease mortality in making a successful rat chronic heart failure model.Increased calcium-binding protein S100A1 expression in chronic heart failure rats by transfer recombinant adeno-associated viral1/2 vector containing calcium-binding protein S100A1 gene can improve cardiac function. Transplant the mixtures of mesenchymal stem cells and rAAV1/2-S100A1 can get better effects than giving rAAV1/2-S100A1 or MSCs only.Increased calcium-binding protein S100A1 expression can inhibit the progress of heart failure.