The Electrophysiological Characteristics Of Cardiomyocyte-like Cells Derived From Bone Marrow Mesenchymal Stem Cells By Four Inductors

BackgroundIschemic heart disease developed from acute myocardial infarctionremains the one of leading cause for heart failure. However, percutaneouscoronary intervention in early times with antiplatelet and lipid lowering therapycould decrease the mortality rates of transmural MI and reduce the risk of arteryartherosclerosis and coronary artery diseases, the necrosis or impairedcardiomyocytes and heart function degraded still remain as a tough problem inthe research field of cardiovascular disease. The cardiac ventricle reconstitutedand heart function predominance deteriorated in the final stage of heart failure,leading to the occurrence of various cardiovascular incidents. The hearttransplantation could be one effective treatment but is greatly limited by theamounts of donators far less than the clinical demands.Transplantation of bone marrow mesenchymal stem cells into heart failurepatients has preliminary obtained ideal clinical trial results. The BMMSCs could be differentiated into cardiomyocytes by many ways, but the electrophysiologyfunction of the cardiomyoctyes which derived from BMMSCs by different wayshas not been identified clearly. In the wake of investigative thorough, we couldfind an effective inducing method to obtain cardiomyocytes derived fromBMMSCs with favorable function status and accordingly to bring into full playof the therapeutic efficacy.Objectives1. The isolation in vitro, primary culture, passage purificatio n andidentification of BMMSCs from SD rats.2. Compared the differences of cardiomyocytes differentiation rates andcardiac specificity protein expression between groups of5-AZA, Ang-II, PFT-α,BMP-2.3. Compared the electrophysiology function of cardiomyocytes derivedfrom BMMSCs after induction by5-AZA, Ang-II, PFT-α，BMP-2.Methods1. The isolation in vitro, primary culture, passage purification andidentification of BMMSCs from SD rats：Took the long bone of the four limbsof SD rats after sacrificed by cervical dislocation and got the whole bonemarrow cells, and then abandon other cells by density gradient centrifugationand difference speed adherence method. Observed the morphology variationevery day during culturing and passage purifying by inversion phase contrastmicroscope, and identified the purity of BMMSCs by detecting the surfacelabeled antigen of CD29(+), CD44(+) and CD45(-) with the flow cytometry.2. Detected the differences of cardiomyocytes differentiation rates andcardiac specificity protein expression between groups of5-AZA, Ang-II, PFT-α, BMP-2：Passaged and purified the BMMSCs until the fourth generation, anddivided them into five groups as follow:5-AZA group (10μmol/L),Ang-Ⅱgroup (0.1μmol/L), PFT-α group (20μmol/L), BMP-2group (10μg/L)and control group (Control). Every experiment group change for the normalcomplete medium after inducing24h, and continue culture for4w. Observedthe morphology variation with inverted microscope and determine the cardiacdifferentiation rate with flow cytometry, the protein of connexin43(Cx43) andcardiac Troponin I (cTn I) by Western Blot, the cardiac Troponin T (cTn T) byimmunofluorescence.3. Detected the electrophysiology function of cardiomyocytes derived fromBMMSCs after inducing by5-AZA、Ang-II、PFT-α and BMP-2: Collected thefourth generation BMMSCs and induced them by different inducers for4w,then determine the fluorescence expression level which reflecting the calciumtransient function of cells by laser scanning confocal microscope and measurethe level of total potassium current of each groups with patch clamp.Results1. The morphology and identification of primary culture and passagepurification of BMMSCs from SD rats in vitro：The primary cells formedirregular astro or short fusiform shape, and tubercle of cells connected with eachother. After7days culture, primary BMMSCs become long spindle shape andproliferated rapidly and formed colonies.3-4generation passaged, cells volumegrew larger than primary cells and grew arranged trend to coincidence. The flowcytometry determined the surface specific antigen CD44and CD29of BMMSCs,and the negative antigen CD45, too. The positive expression rate of CD44is(91.4±1.2)%and CD29is (89.6±2.7)%， and the CD45expressed only(1.8±1.3)%of BMMSCs. 2. The differences of cardiomyocytes differentiation rates and cardiacspecificity protein expression between different groups：The flow cytometrydetected the cardiac differentiation rate of each group and results as follows:5-AZA group is (23.7±2.1)%; Ang-II group is (23.5±1.6)%; BMP-2group is(17.5±2.3)%; PFT-α group is (28.9±0.9)%, and the control group is (1.5±1.4)%.The differentiation rate had no significant differences between5-AZA group andAng-II group (P>0.05), but the rates between other groups certainly hadsignificant differences (P<0.05). The immunofluorescence staining determinedthe expression of cTn T protein of each groups, and the results showed thatweak expression have found after1w of induction, and strong expression after4w of induction in every experiment groups, and the control group only havesprinkle expression after cultured for1w and4w. Western Blot had shown thateach group expressed the protein of Cx43after4w of induction; the controlgroup expression significantly lower than other groups (P<0.01); the5-AZAgroup expressed significantly more than other groups (P<0.05) and the group ofPFT-α expressed more than BMP-2and Ang-II group (P<0.05); the Ang-IIgroup significantly expressed less than other experiment groups (P<0.05).Simultaneously all experiment groups expressed protein of cTn I after4w ofinduction; the PFT-α group expressed significantly more than other groups(P<0.05) and the group of5-AZA and Ang-II had no difference with each other(P>0.05); the BMP-2group significantly expressed less than other experimentgroups (P<0.05); the control group expression significantly lower than othergroups (P<0.01).3. The electrophysiology function of cardiomyocytes derived from BMMSCsafter inducing by5-AZA, Ang-II, PFT-α and BMP-2: Laser scanning confocalmicroscope detected the fluorescence intensity of flou3, and results showed that in5-AZA group is (1686.177±499.122); Ang-II group is (2627.666±407.329);PFT-α group is (2156.606±385.355); BMP-2group is (1301.910±338.681), andthe control group is (824.928±222.399). The intensity of fluorescence is couldbe arranged as follows: Ang-II> PFT-α>5-AZA> BMP-2> Control (P<0.01).The total potassium channel current density level was detected by patch clamp.The steady state current level (pA/pF) of+70mv in5-AZA group is(24.931±1.421); Ang-II group is (25.134±1.481); PFT-α group is (27.102±1.321);BMP-2group is (20.485±1.236); Control group is (18.053±1.241). The PFT-αgroup expressed significantly more(P<0.01, beside of PFT-α group vs. ANG-IIgroup(P<0.05). And the group of5-AZA and Ang-II had no difference with eachother (P>0.05); the BMP-2group significantly expressed less than otherexperiment groups (P<0.01); the control group expression significantly lowerthan other groups (P<0.01).Conclution1. Primary BMMSCs could be purified by density gradient centrifugationand differential adhesion rate, and the purity of primary BMMSCs could beidentification by surface specific antigen of CD29(+), CD44(+) and CD45(-).2. BMMSCs could differentiate into cardiomyocyte-like cells after inducedby5-AZA (10μmol/L), BMP-2(10μg/L), PFT-α (20μmol/L) and Ang-II (0.1μmol/L) respectively, and the differentiate rate highest in PFT-α group (P<0.05),5-AZA group had no significant differences with Ang-II (P>0.05), BMP-2grouphad the lowest rate than other experiment groups (P<0.05).3. The intensity of fluorescence is could be arranged as follows: Ang-II>PFT-α>5-AZA> BMP-2> Control. The reason may be that different inductorsimpact different metabolism pathway of cells, and cause diversity of cellfunction. 4. The current destity of PFT-α group expressed highest, and the group of5-AZA and Ang-II secondly; the BMP-2group significantly expressed less thanother experiment groups. All experiment groups expressed higher currentdensity than control group, which indicating that the number of differentiationinto maturity available patency potassium channels increased and thus enhancedthe current density.