Experimental Study On The Induction Of Fibroblasts Into "Cardiac-like Myocytes" By Transfection Of Both MyoD And Cx43 Genes And Its Effects After Transplantation Into Postinfarcted Myocardium In Rats

Backgrounds:The dogma of the heart as an organ composed of terminally differentiated myocytes incapable of regeneration, which endogenous repair mechanisms are overwhelmed in patients with acute myocardial infarction (AMI), advanced coronary artery disease, and chronic heart failure, is being challenged. Cellular cardiomyo-plasty (CCM) by cell transplantation is a promising strategy that offers the creation of new functional tissue to replace lost or failing myocardium. A major barrier for the clinical application of cell transplantation is obtaining sufficiently large quantities of suitable cells. Several cell sources have been tested, although each of them entails considerable limitations. The myogenic determination (MyoD, a skeletal myogenic determination factor) as unique master genes that are able to prompt myogenesis in a variety of cells including fibroblasts. Transformation of cardiac fibroblasts in situ into skeletal myocytes in myocardial infarct scar tissue could add to postinfarction adaptation. However, attempts to convert the infarcted myocardium into contractile tissue in situ by direct injection of viral vectors encoding the MyoD required a high dose of recombinant adenovirus, and yielded disappointing results. The essential physiological properties of between cardiomyocytes and grafted cells supporting efficient myocardial impulse propagation are an excitable cell membrane and capacity for gap junctional intercellular communication (GJIC), and the capacity for GJIC through expression of connexin43 (Cx43), a gap junction protein. Evidence has been presented that fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling, and the dermal fibroblast (DFs) from skin was an alternative source of autologous cells. Ex vivo cellular cardiomyoplasty is an alternative myocardial repair strategy that combines the advances in genetic and tissue engineering. This approach may provide an alternative source of autologous, transplantable, myogenic cells by ex vivo genetic manipulation of the fibroblasts. The aim of our study was, therefore, to address the issue of whether the phenomenon of myogenic conversion of autologous DFs with both MyoD and Cx43 genes could be exploited in practical terms to provide an alternative source of myogenic cells for transplantation.Objective:The purpose of our study was to determine whether the phenotype of both MyoD and Cx43 genes could induce autologous DFs to produce myoblasts capable of gap junction coupling -- "cardiac-like myocytes" ex vivo, and the influence of the converted cells on myocardial structure and cardiac function after being implantated into acute infarcted myocardial site in rats in a clinically relevant approach.Methods:(1) Explore the biological characteristics of DFs isolated and cultured from the skin dermal tissue of Sprague-Dawley (SD) rats by digesting with collagenase type II ex vivo;(2) Construct efficient eukaryotic expression plasmid lentiviral vector pLenti6/v5-DEST-MyoD and pLenti6/v5-DEST-Cx43 encoding exogenetic rat MyoD cDNA or Cx43 cDNA respectively, which were identified with the agarose electrophoresis and sequent examination;(3) The cultured rat DFs were transfected with the recombinant eukaryotic expression lentiviral vector with lipotransfection method according to lentiviral expression system protocol;(4) The mophological and ultrastructure changes of these transfected cells by MyoD &/or Cx43 genes ex vivo were observed by microscopy, and the expression of MyoD and Cx43 genes in the transfected cells was detected by immunocyto-chemical methods;(5) Rat AMI models were established by ligating the middle-proximal 1/3 segment of left anterior descending coronary artery (LAD);(6) The converted cells (2.0×10~6) were autologous transplanted into the infarcted myocardium four weeks after coronary artery occlusion;(7) The changes of electrocardiogram (ECG) was observed by multiple peripheral adapter (MPA) physiologic recorder system, and plasma B-type natriuretic peptide (BNP) concentration were measured during perioperation of cell transplantation;(8) Four weeks later after cell transplantation, cardiac function of left ventricular were evaluated by Langendorff perfusion of the isolated heart instrument with MPA physiologic recorder system, and myocardial infarction size (MIS) were be detected by 2,3,5-triphenyltetrazolium chloride (TTC) method;(9) The bromodeoxyuridine (BrdU)-labeled immunohistochemistry was used to observe grafted cells differentiation and development in vivo, and connexin43 immunohistochemistry studies were performed to assess the function of these implantated cells;(10) Ultrastructure and typical cross gap junction of the grafted cells in infarcted myocardium were characterized by transmission electron microscopy.Results:(1) The model of rat DFs culture was successfully established with high purity(> 95%) ex vivo, and immunostaining of vimentin was positive in cultured DFs;(2) The MyoD cDNA or Cx43 cDNA were cloned into Lentiviral vector respectively, and the cDNA sequencing results of pLenti6/v5-DEST-MyoD and pLenti6/v5- DEST-Cx43 were identified with the agarose electrophoresis and sequent examination;(3) These transfected cells were detected out the expression of MyoD and Cx43 genes by RT-PCR and Western blot;(4) The efficiented transduced DFs with the MyoD and Cx43-encoding vector underwent myogenic conversion, as evidenced by the positive immunostaining of immunocytochemical methods for desmin and alpha-actin, and detection of typical cross gap junction coupling in these adjacent converted DFs by electron microscopy;(5) A standard rat AMI model, which MIS was invariableness could be created by surgical ligation of LAD;(6) By four weeks after the converted DFs by MyoD and Cx43 genes myocardial transplantation, the MIS and BNP in accepted myocardial transplantation group were lower than that in other control groups, and the dysfunctional ECG and cardial function also improved in the same myocardial transplantation group;(7) Histological study showed that grafted cells in infarcted myocardium were detected in all host hearts and identified by BrdU-labeled immunocytochemistry staining in the trial groups, and the gap junctions protein could be detected by connexin43 immunocytochemistry;(8) After myocardial transplantation, DFs converted by both MyoD and Cx43 genes could be confluenced cardiac myocytes in original myocardium tissue, which gap junctional coupling emerged, were observed by electron microscopy.Conclusions:(1) DFs can be genetically modified by both MyoD and Cx43 genes to differentiate into "cardiac-like myocytes" capable of gap junction coupling ex vivo and to create a vast source of autologous myogenic cells for CCM;(2) The DFs converted by both MyoD and Cx43 genes implantation can survive in the hosts' myocardial scar tissue, and be believed to attenuate the ventricular remodeling process and improve heart function significantly after auto-transplantation in AMI rats, which is a safe and has comprehensive curative effective method.