Researches On Treatment Of Acute Myocardial Infarction Through Transplanting Skeletal Myoblasts Preprocessed By Hypoxia & Infected By HIF-1α Gene In A Rat Model

Myocardial infarction (MI) and heart failure induced by MI are the most important cause of adult death. Recently, transplantation with some kinds of adult stem cells into regional ischemia myocardium shows satisfactory application perspective. However, there are still some problems about transplantation and stem cell remaining unsolved. The examples include how to enlarge the quantity of stem cell in vitro and how to improve the efficacy of transplantation. To find answers for these questions, we chose hypoxia as the way to proliferate skeletal myoblasts (SkMs) in vitro so as to obtain enough transplanting cells. We transplanted SkMs preprocessed by hypoxia into Rat's infarct myocardium and then estimated the heart function and had a discussion on the feasibility and validity of hypoxia as amplification method. In addition, hypoxia inducing factor-1α (HIF-1α ) plays an important role in angiogenesis. We observed the repairing effect with SkMs infected by HIF-1α gene on rat's MI and studied the possible mechanism on the change of the heart function.Firstly, we examined the isolation, culture and identification of SkMs. Wistar rat was executed through dislocation of cervical vertebra and about 1cm³ muscle tissue was acquired from hamstring muscles in asepsis environ. SkMs were isolated and purified from muscle tissue by enzymatic digestion and continuous adherence. Cells were identified by immunohistochemistry with specific marker of SkMs —Desmin. The results show that over 90% cells are Desmin positive. So a stable culture system was established in vitro and we could perform the following experiments with these cells.Secondly, we investigated the effects of hypoxia on the proliferation of SkMs. SkMs were exposed normoxia (20%O₂) or hypoxia (3%, 10%O₂) for 3 days. Wecounted the numbers of Desmin positive cells. The result showed the average numbers were 416+10,633+32 and 1043+67 after a 3-day culture under 20%, 3%, 10%O₂ conditions. The numbers of SkMs cultured in 3%, 10%O₂ were found to be 1.5 and 2.5 times as in normal condition. Low oxygen concentration especially 10%O₂ obviously enhanced the proliferation of SkMs in vitro.To study the possible mechanism of enhancing the proliferation by hypoxia, we chose stable mouse SkM line — C₂C₁₂ cell which showed best the characteristics of SkMs. Cells cultured in 3%, 6%, 10% or 20%O₂ for 24, 48, 72h. The proliferation of C₂C₁₂ cells was investigated by cells counting, BrdU administration and flow cytometric analyses. The expression of HIF-1α mRNA and protein was determined by RT-PCR and Western blot. HIF-1α protein level in endochylema and nucleus measured by Western blot. Results showed that hypoxia enhanced the proliferation of C₂C₁₂ cell, especially 10%O₂. From the results of RT-PCR and Western blot, we were surprised to find that C₂C₁₂ cells expressed HIF-1α mRNA and protein in normoxia and hypoxia (10%O₂). The detection showed no difference between in normoxia and in hypoxia. When the levels of HIF-1α protein in endochylema and nucleus were measured separately by Western blot, however, an interesting phenomenon would be found easily. Under normoxia the levels of HIF-1α protein in endochylema of C₂C₁₂ cells were noticeably higher than those in nucleus, butjhe result truns out to be the opposite under hypoxia. These findings suggest that the happening of nuclear translocation of HIF-1α regulated by the specificity of oxygen concentration would enhance the proliferation of C₂C₁₂ cells under hypoxia. During low oxygen concentration, cytoplasmic HIF-1α was imported into the nucelus, generating a DNA binding complex together with HIF-1β , and upregulated target gene encoding cell's proliferation.Thirdly, we assessed the feasibility and validity of treatment of acute myocardial infarction through transplanting SkMs preprocessed by hypoxia & infected by HIF-1α gene in a rat model. The way to preprocess SKMs withhypoxia was to put SkMs into the incubator with 10%O₂ 48h before transplantation. We infected SkMs with adenovirus vector encoding HIF-1α gene (Ad/HIF-1α ). The rats were randomized into six groups: sham operation group, receiving around-ligation injections of culture medium, skeletal myobalsts alone, skeletal myobalsts preprocessed by hypoxia, skeletal myobalsts infected by an empty vector or skeletal myobalsts infected by adenovirus-encoded HIF-1α . A myocardial infarction was created by means of coronary artery ligation liquation and the transplantation was made in half an hour after infarction confirmed by ECG. One month later, left ventricular function was assessed by left ventricular systolic pressure (LVSP), LV peak velocities of contraction and relaxation (dP/dtmax). Angiogenesis was assessed by immunohistochemistry and reduction of collagen was displayed by Picrosirius red stain. On days 1, 4, 7, 14 and 28 post-transplantation, the expressions of vascular endothelial growth factor (VEGF) and it's receptor (Flk-1) were analyzed by RT-PCR. Results showed that there was no difference on left ventricular function, reduction of collagen and angiogenesis between transplantation with skeletal myobalsts alone and with skeletal myobalsts preprocessed by hypoxia. SkMs modified by HIF-1α implantation obviously improved the function of impaired myocardium, including reduction of collagen I , increasing the number of capillaries compared to other groups (P<0.05) . Hemodynamics assay showed that left ventricular end-diastolic pressure (LVEDP) decreased but LVSP and dP/dtmax remarkably increased, indicating that both systolic and diastolic functions were best preserved in the SkMs modified by HIF-1α group after myocardial infarction. RT-PCR also revealed that mRNA expression levels of VEGF and Flk-1 in SkMs modified by HIF-1α group were significantly higher than other groups. The expression of Flk-1 could still be detected at 14d post-transplantation. These findings indicate that hypoxia can be a safe and convenient way to increase the numbers of SkMs in vitro. Transplantation with SkMs modified by HIF-1α improves post-infarction myocardium function greatly. Induction of angiogenesis by HIF-1α is aneffective means of increasing the functional benefits of myoblast transplantation. Our researches provide experimental foundations for clinical treatment of organ or tissue injury by stem cells modified with certain genes.