Bcl-2 Expression Enhances Myoblast Sheet Transplantation Therapy for Acute Myocardial Infarction

Abstract

Myoblast sheet transplantation is a promising novel treatment modality for heart failure after an ischemic insult. However, low supply of blood and nutrients may compromise sheet survival. The aim of this study was to investigate the effect of mitochondria-protective Bcl-2-modified myoblasts in cell sheet transplantation therapy. In the Bcl-2-expressing rat L6 myoblast sheets (L6-Bcl2), increased expression of myocyte markers and angiogenic mediators was evident compared to wild-type (L6-WT) sheets. The L6-Bcl2 sheets demonstrated significant resistance to apoptotic stimuli, and their differentiation capacity in vitro was increased. We evaluated the therapeutic effect of Bcl-2-modified myoblast sheets in a rat model of acute myocardial infarction (AMI). Sixty-four Wistar rats were divided into four groups. One group underwent AMI (n = 22), another AMI and L6-WT sheet transplantation (n = 17), and a third AMI and L6-Bcl2 sheet transplantation (n = 20). Five rats underwent a sham operation. Echocardiography was performed after 3, 10, and 28 days. Samples for histological analysis were collected at the end of the study. After AMI, the Bcl-2-expressing sheets survived longer on the infarcted myocardium, and significantly improved cardiac function. L6-Bcl2 sheet transplantation reduced myocardial fibrosis and increased vascular density in infarct and border areas. Moreover, the number of c-kit-positive and proliferating cells in the myocardium was increased in the L6-Bcl2 group. In conclusion, Bcl-2 prolongs survival of myoblast sheets, increases production of proangiogenic paracrine mediators, and enhances the therapeutic efficacy of cell sheet transplantation.

Bcl-2 Improves Myoblast Sheet Therapy in Rat Chronic Heart Failure

“…9 The pBabepuro-bcl2 retroviral vector was a kind gift from Dr. Juha Klefströ m, University of Helsinki. 10 Cells were transfected by incubation for 48 h in the presence of retroviral vector and 8 mg/mL polybrene (Sigma-Aldrich).…”

“…9 The L6 rat skeletal myoblast cell line was from the American Type Culture Collection (CRL-1458) with cells at passages 5 to 15 used for experiments. Myoblast cell sheets were formed by plating 6Â10 6 myoblasts on thermoreactive cell culture dishes (CellSeed) with 15% serum and 10 mM ascorbic acid for 16 h. Intact myoblast sheets detached spontaneously from culture dishes at room temperature and were harvested for transplantation.…”

“…Vascular density and number of proliferating cells were assessed with ImageJ (National Institutes of Health, http://rsb.info.nih.gov/ ij) as described. 9 Data were collected separately for infarcted, border, and remote areas. To determine whether the proliferating cells are myocytes, we manually counted the Ki67-tropomyosin double-positive cells from six slides in each group.…”

“…The thickness of a single three-dimensional myoblast sheet was approximately 50 mm or 5 cell layers. 9 Human umbilical vein endothelial cells (HUVECs) were acquired from Clonetics (Lonza) and were cultured in endothelial growth medium (EGM) supplemented with 2% fetal bovine serum, bovine brain extract, epidermal growth factor, hydrocortisone, and antibiotics (all from Clonetics). Cells at passages 2 to 8 were used in the study.…”

“…Moreover, in our recent study we showed that introduction of antiapoptotic bcl2 gene into myoblast sheets increased their production of potent paracrine proangiogenic meditors vascular endothelial growth factor-A (VEGF-A) and placental growth factor (PlGF). 9 Furthermore, we demonstrated that bcl2 gene therapy enhanced the efficacy of myoblast sheet therapy and promoted sheet survival in a rat model of acute MI.…”