Abstract 15473: Mitochondrial Transgenic Expression of Connexin 43 Confers Cytoprotection to he Stem Cells and Cardiomyocytes in Ischemic Myocardium
Background. Mitochondrial translocation of connexin 43 (Cx43) is an integral part of prosurvival signaling during preconditioning of stem cells. We hypothesized that transgenic expression of mitochondrial Cx43 (mito-Cx43) would simulate prosurvival effects of preconditioning against ischemic injury.
Methods and results. Adenoviral (Ad) vector encoding Cx43 or GFP with mitochondrial targeting sequence were constructed for transduction of bone marrow Sca-1+ cells and neonatal cardiomyocytes (>90% transduction efficiency). Double fluorescence immunostaining for cytochrome c and Cx43 showed mitochondrial expression of Cx43 which was confirmed by Western blot on mitochondrial protein lysates. Cells expressing mito-Cx43 showed improved survival under lethal oxygen and glucose deprivation (OGD) as determined by LDH release and TUNEL assay. Mitochondrial fraction of mito-Cx43 transduced cells showed reduced cytochrome c release into cytosol with concomitantly reduced caspase-3 activity and mitochondrial Bak levels. In vivo study was carried out in 2 sets. In first set, 2x106 mito-Cx43 or mito-GFP transduced male Sca-1+ cells were injected in a female rat model of acute myocardial infarction (n=6/group). Sry gene analysis at 7 days post-transplantation showed 4.3 fold higher survival in mito-Cx43 Sca-1+ cells as compared to mito-GFP Sca-1+ cells. Four weeks later, mito-Cx43 Sca-1+ cells transplanted animals showed improved heart function (EF: 47.6% vs 40.2%, p<0.05; FS: 27.7% vs 20.0%, p<0.05). In second set of experiments, Ad-mito-Cx43 or Ad-mito-GFP virus (1x109 particles/ 50μl) were directly injected into infarcted heart of young females rats (n=6/ group) and the heart tissue samples were harvested on day 7 post-surgery. Mitochondrial fractionation of left ventricle tissue showed significantly higher Cx43 level in Ad mito-Cx43 treated animals. Protein lysates from left ventricle showed reduced active caspase-3 determined by westernblot in Ad mito-Cx43 treated animals.
Conclusions. Transgenic mito-Cx43 conferred cytoprotection of stem cells and cardiomyocytes via reducing mitochondrial Bak protein levels and cytochrome c release into cytosol. Ex vivo as well as direct targeting of mtio-Cx43 improved infarcted heart function.
- © 2011 by American Heart Association, Inc.