Subcellular preconditioning of stem cells: mito-Cx43 gene targeting is cytoprotective via shift of mitochondrial Bak and Bcl-xL balance

Aim: To achieve mitochondria-specific expression of connexin-43 (Cx43) transgene for mitochondrial preconditioning in stem cells to improve their survival post-transplantation during heart cell therapy. Methods & results: Cx43- or GFP-encoding adenoviral vectors with a mitochondrial targeting sequence were constructed for transduction of bone marrow Sca-1(+) cells (>90% transduction efficiency). Double-fluorescence immunostaining for cytochrome-c and Cx43 supported by western blotting confirmed mitochondria-specific Cx43 expression in adenoviral-mito-Cx43-transduced cells ((Cx43)Sca-1(+)). (Cx43)Sca-1(+) showed improved survival under lethal oxygen glucose deprivation culture conditions. (Cx43)Sca-1(+) showed an increased mitochondrial Bcl-xL:Bak ratio and reduced cytochrome-c release into cytosol with concomitantly abolished caspase-3 activity. An in vivo study was performed such that 2 x 10(6) male (Cx43)Sca-1(+) or (GFP)Sca-1+ cells were injected into a female rat model of acute myocardial infarction. DMEM-injected rats served as controls. On day 7 post-transplantation, 4.3-fold higher survival of (Cx43)Sca-1(+) cells (p < 0.05 vs control) and reduced terminal deoxynucleotidyl transferase dUTP nick end labeling positivity in the left ventricle (LV) were observed. In comparison, LV ejection fraction (40.2 +/- 0.9%), LV fractional shortening (20.0 +/- 1.6%) and LV end diastolic dimension (6.5 +/- 0.3 mm) were observed in (GFP)Sca-1(+), and treatment with (Cx43)Sca-1(+) cells improved these parameters (47.6 +/- 2.5%, p <0.05; 27.7 +/- 1.2%, p < 0.05; and 5.6 +/- 0.1 mm, p < 0.05, respectively), along with concomitant reductions in infarction size (33.7 +/- 2.9% vs 39.8 +/- 1.4%; p <0.05). Conclusion: Mitochondria-targeted Cx43 expression is a novel approach to improve stem cell survival in the infarcted heart.