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 ( ^Cx43Sca-1 ⁺). ^Cx43Sca-1 ⁺ showed improved survival under lethal oxygen-glucose deprivation culture conditions. ^Cx43Sca-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 × 10 ⁶ male ^Cx43Sca-1 ⁺ or ^GFPSca-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 ^Cx43Sca-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 ^GFPSca-1 ⁺, and treatment with ^Cx43Sca-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.