title: Subcellular preconditioning of stem cells: mito-Cx43 gene targeting is cytoprotective via shift of mitochondrial Bak and Bcl-xL balance.
abstract: OBJECTIVE
  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.
  
  
  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 × 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).
  
  
  CONCLUSIONS
  Mitochondria-targeted Cx43 expression is a novel approach to improve stem cell survival in the infarcted heart.
authors.affiliation: Department of Pathology & Laboratory Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Lu
authors.forename: Shujia
authors.initials: S
authors.lastname: Jiang
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ashraf
authors.forename: Khawaja Husnain
authors.initials: KH
authors.lastname: Haider
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000950:Antigens, Ly / Q000378:metabolism; D002470:Cell Survival; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism; D019610:Cytoprotection; D005260:Female; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics; D005947:Glucose / Q000172:deficiency; D006334:Heart Function Tests; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism*; D009203:Myocardial Infarction / Q000473:pathology / Q000503:physiopathology / Q000628:therapy; D009206:Myocardium / Q000473:pathology; D010100:Oxygen; D051381:Rats; D011916:Rats, Inbred F344; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism*; D051020:bcl-X Protein / Q000378:metabolism*
chemical_list: D000950:Antigens, Ly; D018031:Connexin 43; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein; D005947:Glucose; D010100:Oxygen