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

Gang Lu, Shujia Jiang, Muhammad Ashraf, Khawaja Husnain Haider

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)323-334
Number of pages12
JournalRegenerative Medicine
Volume7
Issue number3
DOIs
StatePublished - May 1 2012

Fingerprint

Connexin 43
Mitochondria
Gene Targeting
Stem cells
Stem Cells
Genes
Heart Ventricles
Rats
Proteins
Cytochromes c
Labeling
Glucose
Bone
Fluorescence
Oxygen
DNA Nucleotidylexotransferase
Heart Transplantation
Cell- and Tissue-Based Therapy
Transgenes
Caspase 3

Keywords

  • survival

ASJC Scopus subject areas

  • Biomedical Engineering
  • Embryology

Cite this

Subcellular preconditioning of stem cells : Mito-Cx43 gene targeting is cytoprotective via shift of mitochondrial Bak and Bcl-xL balance. / Lu, Gang; Jiang, Shujia; Ashraf, Muhammad; Haider, Khawaja Husnain.

In: Regenerative Medicine, Vol. 7, No. 3, 01.05.2012, p. 323-334.

Research output: Contribution to journalArticle

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abstract = "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.",
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N2 - 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.

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