Mitochondria-specific transgenic overexpression of connexin-43 simulates preconditioning-induced cytoprotection of stem cells

Gang Lu, Husnain Kh Haider, Aleksey Porollo, Muhammad Ashraf

Research output: Contribution to journalArticle

42 Scopus citations

Abstract

Aims We previously reported that preconditioning of stem cells with insulin-like growth factor-1 (IGF-1) translocated connexin-43 (Cx-43) into mitochondria, causing cytoprotection. We posit that these preconditioning effects could be simulated by mitochondria-specific overexpression of Cx-43. Methods and resultsDuring IGF-1-induced preconditioning of C57black/6 mouse bone marrow stem cell antigen-1+ (Sca-1+) cells, Cx-43 was mainly translocated onto the mitochondrial inner membrane, which was abrogated by an extracellular signal-regulated kinases 1 and 2 (ERK1/2) blocker PD98059. To investigate the role of mitochondrial Cx-43, we successfully designed a vector coding for full-length mouse Cx-43 with a mitochondria-targeting sequence (mito-Cx-43) and cloned into a shuttle vector (pShuttle-IRES-hrGFP-1) for mitochondria-specific overexpression of Cx-43 (mito-Cx-43). Sca-1+ cells with mito-Cx-43 reduced cytosolic accumulation of cytochrome c, lowered caspase-3 activity, and improved survival during index oxygen-glucose deprivation as determined by terminal deoxynucleotidyl transferase dUTP nick-end labelling and lactate dehydrogenase assays. Computational analysis revealed a B-cell lymphoma-2 (Bcl-2) homology domain-3 (BH3) motif in Cx-43 with a conserved pattern of amino acids consistent with the Bcl-2 family that regulated cytochrome c release. Moreover, computational secondary structure prediction indicated an extended-helix in this region, a known condition for BH3-driven protein-protein interactions.Conclusion Cx-43 translocation into mitochondria during preconditioning was ERK1/2-dependent. Expression of mito-Cx-43 simulated the cytoprotective effects of preconditioning in stem cells. Structural features of Cx-43 were shared with the Bcl-2 family as determined by computational analysis.

Original languageEnglish (US)
Pages (from-to)277-286
Number of pages10
JournalCardiovascular Research
Volume88
Issue number2
DOIs
StatePublished - Nov 1 2010
Externally publishedYes

    Fingerprint

Keywords

  • Apoptosis
  • Connexin-43
  • Heart
  • IGF-1
  • Mitochondria
  • Sca-1

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Cite this