Abstract 3857: Mitochondrial Connexin-43 Shares a Common BH3 Motif With Bcl-2 Family Members to Impart Cytoprotection in Preconditioned Sca-1+ Cells via Regulation of Cytochrome-C Release
Background. We have shown that connexin-43 (Cx43) in preconditioned Sca-1+ cells (PCSca-1+) is cytoprotective and improves their cardiomyogenic differentiation. We investigate the mechanistic participation of Cx-43 in cytoprotection of PCSca-1+.
Methods and Results. Sca-1+ cells preconditioned with 100μM recombinant IGF-1 protein showed improved cell survival under oxygen-glucose deprivation (OGD). Cx43 siRNA in PCSca-1+ induced higher caspase-3 activity under OGD with concomitant loss of preconditioning induced cytoprotection. Confocal imaging of PCSca-1+after immunostaining for Cx43 and cytochrome-c showed predominant mitochondrial translocation of Cx43 in PCSca-1+. Mitochondrial sub-fractionation showed that Cx43 was mainly translocated onto the inner membrane of mitochondria. Cx43 cDNA with a mitochondria targeting sequence (mito-Cx43) was engineered into pCMV/mito/GFP vector for targeted mitochondrial overexpression of Cx43 to study cytoprotective role of mito-Cx43. Sca-1+ overexpressing mito-Cx43 had improved survival under OGD as evidenced by LDH and TUNEL assays. Computational analysis revealed a BH3 motif of Cx43 having a highly conserved pattern of amino acids consistent with Bcl2 family members (Figure-1⇓) which regulate cytochrome-c release. This motif in Cx43 may be sufficient for heterodimerization with Bcl-2 members. More so, the SABLE (http://sable.cchmc.org/) method used for secondary structure prediction showed an extended α-helix at this region, a known condition for BH3-driven protein-protein interactions.
Conclusions. Molecular studies and structural characterization show that Cx43 has structural homology with Bcl-2 family to regulate cytochrome-c release from mitochondria to impart preconditioning induced cytoprotection of stem cells. Figure-1⇓. Left panel: Sequence alignment of BH3 motif with Bcl-2 family. Right panel: Domain organization of Cx43 (top) and ITM2B (bottom). Location of transmembrane domains (TM) was predicted using the MINNOU (http://minnou.cchmc.org/).