Abstract 414: Connexin43 in the Inner Mitochondrial Membrane of Cardiomyocytes Forms Hemichannels that Contribute to Matrix Volume Regulation
Previous immunolabelling-based studies have described that Cx43 is present at the inner mitochondrial membrane of cardiomyocytes (mCx43) and that this localization may be important for preconditioning protection, but the quaternary structure and molecular interactions of mCx43 are unknown.
OBJECTIVE: In the present study we tested the hypothesis that mCx43 forms hemichannels that participate in the regulation of mitochondrial volume.
METHODS AND RESULTS: First, in purified mitochondrial preparations from rat myocardium in which contamination by other cell structures had been ruled out by western blot (WB) analysis, unequivocal evidence of the presence of Cx43 was obtained by mass-spectrometry-based proteomic analysis, and WB analysis demonstrated that the membrane-permeant cross-linking agent DMS induced changes in mobility of immunoreactive bands for Cx43 indicative of the presence of Cx43 oligomers. Second, we analysed uptake of the Cx43 hemichannel-permeant dye Lucifer Yellow (LY) by isolated mitochondria incubated (30min) in substrate-containing buffer with LY 1μM. LY uptake was not modified by CsA, but was markedly reduced (p<0.001) by two chemically unrelated blockers of Cx43 hemichannels, heptanol (up to 10μM) or carbenoxolone (up to 1μM). Third, to analyse the potential influence of mCx43 in mitochondrial volume regulation, we used a KI32 mice in which Cx43 had been replaced by Cx32. Western blot studies in myocardium from homozygous animals demonstrated that Cx32 incorporates to the intercalated disk fraction but, in contrast to Cx43, is virtually absent from the mitochondrial fraction. Mitochondrial swelling induced by low K+concentration (light-scattering at 520nm) was much smaller in mitochondria from Cx32/Cx32 animals than in Cx43/Cx43 controls (p<0.0001). The effect of mCx43 on mitochondrial volume regulation was further confirmed in isolated mitochondria obtained from HL-1 cardiomyocytes stably transfected with either a retrovirus containing the coding sequence of Cx43 (resulting in an about 2 fold overexpression) or an empty vector.
CONCLUSION: These results are consistent with the hypothesis that in cardiomyocytes mCx43 forms connexons that contribute to regulation of mitochondrial volume and function.