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01.03.2009 | Original Contribution

Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria

verfasst von: Kerstin Boengler, Sabine Stahlhofen, Anita van de Sand, Petra Gres, Marisol Ruiz-Meana, David Garcia-Dorado, Gerd Heusch, Rainer Schulz

Erschienen in: Basic Research in Cardiology | Ausgabe 2/2009

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Abstract

Cardiomyocytes contain subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which differ in their respiratory and calcium retention capacity. Connexin 43 (Cx43) is located at the inner membrane of SSM, and Cx43 is involved in the cardioprotection by ischemic preconditioning (IP). The function of Cx43-formed channels is regulated in part by phosphorylation at residues in the carboxy terminus of Cx43. The aim of the present study was (1) to investigate whether Cx43 is also present in IFM, and (2) to characterize its spatial orientation in the inner mitochondrial membrane (IMM). Confirming previous findings, ADP-stimulated respiration was greater in IFM than in SSM from rat ventricles. In preparations from rats and mice not contaminated with sarcolemmal proteins, Cx43 was exclusively detected in SSM, but not in IFM by Western blot analysis (n = 6). SSM were exposed to different proteinase K concentrations to cleave peptide bonds, and Western blot analysis was performed for ATP synthase α (IMM, subunit in the matrix), uncoupling protein 3 (UCP3, IMM, intermembrane space epitope), and manganese superoxide dismutase (MnSOD, matrix). At a proteinase K concentration of 50 μg/ml, immunoreactivities of all the analyzed proteins were completely lost. The use of 5 μg/ml proteinase K resulted in similarly reduced immunoreactivities for Cx43 (19.4 ± 5.8% of untreated mitochondria, n = 6) and UCP3 (23.0 ± 4%, n = 7), whereas the immunoreactivities of ATP synthase α (49.1 ± 6.4%, n = 7) and MnSOD (79.9 ± 17.4%, n = 6) were better preserved, suggesting that the carboxy terminus of Cx43 is directed towards the intermembrane space. The results were confirmed in digitonin-treated mitochondria. Taken together, Cx43 is exclusively localized in SSM, with its carboxy terminus directed towards the intermembrane space. Since loss of mitochondrial Cx43 abolishes IP’s cardioprotection, SSM and IFM apparently differ in their function in the signal transduction of IP.

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Titel: Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria
verfasst von: Kerstin Boengler
Sabine Stahlhofen
Anita van de Sand
Petra Gres
Marisol Ruiz-Meana
David Garcia-Dorado
Gerd Heusch
Rainer Schulz
Publikationsdatum: 01.03.2009
Verlag: D. Steinkopff-Verlag
Erschienen in: Basic Research in Cardiology / Ausgabe 2/2009
Print ISSN: 0300-8428
Elektronische ISSN: 1435-1803
DOI: https://doi.org/10.1007/s00395-009-0007-5

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