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

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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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Acknowledgment

R.S. was the recipient of a grant from the Deutsche Forschungsgemeinschaft (Schu 843/7-1).

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Authors and Affiliations

  1. Institut für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstr. 55, 45122, Essen, Germany

    Kerstin Boengler, Sabine Stahlhofen, Anita van de Sand, Petra Gres, Gerd Heusch & Rainer Schulz

  2. Hospital Vall d’Hebron, Barcelona, Spain

    Marisol Ruiz-Meana & David Garcia-Dorado

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  1. Kerstin Boengler
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  2. Sabine Stahlhofen
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  3. Anita van de Sand
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  4. Petra Gres
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  6. David Garcia-Dorado
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  7. Gerd Heusch
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Correspondence to Rainer Schulz.

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K. Boengler and S. Stahlhofen contributed equally to this work.

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Boengler, K., Stahlhofen, S., van de Sand, A. et al. Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria. Basic Res Cardiol 104, 141–147 (2009). https://doi.org/10.1007/s00395-009-0007-5

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  • DOI: https://doi.org/10.1007/s00395-009-0007-5

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