Interaction between Connexin 43 and nitric oxide synthase in mice heart mitochondria

Mücella Kirca, Petra Kleinbongard, Daniel Soetkamp, Jacqueline Heger, C. Csonka, P. Ferdinándy, Rainer Schulz

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Connexin 43 (Cx43), which is highly expressed in the heart and especially in cardiomyocytes, interferes with the expression of nitric oxide synthase (NOS) isoforms. Conversely, Cx43 gene expression is down-regulated by nitric oxide derived from the inducible NOS. Thus, a complex interplay between Cx43 and NOS expression appears to exist. As cardiac mitochondria are supposed to contain a NOS, we now investigated the expression of NOS isoforms and the nitric oxide production rate in isolated mitochondria of wild-type and Cx43-deficient (Cx43Cre-ER(T)/fl) mice hearts. Mitochondria were isolated from hearts using differential centrifugation and purified via Percoll gradient ultracentrifugation. Isolated mitochondria were stained with an antibody against the mitochondrial marker protein adenine-nucleotide-translocator (ANT) in combination with either a neuronal NOS (nNOS) or an inducible NOS (iNOS) antibody and analysed using confocal laser scanning microscopy. The nitric oxide formation was quantified in purified mitochondria using the oxyhaemoglobin assay. Co-localization of predominantly nNOS (nNOS: 93 ± 4.1%; iNOS: 24.6 ± 7.5%) with ANT was detected in isolated mitochondria of wild-type mice. In contrast, iNOS expression was increased in Cx43Cre-ER(T)/fl mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43Cre-ER(T)/fl mitochondria (0.14 ± 0.02 nmol/min./mg protein) in comparison to wild-type mitochondria (0.24 ± 0.02 nmol/min./mg). These are the first data demonstrating, that a reduced mitochondrial Cx43 content is associated with a switch of the mitochondrial NOS isoform and the respective mitochondrial rate of nitric oxide formation.

Original languageEnglish
Pages (from-to)815-825
Number of pages11
JournalJournal of Cellular and Molecular Medicine
Volume19
Issue number4
DOIs
Publication statusPublished - Apr 1 2015

Fingerprint

Heart Mitochondria
Connexin 43
Nitric Oxide Synthase
Mitochondria
Nitric Oxide
Protein Isoforms
Adenine Nucleotides
Nitric Oxide Synthase Type II
Oxyhemoglobins
Antibodies
Mitochondrial Proteins
Ultracentrifugation
Centrifugation
Cardiac Myocytes
Confocal Microscopy
Gene Expression

Keywords

  • Connexin
  • Heart
  • Mitochondria
  • Nitric oxide

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Medicine

Cite this

Interaction between Connexin 43 and nitric oxide synthase in mice heart mitochondria. / Kirca, Mücella; Kleinbongard, Petra; Soetkamp, Daniel; Heger, Jacqueline; Csonka, C.; Ferdinándy, P.; Schulz, Rainer.

In: Journal of Cellular and Molecular Medicine, Vol. 19, No. 4, 01.04.2015, p. 815-825.

Research output: Contribution to journalArticle

Kirca, Mücella ; Kleinbongard, Petra ; Soetkamp, Daniel ; Heger, Jacqueline ; Csonka, C. ; Ferdinándy, P. ; Schulz, Rainer. / Interaction between Connexin 43 and nitric oxide synthase in mice heart mitochondria. In: Journal of Cellular and Molecular Medicine. 2015 ; Vol. 19, No. 4. pp. 815-825.
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AB - Connexin 43 (Cx43), which is highly expressed in the heart and especially in cardiomyocytes, interferes with the expression of nitric oxide synthase (NOS) isoforms. Conversely, Cx43 gene expression is down-regulated by nitric oxide derived from the inducible NOS. Thus, a complex interplay between Cx43 and NOS expression appears to exist. As cardiac mitochondria are supposed to contain a NOS, we now investigated the expression of NOS isoforms and the nitric oxide production rate in isolated mitochondria of wild-type and Cx43-deficient (Cx43Cre-ER(T)/fl) mice hearts. Mitochondria were isolated from hearts using differential centrifugation and purified via Percoll gradient ultracentrifugation. Isolated mitochondria were stained with an antibody against the mitochondrial marker protein adenine-nucleotide-translocator (ANT) in combination with either a neuronal NOS (nNOS) or an inducible NOS (iNOS) antibody and analysed using confocal laser scanning microscopy. The nitric oxide formation was quantified in purified mitochondria using the oxyhaemoglobin assay. Co-localization of predominantly nNOS (nNOS: 93 ± 4.1%; iNOS: 24.6 ± 7.5%) with ANT was detected in isolated mitochondria of wild-type mice. In contrast, iNOS expression was increased in Cx43Cre-ER(T)/fl mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43Cre-ER(T)/fl mitochondria (0.14 ± 0.02 nmol/min./mg protein) in comparison to wild-type mitochondria (0.24 ± 0.02 nmol/min./mg). These are the first data demonstrating, that a reduced mitochondrial Cx43 content is associated with a switch of the mitochondrial NOS isoform and the respective mitochondrial rate of nitric oxide formation.

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