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Updated: Apr 17, 2026
Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells
Published on: March 16, 2017
Interaction between connexin 43 and nitric oxide synthase in mice heart mitochondria
Mücella Kirca1, Petra Kleinbongard, Daniel Soetkamp
1Physiologisches Institut, Justus-Liebig-Universität, Giessen, Germany; Institute for Pathophysiology, West German Heart and Vascular Center, University Schhool of Medicine Essen, Essen, Germany.
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 (Cx43(Cre-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 Cx43(Cre-ER(T)/fl) mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43(Cre-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.
Insights
Reduced connexin 43 (Cx43) in heart mitochondria alters nitric oxide synthase (NOS) expression and lowers nitric oxide production. This study reveals a novel link between Cx43, mitochondrial NOS isoforms, and cardiac function.
Area of Science:
- Cardiovascular Biology
- Mitochondrial Medicine
- Molecular Cardiology
Background:
- Connexin 43 (Cx43) and nitric oxide synthase (NOS) isoforms have a complex interplay in the heart.
- Cardiac mitochondria are known to contain NOS, but their specific roles and regulation are not fully understood.
Purpose of the Study:
- To investigate the expression of NOS isoforms and nitric oxide production rates in isolated cardiac mitochondria from wild-type and Cx43-deficient mice.
- To elucidate the relationship between Cx43 content, mitochondrial NOS expression, and nitric oxide formation.
Main Methods:
- Isolation and purification of cardiac mitochondria from wild-type and Cx43-deficient mice using differential centrifugation and Percoll gradient ultracentrifugation.
- Immunofluorescence staining for mitochondrial marker ANT and NOS isoforms (nNOS, iNOS) followed by confocal microscopy.
- Quantification of nitric oxide formation using the oxyhaemoglobin assay.
Main Results:
- Predominant co-localization of neuronal NOS (nNOS) with the mitochondrial marker ANT in wild-type mice.
- Increased inducible NOS (iNOS) expression and decreased nNOS expression in mitochondria from Cx43-deficient mice.
- Significantly reduced mitochondrial nitric oxide formation in Cx43-deficient mice compared to wild-type controls.
Conclusions:
- Reduced mitochondrial Cx43 content is associated with a shift in mitochondrial NOS isoform expression.
- This switch in NOS isoforms leads to a decreased rate of mitochondrial nitric oxide formation.
- These findings highlight a novel regulatory mechanism of mitochondrial nitric oxide production involving Cx43.
