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A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Published on: July 13, 2018
Inhibiting nucleolin reduces inflammation induced by mitochondrial DNA in cardiomyocytes exposed to hypoxia and
Lars Henrik Mariero1,2, May-Kristin Torp1,2, Christina Mathisen Heiestad1,2
1Department of Molecular Medicine, Division of Physiology, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway.
Background And Purpose:
Cellular debris causes sterile inflammation after myocardial infarction. Mitochondria constitute about 30 percent of the human heart. Mitochondrial DNA (mtDNA) is a damage-associated-molecular-pattern that induce injurious sterile inflammation. Little is known about mtDNA's inflammatory signalling pathways in cardiomyocytes and how mtDNA is internalized to associate with its putative receptor, toll-like receptor 9 (TLR9).
Experimental Approach:
We hypothesized that mtDNA can be internalized in cardiomyocytes and induce an inflammatory response. Adult mouse cardiomyocytes were exposed to hypoxia-reoxygenation and extracellular DNA. Microscale thermophoresis was used to demonstrate binding between nucleolin and DNA.
Key Results:
Expression of the pro-inflammatory cytokines IL-1β and TNFα were upregulated by mtDNA, but not by nuclear DNA (nDNA), in cardiomyocytes exposed to hypoxia-reoxygenation. Blocking the RNA/DNA binding protein nucleolin with midkine reduced expression of IL-1β/TNFα and the nucleolin inhibitor AS1411 reduced interleukin-6 release in adult mouse cardiomyocytes. mtDNA bound 10-fold stronger than nDNA to nucleolin. In HEK293-NF-κB reporter cells, mtDNA induced NF-κB activity in normoxia, while CpG-DNA and hypoxia-reoxygenation, synergistically induced TLR9-dependent NF-κB activity. Protein expression of nucleolin was found in the plasma membrane of cardiomyocytes and inhibition of nucleolin with midkine inhibited cellular uptake of CpG-DNA. Inhibition of endocytosis did not reduce CpG-DNA uptake in cardiomyocytes.
Conclusion And Implications:
mtDNA, but not nDNA, induce an inflammatory response in mouse cardiomyocytes during hypoxia-reoxygenation. In cardiomyocytes, nucleolin is expressed on the membrane and blocking nucleolin reduce inflammation. Nucleolin might be a therapeutic target to prevent uptake of immunogenic DNA and reduce inflammation.
Linked Articles:
This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc.
Insights
Mitochondrial DNA (mtDNA) triggers sterile inflammation in heart cells after injury. Blocking the protein nucleolin reduces this inflammation, suggesting it as a potential therapeutic target for heart conditions.
Area of Science:
- Cardiovascular Biology
- Immunology
- Molecular Biology
Background:
- Cellular debris, particularly mitochondrial DNA (mtDNA), drives sterile inflammation post-myocardial infarction.
- The precise inflammatory signaling pathways and cellular uptake mechanisms of mtDNA in cardiomyocytes remain largely unknown.
- Mitochondria are abundant in the heart, making mtDNA a significant factor in cardiac injury responses.
Purpose of the Study:
- To investigate the hypothesis that mtDNA can be internalized by cardiomyocytes and elicit an inflammatory response.
- To elucidate the role of nucleolin in mtDNA internalization and subsequent inflammatory signaling.
- To explore nucleolin as a potential therapeutic target for mitigating mtDNA-induced inflammation.
Main Methods:
- Adult mouse cardiomyocytes were subjected to hypoxia-reoxygenation and exposed to extracellular DNA.
- Microscale thermophoresis was employed to assess DNA-nucleolin binding affinity.
- HEK293-NF-κB reporter cells were used to evaluate mtDNA-induced inflammatory signaling.
- Inhibition of nucleolin and endocytosis was performed to study cellular uptake mechanisms.
Main Results:
- Mitochondrial DNA (mtDNA), unlike nuclear DNA (nDNA), upregulated pro-inflammatory cytokines (IL-1β, TNFα) in cardiomyocytes.
- Nucleolin binds mtDNA more strongly than nDNA and is expressed on the cardiomyocyte plasma membrane.
- Inhibition of nucleolin reduced mtDNA-induced inflammation and CpG-DNA uptake, independent of endocytosis.
- mtDNA induced NF-κB activity, indicating activation of inflammatory pathways.
Conclusions:
- mtDNA, not nDNA, induces sterile inflammation in cardiomyocytes during hypoxia-reoxygenation.
- Nucleolin plays a critical role in cardiomyocyte uptake of immunogenic DNA and subsequent inflammation.
- Targeting nucleolin presents a promising therapeutic strategy to prevent DNA uptake and reduce inflammation after cardiac injury.
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