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Extracellular mtDNA activates NF-κB via toll-like receptor 9 and induces cell death in cardiomyocytes

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Abstract

Acute myocardial infarction (AMI) causes sterile inflammation, which exacerbates tissue injury. Elevated levels of circulating mitochondrial DNA (mtDNA) have been associated with AMI. We hypothesized that mtDNA triggers an innate immune response via TLR9 and NF-κB activation, causing cardiomyocyte injury. Murine cardiomyocytes express TLR9 mRNA and protein and were able to internalize fluorescently labeled mouse mtDNA. Incubation of human embryonic kidney cells with serum from AMI patients containing naturally elevated levels of mtDNA induced TLR9-dependent NF-κB activity. This effect was mimicked by isolated mtDNA. mtDNA activated NF-κB in reporter mice both in vivo and in isolated cardiomyocytes. Moreover, incubation of isolated cardiomyocytes with mtDNA induced cell death after 4 and 24 h. Laser confocal microscopy showed that incubation of cardiomyocytes with mtDNA accelerated mitochondrial depolarization induced by reactive oxygen species. In contrast to mtDNA, isolated total DNA did not activate NF-κB nor induce cell death. In conclusion, mtDNA can induce TLR9-dependent NF-κB activation in reporter cells and activate NF-κB in cardiomyocytes. In cardiomyocytes, mtDNA causes mitochondrial dysfunction and death. Endogenous mtDNA in the extracellular space is a danger signal with direct detrimental effects on cardiomyocytes.

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Acknowledgments

Harald Carlsen and Jan Øivind Moskaug, Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, kindly provided NF-κB luciferase reporter mice. The authors acknowledge the expertise of Gerbrand Koster for technical advice (NorMIC imaging cluster, Department of Biosciences, University of Oslo) and technical assistance was expertly performed by Torun Flatebø and Sowmya Sanjeevini.

Funding

This work was supported by the Norwegian Health Association, UNIFOR, the Norwegian Research Council, the University of Oslo, and the Novo Nordisk Foundation. Marte Bliksøen was supported by a grant from South-Eastern Regional Health Trust.

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Authors and Affiliations

  1. Department of Molecular Medicine, Faculty of Medicine, Institute of Basic Medical Sciences, University of Oslo (UiO), Oslo, Norway

    Marte Bliksøen, Lars Henrik Mariero, May Kristin Torp, Anton Baysa, Fred Haugen, Guro Valen & Kåre-Olav Stensløkken

  2. Center for Heart Failure Research, UiO, Oslo, Norway

    Marte Bliksøen, Lars Henrik Mariero, May Kristin Torp, Anton Baysa, Fred Haugen, Ingebjørg Seljeflot, Guro Valen & Kåre-Olav Stensløkken

  3. Cardiovascular Research Group, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway

    Kirsti Ytrehus

  4. Department of Cardiology, Center for Clinical Heart Research, Oslo University Hospital, Oslo, Norway

    Ingebjørg Seljeflot

  5. Department of Emergency Medicine and Intensive Care, Oslo University Hospital, Oslo, Norway

    Marte Bliksøen & Jarle Vaage

  6. Institute of Clinical Medicine, UiO, Oslo, Norway

    Marte Bliksøen, Ingebjørg Seljeflot & Jarle Vaage

  7. Division of Physiology, Department of Molecular Medicine, Institute of Basic Medical Science, Postbox 1103, Blindern, 0317, Oslo, Norway

    Kåre-Olav Stensløkken

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  1. Marte Bliksøen
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  2. Lars Henrik Mariero
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Correspondence to Kåre-Olav Stensløkken.

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Professor Guro Valen passed away 26 September 2014.

G. Valen and K.-O. Stensløkken have shared last authorship.

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395_2016_553_MOESM1_ESM.tif

Supplementary material 1 (TIFF 1029 kb) Figure S1 legend: 1 % agarose gel with SYBR Safe DNA gel stain (Invitrogen) showing DNA isolated from murine liver or from mitochondrial extracts from murine liver, eluted in buffer and subjected to sonication for 2 × 30 s or sham treatment with the same sample handling but without sonication. Arrow: heavy DNA band present in unsonicated mitochondrial DNA sample (mtDNA), which is absent from the sonicated samples (mtDNAs), indicating DNA fragmentation. There is evidence of some DNA degradation in the non-sonicated samples, but the mtDNAs sample shows denser smearing in the ~ 300 bp region. mtDNA (mitochondrial DNA), mtDNAs (sonicated mtDNA), tDNA (total DNA), tDNAs (sonicated total DNA) and 1 kb ladder

395_2016_553_MOESM2_ESM.tif

Supplementary material 2 (TIFF 505 kb) Figure S2 legend: Figure shows remaining primary mouse cardiomyocytes after different DNA agonist treatment for 4 and 24 h. There is a decrease in the number of viable cells after 24 h in control, but the number of cells lost is significantly higher in the cardiomyocytes treated either with mtDNA (B) or CpGC (D). Statistical differences was tested with Wilcoxon matched-pairs signed rank test (* p < 0.05)

395_2016_553_MOESM3_ESM.avi

Supplementary material 3 (AVI 43012 kb) Video S3 The video shows a confocal Z-stack with mouse cardiomyocytes exposed to Cy-5 tagged mtDNA. The mtDNA is present in the peri-nuclear area, but also in the cytosol

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Bliksøen, M., Mariero, L.H., Torp, M.K. et al. Extracellular mtDNA activates NF-κB via toll-like receptor 9 and induces cell death in cardiomyocytes. Basic Res Cardiol 111, 42 (2016). https://doi.org/10.1007/s00395-016-0553-6

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