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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.

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

Aquaporins (AQPs) are channel-forming membrane proteins highly permeable to water. AQP4 is found in mammalian hearts; however, its expression sites, regulation and function are largely unknown. The aim was to investigate cardiac AQP4 expression in humans and mice, its regulation by ischemia and hypoxia, and in particular its role in cardiac ischemic injury using AQP4 knockout (KO) mice. Comparable levels of AQP4 were detected by Western blot and qPCR in biopsies from human donor hearts and wild type C57Bl6 mouse hearts. In mice, AQP4 was expressed on cardiomyocyte plasmalemma (qPCR, Western blot, immunogold), and its mRNA decreased following ischemia/reperfusion (isolated hearts, p = 0.02) and after normobaric hypoxia in vivo (oxygen fraction 10 % for 1 week, p < 0.001). Isolated hearts from AQP4 KO mice undergoing global ischemia and reperfusion had reduced infarct size (p = 0.05) and attenuated left ventricular end-diastolic pressure during reperfusion (p = 0.04). Infarct size was also reduced in AQP4 KO mice 24 h after left coronary artery ligation in vivo (p = 0.036). AQP4 KO hearts had no compensatory change in AQP1 protein expression. AQP4 KO cardiomyocytes were partially resisted to hypoosmotic stress in the presence of hypercontracture. AQP4 is expressed in human and mouse hearts, in the latter confined to the cardiomyocyte plasmalemma. AQP4 mRNA expression is downregulated by hypoxia and ischemia. Deletion of AQP4 is protective in acute myocardial ischemia-reperfusion, and this molecule might be a future target in the treatment of acute myocardial infarction.

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