Inhibiting nucleolin reduces inflammation induced by mitochondrial DNA in cardiomyocytes exposed to hypoxia and reoxygenation

Abstract

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

Molecular Basis of Inflammation in the Pathogenesis of Cardiomyopathies

“…In particular, platelets activated by NETs release polyphosphates, which are prothrombotic and cause cardiac injury. Mitochondrial DNA also represents a trigger for inflammation after its internalization by cardiomyocytes, upregulating proinflammatory signaling pathways [57].…”

Release of Mitochondrial and Nuclear DNA During On-Pump Heart Surgery: Kinetics and Relation to Extracellular Vesicles

“…Like bacterial DNA, mitochondrial DNA (mtDNA) is circular and enriched in CpG motifs (53). Our group demonstrated that circulating mtDNA increases in patients after myocardial infarction (54), extracellular mtDNA induced death of mouse cardiomyocytes via TLR9-dependent activation of NF-kB (55) and that inhibition of extracellular mtDNA uptake reduces inflammation in cardiomyocytes challenged with hypoxia/reperfusion (56). Accumulation of mtDNA in the heart promotes heart failure in a mouse model of pressure overload (53).…”

“…Interestingly, AGER is involved in p66ShcA-dependent oxidative stress under some pathological conditions (140). Another mechanism of mtDNA delivery into cardiomyocytes is by the nucleolin (56). Our group has recently shown that nucleolin is expressed on cardiomyocyte membrane (56) and has an affinity to extracellular mtDNA and nDNA (141).…”

CpG‐ODN‐mediated TLR9 innate immune signalling and calcium dyshomeostasis converge on the NFκB inhibitory protein IκBβ to drive IL1α and IL1β expression

“…The ability of CpG‐rich mitochondrial DNA to trigger TLR9 activation has led to the appropriate use of CpG‐ODN for the study of multiple disease states where mitochondrial stress and injury are believed to play a pathogenic role 51–56 . These include atherosclerosis, 10–14 ischaemia‐reperfusion, 11,15–19,94 infection/sepsis, 20–37 and various autoimmune diseases 38–50 . Due to the direct link between mitochondrial stress and intracellular calcium dyshomeostasis, 84–86 the effect of intracellular calcium on CpG‐ODN‐mediated innate immune signalling is relevant to the study of these disease states associated with sterile inflammation.…”