Mitochondrial DNA activates the inflammasome in cardiac fibroblasts and promotes adverse cardiac remodeling

Abstract Introduction Cardiac inflammation is involved in the genesis of heart failure. We reported that pressure overload induces the binding of mitochondrial DNA (mtDNA) with Toll-like receptor 9 in cardiomyocytes, leading to cardiac inflammation and dysfunction. Oxidized mtDNA is bound to a multimeric protein complex – NLRP3 inflammasome – in inflammatory cells to induce inflammation by producing IL-1β. The in vivo role of NLRP3 inflammasome activated in cardiac fibroblasts during cardiac remodeling remains unknown. Methods To examine the binding of oxidized mtDNA to NLRP3 under the activation of inflammasome in cardiac fibroblasts in vitro, cardiac fibroblasts were isolated from wild-type mouse hearts and stimulated with lipopolysaccharide and ATP as reported before. The cell homogenates were immunoprecipitated with an anti-NLRP3 antibody. The amount of 8-hydroxy-2’-deoxyguanosine (8-OHdG) – a marker of DNA nucleoside oxidation - was measured using an ELISA kit following isolation of DNA from the immunoprecipitates. Cardiac fibroblast-specific NLRP3–deficient mice (CF-KO) were generated by crossing the homozygous floxed Nlrp3 mice with transgenic mice expressing Cre recombinase driven by the periostin promoter. The mice and the control littermates (WT) were subjected to pressure overload employing transverse aortic constriction (TAC) to induce heart failure. To determine whether the inhibition of the binding of oxidized mtDNA to NLRP3 prevents the progression of cardiac remodeling, C57BL/6J mice received an intraperitoneal injection of 8-OHdG, which works as a competitive inhibitor of that binding or phosphate-buffered saline (PBS) before and daily after TAC surgery for one week. Cardiac remodeling was assessed by echocardiography and histological and molecular analyses 1 week after operation. Inflammatory cell infiltration was examined by immunostaining. Results An 8-OHdG amount bound to NLRP3 increased after the stimulation. CF-KO showed no cardiac phenotypes under baseline conditions. Cardiac fibroblast-specific deletion of NLRP3 attenuated cardiac dysfunction indicated by left ventricular fractional shortening (LVFS) in response to pressure overload (WT 23% vs. CF-KO 38%). The area of fibrosis n TAC-operated CF-KO hearts was lower than in TAC-operated WT hearts. TAC-operated CF-KO hearts showed smaller macrophage F4/80-positive cells than TAC-operated WT hearts (WT 980 cells/10000 nuclei vs. CF-KO 260 cells/10000 nuclei). Administering 8-OHdG significantly improved LVFS in TAC-operated C57BL/6J mice (PBS 16% vs. 8-OHdG 28%). The number of F4/80-positive cells was reduced considerably in 8-OHdG-treated C57BL/6J hearts than in PBS-treated C57BL/6J hearts (PBS 1550 cells/10000 nuclei vs. 8-OHdG 980 cells/10000 nuclei). Conclusions The binding of oxidized mtDNA to NLRP3 in cardiac fibroblasts can be a novel therapeutic target for heart failure.