The role and mechanisms of ambient fine particulate matter exposure in cardiac dysfunction in mice
Authors:   , Liu Xuanyou 
Published: February 2023

[EMBARGOED UNTIL 8/1/2023] Ambient fine particulate matter (PM) exposure associates with an increased risk of cardiovascular diseases (CVDs). Oxidative stress and inflammation are considered important mechanisms for the development and progression of CVDs. Cytokines including tumor necrosis factor- [alpha] (TNF-[alpha]) play an important role in reactive oxygen species (ROS) production, oxidative stress, and inflammation. Caspase recruitment domain-containing protein 9 (CARD9) signaling is critically involved in the regulation of cytokines production, and thus oxidative stress. Major sex differences exist in epidemiology, pathophysiology, and outcome of CVDs with females being protected against premature CVDs with largely undefined mechanisms. The present project was designed to test the hypothesis that PM exposure impairs cardiac function in male mice due to increased ROS production through CARD9-mediated TNF-[alpha] production. The aims were: 1) to determine if PM exposure leads to cardiac dysfunction in mice; 2) to define the role of oxidative stress in PM exposure-induced cardiac dysfunction in mice; 3) to examine if PM exposure-induced cardiac dysfunction is through TNF-[alpha]-mediated oxidative stress in mice; 4) to determine if CARD9-mediated TNF-[alpha] production is critical for PM exposure-induced ROS production and cardiac dysfunction in mice; and 5) to demonstrate if a significant sex difference in PM exposure-induced cardiac dysfunction is present, and the role of oxidative stress. Age-matched (8-12 weeks) male and female wild-type (WT) C57BL/6 mice, TNF-[alpha] knockout (KO) mice, CARD9 KO mice, and transgenic (TG) mice with concomitant overexpression of superoxide dismutase (SOD)1, SOD3, and glutathione peroxidase-1 (GPX-1) were exposed to PM for 6 weeks. Echocardiography and cardiac MRI were used to detect cardiac structure and function. DHE staining, Masson's trichrome staining, and Western blotting were used to determine ROS production, collagen level, and expression of antioxidant enzymes in cardiac tissues, respectively. ELISA was used to measure the levels of plasma cytokines, including interleukin (IL)-6, IL-[alpha], and TNF-[alpha]. Experiments were repeated in WT male mice when ROS or TNF-[alpha] signaling was blocked using antioxidants N-acetylcysteine (NAC), Tempol, or anti- TNF-[alpha] antibody to determine the role of ROS and TNF-[alpha] in PM-induced cardiac dysfunction. To define the role of estrogen in PM-induced cardiac dysfunction, experiments were conducted with female mice with and without ovariectomy. The data from the present study demonstrated that PM exposure significantly decreased both systolic and diastolic cardiac function with increased cardiac fibrosis and increased ROS production in cardiac tissues selectively in male mice, not in females, in association with decreased expressions of SOD1 and GPX-1. Treatment of WT male mice with NAC or Tempol or concomitant overexpression of SOD1 and GPX-1 effectively prevented PM exposure-induced ROS production, cardiac fibrosis, and cardiac dysfunction. Plasma TNF-[alpha] level was significantly increased in male mice, not in females, with PM exposure. Treatment of WT male mice with TNF-[alpha] produced similar level of cardiac ROS production and decreased expressions of SOD1 and GPX-1 as male mice with PM exposure. TNF-[alpha] deficiency using TNF-[alpha] KO mice or treatment with anti-TNF-[alpha] antibody also effectively attenuated PM exposure-induced ROS production, cardiac fibrosis, and cardiac dysfunction in male mice. PM exposure significantly increased the protein expression of CARD9 in macrophages in male mice with increased levels of plasma TNF-[alpha] and cardiac ROS. CARD9 deficiency completely prevented PM exposure-induced increases in plasma TNF-[alpha] and cardiac ROS, as well as cardiac fibrosis, and preserved cardiac function in male mice. No changes in cardiac ROS production and cardiac function were observed in female mice with or without ovariectomy after PM exposure. It is concluded that PM exposure induces cardiac fibrosis and cardiac dysfunction selectively in male mice due to increased ROS formation through CARD9-mediated TNF-[alpha] production. Cardiac structure and function were preserved in female mice with PM exposure independent of estrogen.