Effects And Mechanism Of CARD9 On Protection From Myocardial Ischemia/reperfusion Injury

BackgroundAccording to 2013 China health statistics yearly report, cardiovascular disease is the primary lethal cause of Chinese female and its mortality is 131.64/100,000 in 2012, accounting for 21.45% total mortality in China. The mortality of ischemia cardiovascular disease accounts for 93.17% in the mortality of total cardiovascular disease. The current revascularization therapeutic strategies for ischemic heart disease include thrombolytic medication therapy, percutaneous coronary intervention and coronary artery bypass grafting, etc. These current therapeutic strategies have greatly improved survival rate significantly in acute myocardial infarction patients. However, the myocardial ischemia-reperfusion injury might result in increasing of MACE and heart failure during and post revascularization therapy. The mortality caused by ischemia-reperfusion injury during the hospitalization remains stubbornly higher. Therefore, there is an urgent need for new strategies and novel molecular target to improve the coronary heart disease prevention. Inflammation plays a key role in MIRI. The innate immune response induced by MIRI will lead to excessive myocardial inflammation response. The inflammation exacerbates myocardial injury through inducing ROS production, immune cell infiltration, and vascular endothelial injury. Many clinical studies revealed that the efficacy of the treatment which took inflammatory response as treatment target have been proved to be negative. The non-specific blocking or inhibiting the inflammatory response may be the possible reason for it. CARD9, namely as caspase recruitment domain containing protein 9, is one of the most important immune proteins. Generally it exists in neutrophils, macrophages and other myeloid cells-derived immune cells. Previous studies including ours indicated that CARD9 defects on human and mice may present with loss of ability of resistance to pathogens such as fungal. Recent studies show that cytokine secretion from macrophages is reduced in CARD9 knockout mice. In hypertensive heart disease model induced by angiotensin II, CARD9-knockout can reduce the effect of cardiac function from angiotensin II. Our previous studies demonstrate that CARD9-knockout can ameliorate myocardial dysfunction associated with high fat diet-induced obesity through weakening the inflammatory response. To date, the role of CARD9 in myocardial ischemia/reperfusion injury is still unclear. Therefore, this present study planned to elucidate the possible relationship between CARD9 and MIRI and the mechanism involved.Aims1. To determine whether CARD9 affects the cardiac function during MIRI. 2. To investigate how CARD9 is involved in MIRI. 3. To elucidate the possible pathway that is involved in the regulation of inflammation by CARD9 during MIRI.Methods1. Observe whether knockout CARD9 affects MI/R injury.a) Genotype of the CARD9 knockout mice b) Establish the MIRI model c) Determine CARD9 expression by Western blot d) Test the cardiac function via echo and hemodynamics e) Determine infarct size via Evans blue/TTC double staining f) Test cardiac apoptosis via TUNEL staining 2. Investigate how CARD9 involves in MIRI. a) Establish the MIRI model b) Determine neutrophils via Gr-1 immunofluorescent staining c) Test the content of ROS via DCF d) Test the content of TNF-α, IL-6, MCP-1 and CXCL-1 via ELISA kits e) Determine the expression of p38, p65, and their phosphorylation by Western blot 3. The mechanism of CARD9 involving in NOD2/MAPK. Isolate peritoneal neutrophils from two type mice Culture the neutrophils and H9c2 cell line with and without MDP a) Test H9c2 apoptosis via TUNEL staining b) Test the content of TNF-α, IL-6, MCP-1 and CXCL-1 via ELISA kits c) Determine the expression of p38, p65 and CARD9 and their phosphorylation by Western blotResults1. Card9 Ko can reduce the injury from MIRI a) MIRI elevated CARD9 expression b) Card9 Ko ameliorated the cardiac function after MIRI c) Card9 Ko ameliorated the LVEF, LVEDP, +dp/dt max, and-dp/dt max after MIRI d) Card9 Ko reduced the infarct size and cardiac apoptosis after MIRI 2. Card9 Ko can reduce the inflammation from MIRI a) Card9 Ko reduced the infiltration of neutrophils after MIRI b) Card9 Ko reduced the ROS after MIRI c) Card9 Ko reduced the content of TNF-α, IL-6, MCP-1 and CXCL-1 in heart tissue and serum after MIRI d) Card9 Ko reduced the expression of phos-p38 and phos-p65 after MIRI 3. Card9 involves in inflammation via NOD2/MAPK a) Card9 Ko reduced the cardiac apoptosis by treatment with MDP b) Card9 Ko reduced the content of TNF-α, IL-6, MCP-1 and CXCL-1 by treatment with MDP c) Treatment with MDP induced an increase of Card9 expression. CARD9 Ko reduced the expression of phos-p38 by treatment with MDPConclusion1. After MIRI, the expression of CARD9 is up-regulated, which is associated with infiltration of neutrophil. Down-regulation of CARD9 gene expression improves the heart function and prevents from the ischemic-reperfusion injury. 2. CARD9 may mediate the PRRs related inflammatory response during MIRI associating with neutrophils through NOD2/ MAPK signal pathway.