Caspase recruitment domain-containing protein 9 (CARD9) knockout reduces regional ischemia/reperfusion injury through an attenuated inflammatory response

<div><p>Ischemic heart disease remains a leading cause of morbidity and mortality in the United States. Interventional reperfusion induces further damage to the ischemic myocardium through neutrophil infiltration and acute inflammation. As caspase recruitment domain-containing protein 9 (CARD9) plays a critical role in innate immune response and inflammation, we hypothesized that CARD9 knockout would provide protection against ischemia and reperfusion (I/R) injury through attenuation of acute inflammatory responses. C57BL/6 wild-type (WT) and CARD9^-/- mice were subjected to 45 min left anterior descending (LAD) coronary artery occlusion followed by 24-h reperfusion. Area at risk (AAR) and infarct size were measured by Evans blue and triphenyltetrazolium chloride (TTC) staining. Frozen heart sections were stained with anti-mouse GR-1 antibody to detect infiltrated neutrophils. Concentrations of cytokines/chemokines TNF-α, IL-6, CXCL-1 and MCP-1 were determined in heart tissue homogenate and serum by ELISA assay. Western immunoblotting analyses were performed to measure the phosphorylation of p38 MAPK. Our results indicate that following I/R, infarct size was significantly smaller in CARD9^-/- mice compared to WT. The number of infiltrated neutrophils was significantly lower in CARD9^-/- mice compared to WT. Levels of TNF-α, IL-6, CXCL-1 and MCP-1 were significantly reduced in heart tissue and serum from CARD9^-/- mice compared to WT. CARD9^-/- mice also exhibited significantly lower levels of phosphorylated p38 MAPK. Taken together, our results suggest that CARD9 knockout protects the heart from ischemia/reperfusion (I/R) injury, possibly through reduction of neutrophil infiltration and attenuation of CARD9-associated acute inflammatory signaling.</p></div

Measurement of cytokine/chemokine production in response to neutrophil activation in vitro.

<p>Neutrophils were isolated from WT and CARD9^-/- mice and co-cultured with H9C2 cells. MDP was used as an agonist to activate the CARD9 signaling. Cytokine/chemokine production was measured with commercial ELISA kits. The concentrations of IL-6 (A) and CXCL-1 (B) in the supernatant after 24 h co-culturing of the neutrophils with the H9C2 cells. Mean ± SEM, n = 6/group, *<i>p</i> < 0.05, **<i>p</i> < 0.01 vs. non-MDP treatment in the WT neutrophils; ^###<i>p</i> < 0.001 vs. WT.</p

Measurement of chemokine production in the heart and serum following I/R injury.

<p>After 24 h reperfusion, heart tissue and blood were harvested from the WT and CARD9^-/- mice. Monocyte chemotactic protein 1 (MCP-1) and chemokine C-X-C motif ligand 1 (CXCL-1) in heart tissue homogenate and serum were measured using commercial ELISA kits. A, Concentrations of MCP-1 and CXCl-1 in the heart tissue; B, concentrations of MCP-1 and CXCL-1 in serum. CARD9 deficiency significantly reduced concentrations of chemokines MCP-1 and CXCL-1 in the heart tissue and serum following I/R injury. Mean ± SEM, n = 4 /group, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 vs. non-I/R; ^#<i>p</i> < 0.05, ^##<i>p</i> < 0.01, ^###<i>p</i> < 0.001 vs. WT.</p

Measurement of cytokine production in heart tissue and serum in response to I/R injury.

<p>Following I/R injury, heart tissue and blood were harvested from WT and CARD9^-/- sham controls, WT+I/R and CARD9^-/-+I/R mice. TNF-α and IL-6 were measured using commercial ELISA kits. A, Concentrations of TNF-α and IL-6 in heart tissue; B, concentrations of TNF-α and IL-6 in serum. I/R injury significantly increased the production of TNF-α and IL-6 compared to sham controls. CARD9 knockout significantly attenuated the levels of these two cytokines in heart tissue and serum following I/R injury. Mean ± SEM, n = 4/group, ***<i>p</i> < 0.001 vs. sham controls; ^##<i>p</i> < 0.01, ^###<i>p</i> < 0.001 vs. WT.</p

Immunofluorescence staining of neutrophils infiltrated in the heart tissue.

<p>Following 45 min LAD occlusion and 24-h reperfusion, hearts from WT and CARD9^-/- mice were cryo-sectioned (7 μm) and stained with antibodies against GR-1 for neutrophils (red) and DAPI for nuclei (blue). A, Representative heart sections showing GR-1, DAPI and merged staining images of neutrophils and nuclei; B, Analyses of the number of neutrophils as percentage of the total cell nuclei in the section. The number of infiltrated neutrophils in the WT mouse heart was significantly higher than that in the CARD9^-/- mouse heart. Mean ± SEM, n = 3/group, **<i>p</i> < 0.01 vs. WT.</p

Measurement of myocardial ischemia and reperfusion (I/R) injury.

<p>C57BL/6 Wild-type (WT) and CARD9^-/- mice were subjected to 45 min occlusion of left anterior descending (LAD) coronary artery followed by 24 h of reperfusion. A: Representative heart sections stained with Evans blue and TTC to determine the total area of left ventricle (LV), area at risk (AAR, free of blue color) and infarct area (pale color); B: Analyses of the ratio of AAR/LV and infarct size (the ratio of infarct area/AAR) in the hearts of WT and CARD9^-/- mice. There was no significant difference in AAR/LV between the two groups, however the infarct size was significantly smaller in the CARD9^-/-+I/R group than that in the WT+I/R group. Mean ± SEM, n = 5/group, **<i>p</i> < 0.01, CARD9^-/- vs. WT.</p

Myography of vasomotor tone in aortic rings.

<p>Vasomotor tone changes from control mice transfected with Ad-Kir6.1AAA-GFP and Ad-GFP in response to A: phenylephrine (PE) and B: acetylcholine (ACh); Vasomotor tone changes from the exercised and control mice in response to C: diazoxide (Diaz) and D: glibenclamide pre-treatment and Diaz. **p<0.01, ***p<0.001, N = 5/group.</p

The effect of Kir6.1AAA transfection on myocardial I/R injury in the exercised mice.

<p>After 1 week of Ad-Kir6.1AAA-GFP transfection, the exercised mice were subjected to LAD occlusion followed by 24 h reperfusion. AAR (A) and infarct size (B) were measured with Evans blue and TTC staining. As shown, there was no significant difference in the AAR, but infarct size was increased significantly (∼36%) in the Ad-Kir6.1AAA-GFP group compared to that of the Ad-GFP transfected mice. N = 5/group, *p<0.05.</p

The effect of 8-week endurance exercise on heart and body weight.

<p>Data are expressed as mean ± SEM (n), *p<0.05 vs. control.</p><p>The effect of 8-week endurance exercise on heart and body weight.</p

Coronary artery transfection with Kir6.1AAA.

<p>Fluorescence microscopy was performed on heart sections containing coronary arteries. Sections were from Control (row 1) and Ad-Kir6.1AAA-GFP transfected (row 2) hearts. Column 1 shows GFP images, column 2 shows the respective bright field images of the vessels, and column 3 shows the merged images. Magnification is 400X. Green fluorescence representing the expression of Kir6.1AAA is clearly evident in the coronary vessel section, indicating successful transfection with Kir6.1AAA.</p