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

Suppression of Induced microRNA-15b Prevents Rapid Loss of Cardiac Function in a Dicer Depleted Model of Cardiac Dysfunction

<div><p>Background</p><p>Dicer endonuclease, critical for maturation of miRNAs, is depleted in certain forms of cardiomyopathy which results in differential expression of certain microRNAs. We sought to elucidate the mechanisms underlying the rapid loss of cardiac function following cardiac-specific Dicer depletion in adult mice.</p><p>Results</p><p>Conditional Dicer deletion in the adult murine myocardium demonstrated compromised heart function, mitochondrial dysfunction and oxidant stress. Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function. Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy.</p><p>Conclusions</p><p>Anti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.</p></div

miR-15b over expression in HL-1 cells compromises mitochondrial membrane potential.

<p>(<b>A</b>,<b>B</b>) HL-1 cells were transfected (72 hours) with mimic negative control or miRNA-15b mimic. Untransfected cells were used as negative control and cells transfected with carbonyl cyanide m-chlorophenylhydrazone (CCCP) were used as positive control (left panel –8 nM tetramethylrhodamine methyl ester (TMRM); center panel - 0.5 µl/ml plasma membrane potential indicator (PMPI); right panel - merged image). Bar graph shows significant decrease in TMRM with no change in PMPI. Solid bars represent mimic negative control while open bars represent miRNA-15b mimic. (<b>C</b>,<b>D</b>) Loss of mitochondrial membrane potential in HL-1 cells transfected with miRNA-15b mimic, as assessed by JC-1 flow cytometry 72 h post-transfection. Cells were transfected with a (i) mimic control, (ii) miRNA-15b mimic or (iii) treated with CCCP Arrows (K gate) indicate cells containing JC-1 aggregates resulting from intact mitochondria; M gate indicates cells with low or collapsed mitochondrial membrane potential. Ratio of polarized to depolarized cells calculated as a ratio between cells in K gate to M gate. Data indicate decrease in membrane potential upon up-regulation of miRNA-15b. (n = 3).</p

Dicer deletion in the adult heart leads to oxidative stress.

<p>(<b>A</b>) Representative EPR imaging of nitroxide radical decay in (A) Dicer^+/+ and (B) Dicer^−/− hearts. (<b>B</b>) Time course of average % signal change in the region of interest (ROI). Logarithmic values of signal change (normalized to the initial signal at time = 0) in the ROIs are plotted with respect to time. Decay rate constants were obtained from the slope of linear decay after peak. Line with black circles represent Dicer^+/+ and line with grey circles represent Dicer^−/−. (<b>C</b>) Bar-graph showing the measured rate constants of nitroxide reduction in the tissues. (<b>D</b>) Thiobarbituric acid–reactive substances (TBARS), an indicator of lipid peroxidation was measured from Dicer^+/+ and Dicer^−/− hearts and was significantly higher in the later. (<b>E</b>) Total GSSG to GSH ratio in Dicer^+/+ mice heart compared to Dicer^−/− heart. (<b>F</b>) Lactate levels measured in Dicer^+/+ and Dicer^−/− mice hearts.</p

Cardiac specific <i>Dicer</i> deletion leads to early onset changes in some miRNAs including miRNA-15b.

<p>(<b>A</b>) Heat map generated for the differentially expressed miRNAs in Dicer^+/+ and Dicer^−/− mice. (<b>B</b>)Validation of selected miRNAs by real-time PCR. (<b>C</b>) miR-15b expression in laser captured cardiomyocytes from Dicer^+/+ and Dicer^−/− tissues.</p