Colchicine Ameliorates Dilated Cardiomyopathy Via SIRT2-Mediated Suppression of NLRP3 Inflammasome Activation

Abstract

Background Dilated cardiomyopathy remains a leading cause of heart failure worldwide. Immune inflammation response is recognized as a significant player in the progression of heart failure; however, immunomodulatory strategies remain a long-term challenge. Colchicine, a potent anti-inflammatory drug, has many benefits in ischemic cardiovascular events, but its role in nonischemic heart failure remains unclear. Methods and Results Doxorubicin administration was used to establish a murine dilated cardiomyopathy model, and colchicine or saline was orally given. At the end point, cardiac function and fibrosis were measured to investigate the effects of colchicine. Inflammatory cytokine levels, neutrophil recruitment, and NLRP3 (NOD-like receptor protein 3) inflammasome activation were detected to evaluate the inflammatory response. Furthermore, to examine the downstream target of colchicine, SIRT2 (Sirtuin 2) was pharmacologically inhibited in vitro; thus, changes in the NLRP3 inflammasome were detected by immunoblotting. These results showed that murine cardiac function was significantly improved and fibrosis was significantly alleviated after colchicine treatment. Moreover, the infiltration of neutrophils and the levels of inflammatory cytokines in the failing myocardium were both decreased by colchicine treatment. Mechanistically, colchicine upregulated the expression of SIRT2, leading to the inactivation of the NLRP3 inflammasome in an NLRP3 deacetylated manner. Conversely, the inhibition of SIRT2 attenuated the suppressive effect of colchicine on NLRP3 inflammasome activation. Conclusions This study indicated that colchicine could be a promising therapeutic candidate for dilated cardiomyopathy and other nonischemic heart failure associated with the inflammatory response.

Keywords: NLRP3 inflammasome; SIRT2; colchicine; dilated cardiomyopathy; inflammation.

Figures

Figure 1. Colchicine treatment significantly improved murine cardiac functions.

A through C, Cardiac function is analyzed by 2‐dimensional murine echocardiography, left ventricular (LV) ejection fraction, LV fractional shortening, LV internal dimensions at end‐diastole, and LV internal dimensions at end‐systole values are measured at the end point of experiment (n=10 each group). D, Kaplan‐Meier survival curve for each group mice followed for 4 weeks with or without doxorubicin challenge (n=25 each group). E, The changes of body weight from beginning to end (n=6 each group). F, Relative expression of ANP (atrial natriuretic peptide) mRNA and BNP (brain natriuretic peptide) mRNA are normalized to GAPDH mRNA in murine cardiac tissues at the end point of experiment. G and H, The representative hematoxylin and eosin staining (G) and Sirius red staining (H) of hearts in each group are shown. Quantification of interstitial fibrosis (% area) in hearts is shown on the right; n=8 each group. Five images per mouse are evaluated. Scale bar=50 μm. Data are shown as mean±SEM. Data of (B, C, and E through G) are analyzed by 1‐way ANOVA (Tukey post‐test). Data of (D) is analyzed by Mantel‐Cox test. ANP indicates atrial natriuretic peptide; BNP, brain natriuretic peptide; DCM, dilated cardiomyopathy; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVIDd, left ventricular internal dimensions at end‐diastole; and LVIDs, left ventricular internal dimensions at end‐systole. *P<0.05 between groups; **P<0.01 between groups; ***P<0.005 between groups; and ****P<0.001 between groups. Col indicates colchicine.

Figure 2. Colchicine reduced the recruitment of neutrophils.

A, ELISA analysis of inflammatory factor interleukin‐1, interleukin‐6, and tumor necrosis factor‐α in murine plasma at the end point of experiment (n=8–12 each group). B, ELISA analysis of GDF15 in murine plasma at the end point of experiment (n=8–12 each group). C, Relative expression of inflammatory cytokine interleukin‐1 mRNA, interleukin‐6 mRNA, and tumor necrosis factor‐α mRNA are normalized to GAPDH mRNA in murine cardiac tissues at the end point of experiment (n=5 each group). D and E, Representative flow cytometric and statistical analysis of CD11b+Ly6G+ neutrophil in the blood (D) and CD45+Ly6G+ neutrophil in the heart € (n=6 each group). F, Representative immunofluorescence images of cardiac tissues staining with Ly6G (green), cTNT (cardiac troponin T) (red), and DAPI (blue). Frequency of Ly6G‐positive neutrophils is shown on the right (n=6–8 each group). Five images per mouse are evaluated. Scale bar=50 μm. Data are shown as mean±SEM. All data statistical significance are determined by 1‐way ANOVA (Tukey post‐test). cTNT indicates cardiac troponin T; DCM, dilated cardiomyopathy; GDF15, growth and differentiation factor 15; IL‐1, interleukin‐1; IL‐6, interleukin‐6; and TNF‐α, tumor necrosis factor‐α. *P<0.05 between groups; **P<0.01 between groups; ***P<0.005 between groups; and ****P<0.001 between groups. Col indicates colchicine; Ly6G, lymphocyte antigen 6 complex locus G6D.

Figure 3. NLRP3 (NOD‐like receptor protein 3) inflammasome pathway was inhibited by colchicine treatment.

A and B, Representative immunoblots and the corresponding analysis of NLRP3 inflammasome pathway in cardiac tissues at the end point of experiment. GAPDH shows as loading control. C, Representative immunofluorescence images of cardiac tissues staining with ASC (green), cTNT (cardiac troponin T) (red), and DAPI (blue). Frequency of ASC speck is shown on the right (n=6–8 each group). Five images per mouse are evaluated. Scale bar=50 μm. Data are shown as mean±SEM. All data statistical significance are determined by 1‐way ANOVA (Tukey post‐test). cTNT indicates cardiac troponin T; DCM, dilated cardiomyopathy; IL‐1β, interleukin‐1β; and NLRP3, NOD‐like receptor protein 3. *P<0.05 between groups; **P<0.01 between groups; ***P<0.005 between groups; and ****P<0.001 between groups. AIM2 indicates absent in melanoma 2; ASC, apoptosis‐associated speck‐like protein with CARD domain; Col, colchicine.

Figure 4. SIRT2 (Sirtuin 2) is involved in the anti‐inflammation effect of colchicine.

A, Acetylated NLRP3 (NOD‐like receptor protein 3) levels in the cardiac tissues of mice at the end point of experiment. Immunoglobulin G is the negative control and GAPDH is the input loading control. Data are representative of 3 independent experiments. Western blot analysis on expression of SIRT2 in cardiac tissues at the end of experiment (n=6 each group) on the right. B, Relative expression of SIRT2 mRNA is normalized to GAPDH mRNA in cardiac tissues (n=4 each group). C, Relative expression of inflammatory cytokine interleukin‐1 mRNA and NLRP3 mRNA are normalized to GAPDH mRNA in primary neutrophil that are treated +/− colchicine (10 μmol/L, 2 hours), +/− SIRT2 inhibitor AGK2 (10 μmol/L, 2 hours) or challenged with doxorubicin (5 μmol/L, 2 hours) and inflammasome activators nigericin (10 μmol/L, 2 hours); n=6 each group. D, Western blot analysis on expression of NLRP3 inflammasome pathway in primary neutrophil that are treated +/− colchicine (10 μmol/L, 2 hours), +/− SIRT2 inhibitor AGK2 (10 μmol/L, 2 hours) or challenged with doxorubicin (5 μmol/L, 2 hours) and inflammasome activators nigericin (10 μmol/L, 2 hours). GAPDH shows loading control. Data are representative of 3 independent experiments. Data are shown as mean±SEM. Data of (A and B) are analyzed by 1‐way ANOVA (Tukey post‐test). Data of (C and D) are analyzed by 2‐way ANOVA (Bonferroni post‐test). DCM indicates dilated cardiomyopathy; IL‐1β, interleukin‐1β; NLRP3, NOD‐like receptor protein 3; and SIRT2, Sirtuin 2. *P<0.05 between groups; **P<0.01 between groups; ***P<0.005 between groups; and ****P<0.001 between groups. AGK2 indicates a reversible inhibitor of SIRT2; AIM2, absent in melanoma 2; ASC, apoptosis‐associated speck‐like protein with CARD domain; Col, colchicine; IB, immunoblot; IP, immunoprecipitation; Ly6G, lymphocyte antigen 6 complex locus G6D.

Figure 5. Colchicine promotes the deacetylation of NLRP3 (NOD‐like receptor protein 3) by SIRT2 (Sirtuin 2).

A, Representative immunofluorescence images of cardiac tissues staining with NLRP3 (green), SIRT2 (red), and DAPI (blue). (n=6–8 each group). Five images per mouse are evaluated. Scale bar=50 μm. B, Representative immunofluorescence images of primary neutrophils that are treated +/− colchicine (10 μmol/L, 2 hours), +/− SIRT2 inhibitor AGK2 (10 μmol/L, 2 hours) or challenged with doxorubicin (5 μmol/L, 2 hours) and inflammasome activators nigericin (10 μmol/L, 2 hours). NLRP3 (green), SIRT2 (red) and DAPI (blue). Scale bar=20 μm. Data are representative of 3 independent experiments. C, The effect of SIRT2 on the deacetylation of NLRP3 is evaluated by coimmunoprecipitation. NLRP3‐Flag plasmid is transfected into NB4 cell line, then NB4 cell line is treated with +/− colchicine (10 μmol/L, 2 hours), +/− SIRT2 inhibitor AGK2 (10 μmol/L, 2 hours) or challenged with doxorubicin (5 μmol/L, 2 hours) and inflammasome activators nigericin (10 μmol/L, 2 hours). Cell lysates are immunopurified with anti‐Flag antibody. Then the expression of acetylated lysine is measured by IP. GAPDH is the input loading control. Data are representative of 3 independent experiments. DCM indicates dilated cardiomyopathy; IL‐1β, interleukin‐1β; NLRP3, NOD‐like receptor protein 3; and SIRT2, Sirtuin 2. AGK2, a reversible inhibitor of SIRT2; Col, colchicine; IB, immunoblot; IP, Immunoprecipitation.

Figure 6. Inhibition of SIRT2 (Sirtuin 2) abrogates the beneficial effects of colchicine on dilated cardiomyopathy mice.

A through C, Cardiac function is analyzed by 2‐dimensional murine echocardiography, left ventricular (LV) ejection fraction, LV fractional shortening, LV internal dimensions at end‐diastole, and LV internal dimensions at end‐systole values are measured at the end point of experiment (n=10 each group). D, Kaplan–Meier survival curve for each group mice followed for 4 weeks with or without doxorubicin challenge (n=25 each group). E, The changes of body weight from beginning to end (n=10 each group). F, Relative expression of ANP (atrial natriuretic peptide) mRNA and BNP (brain natriuretic peptide) mRNA are normalized to GAPDH mRNA in murine cardiac tissues at the end point of experiment. G and H, The representative hematoxylin and eosin staining (G) and Sirius red staining (H) of hearts in each group are shown. Quantification of interstitial fibrosis (% area) in hearts is shown on the right; n=8 each group. Five images per mouse are evaluated. Scale bar=50 μm. Data are shown as mean±SEM. Data of (B, C, and E through G) are analyzed by 1‐way ANOVA (Tukey post‐test). Data of (D) is analyzed by Mantel‐Cox test. ANP indicates atrial natriuretic peptide; BNP, brain natriuretic peptide; DCM, dilated cardiomyopathy; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVIDd, left ventricular internal dimensions at end‐diastole; and LVIDs, left ventricular internal dimensions at end‐systole. *P<0.05 between groups; **P<0.01 between groups; ***P<0.005 between groups and ****P<0.001 between groups. AGK2 indicates a reversible inhibitor of SIRT2; Col, colchicine.