TAK1 Activation by NLRP3 Deficiency Confers Cardioprotection Against Pressure Overload-Induced Cardiomyocyte Pyroptosis and Hypertrophy

Figures

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  Figure 1. NLRP3 Deficiency Attenuates Left Ventricular Dilation and Preserves Cardiac Function in Pressure Overload Hearts (A) Representative M-mode echocardiograms and relevant quantification of left ventricle in wild-type (WT) or NLRP3 knockout (KO) mice 4 weeks after transverse aortic constriction (TAC) or sham operation (n = 5 to 11 mice per group). Vertical scale bar, 2 mm, horizontal scale bar, 0.1 seconds. (B) Schematic presentations and quantification of left ventricular pressure, schematic presentations and quantification of left ventricular contraction velocity and relaxation velocity (n = 5 to 11 mice per group). Vertical scale bar, 100 mm Hg/s for pressure, 10,000 mm Hg/s for dp/dt; horizontal scale bar, 0.5 seconds for pressure and dp/dt. (C) Upper to lower, representative images of heart, cardiomyocytes stained by hematoxylin and eosin (scale bar, 20 μm), interstitial fibrosis stained by Masson’s trichrome (scale bar, 50 μm), perivascular fibrosis stained by Masson’s trichrome (scale bar, 50 μm), quantification of heart weight to body weight ratio (HW/BW), cross-sectional areas (CSA) of cardiomyocyte (n = 100 cells per group), and fibrotic ratio expressed as % area (n = 10 to…

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  Figure 2. Caspase-1 Mediated Aggravation of Pyroptosis Abrogates the Cardioprotection Conferred by NLRP3 Deficiency Under Pressure Overload (A) In vivo experimental overall schematic to explore the effects of cardiac caspase-1 overexpression in NLRP3 KO mice subjected to TAC. Male NLRP3 KO mice were administered a tail vein injection of adeno-associated virus type 9 (AAV9) vector carrying recombinant caspase-1, with the cardiac troponin T (cTnT) promoter. Vector virus AAV9-EGFP was used as negative control. Three weeks later, these mice were subjected to TAC surgery or sham operation for 4 weeks, followed by echo, hemodynamic, and tissue analysis. (B) Representative Western blot images and quantification showing the cleaved- and pro-forms of caspase-1, GSDMD, IL-1β, and IL-18 in left ventricular samples from NLRP3 KO mice with AAV9 transfected caspase-1 (Oe-caspase-1) or enhanced green fluorescent protein (EGFP) after 4 weeks of TAC or sham operation (n = 4 independent experiments). (C) Representative M-mode echocardiograms and quantification of left ventricle (n = 4 mice per group). Vertical scale bar, 2 mm, horizontal scale bar, 0.1 second. (D) Schematic presentations and quantification…

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  Figure 3. GSDMD Execution of Pyroptosis Decompensates Cardiac Function in NLRP3 Deficiency Mice Under Pressure Overload (A) In vivo experimental overall schematic to explore the effects of cardiac GSDMD overexpression in NLRP3 KO mice subjected to TAC. Male NLRP3 KO mice were administered a tail vein injection of AAV9 vector carrying recombinant GSDMD, with the cTnT promoter. Vector virus AAV9-EGFP was used as negative control. 3 weeks later, these mice were subjected to TAC surgery or sham operation for 4 weeks, followed by echo, hemodynamic and tissue analysis. (B) Representative Western blot images and quantification showing the cleaved- and pro-forms of GSDMD, IL-1β, IL-18 in left ventricular samples from NLRP3 KO mice with AAV9-transfected GSDMD (Oe-GSDMD) or EGFP after 4 weeks of TAC or sham operation (n = 4 independent experiments). (C) Representative M-mode echocardiograms and quantification of left ventricle (n = 4 to 6 mice per group). Vertical scale bar, 2 mm, horizontal scale bar, 0.1 second. (D) Schematic presentations and quantification of left ventricular pressure, schematic presentations and quantification of left ventricular contraction velocity and relaxation velocity (n…

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  Figure 4. NLRP3 Deficiency Enhanced Activation of TAK1 in Hypertrophic Hearts (A) Representative Western blot images and quantification showing phosphorylation levels of TAK1 in left ventricular samples from NLRP3 KO mice after 4 weeks of TAC or sham operation (n = 4 independent experiments). (B) Representative Western blot images and quantification showing phosphorylation levels of TAK1 from MCC950 incubated neonatal mouse ventricular myocytes (NMVMs) after treatment with Ang II or phosphate-buffered saline for 48 hours (n = 4 independent experiments). (C) String database identifies protein-protein interaction of NLRP3 and TAK1. (D) Structure-based interacting surface of NLRP3 (in red) and TAK1 (in blue) modeled by PRISM. The key interacting peptides are labeled in maroon. (E) Coimmunoprecipitation analysis and quantification of NLRP3 and TAK1 in NMVMs. Mean ± SEM. ∗∗∗∗P < 0.0001 vs sham, ††††P < 0.0001 vs WT in A. ∗∗∗∗P < 0.0001 vs PBS, ††P < 0.01 vs PBS+Ang II in B. ∗∗∗∗P < 0.0001 vs PBS in E. Abbreviations as in Figure 1.

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  Figure 5. TAK1 Deficiency Aggravates Cardiac Remodeling in Both WT and NLRP3 KO Mice Under Pressure Overload (A) In vivo experimental overall schematic to explore the effects of cardiac TAK1 knockdown in WT and NLRP3 KO mice subjected to TAC. Male WT and NLRP3 KO mice were administered an ultrasound-guided closed-chest intramyocardial injection of AAV9 carrying small hairpin RNA against TAK1, in line with its corresponding negative control. Three weeks later, these mice were subjected to TAC surgery or sham operation for 4 weeks, followed by echo, hemodynamic, and tissue analysis. (B) Representative M-mode echocardiograms and quantification of left ventricle in WT or NLRP3 KO mice 4 weeks after TAC or sham operation, either with pretreatment of AAV9 transfected small hairpin RNA against TAK1 (shTAK1) or EGFP (n = 4 to 13 mice per group). Vertical scale bar, 2 mm, horizontal scale bar, 0.1 second. (C) Quantification of echocardiographic parameters in B. (D) Schematic presentations and quantification of left ventricular pressure, schematic presentations and quantification of left ventricular contraction velocity and relaxation velocity (n = 4 to 13 mice per group). Vertical scale bars, 100…

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  Figure 6. TAK1 Deficiency Exacerbates Ang II-Induced Cardiomyocyte Hypertrophy Under PBS and MCC950 Treatment (A) Representative immunostaining images and quantification of NMVMs transfected with adenovirus harboring TAK1 (Ad-shTAK1) or with adenovirus containing EGFP (Ad-shRNA), treated with angiotensin II (Ang II, 1 μmol/L) or PBS for 48 hours, incubated with or without MCC950 (10 μmol/L) for 48 hours, followed by double staining of cardiac troponin T (cTnT, red) and 4'-6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 μm (n = 100 cells per group). (B) Real-time quantitative PCR analyses for the mRNA levels of Nppa, Nppb, and Myh7 in NMVMs (n = 4 independent experiments). (C) Representative Western blot images and (D) quantification showing the phosphorylation and/or total protein levels of TAK1, the cleaved- and pro-forms of caspase-1, GSDMD, IL-18, and caspase-18 in NMVMs (n = 4 independent experiments). Mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs corresponding PBS, †P < 0.05, ††P < 0.01, †††P < 0.001, ††††P < 0.0001 vs Ad-shRNA+PBS, ‡P < 0.05, ‡‡P < 0.01, ‡‡‡P < 0.001, ‡‡‡‡P < 0.0001 vs Ad-shRNA+MCC950, §P < 0.05, §§P < 0.01, §§§P < 0.001, §§§§P < 0.0001 vs…