Deubiquitinase Ubiquitin‐Specific Protease 29 Ameliorates Pathological Cardiac Hypertrophy through Inhibiting Transforming Growth Factor β‐Activated Kinase 1

Figure 1. The protein expression of USP29 is upregulated in murine hypertrophic hearts and cardiomyocytes. A, Statistical results of HW, HW/BW ratios, LVEDd, LVESd, EF%, and FS% values (C) in WT mice subjected to sham or 4 weeks of TAC surgery (n=5). B, wheat germ agglutinin and DAPI‐stained LV myocardium, and statistical results of cardiomyocyte cross‐sectional area in the indicated groups. Scale bars, 20 μm. (n=60 cells per group). D, E USP29 mRNA (D) and protein (E) levels in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n=3 for mRNA determination, n=5 for protein determination). F, G USP29 mRNA (F) and protein (G) levels in NRCMs treated with PBS or 24 hours of phenylephrine (50 μM) (n=3). *P<0.05, **P<0.01, ***P<0.001 vs sham or PBS. Data are presented as mean ± SD. Significant differences were assessed by 2‐tailed Student t test. EF indicates ejection fraction; HW/BW, heart weight/ body weight; FS, fractional shortening; LVEDd, left ventricle end‐diastolic diameter; LVESd, left ventricle end‐systolic diameter; NRCM, neonatal rat cardiomyocytes; n.s., no significance; PE, phenylephrine; TAC, transverse aortic constriction; USP29, ubiquitin‐specific protease…

Figure 2. Ablation of USP29 potentiates TAC‐induced cardiac hypertrophy. A, Strategy for the construction of USP29 knockout mice. B, The cardiac protein levels of USP29 in WT and knockout mice (n=3). C, D Statistical results of HW, HW/BW, LW/BW, HW/TL (C), and LW/BW (D) ratios in WT and knockout mice subjected to sham or 4 weeks of TAC surgery (n=10). E, Gross hearts, H&E‐stained LV myocardium, and statistical results of cardiomyocyte cross‐sectional area in the indicated groups. Scale bars, 0.3 cm, and 50 μm, respectively. (n=6). F, Statistical results of the LVESd, LVEDd, LV mass cor, IVSd, LVPWd, FS%, EF%, E/A, and E/E' values in WT and knockout mice subjected to sham or 4 weeks of TAC surgery (n=10). G, mRNA levels of the hypertrophic markers in the indicated groups (n=4). H, PSR‐stained LV myocardium and statistical results of LV collagen volume in the indicated groups. Scale bars, 50 μm. (n=6). I, mRNA levels of the fibrotic markers in each group (n=4). # P<0.05, ## P<0.01, ### P<0.001 vs WT sham, *P<0.05, **P<0.01, ***P<0.001 vs WT TAC. Data are presented as mean ± SD. Significant differences were assessed by 1‐way ANOVA with Bonferroni post hoc test (C–I). Anp indicates atrial…

Figure 3. Bioinformatics analysis based on RNA‐seq data from the LV myocardium of USP29‐knockout and WT mice subjected to 4 weeks of TAC. A, Diagram of experimental design for RNA‐seq experiment. B, Principal component analysis exhibiting a global gene expression distribution between groups. C, GSEA of the biological process involved in cardiac hypertrophy, protein processing, and cardiac fibrosis. D, Heatmaps exhibiting the significantly differentially expressed genes involved in cardiac hypertrophy, protein processing, and cardiac fibrosis. BP indicates biological process; GO, Gene Ontology; GSEA, gene set enrichment analysis; KO, knockout; NES‚ normalized enrichment score; TAC, transverse aortic constriction; and WT, wild‐type.

Figure 4. Overexpression of USP29 blunts TAC‐induced cardiac hypertrophy. A, The cardiac protein levels of USP29 in mice infected with AAV9‐GFP or AAV9‐USP29 (n=4). B, C Statistical results of HW, HW/BW, LW/BW, HW/TL (B), and LW/BW (C) ratios in AAV9‐GFP or AAV9‐USP29 infected mice subjected to sham or 4 weeks of TAC surgery (n=10). D, Gross hearts, H&E‐stained LV myocardium, and statistical results of cardiomyocyte cross‐sectional area in the indicated groups. Scale bars, 0.3 cm, and 50 μm, respectively. (n=6). E, Statistical results of the LVESd, LVEDd, LV mass cor, IVSd, LVPWd, FS%, and EF% values in the indicated groups (n=10). F, mRNA levels of the hypertrophic markers in the indicated groups (n=4). G, Picrosirius red‐stained LV myocardium and statistical results of LV collagen volume in the indicated groups. Scale bars, 50 μm. (n=6). H, mRNA levels of the fibrotic markers in each group (n=4). # P<0.05, ## P<0.01, ### P<0.001, *P<0.05, **P<0.05, ***P<0.001. Data are presented as mean ± SD. Significant differences were assessed by 2‐tailed Student t test (A) or 1‐way ANOVA with Bonferroni post hoc test (B–H). AAV9 indicates adeno‐associated virus 9; Anp, atrial natriuretic peptide;…

Figure 5. USP29 exacerbates phenylephrine‐induced cardiomyocyte hypertrophy. A, The mRNA levels of USP29 in cultured NRCMs infected with AdshRNA or AdshUSP29 (n=3). B, The protein levels of USP29 in cultured NRCMs infected with AdshRNA or AdshUSP29 (n=3). C, Representative images and statistical results of cell surface areas of actinin‐stained NRCMs infected with AdshRNA or AdshUSP29 and administrated with PBS or 24 hours of phenylephrine (n=60 cells per group). Scale bar, 20 μm. D, The mRNA levels of the hypertrophic markers in cultured NRCVs of each group. (n=3). E, The mRNA levels of USP29 in cultured NRCMs infected with AdGFP or AdUSP29 (n=3). F, The protein levels of USP29 in cultured NRCMs infected with AdGFP or AdUSP29 (n=3). G, Representative images and statistical results of cell surface areas of α‐actinin‐stained NRCMs infected with AdGFP or AdUSP29 and administrated with PBS or 24 hours of phenylephrine (n=60 cells per group). Scale bar, 20 μm. H, The mRNA levels of the hypertrophic markers in cultured NRCVs of each group. (n=3). # P<0.05, ## P<0.01, ### P<0.001, *P<0.05, **P<0.05, ***P<0.001. Data are presented as mean ± SD. Significant differences were assessed by 2‐tailed…

Figure 6. USP29 suppresses hypertrophic stress‐induced activation of the JNK/p38 cascade. A, KEGG pathway enrichment analysis of the DEGs based on RNA‐seq data from the heart of WT and USP29 knockout mice under 4 weeks of TAC surgery (n=3). B, Immunoblot analyses and statistical protein levels of the total and phosphorylated (p‐)TAK1, ERK1/2, p38, and JNK in heart tissue from WT and knockout mice subjected to 4 weeks of TAC surgery (n=3). C, Immunoblot analyses and statistical protein levels of the total and phosphorylated TAK1 and MAPK cascade in heart tissue of AAV9‐GFP and AAV9‐USP29 mice subjected to 4 weeks of TAC surgery (n=3). D, Immunoblot analyses and statistical protein levels of the total and phosphorylated TAK1 and MAPK cascade in cultured NRCMs infected with AdshRNA or AdshUSP29 and administrated with 24 hours of phenylephrine (n=3). E, Immunoblot analyses and statistical protein levels of the total and phosphorylated TAK1 and MAPK cascade in cultured NRCMs infected with AdGFP or AdUSP29 and administrated with 24 hours of phenylephrine (n=3). # P<0.05, ## P<0.01, ### P<0.001, and n.s. Data are presented as mean ± SD. Significant differences were assessed by 2‐tailed Student t…

Figure 7. The cardioprotective effect of USP29 depends on TAK1 signaling. A, Immunoblot analyses and statistical protein levels of USP29, total and phosphorylated TAK1 in NRCMs infected with AdshRNA or AdshUSP29 and treated with DMSO (control) or NG52 (iTAK1, 2.5 μmol/L, 24 hours) and phenylephrine (24 hours). ### P<0.001, •••• P<0.001, ***P<0.001 and n.s. B, Representative images and statistical results of cell surface areas of actinin‐stained NRCMs in the indicated groups (n=60). # P<0.05, *P<0.05 and n.s. C, The mRNA levels of hypertrophic markers in cultured NRCMs of each group (n=3). ## P<0.01, ### P<0.001, ***P<0.05 and n.s. Data are presented as mean ± SD. Significant differences were assessed by 1‐way ANOVA with Bonferroni post hoc test. AdshRNA indicates adenovirus encoding empty short hairpin RNA; AdUSP29, adenoviral vector carrying short hairpin RNA against rat USp29; Anp, atrial natriuretic peptide; α‐MHC, α‐myosin‐heavy‐chain; Bnp, B‐type natriuretic peptide; β‐MHC, β‐myosin‐heavy‐chain; CT, control; DMSO, dimethylsulfoxide; iTAK1, specific inhibitor targeting TAK1; KO, knockout; NRCMs, neonatal rat cardiomyocytes; n.s., no significance; PE, phenylephrine; TAK1, transforming…

Figure 8. USP29 interacts with and negatively regulates TAK1 activation through K63 deubiquitination. A, Coimmunoprecipitation of TAK1 was performed with anti‐HA and probed by Immunoblot with anti‐Flag (left); Coimmunoprecipitation of USP29 was performed with anti‐Flag and probed by Immunoblot with anti‐HA (right). B, Coimmunoprecipitation of exogenous USP29 was performed with anti‐Flag and probed by immunoblot with anti‐TAK1 in cultured NRCMs; IgG antibody was used as a negative control. C, GST pulldown assays for the interaction of purified Flag‐TAK1 and GST‐HA‐USP29 (left); GST pulldown assays for the interaction of purified Flag‐USP29 and GST‐HA‐TAK1 (right). D, Immunoblot analyses of total and phosphorylated TAK1 in NRCMs infected with different amounts of AdFlag‐USP29 and treated with phenylephrine. E, The ubiquitination of TAK1 was analyzed by immunoblot in HEK‐293T cells transfected with Flag‐USP29, Myc‐Ub, and HA‐TAK1 plasmids. F, The ubiquitination of TAK1 in HEK‐293T cells transfected with Flag‐USP29, Myc‐WT‐Ub, and Myc‐mutant ubiquitin (K6O, K11O, K27O, K29O, K33O, K48O, and K63O) plasmids. G, The ubiquitination of TAK1 in HEK‐293T cells transfected with Flag‐USP29, Myc‐WT‐Ub,…

Figure 9. The region (aa 284 to 922) of USP29 is essential for its antihypertrophic effects based on deubiquitination. A, Schematic representation of the WT and deletion mutants of USP29 (upper left); coimmunoprecipitation of the WT and deletion mutants of USP29 was performed with anti‐Flag and probed by immunoblot with anti‐HA (lower left); schematic representation of the WT and deletion mutants of TAK1 (upper right); coimmunoprecipitation of the WT and deletion mutants of TAK1 was performed with anti‐HA and probed by immunoblot with anti‐Flag (lower right). B, The ubiquitination of TAK1 was analyzed by immunoblot in HEK‐293T cells transfected with Myc‐Ub, HA‐TAK1, Flag‐WT USP29, or Flag‐mutant USP29(1–283) plasmids. C, Immunoblot analyses of total and phosphorylated TAK1 in NRCMs that were infected with AdVector, AdFlag‐USP29m or AdFlag‐mutant USP29(1–283) and treated with phenylephrine. D, Representative images and statistical results of cell surface areas of actinin‐stained NRCMs infected with AdVector, AdUSP29, or AdUSP29(1–283) and administrated with 24 hours of phenylephrine (n=60 cells per group). Scale bar, 20 μm. E, The mRNA levels of the hypertrophic markers in cultured NRCVs of…

Figure 10. Schematic of the molecular mechanisms underlying the antihypertrophic effects of USP29. Hypertrophic stimuli upregulate USP29, which deubiquitinates TAK1 in K63‐linked polyubiquitination chains to blunt phosphorylated activation of TAK1. Deub indicates deubiquitination; E3, ubiquitin ligase, JNK, c‐JunN‐terminal kinase; PE, phenylephrine; TAK1, transforming growth factor‐β‐activated kinase 1; UB, ubiquitination; and USP29, ubiquitin‐specific protease 29.