Abstract Background Pathological cardiac hypertrophy, characterized by the involvement of multiple regulators, ultimately leads to heart failure in the absence of effective interventions. The identification of key factors involved is crucial for exploring novel treatments for heart failure. However, the function and pathological implications of USP29 (ubiquitin‐specific protease 29) in cardiomyocytes remain unknown. Methods and Results The impacts of USP29 on pathological cardiac hypertrophy were investigated through the use of knockout/overexpression mice and overexpression/knockdown cardiomyocytes, accompanied by bioinformatic analysis and multiple molecular biological techniques to elucidate the underlying mechanisms. We observed upregulation of USP29 protein levels in both transverse aortic constriction‐induced hypertrophic hearts (upregulated by 159.8%) and phenylephrine‐induced hypertrophic cardiomyocytes (upregulated by 184.6%). Moreover, genetic knockout of USP29 in mice exacerbated transverse aortic constriction‐induced heart hypertrophy, dysfunction, and fibrosis, whereas overexpression of USP29 in cardiomyocytes using adeno‐associated virus 9 effectively attenuated the hypertrophic response. Similarly, USP29 alleviated phenylephrine‐induced hypertrophy of primary neonatal rat cardiomyocytes. Mechanistically, the cardioprotective effects mediated by USP29 were attributed to its suppression of TAK1 (transforming growth factor β‐activated kinase 1) activation. Further molecular analysis revealed that USP29 directly interacts with TAK1 through amino acids 284 to 922 of USP29 and amino acids 1 to 306 of TAK1, subsequently inhibiting TAK1 activation via K63‐linked deubiquitination, which is indispensable for regulating cardiac hypertrophy by USP29. Conclusions Here, we have identified USP29 as a novel negative regulator of pathological cardiac hypertrophy. Our findings suggest that targeting either USP29 or its interaction with TAK1 could represent an innovative therapeutic strategy for treating heart failure and cardiac hypertrophy. Keywords: deubiquitination, pathological cardiac hypertrophy, TAK1, USP29 Subject Categories: Heart Failure, Hypertrophy, Remodeling __________________________________________________________________ Nonstandard Abbreviations and Acronyms DUB deubiquitinating enzyme JNK c‐Jun N‐terminal kinase MAPK mitogen‐activated protein kinase NRCM neonatal rat cardiomyocyte PCH pathological cardiac hypertrophy TAC transverse aortic constriction TAK1 transforming growth factor‐β‐activated kinase 1 WT wild‐type Clinical Perspective. What Is New? * USP29 (ubiquitin‐specific protease 29) protein expression levels were markedly upregulated in the left ventricles in response to pressure overload. * USP29 plays a protective role in pathological cardiac hypertrophy after pressure overload and protects against pathological cardiac hypertrophy by potentiating the K63‐linked deubiquitination of TAK1 (transforming growth factor‐β‐activated kinase 1) to block the TAK1‐dependent signaling pathway. What Are the Clinical Implications? * Our findings suggest that targeting either USP29 or its interaction with TAK1 could represent an innovative therapeutic strategy for treating heart failure and cardiac hypertrophy. After a period of hypertrophic stress (eg, hypertension, myocardial infarction, aortic stenosis, and aortic regurgitation), pathological cardiac hypertrophy (PCH) occurs with characteristics of the increments in heart size and cardiomyocyte size, extracellular fibrosis, functional deterioration of the myocardium, and abnormal cardiac gene expression.[39] ^1 PCH is a common harbinger of heart failure, predicting adverse cardiovascular events such as arrhythmias and sudden death for the majority of patients.[40] ^2 Despite recent advancements in this field, fully elucidating the molecular mechanisms underlying PCH and heart failure remains challenging. The limited understanding of these mechanisms hinders the development of effective treatments for heart failure. The ubiquitin proteasome system, comprising ubiquitin‐activating enzymes, ubiquitin‐conjugating enzymes, E3s (ubiquitin ligases), proteasomes, and deubiquitinating enzymes (DUBs), regulates proteasomal degradation of proteins, chromatin remodeling, DNA repair, endocytosis, kinase activation, and transcription regulation.[41] ^3 , [42]^4 Ubiquitination is a posttranslational modification process wherein ubiquitin molecules are activated, conjugated, and ligated to substrate proteins by the aforementioned enzymes. The ubiquitin molecule consists of 7 lysine residues and 1 methionine residue. Through interlinking different residues, ubiquitin molecules can form diverse types of polyubiquitinated chains (K6, K11, K27, K29, K33, K48, K63, and M1).[43] ^5 , [44]^6 Various polyubiquitinated chains result in distinct fates for substrate proteins. For instance, K11 and K48‐related polyubiquitinated chains often target protein for proteasomal degradation,[45] ^3 , [46]^4 K63‐linked polyubiquitinated chains may activate kinases or facilitate intracellular trafficking of modified proteins.[47] ^7 On the other hand, DUBs catalyze deubiquitination by removing ubiquitinated chains from ubiquitinated protein to regulate the activity, stability, and subcellular localization of target substrates.[48] ^8 , [49]^9 Strikingly, the recent emergence of roles played by DUBs superfamily in PCH has expanded our knowledge.[50] ^10 , [51]^11 However, more than 100 DUBs have been discovered in humans,[52] ^12 and our understanding regarding the pathophysiological implications associated with other DUBs in these afflictions is still in infancy. USP29 (ubiquitin‐specific protease 29) is a conventional deubiquitinase that belongs to the largest DUB subfamily.[53] ^13 Multiple biological functions and complex disease pathways have been found to be regulated by USP29. For example, USP29 deubiquitinates cGAS (cyclic GMP‐AMP synthase) in K48‐linked polyubiquitin chains and stabilizes cGAS to enhance cellular antiviral responses and autoimmunity in response to herpes simplex virus 1 infection.[54] ^14 Conversely, USP29 suppresses ORF9b (open reading frame 9b) degradation through its deubiquitinase activity to promote SARS coronavirus‐2 infection.[55] ^15 In addition to its involvement in antiviral responses and autoimmunity, USP29 is well known for its role in tumorigenesis.[56] ^16 , [57]^17 , [58]^18 , [59]^19 , [60]^20 Qian et al. demonstrated that USP29 cooperates with phosphatase SCP1 (small C‐terminal domain phosphatase 1) to promote gastric cancer cell migration by deubiquitinating and stabilizing Snail protein.[61] ^16 Furthermore, USP29 has been implicated in sorafenib resistance in hepatocellular carcinoma.[62] ^20 Moreover, USP29 has been reported to participate in the cellular response mechanisms related to DNA damage caused by DNA replication and oxidative stress, respectively by deubiquitinating and stabilizing Claspin[63] ^21 and p53.[64] ^22 Additionally, USP29 has been implicated in the regulation of repair processes following traumatic spinal cord injury[65] ^23 and cerebral ischemia/reperfusion injury.[66] ^24 However, less is known regarding the role of USP29 in PCH. Interestingly, several tumor regulatory factors play a pivotal role in modulating PCH,[67] ^25 , [68]^26 , [69]^27 and various USPs have been reported to contribute to the pathogenesis of PCH.[70] ^10 , [71]^28 , [72]^29 Therefore, it is reasonable to hypothesize that USP29 may be functionally involved in PCH; however, there is currently insufficient evidence supporting this hypothesis. Thus, we conducted comprehensive in vivo and in vitro studies investigating the effects of manipulating USP29 expression levels on PCH development. Methods All data and more detailed information on materials and methods will be made available to researchers at any time upon request. Animals This study's animal experiments were authorized by Shengjing Hospital and China Medical University's Ethics Committee (Approval No. 2022PS831K). All animal experiments were conducted according to the National Institutes of Health G uidelines for Laboratory Animal Care and Use. The experimental procedures employed in this study adhered strictly to the principle of randomization. Production of USP29‐Knockout Mice Guidance sequence targeting the ninth exon of the mouse USP29 gene (sgRNA1: TGATAATGTTACAGGCGTAG, sgRNA2: AGTGGTATTCAGGGATCCAC) was predicted using a CRISPR‐based design online tool ([73]http://chopchop.cbu.uib.no/). The pUC57‐sgRNA (Addgene, 51 132) and aforementioned sgRNAs were used to create the USP29‐sgRNA expression vector. A mixture of in vitro transcribed Cas9 expression vector products pST1374‐Cas9 (Addgene 44 758) and sgRNA vector was injected into single‐cell fertilized eggs from C57BL/6 mice using a FemtoJet 5247 microinjection system. F0 generation mice were obtained after implanting the fertilized eggs into the oviducts of pseudopregnant foster mothers for a period of 19 to 21 days. DNA was isolated from the mice's toe tissue 2 weeks following birth, and mice were genotyped using these primers: USP29‐check F1: 5′‐ TGGCTCACCTAAAGATAAATGG‐3′, USP29‐check R1: 5′‐ TCACAGCTTTCTCTGCTTTG‐3′. Adeno‐Associated Virus 9 Virus‐Based Gene Overexpression in Mice As previously described,[74] ^30 adeno‐associated virus 9 (AAV9)‐USP29 and AAV9 control vectors (AAV9‐GFP [green fluorescent protein]) were created. Using infusion recombination at NheI and MluI restrictive splice sites, the mouse USP29 coding sequence was cloned into a pAAV vector that harbors a cTnT (cardiac troponin T) promoter. After that, cotransfection of a recombinant plasmid, pAAV‐RC, and Helper was performed in HEK293T cells. After 3 days of purification, AAV9‐USP29 and AAV9‐GFP virus titers were determined by quantitative real‐time (RT) fluorescence polymerase chain reaction (PCR). C57BL/6J male mice were randomly selected for virus injection 2 weeks before transverse aortic constriction (TAC) surgery, in which 7.5 × 10[75] ^11 vg (virus particles) AAV9‐USP29 or AAV9‐GFP virus was administered into the lateral tail vein of the mice. The relevant primers: Gene Primer sequence(5′‐3′) AAV9‐USP29 F CACGCTTAACTAGCTAGCGCCACCATGGCTCACCTAAAGATAAATGGTTTGG R ATCCTTGTAATCACGCGTGAATTCTTCCAGGATGTATGTAAGGCC [76]Open in a new tab Mouse Transverse Aortic Constriction Surgery Male mice aged between 9 and 11 weeks old with genetic modification (USP29‐knockout) and their littermate wild‐type (WT), weighing approximately 25.5 to 27 g, were chosen at random for this study. Following administration of anesthesia without any noticeable response from their toes, the mice were carefully positioned on an automatically regulated heating pad set at 37 °C. The procedure entailed creating an obstruction at the aortic arch level, between the brachiocephalic artery and left common carotid artery, by ligating the mouse's aorta using 7–0 silk sutures around a 26‐gauge blunt needle. Sham control group consisted of mice subjected to all surgical steps except for actual constriction of their thoracic arteries. All surgical procedures described were performed in a blinded manner. Echocardiography was employed post‐TAC surgery to confirm the pressure gradient across the aortic stenosis. The efficacy of the modeling approach was further substantiated in subsequent phenotypic assessment. Echocardiographic Analyses Echocardiographic analysis was performed after 4 weeks of surgery. After inhaling 1.5% to 2% isoflurane, mice were rendered unconscious and placed supine on a thermostat plate. Using small animal ultrasonography (VEVO2100, FUJIFILM) and a probe (MS400, 30‐MHz), left ventricular (LV) volume and LV posterior wall diastolic thickness were obtained from the papillary muscles in 3 successive cardiac cycles across M‐mode. Then, LV end‐systolic diameter, interventricular septal thickness, and LV end‐diastolic diameter were determined, respectively. The peak early diastolic transmitral flow velocity (E), late diastolic transmitral flow velocity (A), and mitral annular early diastolic velocity (E') was measured using pulsed‐wave Doppler and tissue Doppler imaging in the apical 4‐chamber view. The evaluation of cardiac function involved the computation of fractional shortening, ejection fraction, E/A ratio, and E/E' ratio. Animal Sampling Mice were euthanized 4 weeks following TAC surgery and weighed to record body weight. The hearts of the mice were removed and weighed, then placed in a solution containing 10% KCl to cease heartbeats during the diastolic phase. Subsequently, heart tissues were stored in liquid nitrogen or processed with 10% formalin and embedded in paraffin. After they were excised, the weight of lungs and the length of tibias were measured, respectively. Histological Analyses After undergoing a 72‐hour fixation in paraffin, the paraffin blocks were sliced into sections that were 7 micrometers thick. These sections were then subjected to staining using hematoxylin (G1004, Servicebio) and eosin (BA‐4024, Baso), wheat germ agglutinin ([77]W11261, Invitrogen), DAPI (BMU107‐CN, Abbkine), or picrosirius red (26357–02, Hedebiotechnology) for analyzing morphology and collagen deposition detection respectively. Image‐Pro Plus 6.0 was used to analyze collagen fiber content and cardiomyocyte cross‐sectional area. Western Blot Assay Briefly, to extract proteins from LV tissue or cell samples, a lysis buffer consisting of RIPA buffer, complete protease inhibitor cocktail, phenylmethylsulfonyl fluoride, Phos‐stop, NaF, and Na3VO4 was used. The supernatant was assessed for total protein content using BCA protein reagent. Protein extracts were separated through 10% SDS‐PAGE and transferred to polyvinylidene fluoride membranes that were blocked with skim milk powder. Primary antibodies were incubated overnight at 4 °C followed by secondary antibodies and visualization with ECL reagent. Standardization was done using GAPDH and data analysis was performed using Imaging Lab software. Relevant antibody data can be found in Table [78]S1. Real‐Time Polymerase Chain Reaction TRIzol reagent (15596–026, Invitrogen) was used to isolate total RNA from LV tissues or cell samples. Two micrograms RNA were reverse‐transcribed into first‐strand cDNA using Transcriptor 1st Strand cDNA Synthesis reagent (04896866001, Roche). Subsequently, PCR amplification was conducted usingSYBR Green Mix PCR Master (04887352001, Roche). Target gene expression levels were standardized against GAPDH, and Table [79]S2 presents the related gene primer sequences. Adenovirus Vector Construction The full‐length cDNA of USP29 from rats was inserted into a replication‐deficient adenoviral vector containing a cytomegalovirus promoter (AdUSP29), and AdGFP (adenoviral vector expressing green fluorescent protein) was used as a control. To silence the expression of the USP29 gene, an adenoviral vector carrying short hairpin RNA against rat USp29 (AdshUSP29) was employed, and AdshRNA served as a nontargeting control. The multiplicity of infection 50 particles/cell adenovirus was used to infect cardiomyocytes for 24 hours, and then the expression of USP29 was detected by western blotting. The virus primers were as follows: AdshUSP29‐rat‐F: CCGGGCGCAACAATTGGAAGCTTGACTCGAGTCAAGCTTCCAATTGTTGCGCTTTTTG. AdshUSP29‐rat‐R: AATTCAAAAAGCGCAACAATTGGAAGCTTGACTCGAGTCAAGCTTCCAATTGTTGCGC. AdUSP29‐rat‐F: GGCTAGCGATATCGGATCCGCCACCATGGCTCACCTAAAGATACATGGTTTG. AdUSP29‐rat‐R: CGTCCTTGTAATCACTAGTGAACTCTTCTAGGATATATGTAAGGCCAT. Plasmid Constructs Using mouse cDNA as a template, the CDS sequences of USP29 and TAK1 genes were identified through PCR‐based cloning and were then constructed into mammalian overexpression vectors pcDNA5‐Flag, pcDNA5‐Myc, pcDNA5‐HA, and pcDNA5‐GST (glutathione S‐transferase)‐HA (hemagglutinnin) via recombination. pcDNA5‐Myc‐ubiquitinated and USP29/TAK1 gene truncated plasmids (PcDNA5‐Flag‐USP29(1‐283aa), pcDNA5‐Flag‐USP29(284‐922aa), pcDNA5‐HA‐TAK1 (1‐306aa), pcDNA5‐HA‐TAK1 (307‐480aa), pcDNA5‐HA‐TAK1 (481‐579aa)) were also designed and constructed in the same way. Table [80]S3 lists these construct primers. All plasmids were validated through sequencing. Primary Neonatal Rat Cardiomyocytes Culture After removing vascular components, neonatal Sprague–Dawley rats hearts (1–2 days old) were dissected into 1 to 2 mm^3, and then 0.125% trypsin was used for digestion. Neonatal rat cardiomyocytes (NRCMs) were cultivated for 24 hours in DMEM/F12 ([81]C11330, Gibco) medium, and cardiomyocytes were then infected with adenovirus. Six hours after infection, starvation of cells occurred in serum‐free medium for 12 hours, followed by stimulation with 50 μM phenylephrine (PHR1017, Sigma) or TAK1 inhibitor (iTAK1)‐NG52 (2.5 μmol/L, HY‐15434, MCE) for 24 hours. The control group received the same volume of PBS. The entire cell culture process was carried out under conditions of 5% CO[2] and a temperature of 37 °C. Cardiomyocytes Immunofluorescence Staining NRCMs were inoculated on slides placed in a 24‐well plate. Following treatment with phenylephrine or PBS for 24 hours, the cardiomyocytes were fixed using 4% formaldehyde (G1101, Servicebio) for a duration of 30 minutes. Subsequently, permeabilization was carried out using 0.2% Triton X‐100 and blocking was performed at 37 °C using an 8% goat serum (BMS0050, Abbkine). The cardiomyocytes were then subjected to pretreatment with α‐actinin antibody at a dilution of 1:100 (05–384, Merck Millipore), followed by incubation with donkey antimouse IgG [H + L] secondary antibody at a dilution of 1:200 (A21202, Invitrogen). Finally, the slides containing the aforementioned cardiomyocytes were covered with a cover glass containing DAPI (nuclear staining), and Image‐Pro Plus 6.0 assessed the cardiomyocyte surface area. RNA Sequencing and Analysis Three mice were randomly selected from each group. We isolated total RNA from cardiac tissues of USP29‐knockout and WT mice that were subjected to 4 weeks of TAC. Subsequently, a cDNA library was generated and sequenced using the MGISEQ‐2000 RS system with 50‐bp single‐end reads. To align the sequence fragments to the reference genome for mice (mm10/GRCm38), we employed HISAT2 software. The resulting files were then converted to binary BAM format using SAMtools. Following this, StringTie was used with default parameters to calculate fragments per kilobase of exon model per million mapped fragments for identified genes. DESeq2 was applied to identify differentially expressed genes based on 2 criteria: (1) fold change >2; and (2) adjusted P <0.05. The RNA‐seq data have been deposited in the National Center for Biotechnology Information Sequence Read Archive database (on February 28, 2024) and Gene Expression Omnibus database (on April 15, 2024), the BioProject accession is PRJNA1081174 and the Gene Expression Omnibus accession number is [82]GSE263965. Principal Component Analysis Principal component analysis is a linear dimensionality reduction algorithm and a commonly used data preprocessing method. Using the fast.prcomp function in the R‐package, principal component analysis was performed to analyze the gene expression differences in heart tissues of USP29‐KO and WT treated with TAC, the result of which was visualized with R‐package GGPLOT2. Gene Set Enrichment Analysis Changes in signaling pathways involved in biological processes in heart tissues between USP29‐knockout and WT mice under TAC treatment were analyzed using gene set enrichment analysis GSEA. The aforementioned RNA‐seq data were applied to gene set enrichment analysis and calculated by enrichment score values. The Gene Ontology set in the National Center for Biotechnology Information database was conducted on gene set enrichment analysis java program, P value <0.05 and false discovery rate <0.25 was regarded as statistically significant. Kyoto Encyclopedia of Genes and Genomes Analysis To further understand the enrichment pathways of differentially expressed genes, USP29 ablation induced 362 differentially expressed genes were subjected to pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes database. Significantly enriched pathways were defined as a P value <0.05 and visualized with the R‐package GGPLOT2. Coimmunoprecipitation Briefly, cotransfection of indicated plasmids into cultured HEK293T cells or coinfection of NRCMs with corresponding adenovirus. After 24 hours, the cells were lysed using immunoprecipitation lysis buffer (containing 20 mM Tris–HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; and 1% NP‐40). The resulting supernatants were collected by high‐speed centrifugation and then incubated overnight at 4 °C with protein G agarose beads and specific antilabel antibodies as indicated. Subsequently, the beads were pelleted by centrifugation at 1000g at 4≤°C and washed 3 times with buffers containing either 300 or 150 mM NaCl. Finally, the beads were resuspended in boiling SDS loading buffer (2×) at a temperature of 95 °C for a duration of approximately 5 to 10 minutes. Ultimately, immunoblotting was performed to test and analyze the proteins obtained from this procedure. Table [83]S3 presents the plasmid primer information. Ubiquitination Assay Co‐transfection of the indicated plasmids into HEK293 T cells, which were lysed in lysis buffer (a solution composed of 80 μL of 150 mM immunoprecipitation buffer and 10 μL of 10% SDS) and subsequent denaturation for 10 minutes (95 °C). Subsequently, the lysates were added to 900 μL of 150 mM IP lysis buffer. Through sonicating and centrifuging for 15 minutes (12 000g), the supernatants were collected and incubated with specific antibodies and protein G‐agarose beads at a temperature of 4 °C for an interval spanning over 3 hours. Next, using 500 mM immunoprecipitation lysis buffer (20 mM Tris–HCl, pH 7.4; 1% NP‐40: 500 mM NaCl; and 1 mM EDTA), beads were rinsed 3 times and centrifugated for 2 minutes at 1000g. Finally, immunoblotting analysis was performed on the beads after heating them in 2× SDS loading buffer at 95 °C for 10 minutes. GST Pulldown The fusion proteins Flag‐USP29, GST‐HA‐TAK1, Flag‐TAK1, and GST‐HA‐USP29 that were overexpressed in HEK293T cells underwent lysis using a lysis buffer (composed of 50 mM Na2HPO4 at pH 8.0, 300 mM NaCl, 1% TritonX‐100, and a cocktail). Following purification with GST beads, the purified Flag‐USP29 and GST‐HA‐TAK1 or Flag‐TAK1 and GST‐HA‐USP29 were combined and incubated overnight at 4 °C. Subsequently, the immunocomplex was subjected to 3 washes using buffers containing 20 mM Tris–HCl, 150 mM NaCl, and 0.2% TritonX‐100. The resuspended immunocomplex was then mixed with a solution of 2× SDS loading buffer before being heated for a duration of 5 to 10 minutes at a temperature of 95 °C. Finally, protein analysis was conducted through immunoblotting. Statistical Analysis Data analysis was conducted using SPSS (version 25.0). Data are described as mean ± SD and visualized using GraphPad Prism 8.0. In animal experiments, n ≥ 6 were used for pathology studies or functional analysis. In the cell experiments, at least 3 independent replications were performed. All data were subjected to the normality test. The means of 2‐group samples were compared using 2‐tailed Student's t tests (when data are normal distribution). One‐way ANOVA with Bonferroni post hoc test (for data with homogeneous variances, normal distribution) and Kruskal–Wallis test (for data with skewed distribution) were used to test differences among more than 2 groups. P<0.05 was considered to indicate statistical significance. Results The USP29 Protein Expression Is Upregulated in Murine Hypertrophic Hearts and Cardiomyocytes To investigate the association between USP29 and PCH, our study commenced by examining the expression of USP29 in hypertrophic hearts and cardiomyocytes. WT mice were subjected to either TAC or a sham procedure for 4 weeks. Cardiac hypertrophy was evaluated through measurements of heart weight (HW), HW/body weight (BW) ratio, cross‐sectional area values of cardiomyocytes, LV end‐diastolic diameter, LV end‐systolic diameter, LV fractional shortening, and ejection fraction (Figure [84]1A through [85]1C). RT‐PCR analysis revealed no significant changes in USP29 transcription levels between TAC‐induced hypertrophic mouse hearts and sham group mouse hearts, as well as between NRCMs treated with phenylephrine (phenylephrine; 50 μM, 24 hours) and PBS. (Figure [86]1D through [87]1F). Interestingly, western blotting data demonstrated elevated levels of USP29 protein in TAC‐induced hypertrophic mouse hearts compared with sham group hearts (Figure [88]1E). Similarly, phenylephrine treatment significantly increased USP29 protein levels in NRCMs compared with PBS‐treated NRCMs (Figure [89]1G). Taken together, these expression alterations imply that Usp29 may participate in the development of PCH. Figure 1. The protein expression of USP29 is upregulated in murine hypertrophic hearts and cardiomyocytes. Figure 1 [90]Open in a new tab 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 29; and WT, wild type. Ablation of USP29 Potentiates TAC‐Induced Cardiac Hypertrophy To investigate the impact of USP29 on cardiac hypertrophy, we generated a mouse strain with a global knockout of USP29 (knockout) (Figure [91]2A) and confirmed the absence of USP29 protein in this strain (Figure [92]2B). Subsequently, both knockout and control mice (littermate WT mice) were subjected to either TAC or sham procedure, followed by comprehensive evaluation of the hypertrophic phenotype in the respective groups. Notably, no detectable abnormalities were observed in USP29‐deficient mice after sham operation (Figure [93]2C through [94]2I). However, increased HW, HW/BW ratio, HW/tibia length ratio, gross heart sizes, and cross‐sectional area values of cardiomyocytes indicated that USP29 deficiency accentuated TAC‐induced increment of heart weight and cardiomyocyte size (Figure [95]2C and [96]2E). Echocardiography data further substantiated severe ventricular hypertrophy characterized by wall thickening, and ventricular expansion as evidenced by increased interventricular septal thickness, LV posterior wall diastolic thickness, LV end‐diastolic diameter, and LV end‐systolic diameter in Usp29‐knockout mice compared with WT mice after 4 weeks of TAC (Figure [97]2F). Additionally, an anabatic lung weight/body weight ratio indicative of aggravated pulmonary congestion resulting from LV dysfunction was observed in Usp29‐knockout mice relative to WT controls (Figure [98]2D). This finding is consistent with worsened TAC‐induced impairment of LV fractional shortening and ejection fraction in Usp29‐knockout mice compared with WT controls (Figure [99]2F). LV diastolic function was evaluated by assessing the mitral inflow early‐to‐late diastolic flow (E/A) ratio, and the ratio of early diastolic transmitral flow velocity to tissue Doppler mitral annular early diastolic velocity (E/E'). After 4 weeks of TAC, Usp29‐knockout mice exhibited increased E/A ratio and E/E' ratio compared with WT mice, indicating that USP29 knockout exacerbates the impairment of LV diastolic function (Figure [100]2F). At the molecular level, knockout of USP29 markedly augmented mRNA expression levels of Anp (atrial natriuretic peptide), Bnp (B‐type natriuretic peptide), and β‐MHC (myosin heavy chain beta; hypertrophic markers), while reducing α‐MHC (antihypertrophic marker) expression levels (Figure [101]2G). Figure 2. Ablation of USP29 potentiates TAC‐induced cardiac hypertrophy. Figure 2 [102]Open in a new tab 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 natriuretic peptide; α‐MHC, α‐myosin‐heavy‐chain; Bnp, B‐type natriuretic peptide; β‐MHC, β‐myosin‐heavy‐chain; Col1a1, collagen 1α1; Col3a1, collagen 3α1; Col8a1, collagen 8α1; Ctgf, connective tissue growth factor; E/A, early‐to‐late diastolic flow; E/E', ratio of early diastolic transmitral flow velocity to tissue Doppler mitral annular early diastolic velocity; EF, ejection fraction; FS, fractional shortening; H&E, hematoxylin–eosin; HW/BW, heart weight/body weight; HW/TL, heart weight /tibia length; IVSd, interventricular septal thickness; KO, knockout; LVEDd, left ventricle end‐diastolic diameter; LVESd, left ventricle end‐systolic diameter; LVPWd, left ventricular posterior wall diastolic thickness; LW/BW, lung weight/body weight; n.s., no significance; PSR, picrosirius red; TAC, transverse aortic constriction; USP29, ubiquitin‐specific protease 29; and WT, wild‐type. Fibrosis is a prominent characteristic of PCH. In the current study, cardiac fibrosis caused by pressure overload was explored by picrosirius red staining. Figure [103]2H demonstrates that Usp29‐knockout mice exhibited elevated LV perivascular and interstitial collagen volumes compared with WT mice after 4 weeks of TAC. Consistently, the transcriptional level of the cardiac fibrosis markers including collagen 1α1, collagen 3α1, collagen 8α1, and Ctgf (connective tissue growth factor) were upregulated in Usp29‐knockout mice relative to WT mice after TAC (Figure [104]2I). Four weeks post TAC surgery, RNA‐seq analyses were performed to characterize gene expression changes in the LV myocardium of USP29‐knockout and WT mice (Figure [105]3A). Principal component analysis of transcriptomes revealed distinct clustering of gene expression profiles between hearts of USP29‐knockout and WT mice, indicating a global shift in gene expression patterns (Figure [106]3B). Applying fold change (>1.5) and adjusted P value (<0.05) as thresholds, we identified 222 upregulated genes and 140 downregulated genes specifically in the hearts of USP29‐knockout mice compared with those in WT mice. Furthermore, gene set enrichment analysis (Figure [107]3C) along with heat map visualization (Figure [108]3D), confirmed significant upregulation of cardiac hypertrophy‐related genes, protein processing‐associated genes, and fibrosis‐related genes in the hearts of USP29‐knockout mice. These findings align with our previously reported RT‐PCR data on hypertrophic markers and fibrotic markers (Figure [109]2G and [110]2I). Taken together, these data suggested that USP29 deficiency sensitizes TAC‐treated hearts to hypertrophy while indicating its protective role against pressure overload‐induced myocardial hypertrophy. 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. Figure 3 [111]Open in a new tab 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. Overexpression of USP29 Blunts TAC‐Induced Cardiac Hypertrophy To further substantiate the cardioprotective effect of USP29 in cardiac hypertrophy, we generated cardiac‐specific USP29 overexpression mice using AAV9 vector carrying the cTnT promoter‐controlled USP29 gene (AAV9‐USP29). Control mice were infected with AAV9 vector expressing GFP (AAV9‐GFP). Western blot verified the overexpression of USP29 in AAV9‐USP29 mice (Figure [112]4A). Figure 4. Overexpression of USP29 blunts TAC‐induced cardiac hypertrophy. Figure 4 [113]Open in a new tab 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; α‐MHC, α‐myosin‐heavy‐chain; Bnp, B‐type natriuretic peptide; β‐MHC, β‐myosin‐heavy‐chain; Col1a1, collagen 1α1; Col3a1, collagen 3α1; Col8a1, collagen 8α1; Ctgf, connective tissue growth factor; E/A, early‐to‐late diastolic flow; E/E', ratio of early diastolic transmitral flow velocity to tissue Doppler mitral annular early diastolic velocity; EF, ejection fraction; FS, fractional shortening; GFP, green fluorescent protein; H&E, hematoxylin–eosin; HW/BW, heart weight/body weight; HW/TL, heart weight/tibia length; IVSd, interventricular septal thickness; LVEDd, left ventricle end‐diastolic diameter; LVESd, left ventricle end‐systolic diameter; LVPWd, left ventricular posterior wall diastolic thickness; LW/BW, lung weight/body weight; NRCM, neonatal rat cardiomyocytes; n.s., no significance; TAC, transverse aortic constriction; and USP29, ubiquitin‐specific protease 29. Neither AAV‐USP29 transfected mice nor AAV9‐vector transfected mice showed any discernible differences in cardiac phenotype at the basal level. However, following 4 weeks of TAC‐induced stress, USP29 overexpression significantly ameliorated cardiac hypertrophy and pulmonary congestion caused by pressure overload, as evidenced by reductions in HW, HW/tibia length ratio, HW/BW ratio, lung weight/BW ratio (Figure [114]4B), gross heart sizes, cardiomyocyte cross‐sectional area (Figure [115]4D) and lung weight/BW ratio (Figure [116]4C) relative to control (AAV9‐GFP) group. Moreover, the observed decrease in interventricular septal thickness, LV posterior wall diastolic thickness, LVEDd, LV end‐systolic diameter, E/A ratio, and E/E' ratio, along with increased fractional shortening, ejection fraction in AAV‐USP29 mice compared with control mice indicated that USP29 overexpression significantly attenuated ventricular hypertrophy, ventricular enlargement, and preserved cardiac contractility and diastolic function induced under pressure overload conditions (Figure [117]4E). We then sought to explore USP29 overexpression's impact on cardiac fibrosis. Picrosirius red staining of heart sections revealed reduced interstitial fibrosis in AAV‐USP29 mice after TAC compared with controls (Figure [118]4G). Consistently supported by RT‐PCR analysis findings demonstrating markedly decreased mRNA levels of cardiac hypertrophic markers (Figure [119]4F) and fibrotic markers (Figure [120]4H), while augmented levels of the antihypertrophic marker α‐MHC upon USP29 overexpression after TAC treatment were observed. USP29 Exacerbates Phenylephrine‐Induced Cardiomyocyte Hypertrophy Gain and loss of function assays were performed in cultured NRCMs to further investigate the role of USP29 in cardiomyocyte hypertrophy. USP29 knockdown and overexpression were achieved by AdshUSP29 and adenoviral USP29 (AdUSP29) infection, respectively. Adenovirus encoding empty short hairpin RNA (AdshRNA) and GFP (AdGFP) were used as controls. The knockdown and overexpression efficiency of USP29 in NRCMs was confirmed by RT‐PCR and western blot analysis (Figure [121]5A, [122]5B, [123]5E and [124]5F). Cardiomyocytes were pretreated for a day with PBS or phenylephrine (50 μM). Subsequently, immunostaining with α‐actinin antibody was performed to assess cell surface areas, and mRNA levels of hypertrophic markers were measured after extraction from the cells. Under basal conditions (PBS treatment), no differences in terms of cell surface area (Figure [125]5C and [126]5G) or mRNA levels of hypertrophic markers (Figure [127]5D and [128]5H) were observed between the groups. However, USP29 knockdown markedly worsened phenylephrine–mediated cardiomyocyte hypertrophy compared with control‐infected NRCMs after phenylephrine treatment, which was reached based on increased cell surface area, enhanced hypertrophic markers expression, and reduced antihypertrophic marker expression in AdshUSP29‐infected NRCMs (Figure [129]5C and [130]5D). Conversely, USP29 overexpression attenuated phenylephrine‐induced cardiomyocyte hypertrophy (Figure [131]5G and [132]5H). These findings demonstrated that USP29 protects from phenylephrine‐induced cardiomyocyte hypertrophy in vitro. Figure 5. USP29 exacerbates phenylephrine‐induced cardiomyocyte hypertrophy. Figure 5 [133]Open in a new tab 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 Student t test (A, B, E, F) or 1‐way ANOVA with Bonferroni post hoc test (C, D, E, H). AdGFP indicates adenoviral vector expressing green fluorescent protein; AdshRNA, 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; GFP, green fluorescent protein; NRCM, neonatal rat cardiomyocytes; n.s., no significance; PE, phenylephrine; and USP29, ubiquitin‐specific protease 29. USP29 Suppresses Hypertrophic Stress‐Induced Activation of the c‐Jun N‐Terminal Kinase /p38 Cascade To investigate the potential mechanisms underlying the protective impact of USP29 in cardiac hypertrophy, we performed a Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis using the aforementioned RNA‐seq data set. The results revealed that USP29 ablation primarily influenced the MAPK (mitogen‐activated protein kinase), PI3K‐Akt, and estrogen signaling pathways (Figure [134]6A). Considering that MAPK signaling pathways are recognized key hubs in PCH,[135] ^31 , [136]^32 we subsequently determined the expression and activity of MAPK signaling molecules, such as P38, ERK1/2 (extracellular signal‐regulated kinase 1/2), and JNK (c‐Jun N‐terminal kinase). In vivo experiments demonstrated significantly increased levels of phosphorylated p38 and JNK proteins in hearts of USP29‐knockout mice compared with WT mice after 4 weeks of TAC‐induced stress; however, there was no statistically significant difference observed in ERK1/2 phosphorylation between groups (Figure [137]6B and [138]6C). Consistently, knockdown of USP29 enhanced phenylephrine‐induced activation of p38 and JNK in cardiomyocytes whereas overexpression of USP29 markedly attenuated their activation; however, no evident alteration was observed in ERK1/2 phosphorylation levels (Figure [139]6D and [140]6E). These results suggested that USP29 may exert protective effects on PCH through negative regulation of JNK/p38 signaling. Figure 6. USP29 suppresses hypertrophic stress‐induced activation of the JNK/p38 cascade. Figure 6 [141]Open in a new tab 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 test (B, C, D, E). AdGFP indicates adenoviral vector expressing green fluorescent protein; AdshRNA, adenovirus encoding empty short hairpin RNA; AdUSP29, adenoviral vector carrying short hairpin RNA against rat USp29; AAV9, adeno‐associated virus 9; DEG, differentially expressed gene; ERK, extracellular signal‐regulated kinase; GFP, green fluorescent protein; HIF‐1, hypoxia‐inducible factor 1; JNK, c‐Jun N‐ terminal kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; KO, knockout; MAPK, mitogen‐activated protein kinase; NF‐κB, nuclear factor kappa; NRCM, neonatal rat cardiomyocytes; n.s., no significance; PE, phenylephrine; TAC, transverse aortic constriction; TAK1, transforming growth factor‐β‐activated kinase 1; USP29, ubiquitin‐specific protease 29; and WT, wild‐type. Cardioprotective Effect of USP29 Depends on TAK1 Signaling Previous studies have reported that TAK1 acts as an upstream kinase, activating JNK1/2 and p38 in response to various stresses, including hypertrophic stress.[142] ^33 , [143]^34 , [144]^35 , [145]^36 Therefore, we speculated that USP29 may modulate JNK/p38 signaling through its interaction with TAK1 during PCH development. Consistent with our hypothesis, USP29 deficiency resulted in increased phosphorylation levels of TAK1 in hypertrophied hearts relative to the WT controls, whereas overexpression of USP29 significantly reduced TAK1 phosphorylation (Figure [146]6B and [147]6C). These findings were further supported by in vitro experiments demonstrating the suppressive effects of USP29 on the activation of TAK1, p38, and JNK in PCH (Figure [148]6D and [149]6E). We then investigated whether inhibition of TAK1 signaling is crucial for the antihypertrophic effect mediated by USP29. To address this question, we employed NG52 as a specific inhibitor targeting TAK1 activity (with DMSO serving as a control) for subsequent experiments. After treating NRCMs with NG52 or DMSO for 24 hours and transfecting them with AdshUSP29 or AdshRNA for 6 hours followed by phenylephrine treatment for another 24 hours; western blot analysis confirmed successful inhibition of TAK1 activity by NG52 even after knockdown of USP29 expression (Figure [150]7A). Furthermore, USP29 silencing augmented phenylephrine‐induced cardiomyocyte enlargement and the deterioration of hypertrophic markers, nevertheless, treatment with the TAK1 inhibitor NG52 remarkably attenuated these hypertrophic responses (Figure [151]7B and [152]7C). Interestingly, TAK1 blockage effectively countervailed the auxo‐action of USP29 knockdown on the hypertrophic phenotype. Collectively, these data demonstrated that activation of TAK1 signaling is essential for the antihypertrophic effect mediated by USP29. Figure 7. The cardioprotective effect of USP29 depends on TAK1 signaling. Figure 7 [153]Open in a new tab 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 growth factor‐β‐activated kinase 1; USP29, ubiquitin‐specific protease 29; and WT, wild‐type. USP29 Interacts With and Negatively Regulates TAK1 Activation Through K63‐Deubiquitination We then further investigated the mechanism by which USP29 negatively regulates TAK1 activation. First, we examined whether USP29 directly inhibited TAK1 activation. Coimmunoprecipitation assays were conducted in HEK293T cells transfected with HA‐tagged TAK1 and Flag‐tagged USP29 plasmids, confirming a physical interaction between USP29 and TAK1 (Figure [154]8A). Additionally, protein interactions between endogenous TAK1 and exogenous USP29 in cultured NRCMs were validated through additional coimmunoprecipitation experiments (Figure [155]8B). Subsequently, GST pulldown assays were performed to rule out the possibility of indirect binding between USP29 and TAK1. Figure [156]8C illustrates that Flag‐tagged TAK1 in cell lysate could be specifically bound and eluted by GST‐USP29 fusion protein but not GST alone. Consistently, Flag‐tagged USP29 could be reciprocally bound and eluted by GST‐TAK1. Figure 8. USP29 interacts with and negatively regulates TAK1 activation through K63 deubiquitination. Figure 8 [157]Open in a new tab 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, and Myc‐mutant ubiquitin (K63O or K63R) plasmids. GST‐HA indicates glutathione S‐transferase‐hemagglutinnin; IP:HA, immunoprecipitation:hemagglutinnin; Myc‐Ub, Myc‐ubiquinated; NRCM, neonatal rat cardiomyocytes; n.s., no significance; PD, Pull down; TAK1, transforming growth factor‐β‐activated kinase 1; USP29, ubiquitin‐specific protease 29; and WT, wild‐type. As mentioned, USP29 is a deubiquitinase that plays an important role in a variety of pathophysiological processes through the process of deubiquitination.[158] ^14 , [159]^16 , [160]^17 , [161]^23 Additionally, it has been established that K63‐linked ubiquitination of TAK1 is essential for its activation.[162] ^37 , [163]^38 Therefore, we speculate that USP29 may inhibit the activation of TAK1 in hypertrophic cardiomyocytes by cleaving K63‐polyubiquitin chains from it. To test this hypothesis, we overexpressed Flag‐tagged USP29 to varying degrees in cultured NRCMs using adenovirus infection and subsequently assessed the activity of TAK1 after treating with phenylephrine for 24 hours. The immunoblot analysis revealed a gradual decrease in phosphorylated TAK1 protein levels with increasing extent of USP29 overexpression (Figure [164]8D). Furthermore, cotransfection experiments conducted in HEK293T cells with Myc‐ubiquitinated and HA‐TAK1 demonstrated that introduction of Flag‐USP29 significantly reduced the level of TAK1 polyubiquitination (Figure [165]8E). In order to elucidate the molecular mechanism underlying USP29‐mediated regulation of TAK1 ubiquitination, we transfected Myc‐tagged K63‐only ubiquitin (K63O ubiquitin variants that contain only K63, whereas all other lysines are mutated to arginines), K6O (K6‐only ubiquitin), K11O (K11‐only‐ubiquitin), K27O (K27‐only‐ubiquitin), K29O (K29‐only‐ubiquitin), K33O (K33‐only‐ubiquitin), K48O (K48‐only‐ubiqutin), K63R (K63‐resistant ubiquitin, ubiquitin variants that K63 are mutated to arginine), Myc‐tagged WT ubiquitin plasmids along with HA‐tagged TAK1 and Flag‐tagged USP29 plasmids into HEK293T cells. The results indicated that only K63‐linked ubiquitination of TAK1 was mitigated by USP29 overexpression, indicating that USP29‐mediated deubiquitination negatively regulates TAK1 activation by targeting its K63‐linked chains (Figure [166]8F and [167]8G). The Region (Aa 284 to 922) of USP29 Is Essential for Its Antihypertrophic Effects Based on Deubiquitination To delineate the binding domains responsible for their association, we generated a panel of deletion mutants for both USP29 and TAK1. Our findings revealed that the region spanning amino acids 284 to 922 in USP29 and amino acids 1 to 306 in TAK1 were indispensable for their protein interaction (Figure [168]9A). Subsequent data demonstrated that the absence of the region spanning amino acids 284 to 922 in USP29 resulted in an inability to attenuate polyubiquitination and phosphorylation levels of TAK1 (Figure [169]9B and [170]9C). Furthermore, it was observed that the mutant form of USP29 failed to confer protection against phenylephrine‐induced cardiomyocyte hypertrophy (Figure [171]9D and [172]9E). In summary, our results indicate that the antihypertrophic effects mediated by USP29 are contingent upon its deubiquitinating activity within the region spanning amino acids 284 to 922. Figure 9. The region (aa 284 to 922) of USP29 is essential for its antihypertrophic effects based on deubiquitination. Figure 9 [173]Open in a new tab 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 each group. (n=3). *P<0.05, **P<0.05, ***P<0.001. Data are presented as mean ± SD. Significant differences were assessed by Kruskal–Wallis test (D) or 1‐way ANOVA with Bonferroni post hoc test (E). Anp indicates atrial natriuretic peptide; α‐MHC, α‐myosin‐heavy‐chain; Bnp, B‐type natriuretic peptide; β‐MHC, β‐myosin‐heavy‐chain; IP:HA, immunoprecipitation:hemagglutinnin; Myc‐Ub, Myc‐ubiquinated; NRCM, neonatal rat cardiomyocytes; n.s, no significance; PE, phenylephrine; PTM, posttranslational modification; TAK1, transforming growth factor‐β‐activated kinase 1; USP29, ubiquitin‐specific protease 29; and WT, wild‐type. Discussion In this study, we have identified USP29 as a negative regulator of PCH. The USP29 protein expression was significantly upregulated in hypertrophic hearts and cardiomyocytes, suggesting its potential role in cardiac hypertrophy. Further exploration uncovered that the absence of USP29 remarkably aggravated cardiac hypertrophy, fibrosis, and LV dysfunction induced by TAC, as well as phenylephrine‐induced cardiomyocyte hypertrophy, and USP29 overexpression yielded the opposite response. Mechanistically, we substantiated that after TAC or phenylephrine treatment, USP29 exerts its antihypertrophic effects by suppressing TAK1‐JNK/p38 signaling. Specifically, in the context of cardiac hypertrophy, USP29 directly interacts with TAK1 through its binding region (aa 284 to 922), deubiquitinating TAK1 in its K63‐linked polyubiquitination chain within the region (aa 1 to 306) of TAK1 itself. This interaction inhibits the activation of TAK1 autophosphorylation and ultimately represses JNK/p38 signaling. Collectively, our findings provide novel evidence for the pivotal role played by USP29 in PCH development and suggest reintroducing endogenous USP29 as a potential therapeutic strategy for heart failure. A previous study by Liu et al. reported that endogenous JTV1 migrates into the nucleus and associates with the nuclear FBP (far upstream element binding protein) to initiate USP29 transcription and then upregulate its protein level in response to oxidative stress.[174] ^22 However, our study observed an upregulation of USP29 protein expression levels as a negative regulator of cardiac hypertrophy in response to hypertrophic stimuli, without any concurrent alteration in USP29 mRNA levels. This phenomenon raises 2 noteworthy points. First, the upregulation of USP29 is consistent with other negative cardiac regulators of PCH[175] ^27 , [176]^39 , [177]^40 and may be interpreted as a compensatory defensive response of the heart against excessive hypertrophic stress. Second, the discrepancy between the USP29 protein expression and transcription suggests that hypertrophic stimuli significantly enhanced USP29 protein stability, through posttranscriptional modifications induced by pathological stimuli. Recent research by Hou et al. demonstrated that USP29 self‐stabilizes through deubiquitination to protect itself from proteasomal degradation.[178] ^41 Therefore, it is possible that the upregulation of USP29 during cardiac hypertrophy results from enhanced self‐stabilizing regulation induced by hypertrophic stress, however, these hypotheses still warrant further verification. In investigating the underlying mechanisms by which USP29 restrains cardiac hypertrophy, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and western blotting confirmed the involvement of JNK/p38 signaling in the antihypertrophic role of USP29. We then sought to explore the upstream kinases of p38 and JNK1/2 in this molecular event. TAK1, a vital member of MAPKKK (MAPK kinase kinase) and a common upstream activator of P38 and JNK, has been implicated in regulating PCH.[179] ^42 Furthermore, upon stimulation, TAK1 can phosphorylate MAPKKs (MKK3/6, MKK4/7) and eventually leading to the activation of P38 and JNK.[180] ^43 Therefore, we determined that the activity of TAK1 was enhanced by USP29‐deficient in response to TAC or phenylephrine treatment but abated by USP29‐overexpression. Subsequently, additional rescue experiments using the TAK1 inhibitor NG52 supported the requirement for TAK1–dependent signaling for the antihypertrophic property of USP29. In line with previous research studies,[181] ^42 , [182]^44 , [183]^45 , [184]^46 our findings also demonstrate that TAK1 activation is essential as well as sufficient for the pathological progression of cardiac hypertrophy. We then investigated how USP29 regulates TAK1 through coimmunoprecipitation and GST‐pulldown experiments demonstrating direct interaction between them. Further mapping experiments presented evidence that aa 284 to 922 of USP29 and aa 1 to 306 of TAK1 are responsible for their interaction with each other respectively. Recently, many studies have corroborated the ubiquitination's indispensable role in regulating activation related to TAK1.[185] ^38 For instance, the activated E3 ligase TRAF6 collaborates with E2 enzymes Ubc13/Uev1A facilitate K63‐related polyubiquitin chain formation, recruiting TAB2 and TAB3, resulting in autophosphorylation of TAK1.[186] ^38 , [187]^47 Chen et al. revealed that K63‐linked polyubiquitination of TAK1 is necessary for its phosphorylation and contributes to the activation of P38 and JNK1/2.[188] ^38 On the contrary, deubiquitination in K63‐related polyubiquitin chains can block the phosphorylation and activation of TAK1. USP18 negatively regulates TAK1 activity during Th17 differentiation by interacting with and removing K63‐related ubiquitin moieties from TAK1.[189] ^48 Cyclindromatosis dampens TAK1 ubiquitination and autoactivation, thereby blocking JNK and IKK signaling in T cells.[190] ^49 In addition to K63‐related ubiquitination‐mediated TAK1 activation, several studies have reported a negative regulatory effect of K48‐linked polyubiquitination on TAK1 activation.[191] ^38 The E3 ubiquitin ligase itchy targets TAK1 for K48‐linked polyubiquitylation and degradation.[192] ^50 Xia et al. found that an innovative E3 ubiquitin ligase FBXW2 mediates K48‐related polyubiquitination of TAK1 and leads to its degradation, which attenuates hepatocellular carcinoma progression and sorafenib‐resistant.[193] ^51 Considering that USP29 is a DUB acting as a negative regulator of PCH, we conjectured that USP29 dampens the activity of TAK1 in PCH by removing K63‐linked polyubiquitination of TAK1. Our subsequent ubiquitination assays confirmed this hypothesis, consistent with previous reports on the regulatory mechanism involving TAK1.[194] ^36 Conclusions In conclusion, our present research unveils a previously uncharacterized negative regulator USP29 involved in the progression of PCH. Moreover, the antihypertrophic effect of USP29 is ascribed to its deubiquitinating enzyme activity targeting TAK1 K63‐linked ubiquitination, which dampens phosphorylation of TAK1 and activation of JNK/p38 signaling cascade (Figure [195]10). Therefore, targeting USP29 or its interaction with TAK1 may represent a novel therapeutic strategy for myocardial hypertrophy and heart failure. Figure 10. Schematic of the molecular mechanisms underlying the antihypertrophic effects of USP29. Figure 10 [196]Open in a new tab 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. Sources of Funding This work was supported by grants from the National Natural Science Foundation of China (81700216); The 345 Talent Project of Shengjing Hospital of China Medical University. Disclosures None. Supporting information Tables S1–S3 [197]JAH3-14-e034962-s001.pdf^ (128.2KB, pdf) This article was sent to Julie K. Freed, MD, PhD, Associate Editor, for review by expert referees, editorial decision, and final disposition. Supplemental Material is available at [198]https://www.ahajournals.org/doi/suppl/10.1161/JAHA.124.034962 For Sources of Funding and Disclosures, see page 22. Contributor Information Yufeng Hu, Email: yufenghu21@gmu.edu.cn. Tiesheng Niu, Email: niuts@sj-hospital.org. References