Abstract Pathological myocardial hypertrophy can induce heart failure with high mortality, it is necessary to explore its pathogenesis. Tripartite motif-containing 26 (TRIM26) belongs to the multidomain E3 ubiquitin ligase family. We observed increased expression of TRIM26 in the myocardium of C57BL/6 mice subjected to transverse aortic constriction (TAC) surgery and neonatal rat cardiomyocytes (NRCMs) treated with phenylephrine (PE). To evaluate the role of TRIM26 in pathological cardiac hypertrophy, we generated Trim26 global knockout mice and Trim26 overexpression adenoviruses. Mice with Trim26 deletion showed alleviated cardiomyocyte enlargement, inflammation, fibrosis, and cardiac dysfunction after TAC surgery. In PE-treated NRCMs, Trim26 overexpression promoted cardiomyocyte enlargement and inflammation, while Trim26 knockdown had the opposite effects. RNA sequencing and molecular biology methodologies were performed to identify targets conducive to TRIM26 function. The results showed that TRIM26 activated the transforming growth factor-beta activated kinase 1 (TAK1)-c-Jun N-terminal kinase/p38 signaling pathway in response to hypertrophic stress. Moreover, inhibition of TAK1 activation can reverse the promotion effect of TRIM26 overexpression on cardiomyocyte hypertrophy induced by PE stimulation in vitro. Our study demonstrated that TRIM26 plays an active role in pathological cardiac hypertrophy, and the TRIM26-TAK1 pathway may represent a therapeutic target for treating pathological cardiac hypertrophy and heart failure. Keywords: Tripartite motif containing 26, Pathological cardiac hypertrophy, TGF-Beta activated kinase 1, Transverse aortic constriction 1. Introduction When the heart receives stress stimuli, such as injury, volume overload, or ischemia, it exhibits adaptive hypertrophic growth, characterized by increased heart afterload and weight, and an enlarged volume of myocardial cells, which play a protective role [[41]1,[42]2]. However, long-term overload stimulation could cause pathological cardiac hypertrophy, a transformation from compensatory to decompensated hypertrophy [[43]3,[44]4]. At this point, ventricular diastolic and contractile dysfunction occur, eventually leading to adverse events such as arrhythmia, heart failure, and sudden death [[45]5,[46]6]. Pathological cardiac hypertrophy is characterized by irreversible cardiomyocyte hypertrophy, sarcomere disorders, fibrosis, and inflammatory infiltration [[47]3]. Although the molecular mechanisms underlying pathological cardiac hypertrophy have been extensively studied, feasible therapeutic targets and strategies to prevent or reverse its unfavorable progression are still far from satisfactory. The tripartite motif-containing (TRIM) proteins have evolutionarily conserved domain structures, including over 70 distinct proteins in humans [[48]7,[49]8]. TRIM proteins are multidomain E3 ubiquitin ligases characterized by the inclusion of an N-terminal tripartite motif (RBCC domain) consisting of three zinc-binding domains; a RING (R) domain, a B-box (B) domain, and a coiled-coil (CC) domain [[50][9], [51][10], [52][11]]. TRIM proteins are widely involved in many cellular processes, including intracellular signal transduction, protein quality control, apoptosis, response to virus infection, innate and intrinsic immunity, autophagy, and carcinogenesis [[53]12,[54]13]. Although the expression level of this protein family is high in the heart, few studies have focused on function of these proteins in pathological cardiac hypertrophy [[55]14]. As an important member of the TRIM family, TRIM26 is involved in the occurrence and development of various diseases, such as bladder cancer, hepatocellular carcinoma, and liver fibrosis [[56][15], [57][16], [58][17]]. However, the possible functional roles of TRIM26 in pathological cardiac hypertrophy remain unknown. In the present study, we discovered that TRIM26 was upregulated and exacerbated pathological cardiac hypertrophy in vivo and in vitro. The TRIM26-KO mice subjected to transverse aortic constriction (TAC) surgery revealed that TRIM26 depletion alleviated cardiac dysfunction, cardiac hypertrophy and concomitant fibrosis and inflammation. Consistently, TRIM26 overexpression or knockdown in phenylephrine (PE)-induced neonatal rat cardiomyocyte (NRCM) hypertrophy models aggravated or alleviated cardiomyocyte hypertrophy, respectively. Expression profiling by high throughput sequencing and informatics analysis revealed that MAPK signaling was prominent and might mediate TRIM26 function. TAK1 phosphorylation was found to be positively regulated by TRIM26 and inhibition of TAK1 activity abolished the pro-hypertrophic actions of TRIM26. In conclusion, our findings revealed that TRIM26 exacerbated pathological cardiac hypertrophy and may serve as a promising potential therapeutic target in the TRIM26-TAK1 axis. 2. Materials and methods 2.1. Trim26 KO animal construction All animal use protocols were approved by Renmin Hospital of Wuhan University. The procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Through the CRISPR online design tool ([59]http://chopchop.cbu.uib.no/), the guide RNA target sequence: GACAGGCCGGTTCCCCGATAT was chosen and cloned and inserted into the pUC57-sgRNA expression vector (51132, Addgene) to construct the Trim26-sgRNA expression vector. In vitro transcription and purification products of the Cas9 expression vector pST1374-NLS-flag-linker-Cas9 (44758, Addgene) and sgRNA were mixed. The mixture was injected into single-cell fertilized eggs of C57BL/6 mice by a FemtoJet 5247 microinjection system. The injected fertilized egg was transplanted into surrogate female mice, and F0 generation mice were obtained after 19–21 days of gestation. Two weeks after birth, the toe tissues of the mice were collected, and genomic DNA was extracted. The following primers were used to identify the genotypes of the mice: Trim26-check F1: 5′- AAGGAACAGAGACCCCCAAC-3’. Trim26-check R1: 5′- AGCCGTTCAATGTTCTCCAC-3’. 2.2. TAC surgery Male WT and Trim26-KO mice aged 9–11 weeks with a body weight of 25.5–27 g were randomly selected. Before TAC surgery, the mice were anesthetized until no obvious toe reaction was observed after clamping, and their breathing rules were stable and even according to previous work [[60]18]. The mice were placed in the supine position and fixed on a self-adjusting heating pad at 37 °C. After separating the thymus on both sides, the aortic arch was exposed and 7-0 silk thread was passed through the aortic arch. Then a 26-G needle was placed parallel to on the aortic arch, and a narrowing of the aortic arch was ligated and constructed after the needle was pulled out. Mice in the sham groups underwent the same operation without narrowing. After the operation, the incision was sutured and the mice were placed in a 37 °C incubator to wake up. Doppler was used to analyze adequate constriction of the aortic arch and the mice with unsuccessful surgery were removed from the experimental groups. All surgical procedures were performed blindly. 2.3. Ultrasonic examination of mouse heart Mice were anesthetized by inhalation of isoflurane (1.5–2%) and then fixed in a supine position on a thermostatic plate. A small animal ultrasound imaging system (VEVO2100, FUJIFILM VISUALSONICS, Canada) and 30-MHZ (MS400) probe were used for ultrasonic detection. Under the M-mode echocardiography mode, echograms of left ventricular volume and left ventricular wall thickness were obtained from papillary muscle for three consecutive cardiac cycles, and left ventricular end-systolic diameter (LVESd), left ventricular end-diastolic diameter (LVEDd), left ventricular ejection fraction (EF%) and fraction shortening (FS%) were measured. 2.4. Animal sample collection Four weeks after TAC surgery, mice were euthanized and weighed. After the heart was removed, it was quickly placed in 10 % KCl solution to stop the heart and keep it in the diastolic stage. The heart was weighed and fixed in liquid nitrogen or 10 % formalin, and the length of the tibia was also measured. 2.5. Histomorphological staining analysis After soaking in 10 % formalin for 72 h, mouse heart tissues from each group were dehydrated and embedded in paraffin. The cross-cut wax blocks were sectionalized with a thickness of 5 μm. Then, hematoxylin (G1004, Servicebio) and eosin (BA-4024, Baso) (H&E) staining and picrosirius red (26357–02, Hede biotechnology) (PSR) staining were used to measure the cross-sectional area of cardiomyocytes and collagen fiber content, respectively, in cardiac tissue respectively. Images were captured by a digital pathology scanner (Aperio Versa 200, Leica, Wetzlar, Germany). Image-Pro Plus 6.0 software was used for measurement. 2.6. Western blot Mouse heart left ventricular tissues or NRCM samples were collected and added to RIPA buffer (65 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 % NP-40 (N8030, Solarbio), 0.5 % sodium deoxycholate, 0.1 % SDS) with protease inhibitor, followed by lysis using a homogenizer or sonicator. After centrifugation, the supernatant was taken as total protein and quantified by a BCA protein kit (23225, Pierce). Then protein samples were separated by 10 % SDS‒PAGE and transferred to 0.45 μm PVDF (IPVH00010, Millipore) membranes. The membrane was then blocked with 5 % milk in TBST at room temperature for 1 h. The PVDF membrane was washed with TBST every 5 min for 3 times after incubation with the corresponding primary antibody overnight at 4 °C. Then, the corresponding species of secondary antibody (Jackson Immunoresearch) was added and incubated at room temperature for 1 h. Then ECL luminescent substrate (1705062, Bio-Rad) was added, and a ChemiDoc XRS + Imaging System (Bio-Rad) was used for protein signal collection. Image Lab (Version 5.1) software was used to analyze the results, and the corresponding antibody information is shown in [61]Table S1. 2.7. Real-time PCR TRIzol reagent (15596–026, Invitrogen) was used to extract total RNA from the left ventricle of the heart tissues or NRCM samples. Transcriptor First Strand cDNA Synthesis Kit (04896866001, Roche) was used to reverse transcribe 2 μg of RNA into cDNA. Subsequently, SYBR Green PCR Master Mix (04887352001, Roche) was added to the system to measure the expression levels of the genes, which were tested in an RT‒PCR instrument (Roche). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene, and the sequences of primers used were in [62]Table S2. 2.8. Vector construction The mouse Trim26 gene was subcloned and inserted into the replication-deficient adenovirus vector (AdTrim26) driven by the CMV promoter (AdTrim26), achieving Trim26 overexpression in cardiomyocytes, and adenovirus with GFP expression served as a control. An adenovirus vector carrying a short hairpin RNA targeting Trim26 (AdshTrim26) was used to knock down TRIM26, and an AdshRNA adenovirus was used as a control. Adenoviruses were used to infect NRCMs at a multiplicity of infection (MOI) of 50-particle/cell for 12 h, and Western blotting was subsequently performed for identification subsequently. The sequences of the primers are as follows: AdTrim26-FP: GGCTAGCGATATCGGATCCGCCACCATGGCAGTGTCAG CCCCCTTG. AdTrim26-RP: CGTCCTTGTAATCACTAGTGGGTCTCAGCAGAAGGCG TGC short hairpin RNA: GCAGGGCCACCAGTTTCTAAA. AdshTrim26-FP: CCGGGCACTGTGACATTCACCAATGCTCGAGCATTGGTGAATGTCACAGTGCTTTTTG. AdshTrim26-RP: AATTCAAAAAGCACTGTGACATTCACCAATGCTCGAGCATTGGTGAATGTCACAGTGC. 2.9. Isolation and culture of primary NRCMs The hearts of 1- to 2-day-old Sprague‒Dawley rats were isolated and cut into 1 mm^3 tissue blocks after the vascular components of the hearts were removed. Then, the blocks were digested with 0.125 % trypsin to obtain NRCMs. The NRCMs were cultured in DMEM/F12 ([63]C11330, Gibco) medium with 10 % fetal bovine serum, 1 % penicillin/streptomycin, and 0.1 mM 5-bromodeoxyuridine for 24 h. Subsequently, NRCMs were infected with adenovirus for 6 h, and then cultured in serum-free medium for 12 h. Finally, 50 μM PE was added to the medium with NRCMs for 24 h to induce a pathological model. Then, 2.5 μM TAK1 inhibitor (iTAK1) (HY-15434, NG-25, MCE) was added for 12 h to inhibit phosphorylated TAK1, and the control group was treated with the same amount of PBS or DMSO. The whole cell culture was carried out at 37.0 °C and 5 % CO[2]. 2.10. Immunofluorescence staining NRCMs were cultured on the slides of 24-well plates at a density of 1.6–2 × 10^5 cells per well. After the corresponding treatment and culture, NRCMs were fixed with 4 % formaldehyde for 30 min, permeabilized with 0.2 % Triton X-100, and then blocked with 8 % goat serum for 30–60 min at 37 °C. Next, the cells were incubated with α-actinin antibody (05–384, Merck Millipore, 1:100 dilution) and the secondary antibody (donkey anti-mouse IgG [H + L] antibody, a21202, Invitrogen, 1:200 dilution) in sequence. Finally, the slides were sealed with DAPI solution containing anti-fade medium Images were obtained using a fluorescence microscope (BX51, Olympus). The cardiomyocyte surface area was measured using Image-Pro Plus 6.0 software. 2.11. Immunoprecipitation assays For coimmunoprecipitation (Co-IP) assays, the NRCMs were first infected with TRIM26-overexpression adenovirus, and then lysed with IP lysis buffer (20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; and 1 % NP-40). After high-speed centrifugation at 4 °C, the protein-containing supernatant was incubated with protein G agarose beads conjugating Flag antibodies or negative control IgG antibodies overnight at 4 °C. Centrifuge at 3000 rpm at 4 °C to separate the supernatant and beads. The beads were then washed with 300 mM and 150 mM NaCl buffers for 3 times to obtain conjugated protein solution. The supernatant and the isolated coupled protein solution were taken for subsequent Western blot analysis. 2.12. Ubiquitination assay Cultured HEK293T cells were lysed in SDS lysis buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 % SDS) including protease inhibitor cocktail (04693132001, Roche, Basel, Switzerland). Heart tissues were broken at 60 Hz for 120 s by a Tissuelyser-24 (Jingxin, Shanghai). After denaturation at 95 °C for 10 min, the lysates were diluted 10-fold with IP buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA and 1 % Triton X-100). Then, the lysates were centrifuged at 15,000 rpm for 10 min at 4 °C and the supernatants were subjected to IP assays with the indicated antibodies, followed by western blotting. 2.13. Transcriptome analysis For RNA sequencing, the total RNA of mouse heart left ventricular tissue from the WT TAC and KO TAC groups were extracted and the cDNA library was constructed. MGISEQ-2000 RS was used for RNA-sequencing of the single-ended library with a read length of 50 bp. The sequenced fragments were compared to the mouse reference genome (mm10) with HISAT2 software, and the files obtained in the above steps were transformed into binary BAM format, which could store the comparison information through SAM tools. Then, fragments per kilobase of exon model per million mapped fragments (FPKM) of each identified gene was calculated through the default parameters of String Tie. DESeq2 identified differentially expressed genes (DEGs) were selected for further investigation based on the following two criteria: (1) the fold change was greater than 2; and (2) the corresponding corrected p value was less than 0.05. 2.14. Principal component analysis Principal component analysis (PCA) for comparing the differences between 2 groups was implemented using the fast.prcomp function in R and visualized with the ggplot2 package. 2.15. Gene set enrichment analysis Gene set enrichment analysis (GSEA) was applied to sort the genes according to the degree of differential expression with the gene set of the KEGG pathway, and then test whether the gene set was concentrated at the top or bottom of the sorting table to obtain the overall expression changes of these gene sets. The analysis was performed on the Java GSEA platform using the “signal2noise” metric. Gene sets with p < 0.05 and FDR <0.25 were considered statistically significant. 2.16. Reactome enrichment analysis Reactome pathway enrichment analysis was performed for all differentially expressed genes by [64]clusterProfiler package, and p < 0.05 were defined as significantly enriched pathways. 2.17. Statistical analysis Statistical Package for the Social Sciences (SPSS) 25.0 software was applied to analyze the data, and data in this study are displayed as the mean ± SD. The normality of each group was determined by the Shapiro‒Wilk test, and the data in this study were normally distributed. The two-tailed Student's t-test was used for data comparison between the two groups, and the one-way or two-way ANOVA was used for data comparison between multiple groups, which was corrected by Bonferroni correction (assumed homogeneous variance) or Tamhane's T2 (assumed heterogeneous variance). P < 0.05 was considered statistically significant. 3. Results 3.1. TRIM26 protein expression is increased in hypertrophic mouse hearts To investigate the role of TRIM26 in the development of pathological cardiac hypertrophy, we treated NRCMs with PE in vitro, and performed TAC surgery on mice, and the increased mRNA levels of cardiac hypertrophic markers (Anp and Bnp) confirmed the success in the two pathological myocardial hypertrophy models ([65]Fig. 1A–B and D-E). In these two models, the protein levels of TRIM26 were significantly elevated compared to those in the control group, but the mRNA levels showed no significant difference ([66]Fig. 1B–E). These results suggest that TRIM26 may be involved in the development of pathological cardiac hypertrophy. Fig. 1. [67]Fig. 1 [68]Open in a new tab TRIM26 aggravate PE-induced cardiomyocyte hypertrophy in vitro. (A) Representative H&E stained images of mouse hearts for 4 weeks after TAC or sham surgery. Scale bar, 25 μm. (B) mRNA levels of TRIM26, Anp, and Bnp in mouse hearts for 4 weeks after TAC or sham surgery (n = 4). (C) The protein levels of TRIM26, Anp, and Bnp in mouse hearts for 4 weeks after TAC or sham surgery (n = 4 mice/group). (D) Representative α-actinin immunofluorescence images of neonatal rat cardiomyocytes (NRCMs) treated with PBS or PE for 24 h. Scale bar, 20 μm. (E) mRNA levels of Anp, Bnp, and TRIM26 in NRCMs treated with PBS or PE for 24 h (n = 3 independent experiments). (F) The protein levels of TRIM26 in NRCMs treated with PBS or PE for 24 h and statistical results (n = 4 independent experiments). (G, H) mRNA (G) and protein (H) levels of TRIM26 in NRCMs infected with adenovirus overexpressing Trim26 (AdTrim26) or GFP vector (AdVector) and statistical results (n = 3 independent experiments). (I) Representative immunofluorescence images of α-actinin staining and its statistical results in NRCMs infected with AdVector or AdTrim26 and treated for 24 h with PBS or PE and statistical analysis (n > 50 cells per group in each independent experiment). Scale bar, 20 μm. (J) mRNA levels of Anp, Bnp, and Myh7 in NRCMs infected with AdVector or AdTrim26 and treated for 24 h with PBS or PE (n = 3 independent experiments). (K) Protein levels of Anp, Bnp, and Myh7 in NRCMs infected with AdVector or AdTrim26 treated with PBS or PE for 24 h and statistical results (n = 3 independent experiments). (L) mRNA levels of Tnf, Il6, and Il1b in NRCMs infected with AdVector or AdTrim26 and treated for 24 h with PBS or PE (n = 3 independent experiments). GAPDH served as a loading control in C, F, H, K. ∗∗, P < 0.01 vs. PBS, sham group, or AdshRNA PE group. #, P < 0.05, ##, P < 0.01 vs. AdshRNA PBS group. Statistical analysis was conducted using two-tailed Student's t-test (B, C, E-H, and K) or One-way ANOVA (I, J, and L). Next, we infected NRCMs with a Trim26-overexpressing adenovirus (AdTrim26) and its control virus (AdVector) in vitro, and the efficacy was analyzed. As shown, the mRNA and protein levels of TRIM26 drastically increased in AdTrim26-infected NRCMs ([69]Fig. 1G and H). NRCMs were treated with PE or phosphate-buffered saline (PBS), and then the cells in each group were subjected to α-actinin immunofluorescence staining to determine the surface area of cardiomyocytes. The results showed no significant differences in the morphology and surface area of the cardiomyocytes between the PBS groups. However, in the PE-stimulated groups, compared with that in the Advector-infected control, the AdTrim26 infection-mediated TRIM26 overexpression significantly exacerbated PE-induced cardiomyocyte hypertrophy, as demonstrated by the increased-surface area of cardiomyocytes ([70]Fig. 1I). In addition, both the mRNA and protein expression levels of myocardial hypertrophy markers (Anp, Bnp, and Myh7) and inflammation-related genes (Tnf, Il6, and Il1b) were higher in the AdTrim26 group ([71]Fig. 1J-L). In summary, TRIM26 promotes the progression of pathological cardiomyocyte hypertrophy in vitro. 3.2. TRIM26 knockdown alleviates cardiomyocyte hypertrophy in vitro To investigate the role of TRIM26 knockdown, NRCMs were infected with Trim26 knockdown adenovirus (AdshTrim26) or corresponding control virus (AdshRNA), and their efficacy was analyzed. The results showed that the mRNA and protein levels of TRIM26 were drastically decreased in AdshTrim26-infected NRCMs ([72]Fig. 2A and B). Similarly, α-actinin immunofluorescence staining revealed that the knockdown of TRIM26 significantly alleviated PE-induced cardiomyocyte hypertrophy, which was demonstrated by the decreased surface area of cardiomyocytes ([73]Fig. 2C), reduced mRNA and protein levels of hypertrophy markers (Anp, Bnp, and Myh7) ([74]Fig. 2D and E), and lower mRNA expression levels of inflammation-related genes (Tnf. Il6, and Il1b) ([75]Fig. 2F). These data reveal that TRIM26 knockdown relieves cardiomyocyte hypertrophy in vitro. Fig. 2. [76]Fig. 2 [77]Open in a new tab TRIM26 knockdown alleviate PE-induced cardiomyocyte hypertrophy in vitro. (A, B) mRNA (A) and protein (B) levels of TRIM26 in NRCMs infected with adenovirus knock-down Trim26 (AdshTrim26) or control adenovirus (AdshRNA) (n = 3 independent experiments) and statistical analysis. (C) Representative immunofluorescence images of α-actinin staining and its statistical results in NRCMs infected with AdshRNA or AdshTrim26 treated with PBS or PE for 24 h and statistical analysis (n > 50 cells per group in each independent experiment). Scale bar, 20 μm. (D) The mRNA levels of Anp, Bnp, and Myh7 in NRCMs infected with AdshRNA or AdshTrim26 and treated for 24 h with PBS or PE (n = 3 independent experiments). (E) Anp, Bnp, and Myh7 protein levels in NRCMs infected with AdshRNA or AdshTrim26 and treated for 24 h with PBS or PE and statistical results (n = 3 independent experiments). (F) mRNA levels of Tnf,Il6, and Il1b in NRCMs infected with AdshRNA or AdshTrim26 and treated for 24 h with PBS or PE (n = 3 independent experiments). GAPDH was a loading control in B and E. ∗, P < 0.05, ∗∗, P < 0.01 vs. the AdshRNA PE group. #, P < 0.05, ##, P < 0.01 vs. AdshRNA PBS group. Statistical analysis was conducted using two-tailed Student's t-test (A, B, and E) or One-way ANOVA (C, D, and F). 3.3. TRIM26 deficiency improves cardiac dysfunction and cardiomyocyte hypertrophy caused by TAC surgery To evaluate the effect of TRIM26 on cardiac hypertrophy, we established Trim26 knockout (KO) mice ([78]Fig. 3A), and Trim26-KO mice were healthy and fertile. The mice and their age-matched wild-type (WT) littermates underwent TAC or sham surgery. No significant cardiac morphology or functional abnormalities were observed in WT and KO Sham groups before surgery ([79]Fig. 3B–I). However, TRIM26 deficiency significantly repressed TAC-induced cardiac hypertrophy, which was indicated by the reduction in heart weight ([80]Fig. 3B), the ratios of heart weight to body weight ([81]Fig. 3C), the ratios of lung weight to body weight ([82]Fig. 3D), and the ratios of heart weight to tibia length ([83]Fig. 3E) in the Trim26-KO TAC group compared with those in the WT TAC group. Furthermore, compared with that in WT mice after TAC surgery, Trim26-KO mice showed markedly improved cardiac function by reducing the left ventricular end-diastolic diameter (LVEDd) and left ventricle end-systolic dimension (LVESd), and increasing the ejection fraction (EF) and fraction shortening (FS) ([84]Fig. 3F–I). The gross size of the heart and the cardiomyocyte cross-sectional area stained using hematoxylin-eosin and quantitation of mRNA levels of cardiac hypertrophic markers (Anp, Bnp, and Myh7) revealed the same results ([85]Fig. 3J and K). These results indicate that TRIM26 deficiency alleviates cardiac dysfunction and cardiomyocyte hypertrophy induced by TAC surgery. Fig. 3. [86]Fig. 3 [87]Open in a new tab TRIM26 deficiency improves cardiac dysfunction and cardiomyocyte hypertrophy caused by TAC surgery. (A) The protein levels of TRIM26 in WT and Trim26-KO mice (n = 3 mice/group). (B–E) Heart weight (B), HW/BW (C), LW/BW (D), HW/TL (E) of WT and Trim26-KO mice subjected to TAC or sham surgery (n = 10 mice/group). (F–I) Measurements of LVEDd (F), LVESd (G), EF (H), and FS (I) in the indicated groups (n = 10 mice/group). (J) Representative images of gross morphology (Scale bar, 0.3 cm), H&E staining (Scale bar, 50 μm) (n = 6 mice/group), and the statistical result of cell surface area from the indicated groups (n > 300 cells per group). (K) mRNA levels of Anp, Bnp, and Myh7 in the indicated groups (n = 4 mice/group). GAPDH was a loading control in A. ∗, P < 0.05, ∗∗, P < 0.01 vs. WT TAC group. ##, P < 0.01 vs. WT sham group. Statistical analysis was conducted using One -way ANOVA (B-K). 3.4. TRIM26 deficiency ameliorates fibrosis and inflammation in myocardial hypertrophy To further study the effect of TRIM26 deficiency on myocardial hypertrophy in mice, four groups of mouse hearts after TAC surgery were subjected to Picrosirius Red staining analysis of collagen deposition and detection of fibrotic related gene expression. Picrosirius red staining was performed on heart sections to evaluate the extent of fibrosis, and quantified as the left ventricular collagen volume. The results showed that Trim26-KO mice exhibited less cardiac interstitial and perivascular fibrosis than WT mice ([88]Fig. 4A). In parallel, the mRNA expression levels of fibrotic markers (collagen Iα1, collagen IIIα1, and Ctgf) in Trim26-KO mice were decreased compared with those in WT mice after TAC surgery ([89]Fig. 4B). In addition, TRIM26 deficiency significantly downregulated the expression of inflammation-related genes (Tnf, Il6, and Il1b) in the hearts of mice after TAC surgery ([90]Fig. 4C). Accordingly, the phosphorylation of IKKβ (inhibitor of nuclear factor kappa-B kinase subunit beta) and P65 in the NF-κB (nuclear factor kappa-B) inflammation pathway, was significantly downregulated in the Trim26-KO TAC group ([91]Fig. 4D). These data suggest that TRIM26 deficiency significantly reduces fibrosis and inflammation in the heart during myocardial hypertrophy caused by TAC surgery. Fig. 4. [92]Fig. 4 [93]Open in a new tab TRIM26 deficiency ameliorates fibrosis and inflammation in myocardial hypertrophy. (A) Representative images of PSR staining (Scale bar, 50 μm) (n = 6 mice/group), and the statistical result of relative LV collagen volume from the indicated groups (n > 50 images per experimental group). (B, C) mRNA levels of Collagen Iα1, Collagen IIIα1, Ctgf, Tnf,Il6, and Il1b in the indicated groups (n = 4 mice/group). (D) The protein levels of p-IKKβ, IKKβ, p-P65, and P65 from the indicated groups and statistical analysis (n = 3 mice/group). GAPDH was a loading control in D. ∗, P < 0.05, ∗∗, P < 0.01, vs. WT TAC group. #, P < 0.05, ##, P < 0.01, vs. WT sham group. Statistical analysis was conducted using One-way ANOVA (A-C) or a two-tailed Student's t-test (D). 3.5. TRIM26 activates the TAK1-JNK/P38 signaling pathway in cardiomyocytes after TAC or PE stimulation To explore the underlying mechanism by which TRIM26 regulates pathological cardiac hypertrophy, we performed RNA-sequencing (RNA-Seq) analysis of the hearts of WT and Trim26-KO mice after TAC surgery. The RNA-Seq distribution profiles were analyzed by principal component analysis, which showed that Trim26-KO and WT samples were separated into two clusters ([94]Fig. 5A). The Gene set enrichment analysis (GSEA) highlighted that the heart function, protein processing, fibrosis and inflammatory signaling pathways were all suppressed due to the lack of TRIM26 ([95]Fig. 5B). The heatmap results indicated that TRIM26 deficiency inhibited the expression of genes related to heart function, protein processing, fibrosis, and inflammation ([96]Fig. 5C). Reactome database analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the most enriched in the hearts of Trim26-KO and WT mice after TAC surgery ([97]Fig. 5D). TAK1 (transforming growth factor-beta-activated kinase 1) is an important member of the MAPK kinase kinase (MAPKKK) family, and previous studies have shown that TRIM26 catalyzes the K11-linked polyubiquitination of TAB1 to enhance the activation of TAK1 and subsequent nuclear factor kappa-B and MAPK signaling [[98]19,[99]20]. Fig. 5. [100]Fig. 5 [101]Open in a new tab The RNA-Seq results of WT and Trim26-KO mice heart samples after TAC surgery. (A) Principal component analysis (PCA) showing global sample distribution profiles from WT and Trim26-KO mice heart samples subjected to TAC for 4 weeks (n = 4 mice/group). (B) Gene set enrichment analysis (GSEA) analysis showed the molecular events involved in heart function, protein processing, fibrosis, and inflammation in RNA-Seq data (n = 4 mice/group). (C) Heatmap of significantly altered genes related to heart function, protein processes, fibrosis, and inflammation in the WT TAC and Trim26-KO TAC groups (n = 4 mice/group). (D) Top enriched pathways contributing to the Trim26 function were identified using reactome enrichment analysis(n = 4 mice/group). To investigate whether TRIM26 regulates TAK1 activation in cardiac hypertrophy, we analyzed the total and phosphorylated levels of TAK1, ERK, JNK, and p38 in heart tissues of WT and KO mice suffer from Sham and TAC treatment and NRCMs infected with AdVector or AdTRIM26 and stimulated with PBS or PE. Compared with that in the WT mice, the level of p-ERK in Trim26-KO mice did not change in response to TAC surgery, whereas the levels of p-TAK1, p-JNK, and p-p38 were downregulated ([102]Fig. 6A). Consistent results were obtained in vitro when NRCMs were transfected with AdTrim26 adenovirus undergoing PE treatment, and the PE-stimulated phosphorylation of TAK1, JNK and p38 was accelerated after TRIM26 overexpression ([103]Fig. 6B). Endogenous Coimmunoprecipitation in NRCMs infected with Trim26-overexpressing adenovirus revealed that TRIM26 interacts with TAK1 ([104]Fig. 6C). Given that TRIM26 is a ubiquitin ligase, we further investigated whether TRIM26 affected TAK1 ubiquitination using ubiquitin content analysis. The results showed that overexpression of TRIM26 accelerated TAK1 K63-linked ubiquitin, which could activate TAK1 ([105]Fig. 6D and E). In order to rule out that Trim26 affects the activity of TAK1 by modulating TAB1, we measured the protein expression level and ubiquitination of TAB1 in pathological myocardial hypertrophy. According to the [106]Supplementary Fig. S1A, we found that there was no significant change in the expression level of TAB1 protein after TAC surgery. After infection of NRCMs by AdshTRIM26 or AdTRIM26, it was found that changes in TRIM26 expression levels did not affect the expression and ubiquitination levels of TAB1 in NRCMs after PE stimulation (S1B and 1C Fig). Taken together, these results demonstrated that TRIM26 interacted with TAK1 and promoted the activation of TAK1-JNK/P38 by activating the K63 ubiquitination of TAK1. Fig. 6. [107]Fig. 6 [108]Open in a new tab TRIM26 promotes the activation of TAK1-JNK/P38 signaling pathway. (A) Protein levels of total and phosphorylated TAK1, ERK, JNK, and P38 in heart tissues of WT and Trim26-KO mice treated with TAC or sham surgery and statistical analysis (n = 4 mice/group). (B) Protein levels of total and phosphorylated TAK1, ERK, JNK, and P38 in PE-stimulated AdTrim26 and AdVector NRCMs treated with PBS or PE for 24 h and statistical analysis (n = 3 independent experiments). (C) Representative endogenous co-immunoprecipitation (IP) analyses showing the binding of TRIM26 to TAK1 in NRCMs; IgG was used as a control (n = 3 independent experiments). (D) IP analysis of TAK1 ubiquitination in HEK 293T cells transfected with the indicated plasmids (n = 3 independent experiments). (E) IP analysis of TAK1 K63-linked ubiquitination in HEK 293T cells transfected with the indicated plasmids (n = 3 independent experiments). GAPDH was a loading control in E and F. ∗, P < 0.05, ∗∗, P < 0.01, n.s., no significance. Statistical analysis was conducted using One-way ANOVA. 3.6. Inhibition of TAK1 activation represses TRIM26 overexpression-induced cardiac hypertrophy in vitro To further study the importance of TAK1 in Trim26 regulation of cardiomyocyte hypertrophy, we treated PE-stimulated TRIM26-overexpressing cells (AdTrim26) and control cells (AdVector) with a TAK1 inhibitor (iTAK1), and dimethyl sulfoxide (DMSO) was used as a control. The efficiency of iTAK1 was evaluated though the level of p-TAK1 ([109]Fig. 7A). Immunostaining results of α-actinin showed that the TAK1 inhibitor abolished increased cardiomyocyte surface areas resulting from TRIM26 overexpression ([110]Fig. 7B), and the upregulated expression of hypertrophy markers (Anp, Bnp, and Myh7) and inflammation-related genes (Tnf, Il6, and Il1b) in TRIM26-overexpressing cells was also inhibited due to iTAK1 addition ([111]Fig. 7C and D). Fig. 7. [112]Fig. 7 [113]Open in a new tab TRIM26 regulate cardiomyocyte hypertrophy through TAK1 activation. (A) Protein levels of TRIM26, p-TAK1, and TAK1 in NRCMs in the indicated groups (n = 3 independent experiments). (B) Representative immunofluorescence images of α-actinin staining and its statistical results in PE-stimulated AdTrim26 and AdVector NRCMs with iTAK1 or DMSO as control (n > 50 cells per group in each independent experiment). Scale bar, 20 μm. (C, D) mRNA levels of Anp, Bnp, Myh7, Tnf, Il6, and Il1b in PE-stimulated AdTrim26 NRCMs and AdVector with iTAK1 or DMSO as control (n = 3 independent experiments). GAPDH served as a loading control in A. ∗∗, P < 0.01 vs. AdTrim26 CT PE group. #, P < 0.05, ##, P < 0.01, n.s., no significance vs. AdVector CT PE group. Statistical analysis was conducted using One-way ANOVA (A-D). Moreover, we established a TAK1-overexpressing adenovirus (AdTAK1) to further investigate the role of TAK1 in TRIM26-induced cardiomyocyte hypertrophy ([114]Fig. 8A). Consistently, TAK1 overexpression exacerbated the improvement in cardiomyocyte hypertrophy induced by loss of TRIM26 under PE treatment ([115]Fig. 8B–D). Collectively, these data suggest that TRIM26 regulates the occurrence and development of pathological cardiac hypertrophy in a TAK1 activation-dependent manner. Fig. 8. [116]Fig. 8 [117]Open in a new tab TRIM26 regulate cardiomyocyte hypertrophy through TAK1 activation. (A) Protein levels of TRIM26, p-TAK1, and TAK1 in NRCMs in the indicated groups (n = 3 independent experiments). (B) Representative immunofluorescence images of α-actinin staining and its statistical results in PE-stimulated AdshTrim26 and AdshRNA NRCMs infected with AdTAK1 or AdVector (n > 50 cells per group in each independent experiment). Scale bar, 20 μm. (C, D) mRNA levels of Anp, Bnp, Myh7, Tnf, Il6 and Il1b in PE-stimulated AdshTrim26 and AdshRNA NRCMs infected with AdTAK1 or AdVector (n = 3 independent experiments). GAPDH served as a loading control in A. ∗∗, P < 0.01 vs. AdshTrim26 AdVector PE group. ##, P < 0.01, n.s., no significance vs. AdshRNA AdVector PE group. Statistical analysis was conducted using One-way ANOVA. 4. Discussion Pathological cardiac hypertrophy due to pressure overload is a precursor to heart failure, a major cardiovascular disease with increasing morbidity and mortality worldwide [[118]21,[119]22]. Pathological cardiac hypertrophy is an injury reaction when the heart is overloaded. Recently, significant progress has been made in identifying and researching key molecular targets and signaling pathways involved in the process of cardiac hypertrophy [[120][23], [121][24], [122][25], [123][26]]. However, the advancements in the clinical treatment of cardiac hypertrophy, from previous studies, are not satisfactory. Therefore, further research on the mechanisms underlying cardiac hypertrophy is urgently needed. This study reveals the biological function of TRIM26 in the development of cardiac hypertrophy. TRIM26 protein expression levels were significantly increased in TAC-operated mice and PE-stimulated NRCMs, but no significant changes were observed at the transcriptional level. This may be associated with the posttranscriptional regulation of the TRIM26 protein. Based on studies of various in vivo and in vitro models of cardiac hypertrophy, we determined that TRIM26 promotes the development of cardiac hypertrophy related events by activating the TAK1-JNK/P38 signaling pathway. The TRIM family of proteins is involved in the development of many diseases. Recent studies have shown that many members of the TRIM family, such as TRIM8, TRIM 24, TRIM 32, and TRIM 16, play important roles in cardiac hypertrophy and other cardiovascular diseases. In mice, TRIM8 deletion prevents pathological myocardial hypertrophy during stress, whereas TRIM8 overexpression has the opposite effect, indicating that TRIM8 promotes myocardial hypertrophy [[124]27,[125]28]. Another study suggested that TRIM32 protects against pathological cardiac remodeling by blocking the Akt-dependent signaling pathways [[126]29,[127]30]. Liu et al. found that TRIM16 is an important inhibitor of pathological cardiac hypertrophy [[128]18]. Our study found that the expression of TRIM26 was increased in hypertrophic mouse hearts and PE-stimulated NRCMs, so it is speculated that the upregulation of TRIM26 may contribute to the progression of cardiac hypertrophy under pathological stress. To verify this hypothesis, we constructed Trim26-deficient mice that displayed inhibition of cardiac hypertrophy and poor remodeling caused by pressure overload. Similar results were observed in the gain- and loss-of function investigations of TRIM26 using NRCMs in vitro. Therefore, our results provide convincing evidence that TRIM26 plays an adverse role in pathological cardiac hypertrophy. It is important to note that TRIM26 deficiency did not result in significant changes in cardiac function, fibrosis, or inflammation in the Sham group compared to wild-type mice. This observation suggests that under normal physiological conditions, TRIM26 is not essential for maintaining normal cardiac structure and function. Instead, TRIM26 appears to play a more prominent role under pathological stress, such as pressure overload-induced cardiac hypertrophy, where its expression and activity may be upregulated. In the absence of external stressors like pressure overload, TRIM26 likely remains suppressed or expressed at low levels, preserving normal cardiac homeostasis. Furthermore, TRIM26 may mediate its pro-hypertrophic effects through stress-responsive signaling pathways, such as the TAK1-JNK/P38 axis, which are not activated in normal heart tissue. Thus, the pathogenic role of TRIM26 may only become evident under pathological conditions, while it remains inactive under normal physiological states. Therefore, it is important to determine the mechanism by which TRIM26 promotes the progression of pathological cardiac hypertrophy. RNA-Seq analysis revealed that the MAPK signaling pathway plays an important role in the process by which TRIM26 promotes pathological cardiac hypertrophy. In previous studies, ERK1/2, P38 and JNK1/2 were shown to play important roles in cardiac hypertrophy [[129][31], [130][32], [131][33]]. Therefore, we examined the expression of multiple molecules in the MAPK pathway in TRIM26-deficient hearts after TAC surgery. The results showed that the phosphorylation of TAK1, JNK, and P38 was significantly blocked in pressure-overloaded TRIM26-deficient hearts in vivo, whereas the overexpression of TRIM26 accelerated the phosphorylation of TAK1, JNK, and P38 stimulated by PE in NRCMS in vitro. TRIM26 interacts with TAK1 in 293T cells and affects TAK1 activation via K63-linked ubiquitination. Moreover, rescue experiments confirmed that the hypertrophic function of TRIM26 is mediated by the activation of TAK1. Due to time limitations, these experiments were not feasible in the current study. However, based on our in vitro findings in NRCMs, we hypothesize that TRIM26 overexpression in vivo would exacerbate pathological cardiac hypertrophy. Previous studies in TRIM family proteins, such as TRIM8 and TRIM32, suggest that overexpression enhances hypertrophic signaling in mice [[132]29,[133]34]. Therefore, we predict that TRIM26 overexpression would accelerate cardiac hypertrophy via the activation of TAK1-JNK/P38 signaling under pressure overload. Future studies will focus on confirming this hypothesis by performing in vivo experiments with TRIM26 overexpression models. In conclusion, TRIM26 knockout inhibits adverse cardiac remodeling under hypertrophic stress. In addition, our study provides convincing evidence to support the hypothesis that TRIM26 exacerbates the development of cardiac hypertrophy, fibrosis, and inflammation by activating TAK1-dependent signaling through enhancing TAK1 K63-linked ubiquitination to activate TAK1-dependent signaling. Therefore, our results suggest that targeting TRIM26 is a potential therapeutic strategy for preventing or reversing pathological cardiac hypertrophy. CRediT authorship contribution statement Xiaochuang Xia: Writing – review & editing, Methodology, Formal analysis, Data curation. Huajing Shan: Writing – review & editing, Methodology, Formal analysis, Data curation. Zhaoxia Jin: Writing – review & editing, Methodology, Formal analysis, Data curation. Tengfei Ma: Writing – original draft, Methodology, Data curation. Yemao Liu: Writing – original draft, Methodology, Data curation. Jianqing Zhang: Writing – original draft, Methodology, Data curation. Han Tian: Formal analysis, Data curation. Bizhen Dong: Methodology, Formal analysis, Data curation. Chengsheng Xu: Writing – review & editing, Formal analysis. Shaoze Chen: Writing – review & editing, Project administration, Methodology, Data curation, Conceptualization. Ethics approval and consent to participate This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (2023.04.03/WDRM# 20230401D). Availability of data and material The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding No funding was obtained for this study. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Footnotes ^Appendix A Supplementary data to this article can be found online at [134]https://doi.org/10.1016/j.heliyon.2024.e40653. Contributor Information Chengsheng Xu, Email: xuchengsheng@hgyy.org.cn. Shaoze Chen, Email: chenshaoze@hgyy.org.cn. Appendix A. Supplementary data The following are the Supplementary data to this article: Multimedia component 1 [135]mmc1.docx^ (31.6KB, docx) figs1. [136]figs1 [137]Open in a new tab References