Abstract Background Pathological cardiac hypertrophy stands as a pivotal mechanism contributing to diverse cardiovascular diseases, ultimately leading to heart failure. Despite its clinical significance, the intricate molecular mechanisms instigating pathological cardiac hypertrophy remain inadequately understood. In this study, we aim to further reveal its complex pathogenesis by exploring the role of Fas apoptotic inhibitory molecule 2 (FAIM2) in modulating pathological cardiac hypertrophy. Methods and Results We used phenylephrine‐induced hypertrophic cardiomyocytes and also generated cardiac‐specific knockout mice and adeno‐associated virus serotype 9‐Faim2 mice to evaluate the function of FAIM2 in pathological myocardial hypertrophy. Furthermore, unbiased RNA‐sequencing analysis was used to identify the direct target and corresponding molecular events contributing to FAIM2 function. Ultimately, our study revealed a downregulation of FAIM2 expression in phenylephrine‐induced hypertrophic cardiomyocytes and pressure overload–induced hypertrophic hearts. FAIM2 exhibited a significant attenuation of phenylephrine‐induced enlargement of primary neonatal rat cardiomyocytes, whereas FAIM2 knockdown aggravated the hypertrophic response. Furthermore, Faim2 gene knockout significantly exacerbated cardiac hypertrophy and heart fibrosis in vivo. Mechanistic investigations unveiled that FAIM2 exerts its inhibitory effect by suppressing TAK1‐JNK1/2‐p38 MAPK signaling cascades, thereby mitigating cardiac hypertrophy. Conclusions Our findings position FAIM2 as a novel negative regulator of pathological cardiac hypertrophy through its inhibitory action on mitogen‐activated protein kinase signaling activation. This identification of FAIM2's role provides crucial insights that may pave the way for the development of effective therapeutic strategies aimed at mitigating pathological cardiac hypertrophy, addressing a critical need in cardiovascular disease management. Keywords: cardiac hypertrophy, FAIM2, fibrosis, heart failure, therapy Subject Categories: Cardiomyopathy, Hypertrophy, Heart Failure __________________________________________________________________ Nonstandard Abbreviations and Acronyms Acta1 actin α1 AdFaim2 Faim2 gene overexpression adenovirus AdshFaim2 short hairpin RNA against Faim2 adenoviral constructs AKT protein kinase B Anp atrial natriuretic peptide ASK1 apoptosis signal‐regulated kinase Bax BCL2‐associated X protein Bcl2 B‐cell lymphoma 2 BW body weight Col1a1 collagen type I alpha 1 chain Col3a1 collagen type III alpha 1 chain Ctgf connective tissue growth factor ERK1/2 extracellular signal‐regulated kinase 1/2 FAIM2 Fas apoptotic inhibitory molecule 2 GSEA Gene Set Enrichment Analysis HEK293T human embryonic kidney 293T JNK1/2 c‐Jun NH2‐terminal kinase 1/2 KEGG Kyoto Encyclopedia of Genes and Genomes KO knockout LVEDd left ventricular internal diameter at end‐diastole MAPK mitogen‐activated protein kinase Myh7 β‐myosin heavy chain NRVM neonatal rat ventricular myocyte SAMtools sequence Alignment/Map Format Tools SIRT2 sirtuin 2 STEAP3 six‐transmembrane epithelial antigen of prostate 3 TAC transverse aortic constriction TAK1 transforming growth factor β–activated kinase 1 Timp1 tissue inhibitor matrix metalloproteinase 1 TLR4 toll‐like receptor 4 USP25 ubiquitin‐specific peptidase 25 5Z‐7‐ox 5Z‐7‐oxozeaenol Research Perspective. What Is New? * The Fas apoptosis inhibitory molecule 2 (FAIM2) protects against pressure overload–induced cardiac hypertrophy. * FAIM2 suppresses the TAK1‐MAPK signaling pathway by targeting c‐Jun NH2‐terminal kinase 1/2 and p38 and inhibiting cardiac hypertrophy. What Question Should Be Addressed Next? * Further studies are needed to refine the study of FAIM2 overexpression on cardiac hypertrophy in vivo and establish a molecular role for FAIM2 and transforming growth factor β–activated kinase 1 in the regulation of cardiac remodeling and the progression of cardiac hypertrophy. Heart failure (HF) remains a significant contributor to mortality, morbidity, and diminished quality of life.[42] ^1 Myocardial remodeling caused by cardiac hypertrophy stands as a pivotal mechanism in HF.[43] ^2 , [44]^3 , [45]^4 However, the complete understanding of cardiac hypertrophy's mechanism remains elusive. Investigating this mechanism and intervening early to prevent or manage cardiac hypertrophy holds immense clinical significance in the prevention and treatment of HF. The pathophysiological alterations in cardiac hypertrophy encompass a broad spectrum of genetic, cellular, molecular, and metabolic changes.[46] ^5 , [47]^6 Current research has highlighted the involvement of several factors, including USP25 (ubiquitin‐specific peptidase 25),[48] ^7 SIRT2 (sirtuin 2),[49] ^8 and STEAP3 (Six‐Transmembrane Epithelial Antigen of Prostate 3),[50] ^9 , [51]^10 alongside the expression of fetal genes. Various molecular pathways, such as vascular endothelial growth factor and Sammad‐TAK1,[52] ^11 , [53]^12 have also been implicated in the process of cardiac hypertrophy. Nevertheless, none of these factors or pathways singularly elucidate the entirety of the myocardial hypertrophy process. FAIM2 is a transmembrane protein, also known as transmembrane BAX inhibitor motif‐containing protein 2, which belongs to a group of evolutionarily conserved transmembrane proteins known for their cytoprotective and antiapoptotic properties.[54] ^13 Experimental evidence has demonstrated its protective role in both cell cultures and a mouse model of transient vascular ischemia.[55] ^14 , [56]^15 Previous reports suggest that lysosomal membrane protein‐1 (a related family member of FAIM2), can ameliorate pathological cardiac hypertrophy by targeting toll‐like receptor 4 (TLR4) degradation.[57] ^16 Hence, speculation has arisen about the potential association between FAIM2 and cardiovascular diseases. However, the specific function and mechanism of FAIM2 in cardiac hypertrophy remain unreported. Our study revealed a significant reduction in FAIM2 expression in cardiac hypertrophy induced by transverse aortic constriction (TAC), consistent with decreased FAIM2 expression observed in phenylephrine‐induced cardiomyocyte hypertrophy models. Further investigation uncovered that FAIM2 might modulate myocardial hypertrophy via the MAPK‐JNK signaling pathway, while there was no increase in extracellular signal‐regulated kinase 1/2 (ERK1/2) expression. Overall, our findings suggest that FAIM2 suppresses the progression of cardiac hypertrophy by interacting with the mitogen‐activated protein kinase (MAPK) signaling pathway, consequently impeding the activation of this signaling cascade. METHODS The authors declare that all supporting data are available within the article and its online supplementary files. Experimental Animals The purpose of the animal experiment was to test the effects of FAIM2 on cardiac hypertrophy in vivo. All animal procedures conducted in this study adhered to institutional ethical guidelines and received approval from the Animal Care and Use Committee of the First Affiliated Hospital of Gannan Medical University. Faim2‐Flox mice (T016639) were customized from GemPharmatech LLC, in Jiangsu Province, China. Then, Faim2 knockout (KO) mice were bred from the Gannan Innovation and Translational Medicine Institute. All mice were housed in a specific pathogen‐free environment where temperature, humidity, and light cycles (12 hours light, 12 hours dark) were strictly regulated. They had ad libitum access to both food and water. For our experiments, Faim2‐KO mice and their wild‐type (WT) littermates, aged 8 to 10 weeks and weighing 25.5 to 27.0 g, were utilized. All surgeries and subsequent analyses were conducted in a blinded manner. Transverse Aortic Constriction TAC surgery was used to establish the pressure overload–triggered cardiac hypertrophy mice model as described in vivo.[58] ^17 Briefly, the male mice (9 to 11 weeks old) were anesthetized with tribromoethanol (300 mg/kg, [59]T48402, Sigma) via intraperitoneal injection and then fixed on an auto‐adjusting heating pad, which maintained the body temperature to 37 °C as close as possible. The efficiency of the anesthesia was confirmed by a negative response to toe pinch reflex. The index finger of the left hand was placed at the back of the mouse's brain, the thumb and middle finger were placed at the mouse's upper limbs, and then the 3 fingers were put together to expose the mouse's chest. The mouse's chest was shaved with pet electric push clippers, and the shaved area was wiped with medical gauze after shaving. The mice were fixed on the heating plate with medical tape, mainly fixing the head and forelimbs of the mice. The mice were fixed with adhesive tape by pulling the head toward the surgeon and the forelimbs horizontally to the left and right side. The thymus lobes were separated to reveal the transverse aortic arch and 2 carotid arteries. The left side of the chest was opened and the transverse aorta was ligated transversely with a 7‐0 silk thread and a 26‐gauge needle. After the needle was removed, the chest wall was closed using a 6‐0 silk suture with a simple interrupted suture pattern. The mouse was kept in the cage under a heat lamp and monitored for 2 hours until wake up. The sham group underwent a similar procedure without the ligation of the aorta. Echocardiographic Analyses and General Physiological Index Detection After 4 weeks of TAC or sham surgery, heart function was evaluated by echocardiography as previously described.[60] ^18 , [61]^19 To determine cardiac function and structure, a Mylab30CV machine with 15‐MHz linear‐array ultrasound transducer was used to perform echocardiography. The 2‐dimensional M‐mode measurement of the left ventricular (LV) inner diameter was acquired from at least 3 beats and then averaged. The parameters including LV internal diameter at end‐systole, LV internal diameter at end‐diastole (LVEDd), and ejection fractions were measured in each group. Fractional shortening was calculated as follows: fractional shortening (%) = (LVEDd − LV internal diameter at end‐systole) ÷ LVEDd × 100%. After euthanasia, the heart, lung, and tibia of the mice were dissected and measured, and the ratios of heart weight (HW)/body weight (BW) (mg/g), HW/tibia length (mg/mm), and lung weight/body weight (mg/g) were calculated. All operations and subsequent analyses were performed in a blinded fashion. Histological Analyses After 4 weeks of TAC or sham surgery, the hearts of the mice were excised and stopped in diastole by placing them in a 10% potassium chloride solution. After a saline solution wash, the hearts were fixed in 10% formalin, embedded in paraffin, and then sectioned transversely at a thickness of 5 μm. For hematoxylin–eosin staining, paraffin sections were baked in an oven at 60 °C for 60 minutes, and then deparaffinized by 3 dips in xylene. After rehydration in different concentrations of alcohol and water, the heart sections were incubated in hematoxylin stain (Servicebio, G1004) for 3 minutes to stain the nuclei, soaked in 1% hydrochloric acid alcohol for a few seconds, and then rinsed with ddH[2]O. The heart sections were placed in Scott tap water (20 g MgSO[4]‐7 H[2]O and 3.5 g NaHCO[3] dissolved in 1000 mL ddH[2]O) and rinsed with ddH[2]O. The heart sections were then immersed in eosin stain (BASO, BA‐4024) for 5 minutes and rinsed with ddH[2]O. Paraffin sections were rinsed in 70% and 90% alcohol for a few seconds, in 100% alcohol for 1 minute (twice), and in xylene for 2 minutes (3 times). Residual xylene was removed from the surrounding area by blotting it with filter paper and the sections were sealed with sealant (BASO, BA‐7004). Histopathological images were acquired with a light microscope (StrataFAXS P‐S, TissueGnostics). The cross‐sectional areas and LV collagen volume were measured using a quantitative digital analysis imaging system (Image‐Pro Plus 6.0). At least 100 cardiomyocytes in the examined sections were profiled in each group. For picrosirius red staining, heart sections were baked in an oven at 60 °C for 60 minutes, and then deparaffinized by 3 dips in xylene. After deparaffinization and rehydration, heart sections were then soaked in 0.2% phosphomolybdic acid for 2 minutes and dyed with 0.1% picrosirius red staining (HEAD Biotechnology, 26357‐02) for 90 minutes. After staining, the tissue was treated with 0.01 mol/L hydrochloric acid for 3 seconds and then dehydrated according to the standard steps. The residual xylene liquid around the sliced tissue was absorbed and the sections were sealed with a sealing cement (BASO, BA‐7004). Immunohistochemical Staining Cardiac paraffin sections were baked in an oven at 60 °C for 60 minutes, and then dipped in xylene 3 times to remove paraffin. After rehydration, the cardiac sections were treated with pH 9.0 EDTA (MVS‐0099, Maxim) at a high temperature for 20 minutes for antigen extraction. The sections were rinsed with ddH[2]O for 5 minutes (twice) and 3% H[2]O was added for 20 minutes. The sections were then rinsed with PBS for 5 minutes and blocked with 10% BSA at 37 °C for 30 minutes. The BSA was discarded and incubated with murine anti‐Faim2 antibody (1:100 dilution, ab72113, Abcam) for 2 hours at 37 °C. The above steps were repeated and the secondary antibody was incubated at 37 °C for 20 minutes. The sections were rinsed with PBS for 5 minutes (3 times) and then incubated with diaminobenzidine working solution (ZLI‐9018, ZSGB‐BIO). After complete color development, the sections were washed with ddH[2]O for 5 minutes. The sections were then stained with hematoxylin (G1004, Servicebio) for 3 seconds and rinsed with ddH[2]O for 10 minutes. After dehydration in 70% alcohol, the sections were sealed with sealant (BA‐7004, BASO). Neonatal Rat Ventricular Myocytes Culture and Adenovirus Infection To verify the effect of FAIM2 in vitro, we isolated neonatal rat ventricular myocytes (NRVMs) and built a phenylephrine (P6126, Sigma)‐induced hypertrophy in cardiomyocytes with previously described methods.[62] ^20 , [63]^21 Cardiomyocytes were isolated from 0‐ to 3‐day‐old Sprague–Dawley rat hearts and then cultured in DMEM/F12 (PM150323, Procell) containing 10% FBS (BS‐S500, Newzerum) and 1% penicillin/streptomycin. NRVMs were plated onto 6‐well culture plates coated with gelatin at a density of 2.3×10^6 cells per well. After 48‐hour culture, when cells were well attached, the medium was changed into the serum‐free DMEM/F12 and cells were starved for 12 to 18 hours before stimulating with 50 μmol phenylephrine (Sigma, category # PHR1017) in the complete DMEM/F12 medium (10% FBS, 1% penicillin/streptomycin) for cardiomyocyte hypertrophy induction. Cells in the control group were treated with 0.1% dimethyl sulfoxide in the complete medium. To inactivate the phosphorylated activity of transforming growth factor β–activated kinase 1 (TAK1) in vitro, NRVMs were cultured overnight in serum‐free DMEM/F12 and then treated with 5Z‐7‐oxozeaenol (5Z‐7‐ox; MCE, 1 μmol, 24 hours) or dimethyl sulfoxide in plates with or without phenylephrine for a further 24 hours. We used Faim2 gene overexpression adenovirus (AdFaim2) to infect primary cardiomyocytes to overexpress Faim2 in vitro. To knockdown the expression of Faim2, we generated rat short hairpin RNA against Faim2 adenoviral constructs (AdshFaim2). The recombinant adenoviral vector (pENTER‐CMV‐Faim2‐ATG‐Flag, pENTER‐U6‐shFaim2) was linearized by PacI (R0547L, NEB) before co‐transfected into HEK293 cells with a polyethyleneimine transfection reagent. The primary virus was harvested 7 days later, and adenovirus was amplified by HEK293 cells, purified by cesium chloride density gradient centrifugation. The titer was then measured by the 50% tissue culture infective dose method. Adenoviruses‐expressing green fluorescent protein and nontargeting short hairpin RNA were used as the control of overexpression and depletion, respectively. The NRVMs were infected with the indicated adenoviruses at a multiplicity of infection of 30 for 24 hours. The primers used to construct adenoviruses were listed in Table [64]S1. Immunofluorescence Staining To measure the surface area of cardiomyocytes, immunofluorescence assays were performed on NRVMs by α‐actinin staining (A7811, Sigma). NRVMs were seeded on coverslips, infected by indicated adenoviruses for 24 hours and then stimulated by phenylephrine. Cells were fixed with 4% formaldehyde at room temperature for 15 minutes, followed by washing with PBS, 0.2% Triton‐X 100 for 10 minutes, and then blocked with 8% goat serum in PBS for 30 minutes. For α‐actinin staining, NRVMs were incubated at 37 °C for 1 hour with a mouse anti–α‐actinin (1:100 dilutions, A7811, Sigma) antibody and a corresponding anti‐mouse Alexa Fluor plus 568 secondary antibody (1:200 dilution, A11061, Invitrogen) for 1 hour at 37°C. DAPI was used to visualize the nucleus. Images were obtained via a confocal laser scanning microscope (TCS SP8, LEICA). Western Blot Assay To investigate the downstream signal pathways of FAIM2 or protein identification, we used Western blot assay to detect proteins in heart tissue and cardiomyocytes. LV tissues and cultured cardiomyocytes samples were prepared using RIPA lysis buffer (65 mmol Tris–HCl pH 7.5, 150 mmol NaCl, 1 mmol EDTA, 1% Nonidet P‐40, 0.5% sodium deoxycholate, 0.1% SDS, 1×protease inhibitor cocktail and 1×phosphatase inhibitor PhosSTOP). The protein concentration was measured using the BCA Protein Assay Kit, and 50 μg of total protein extracts were loaded and separated by 8% SDS‐PAGE gels and then transferred to polyvinylidene difluoride membranes (IPVH00010, Millipore). After blocking with 5% skim milk in TBST (Tris‐buffered saline and 0.1% Tween‐20) for 1 hour at room temperature and incubating with the indicated primary antibodies overnight at 4 °C, the membranes were maintained with appropriate secondary antibodies for 1 hour at room temperature. The blots were washed 3 times in TBST after each incubation. To visualize Western blot, the blots were incubated in enhanced chemiluminescence reagents and detected and quantified by a ChemiDoc XRS+ System (Bio‐Rad). GAPDH was used as the control in cell and tissue samples. The information of corresponding antibodies is shown in Table [65]S2. Real‐Time Polymerase Chain Reaction To detect changes in gene expression levels, total RNA was extracted from LV tissues and cultured cardiomyocytes using a TRI reagent and reverse‐transcribed into cDNA using Transcriptor HiScript III RT SuperMix for quantitative polymerase chain reaction according to the manufacturer's instructions. The information of primer sequences is shown in Table [66]S3. Immunoprecipitation Assays For immunoprecipitation assays, NRVMs were infected with AdFaim2 and then lysed with cold immunoprecipitation buffer (20 mmol Tris–HCl pH 7.4, 150 mmol NaCl, 1 mmol EDTA, and 1% Triton X‐100) containing 1×protease inhibitor cocktails. Samples were lysed on ice for 20 minutes and centrifuged at 12 000g for 10 minutes. The 800 μL supernatants were incubated with 20 μL protein A/Gagarose beads ([67]AA104307; Bestchrom) and 1 μg corresponding antibody at 4 °C for 3 hours. The beads were then washed 3 times with cold immunoprecipitation low‐salt buffer and boiled with 2×SDS loading buffer (95 °C, 15 minutes) before Western blot analysis. RNA Sequencing and Data Processing To profile the gene expression differences, RNA extracted from the hearts of WT and Faim2‐KO mice 4 weeks after TAC surgery was used to construct the cDNA libraries. Briefly, TransZol Up Plus RNA Kit (ER501‐01, TransGen Biotech), RNAClean XP Kit (A63987, Beckman Coulter Inc) and RNase‐Free DNase Set (79254, QIAGEN GmBH) were used to extract and purify total RNA of left ventricle tissues. The purified total RNA was subjected to mRNA separation, fragmentation, first‐strand cDNA synthesis, second‐strand cDNA synthesis, end repair, and other steps to complete the construction of sequencing sample library. Later, the single‐end library was sequenced with an MGISEQ‐2000 RS sequencer with a read length of 50 bp. Strict quality inspection was performed after each operation to ensure the accuracy of data for further analysis. Hierarchical Indexing for Spliced Alignment of Transcripts 2 software was used to compare the sequence fragments to the mouse reference genome (mm10/GRCm38). The files obtained from the above steps were then converted to binary BAM format by Sequence Alignment/Map Format Tools (SAMtools), which can store the alignment information. Next, the fragments per kilobase of exon model per million mapped fragments values were calculated for each identified gene using StringTie's default parameters. In the hierarchical clustering analysis, the unweighted pair group method with arithmetic mean algorithm was used to establish a hierarchical nested clustering tree by calculating the similarity between different samples, and the hclust function of the R package was used for visualization. Using Gene Set Enrichment Analysis (GSEA), genes were sorted according to the level of differential expression, and gene sets based on the gene ontology database were examined to determine whether they were concentrated at the top or bottom of the sorting list to investigate the overall expression changes. Java GSEA was used to perform GSEA with the “Signal2Noise” metric. Gene sets with P values <0.05 and a false discovery rate <0.25 were considered statistically significant. Method of Cell Surface Examination We selected appropriate representative cells according to the size of the whole group of cardiomyocytes, dragged the picture into Photoshop, circled and filled in the color of the selected cells (which can be circled in more than a few cells in one picture [circling standard is uniform]), and saved the circled and filled in the color for backup. The image was saved in TIF format for subsequent counting. After that, we used Image‐Pro Plus to perform the statistical analysis, dragged the scale image into Image‐Pro Plus, set the name such as “63X‐cardiomyocyte,” set the unit as μm (ie, scale unit), dragged the color‐filled image from Photoshop into Image‐Pro Plus, and began area statistics. In the “Sum” column of the table, the area of cardiomyocytes was counted, the data was pasted into the Excel table, and the values were organized into a whole group of cell areas. Finally, we created a bar chart of the statistics through GraphPad. Statistical Analysis All data were analyzed by SPSS version 21.0 (IBM) and presented as mean±SD. A 2‐tailed Student t test was performed to compare the differences between 2 groups, and 1‐way ANOVA was used to compare the means of >2 groups when the data had a normal distribution. For data meeting homogeneity of variance or showing heteroscedasticity, Bonferroni post hoc analysis or Tamhane T2 analysis was used for multiple comparisons, respectively. Nonparametric statistical analysis was performed if the data showed a skewed distribution. Mann–Whitney U test or Kruskal–Wallis H test was respectively used to compare the differences between 2 groups or >2 groups. A level of P<0.05 was considered to be statistically significant. RESULTS FAIM2 Protein Level Is Decreased in In Vivo and In Vitro Cardiac Hypertrophic Models To investigate the potential role of FAIM2 in pathological cardiac hypertrophy, we first evaluated the expression of FAIM2 in in vivo and in vitro cardiac hypertrophic models, by detecting the mRNA and protein expression levels. Surprisingly, the mRNA levels of FAIM2 exhibited no significant differences between the hearts of mice subjected to TAC and surgery, as well as phenylephrine and PBS‐treated NRVMs (Figure [68]1A and [69]1B). However, notably, the protein levels of FAIM2 displayed a significant decrease in both in vitro and in vivo models of cardiac hypertrophy (Figure [70]1C and [71]1D). We also found that the protein level of FAIM2 was markedly decreased in both the 2‐ and 4‐week time points following TAC (Figure [72]S1), indicating that FAIM2 might be an early marker during the progression of cardiac hypertrophy. Furthermore, immunohistochemical staining of FAIM2 revealed a distinct decline in the hearts of mice undergoing TAC surgery when compared with the sham‐operated group (Figure [73]1E). These findings collectively suggest that FAIM2 may be a biological marker in the pathogenesis of cardiac hypertrophy. Figure 1. Fas apoptotic inhibitory molecule 2 (FAIM2) protein level was decreased in cardiac hypertrophic models in vivo and in vitro. Figure 1 [74]Open in a new tab (A) The mRNA level of FAIM2 in mice hearts after 4 weeks of transverse aortic constriction (TAC) or sham surgery (n=5 mice per group). (B) The mRNA level of FAIM2 in control and phenylephrine‐treated neonatal rat ventricular myocytes (NRVMs; phenylephrine, 50 μmol 24 hours). (C) Western blot (upper) and quantification result (lower) of FAIM2 protein in mice hearts after 4 weeks of TAC or sham surgery (n=3 mice per group). (D) Western blot (upper) and quantification result (lower) of FAIM2 protein in control and phenylephrine‐treated NRVMs. (E) Immunohistochemical staining of FAIM2 in mice hearts after 4 weeks of TAC or sham surgery (n=4 mice per group). Scale bar, 25 μm. **P<0.01. * indicates between‐group differences between the control and experimental groups. Normality test and independent samples test were used for all of the statistical methods. All of the cell experiments were completed in 3 independent experiments. n.s. indicates not significant. Faim2 Knockdown Exacerbates Phenylephrine‐Induced Myocardial Dysfunction and Apoptosis in NRVMs, While Faim2 Overexpression Alleviates These Effects In Vitro To elucidate the role of Faim2 in vitro, NRVMs were isolated, infected with adenovirus, and subjected to phenylephrine treatment to mimic myocardial hypertrophy. We initially confirmed the impact of Faim2 using an adenovirus for overexpression (AdFaim2). First, validation of Faim2 overexpression in NRVMs was performed, as shown in Figure [75]2A. Subsequently, immunofluorescent staining of α‐actinin revealed that although the cell surface area increased because of phenylephrine treatment, Faim2 overexpression markedly reduced this enlargement (Figure [76]2B, Figure [77]S2A), indicating the protective role of Faim2 against cardiac hypertrophy in vitro. In addition, analysis of mRNA expression levels of Anp (atrial natriuretic peptide), Bnp (brain natriuretic peptide), Myh7 (β‐myosin heavy chain), and Acta1 (actin α1) demonstrated that Faim2 overexpression effectively attenuated the increased expression of these genes in phenylephrine‐treated NRVMs (Figure [78]2C). Furthermore, contrasting yet plausible changes in the expression of Bax (BCL2‐associated X protein) and Bcl2 (B‐cell lymphoma 2) genes supported the notion that Faim2 overexpression mitigated phenylephrine‐induced apoptosis in vitro (Figure [79]2D). Figure 2. Fas apoptotic inhibitory molecule 2 (FAIM2) overexpression ameliorated phenylephrine‐induced myocardial dysfunction and apoptosis in vitro. Figure 2 [80]Open in a new tab (A) Western blot analysis showing the overexpression effects of Faim2 gene overexpression adenovirus (AdFaim2) in neonatal rat ventricular myocytes (NRVMs). (B) Representative images (left) and cell surface area's statistical results (right) of NRVMs harboring control or Faim2 overexpression adenovirus subjected to phenylephrine treatment or PBS. α‐actinin (the red signal in the figure) was used to stain the cardiomyocyte skeleton, anti‐Faim2 antibody (the green signal in the figure) was used to detect the expression of Faim2 in cells, and DAPI (the blue signal in the figure) was used to stain the cardiomyocyte nucleus. Scale bar=20 μm. (C) Real‐time quantitative polymerase chain reaction (RT‐qPCR) results of atrial natriuretic peptide (Anp), brain natriuretic peptide (Bnp), β‐myosin heavy chain (Myh7), and actin α1 (Acta1) in NRVMs harboring control or Faim2 overexpression adenovirus subjected to phenylephrine treatment or PBS. (D) RT‐qPCR results of BCL2‐associated X protein (Bax) and B‐cell lymphoma 2 (Bcl2) in NRVMs harboring control or Faim2 overexpression adenovirus subjected to phenylephrine treatment or PBS. The asterisk signs indicate the significant difference between the AdVector PBS group and AdVector phenylephrine group, while the pound signs indicate the significant difference between the AdVector phenylephrine group and the AdFaim2 phenylephrine group. ^# P<0.05, ** or ^## P<0.01. *denotes comparisons between the AdVector PBS group and the AdVector phenylephrine group, and # is a comparison between the AdFaim2 PBS group and the AdFaim2 phenylephrine group. Normality test, χ^2 test, and multiple comparisons were used for all of the statistical methods. All of the cell experiments were completed in 3 independent experiments. On the other hand, we also evaluated the phenotype of Faim2 in gene knockdown cells. The efficacy of Faim2 knockdown adenovirus (AdshFaim2) was validated via Western blot analysis (Figure [81]3A). Subsequently, immunofluorescent staining of α‐actinin revealed that phenylephrine treatment increased the cell surface area of NRVMs compared with the PBS groups, while Faim2 knockdown further augmented the cell surface area (Figure [82]3B, Figure [83]S2B), indicating that Faim2 knockdown promoted myocardial hypertrophy in vitro. In addition, cardiac functional markers including Anp, Bnp, Myh7, and Acta1 were upregulated in response to phenylephrine treatment and were further elevated due to Faim2 knockdown (Figure [84]3C), indicating that Faim2 knockdown exacerbated myocardial dysfunction in vitro. Moreover, the higher expression of the proapoptotic gene Bax and lower expression of the antiapoptotic gene Bcl2 in Faim2 knockdown phenylephrine‐treated NRVMs compared with phenylephrine‐treated control NRVMs demonstrated that Faim2 knockdown also intensified phenylephrine‐induced apoptosis in vitro (Figure [85]3D). Collectively, these findings conclude that Faim2 knockdown exacerbates, while Faim2 overexpression ameliorates, phenylephrine‐induced myocardial hypertrophy, dysfunction, and apoptosis in vitro. Figure 3. Fas apoptotic inhibitory molecule 2 (FAIM2) knockdown aggravated phenylephrine‐induced myocardial dysfunction and apoptosis in vitro. Figure 3 [86]Open in a new tab (A) Western blot analysis showing the knockdown efficiency of short hairpin RNA against Faim2 adenoviral constructs (AdshFaim2) adenovirus in neonatal rat ventricular myocyte (NRVMs). (B) Representative images (left) and cell surface area's statistical results (right) of NRVMs harboring control or Faim2 knockdown adenovirus subjected to phenylephrine treatment or PBS. α‐actinin (the red signal in the figure) was used to stain the cardiomyocyte skeleton, anti‐Faim2 antibody (the green signal in the figure) was used to detect the expression of Faim2 in cells, and DAPI (the blue signal in the figure) was used to stain the cardiomyocyte nucleus. Scale bar=20 μm. (C) Real‐time quantitative polymerase chain reaction (RT‐qPCR) results of atrial natriuretic peptide (Anp), brain natriuretic peptide (Bnp), β‐myosin heavy chain (Myh7), and actin α1 (Acta1) in NRVMs harboring control or Faim2 knockdown adenovirus subjected to phenylephrine treatment or PBS. (D) RT‐qPCR results of BCL2‐associated X protein (Bax) and B‐cell lymphoma 2 (Bcl2) in NRVMs harboring control or Faim2 knockdown adenovirus subjected to phenylephrine treatment or PBS. The asterisk signs indicate the significant difference between the nontargeting short hairpin RNA (AdshRNA) PBS group and AdshRNA phenylephrine group, while the pound signs indicate the significant difference between the AdshRNA phenylephrine group and AdshFaim2 phenylephrine group. ^# P<0.05, ** or ^## P<0.01. *denotes comparisons between the AdVector PBS group and the AdVector phenylephrine group, and # is a comparison between the AdFaim2 PBS group and the AdFaim2 phenylephrine group. Normality test, χ^2 test and multiple comparisons were used for all of the statistical methods. All of the cell experiments were completed in 3 independent experiments. FAIM2 Deficiency Aggravates TAC‐Induced Cardiac Dysfunction In Vivo To investigate FAIM2's role in pathological cardiac hypertrophy Faim2‐KO mice were utilized for further study. Initially, the efficacy of Faim2‐KO was confirmed in mice hearts (Figure [87]4A). Subsequently, WT and Faim2‐KO mice were subjected to either TAC or sham surgery. Interestingly, the BW among all groups, including KO TAC, WT TAC, KO sham, and WT sham groups did not exhibit significant differences (Figure [88]4B), confirming the uniformity of mice and the successful implementation of TAC or sham surgery. However, differences were observed in heart function–related parameters among the groups. Following sham surgery, Faim2 deficiency showed no significant effects on HW, HW/BW ratio, lung weight/BW ratio, and HW/tibia length ratio (Figure [89]4C through [90]4F). Yet, following 4 weeks of TAC surgery, HW, HW/BW, lung weight/BW, and HW/tibia length significantly increased in WT TAC mice compared with WT sham mice (Figure [91]4C through [92]4F), indicating the successful establishment of the TAC‐induced cardiac hypertrophic model. Notably, FAIM2 deficiency further intensified these increases in the KO TAC group compared with the WT TAC group (Figure [93]4C through [94]4F). In addition, echocardiographic assessments showed no significant difference in heart rate among all groups (Figure [95]4G). Although FAIM2 deficiency did not alter LVEDd, LV internal diameter at end‐systole, ejection fraction, fractional shortening, interventricular septum diameter in diastole, LV posterior wall diameter in diastole, and corrected LV mass, FAIM2 deficiency accentuated the TAC‐induced changes in LVEDd, LV internal diameter at end‐systole, interventricular septum diameter in diastole, LV posterior wall diameter in diastole, corrected LV mass, ejection fraction, and fractional shortening in opposite directions (Figure [96]4H through [97]4N). The combined results of body index measurements and echocardiographic analyses indicate that FAIM2 deficiency exacerbates TAC‐induced cardiac dysfunction in vivo. Figure 4. Fas apoptotic inhibitory molecule 2 (FAIM2) depletion aggravated transverse aortic constriction (TAC)–induced cardiac dysfunction in vivo. Figure 4 [98]Open in a new tab (A) Western blot analysis showing the knockout (KO) efficiency of Faim2 in mice hearts (n=3 mice per group). (B–F) Statistical results of body weight (BW; B), heart weight (HW; C), HW/BW (D), lung weight (LW)/BW (E), and HW/tibia length (TL; F) in indicated groups (n=10 mice per group). (G–N) Echocardiography measurements of heart rate (HR; G), left ventricular (LV) internal diameter at end‐diastole (LVEDd; H), LV internal diameter at end‐systole (LVESd; I), ejection fraction (EF; J), fractional shortening (FS; K), interventricular septum diameter in diastole (IVSd; L), LV posterior wall diameter in diastole (LVPWd; M), and corrected LV mass (LV mass cor; N) in indicated groups (n=10 mice per group). The asterisk signs indicate the significant difference between the WT sham group and the WT TAC group, while the pound signs indicate the significant difference between the WT TAC group and Faim2‐KO TAC group. * or ^# P<0.05, ** or ^## P<0.01. *denotes comparisons between the WT sham and WT TAC groups, and # denotes comparisons between the KO sham and KO TAC groups. Normality test, χ^2 test, and multiple comparisons were used for all of the statistical methods. FAIM2 Deficiency Aggravates TAC‐Induced Cardiac Hypertrophy, Fibrosis, and Apoptosis In Vivo To acquire more detailed evidence, we analyzed the characteristics of myocardial cells from different experimental groups. First, evaluation of gross hearts, hematoxylin and eosin–stained images, and subsequent statistical analyses collectively demonstrated that FAIM2 deficiency did not alter heart or myocardial cell size following sham surgery. However, it exacerbated the enlarged heart and myocardial cell size induced by TAC surgery (Figure [99]5A). Second, the expression levels of cardiac hypertrophy–related marker genes, such as Anp, Bnp, Myh7, and Acta1, were significantly upregulated in hearts from the WT TAC group compared with the WT sham group. Notably, the increase in gene expression was even more pronounced in the Faim2‐KO TAC group than in the WT TAC group (Figure [100]5B), indicating exacerbated myocardial cell dysfunction caused by FAIM2 deficiency following TAC surgery. Figure 5. Fas apoptotic inhibitory molecule 2 (FAIM2) deficiency aggravated transverse aortic constriction (TAC)–induced cardiac hypertrophy, fibrosis, and apoptosis in vivo. Figure 5 [101]Open in a new tab (A) Representative images of gross hearts, hematoxylin–eosin staining (left) and statistical results of cardiomyocytes' areas (right) in mice hearts of different groups (n=6 mice per group). Scale bars: 0.3 cm, 25 μm. (B) Real‐time quantitative polymerase chain reaction (RT‐qPCR) results of Anp (atrial natriuretic peptide), Bnp (brain natriuretic peptide), Myh7 (β‐myosin heavy chain), and Acta1 (actin α1) in the mice heart of designated groups (n=3 mice per group). (C) Representative images of picrosirius red staining (left) and statistical results of collagen volume (right) in sections of mice hearts from different groups (n=6 mice per group). Scale bar=50 μm. (D) RT‐qPCR results of collagen type I alpha 1 chain (Col1α1), collagen type III alpha 1 chain (Col3α1), connective tissue growth factor (Ctgf), and tissue inhibitor matrix metalloproteinase 1 (Timp1) in the mice hearts of designated groups (n=3 mice per group). (E) RT‐qPCR results of BCL2‐associated X protein (Bax) and B‐cell lymphoma 2 (Bcl2) in the mice heart of designated groups (n=3 mice per group). The asterisk signs indicate the significant difference between the wild‐type (WT) sham group and WT TAC group, while the pound signs indicate the significant difference between the WT TAC group and the Faim2‐knockout (KO) TAC group. * or ^# P<0.05, ** or ^## P<0.01. *denotes comparisons between the WT sham and WT TAC groups, and # denotes comparisons between the KO sham and KO TAC groups. Normality test, χ^2 test, and multiple comparisons were used for all of the statistical methods. Conversely, picrosirius red staining was employed to visualize collagen fibers in myocardial cells. While the perivascular or interstitial areas of the heart originally exhibited few or no collagen fibers, TAC surgery notably increased fiber content in these regions. Moreover, Faim2‐KO intensified the severity of fibrosis (Figure [102]5C). In addition, the expression levels of several collagen synthesis or regulation‐related genes—such as collagen type I alpha 1 chain (Col1a1), collagen type III alpha 1 chain (Col3a1), connective tissue growth factor (Ctgf), and tissue inhibitor matrix metalloproteinase 1 (Timp1)—were analyzed. Consistently, these genes displayed significant upregulation following TAC surgery, with markedly higher expression levels observed due to Faim2‐KO (Figure [103]5D). These findings substantiate that Faim2‐KO exacerbates myocardial fibrosis in vivo. Furthermore, the increased expression of the proapoptotic gene Bax and decreased expression of the anti‐apoptotic gene Bcl2 induced by TAC surgery were notably amplified in response to Faim2‐KO (Figure [104]5E), indicating that FAIM2 deficiency worsens apoptosis in TAC‐induced cardiac hypertrophy. Taken together, these observations conclude that FAIM2 deficiency exacerbates TAC‐induced myocardial dysfunction, fibrosis, and apoptosis in vivo. FAIM2 Regulates the MAPK Signaling Pathway Through TAK1 Phosphorylation To deepen our understanding of the regulatory mechanism involving FAIM2, hearts from WT and Faim2‐KO mice that underwent TAC surgery were collected for transcriptomic sequencing analysis. First, hierarchical cluster analysis distinctly segregated the samples into 2 distinct subgroups (Figure [105]6A), illustrating pronounced heterogeneity between the Faim2‐KO TAC and WT TAC group. Second, GSEA revealed significant enrichment of genes associated with biological processes in Gene Ontology data sets, particularly those related to heart function, protein processing, and fibrosis, in the Faim2‐KO TAC group (Figure [106]6B). Finally, heatmaps illustrating differentially expressed genes involved in heart function, protein processing, and fibrosis indicated that the majority of listed genes were upregulated in the Faim2‐KO TAC group (Figure [107]6C). These findings suggest that Faim2‐KO elevates the expression of genes linked to heart function and fibrosis following TAC surgery, contributing to the exacerbation of cardiac hypertrophy. Figure 6. RNA‐sequencing (RNA‐Seq) analysis showing that Fas apoptotic inhibitory molecule 2 (FAIM2) deficiency aggravates transverse aortic constriction (TAC)–induced heart dysfunction and fibrosis. Figure 6 [108]Open in a new tab (A) Hierarchical clustering dendrogram showing the distribution of samples from wild‐type (WT) and Faim2‐knockout (KO) mice hearts subjected to TAC surgery. (B) Enrichment analysis of biological processes of Gene Ontology data sets, including heart function, protein processing and fibrosis, in the RNA‐Seq results from WT and Faim2‐KO mice hearts subjected to TAC surgery. (C) Heatmaps showed the differentially expressed genes involved in the heart function, protein processing, and fibrosis in the RNA‐Seq results from WT and Faim2‐KO mice hearts subjected to TAC surgery. To further delve into the downstream mechanisms directly influenced by the FAIM2 gene, differential gene expression identification was utilized (representative top genes are shown in Table [109]S4), followed by enrichment analysis to delineate alterations in signaling pathways. Our study demonstrated nearly identical dysregulation of the mitogen‐activated protein kinase (MAPK) and protein kinase B (AKT) pathways following Faim2‐KO, as revealed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (Figure [110]7A). In addition, quantitative polymerase chain reaction validation of 5 upregulated differentially expressed genes confirmed the reproducibility of our RNA‐sequencing analysis (Figure [111]S3). While both pathways are known to have significant implications for pathological cardiac hypertrophy, prior research suggests that the MAPK signaling pathway primarily induces cardiac hypertrophy through cytokine activation, whereas the AKT signaling pathway is predominantly associated with cardiac hypertrophy induced by physiological stimuli such as exercise. Further analysis of the differential expression genes in 2 pathways reveals their roles in signal transduction. Pathway analysis using pathview demonstrates that some differential genes in the MAPK pathway are direct downstream effectors of MAP cascades, while differential genes in the PI3K‐AKT pathway mainly function as upstream ligands and cell membrane receptors in this signaling cascade (data not shown). Therefore, the gene alterations along the MAPK signaling axis are more extensive, indicating their priority as core mechanisms for experimental validation. Figure 7. Fas apoptotic inhibitory molecule 2 (FAIM2) regulates the mitogen‐activated protein kinase (MAPK) signaling pathway through transforming growth factor β–activated kinase 1 (TAK1) phosphorylation. Figure 7 [112]Open in a new tab (A) Kyoto Encyclopedia of Genes and Genomes analysis of RNA sequencing results from wild‐type (WT) and Faim2‐knockout (KO) mice hearts subjected to transverse aortic constriction (TAC) surgery. (B) Western blot (upper) and quantification results (lower) of representative MAPKs in WT and Faim2‐KO mice hearts subjected to TAC surgery (n=3 mice per group). (C) Western blot (upper) and quantification results (lower) of representative MAPKs in neonatal rat ventricular myocytes (NRVMs) harboring control or Faim2 knockdown adenovirus subjected to phenylephrine treatment. (D) Western blot (upper) and quantification results (lower) of representative MAPKs in NRVMs harboring control or Faim2 overexpression adenovirus subjected to phenylephrine treatment. (E) Western blot (upper) and quantification results (lower) of representative MAPK kinase kinases in WT and Faim2‐KO mice hearts subjected to TAC surgery (n=3 mice per group). (F) Western blot (upper) and quantification results (lower) of representative MAPK kinase kinases in NRVMs harboring control or Faim2 knockdown adenovirus subjected to phenylephrine treatment. (G) Western blot (upper) and quantification results (lower) of representative MAPK kinase kinases in NRVMs harboring control or Faim2 overexpression adenovirus subjected to phenylephrine treatment. ^#＜0.05. ^## P<0.01. # indicates comparison of control and experimental groups. Normality and independent samples tests were used for all of the statistical methods. All of the cell experiments were completed in 3 independent experiments. AdshFaim2 indicates short hairpin RNA against Faim2 adenoviral constructs; AdshRNA, nontargeting short hairpin RNA; AKT, protein kinase B; ASK1, apoptosis signal‐regulated kinase 1; ERK1/2, extracellular signal‐regulated kinase1/2; JNK1/2, c‐Jun NH2‐terminal kinase1/2; n.s., not significant; p‐ASK1, phosphorylated apoptosis signal‐regulated kinase 1; p‐ERK1/2, phosphorylated extracellular signal‐regulated kinase1/2; p‐JNK1/2, phosphorylated c‐Jun NH2‐terminal kinase1/2; p‐AKT, phosphorylated protein kinase B; and p‐TAK1, phosphorylated transforming growth factor β–activated kinase 1. We further detected the activity of the MAPK signaling pathway (3 downstream MAPK, including ERK1/2, c‐Jun NH2‐terminal kinase 1/2 [JNK1/2], and p38) and the PI3K‐AKT signaling pathway. As shown in Western blot assays, only phosphorylated JNK1/2 (Thr183/Tyr185 site) and p‐p38 (Thr180/Tyr182) were significantly increased in the KO TAC group, while phosphorylated ERK1/2 (Thr204/Tyr202) and AKT (p‐AKT‐Ser473) showed no significant changes between KO TAC and WT TAC groups (Figure [113]7B). This suggests that FAIM2 may regulate the activation of JNK1/2‐P38 signal cascades. This regulatory pattern was further validated in NRVMs subjected to phenylephrine treatment, in the absence or presence of FAIM2 (Figure [114]7C and [115]7D). Despite our initial observation of similar dysregulation in both pathways, it is important to note that phosphorylation analysis via Western blot analysis did not detect a significant difference in the AKT pathway after FAIM2 knockdown or overexpression. Altogether, these data further support the conclusion that FAIM2 may regulate the activation of JNK1/2‐P38 signal cascades. Previous research indicates that TAK1 and apoptosis signal‐regulated kinase 1 (ASK1) were the prominent hubs of MAP3K responsible for JNK1/2‐p38 signal cascades in cardiac hypertrophy.[116] ^19 , [117]^21 , [118]^22 , [119]^23 , [120]^24 We thus further detected the phosphorylation levels of TAK1 (p‐TAK1‐Thr184/187) and phosphorylated ASK1 (p‐ASK1‐Thr845) in the hearts of Faim2‐KO TAC mice groups and NRVMs with Faim2 knockdown and overexpression. Data showed that only phosphorylation of TAK1 was affected by FAIM2 (Figure [121]7E through [122]7G). These findings indicate that TAK1, but not ASKT, is the core downstream target of FAIM2. Collectively, these findings provide evidence that FAIM2 potentially regulates the MAPK signaling pathway initiated from TAK1, contributing to the regulation of cardiac hypertrophy. The Exacerbating Role of FAIM2 Knockdown in Cardiac Hypertrophy In Vitro Is Mediated by the Activation of TAK1 To elucidate the relationship between TAK1 and FAIM2, a series of rescue assays were conducted. Initially, an endogenous immunoprecipitation assay in NRVMs overexpressing FAIM2 validated an interaction between FAIM2 and TAK1 (Figure [123]8A). 5Z‐7‐ox is a novel TAK1 inhibitor that exhibits strong efficacy in various diseases, including cancers and cardiovascular diseases.[124] ^25 , [125]^26 5Z‐7‐ox has been reported in some studies to have a significant inhibitory effect against TAK1 both in vitro and in vivo in cardiac hypertrophy.[126] ^19 , [127]^21 , [128]^27 We thus used 5Z‐7‐ox to further validate whether inhibition of TAK1 activity could reverse the effects of Faim2 in vitro. The efficacy of 5Z‐7‐ox was first confirmed by Western blot analysis, demonstrating selective inhibition of the phosphorylation of TAK1 protein levels (Figure [129]8B). Under conditions of phenylephrine treatment, knockdown of Faim2 significantly enlarged the cell surface area of NRVMs. However, 5Z‐7‐ox treatment markedly counteracted the prohypertrophic effects induced by Faim2 knockdown, effectively suppressing myocardial hypertrophy (Figure [130]8C). This evidence supports the notion that inhibition of TAK1 activity mitigates Faim2 knockdown‐mediated myocardial hypertrophy in vitro. Subsequently, the mRNA expression levels of Anp, Bnp, Myh7, and Acta1 were significantly upregulated due to Faim2 knockdown in phenylephrine‐treated NRVMs. Remarkably, on application of the 5Z‐7‐ox, mRNA expressions were notably downregulated, nearly restoring them to control levels (Figure [131]8D). This indicates that Faim2 knockdown‐induced exacerbation of myocardial dysfunction was effectively mitigated by TAK1 inactivation. In addition, a similar trend was observed in the proapoptotic gene Bax, with a contrary trend seen in the antiapoptotic gene Bcl2 (Figure [132]8E). This supports the assertion that inhibition of TAK1 activity counteracted the aggravated effect of Faim2 knockdown on apoptosis induced by phenylephrine treatment in vitro. Altogether, these data suggest that Faim2 knockdown exacerbates cardiac hypertrophy through TAK1 activation in vitro. In conclusion, we describe the important role of FAIM2 in cardiac hypertrophy, which protects the heart from pressure overload–induced hypertrophy, and that FAIM2 has potential as a therapeutic target for cardiac hypertrophy and HF (Figure [133]8F). Figure 8. The exacerbating role of Fas apoptotic inhibitory molecule 2 (Faim2) knockdown in cardiac hypertrophy in vitro is mediated by the activation of transforming growth factor β–activated kinase 1 (TAK1). Figure 8 [134]Open in a new tab (A) Endogenous immunoprecipitation (IP) assays proved the interaction between FAIM2 and TAK1 in cardiomyocytes. (B) Western blot (left) and quantification results (right) verified the inhibitory effects of 5Z‐7‐oxozeaenol (5Z‐7‐ox) on TAK1 activity. The neonatal rat ventricular myocytes (NRVMs) were treated with 1 μmol of 5Z‐7‐ox for 24 hours in the presence of phenylephrine (50 μmol) pressure. (C) Representative images (left) and cell surface area's statistical results (right) of NRVMs harboring nontargeting short hairpin RNA (AdshRNA) and short hairpin RNA against Faim2 adenoviral constructs (AdshFaim2) adenovirus with 5Z‐7‐ox or control (CT) subjected to phenylephrine treatment. α‐actinin was used to stain the cardiomyocyte skeleton, and DAPI was used to stain the cardiomyocyte nucleus. Scale bar=20 μm. (D) Real‐time quantitative polymerase chain reaction (RT‐qPCR) results of Anp (atrial natriuretic peptide), Bnp (brain natriuretic peptide), Myh7 (β‐myosin heavy chain), and Acta1 (actin α1) in NRVMs harboring AdshRNA and AdshFaim2 adenovirus with 5Z‐7‐ox or CT subjected to phenylephrine treatment. (E) RT‐qPCR results of BCL2‐associated X protein (Bax) and B‐cell lymphoma 2 (Bcl2) in NRVMs harboring AdshRNA and AdshFaim2 adenovirus with 5Z‐7‐ox or CT subjected to phenylephrine treatment. (F) Model of FAIM2 function in pressure overload–induced hypertrophic signaling. The asterisk signs indicate the significant difference between the AdshRNA CT phenylephrine group and the AdshFaim2 CT phenylephrine group, while the pound signs indicate the significant difference between the AdshFaim2 CT phenylephrine group and the AdshFaim2 5Z‐7‐ox phenylephrine group. ** or ^## P<0.01. * denotes comparison of the AdshRNA CT phenylephrine group and the AdshFaim2 CT phenylephrine group, and # denotes comparison of the AdshFaim2 CT phenylephrine group and the AdshFaim2 5A‐7‐ox phenylephrine group. Normality test, χ^2 test, and multiple comparisons were used for all of the statistical methods. All of the cell experiments were completed in 3 independent experiments. ERK indicates extracellular signal‐regulated kinase; JNK, c‐Jun NH2‐terminal kinase; MAPK, mitogen‐activated protein kinase; and P‐TAK1, phosphorylated transforming growth factor β–activated kinase 1. DISCUSSION Pathological cardiac hypertrophy, triggered by stressors such as pressure overload, can lead to HF—a prevalent and critical cardiovascular condition. Cardiac hypertrophy often coincides with myocardial fibrosis, characterized by increased cardiac collagen accumulation.[135] ^28 , [136]^29 Understanding the molecular mechanisms underlying this process is crucial for developing effective therapeutic interventions. In this study, we aimed to investigate the role of FAIM2 in the context of pathological cardiac hypertrophy and its subsequent impact on myocardial fibrosis. Previous observations of decreased FAIM2 expression in mouse hearts subjected to TAC and in cardiomyocytes treated with phenylephrine prompted our exploration into its potential role in mitigating or exacerbating cardiac hypertrophy and fibrosis. To elucidate the influence of FAIM2 on cardiac hypertrophy and fibrosis, we employed gain‐ and loss‐of‐function approaches. FAIM2 expression was manipulated to investigate its effects in response to pressure overload–induced cardiac hypertrophy. Specifically, we utilized techniques such as gene KO mice and assessed cardiac hypertrophy markers and fibrotic indicators. The observed effects of FAIM2 deficiency exacerbates fibrosis and hypertrophy, while its overexpression ameliorates these conditions. These findings from our study suggest a pivotal role for FAIM2 in modulating cardiac hypertrophy and fibrosis, highlighting the potential therapeutic significance of targeting FAIM2 in managing HF. FAIM2's ability to mitigate cardiac fibrosis may hold promise for the development of novel treatments aimed at alleviating cardiac hypertrophy and preventing HF progression. Exploring the molecular mechanisms through which FAIM2 regulates pathological cardiac hypertrophy and fibrosis is crucial for identifying novel treatment strategies. Previous studies have shown the protective effect of FAIM2 in both cell cultures and a mouse transient vascular ischemia model.[137] ^14 , [138]^15 In addition, a FAIM2 family member, lysosomal membrane protein‐1, can ameliorate pathological cardiac hypertrophy by targeting TLR4 degradation.[139] ^16 To our knowledge, this is the first study to demonstrate that FAIM2 could alleviate pathological cardiac hypertrophy through the MAPK signaling pathway. Interestingly, we found that the protein level of FAIM2 in the pathogenesis of cardiac hypertrophy was downregulated, while the mRNA level remained unchanged, indicating the posttranslational modification of FAIM2 in cardiac hypertrophy. Previous research has shown that phosphorylation can affect the interaction between Faim2 and E3 ubiquitin ligases. Phosphorylated Faim2 may reduce the affinity for these ligases, leading to decreased ubiquitination and protein degradation.[140] ^30 Similarly, FAIM2 methylation can influence the targeting of the ubiquitin‐proteasome system or autophagy, potentially resulting in reduced degradation, which contributes to its protein stabolity.[141] ^31 Although the mechanism of FAIM2 protein stability in the pathogenesis of cardiac hypertrophy was not fully investigated in our current work, we speculate that FAIM2 might be repressed by the ubiquitin‐proteasome system, which is an interesting question in our further research. The MAPK pathway is a fundamental signal transduction pathway involved in various cellular processes, including proliferation, differentiation, stress responses, inflammation, and apoptosis.[142] ^32 , [143]^33 , [144]^34 Our results reveal increased expression of phosphorylated JNK1/2 and p38, indicating their biological activity, in both in vivo and in vitro pathological cardiac hypertrophy models.[145] ^35 , [146]^36 , [147]^37 The identification of the MAPK signaling pathway as a key mediator through which FAIM2 exerts its effects in cardiac hypertrophy marks a significant advancement in understanding its mechanistic involvement. The activation of JNK1/2 and p38 signaling cascades in response to pathological stimuli aligns with their established roles in cellular stress responses and inflammatory processes. The delineation of this pathway's involvement in FAIM2‐mediated cardioprotection provides crucial insights into potential therapeutic targets for modulating hypertrophic responses. The relevance of the MAPK pathway in cardiovascular diseases has been demonstrated by the impact of drugs such as statins, which exhibit cardioprotective effects partly through MAPK inhibition.[148] ^38 Drawing parallels between these pharmacological interventions and the observed modulation of MAPK signaling by FAIM2 sheds light on potential therapeutic avenues. The activation of JNK1/2 and p38 signaling pathways occurs in response to various stresses, including genotoxic, osmotic, hypoxic, and oxidative stress, as well as proinflammatory cytokines such as tumor necrosis factor α and interleukin 1β.[149] ^39 , [150]^40 Certain drugs, including statins, can reduce cardiovascular events (eg, stroke, myocardial infarction, cardiovascular death) by likely inhibiting the MAPK signaling pathway,[151] ^41 , [152]^42 which could be relevant in the context of myocardial infarction–induced cardiac hypertrophy. Finally, in our study, while the levels of ERK1/2 and ASK1 showed no significant changes in TAC‐induced mouse hearts and phenylephrine‐treated cardiomyocytes, phosphorylated TAK1 was notably increased in the TAC‐induced Faim2‐KO group compared with the WT group. The subsequent inhibition of the MAPK signaling pathway by the TAK1 inhibitor, 5Z‐7‐ox, further strengthens the link between FAIM2 and MAPK pathway regulation. Understanding the interplay between FAIM2 and TAK1‐mediated signaling cascades presents a potential focal point for therapeutic interventions aimed at attenuating cardiac hypertrophy. Future research endeavors should aim to delve deeper into the molecular intricacies of how FAIM2 modulates the MAPK pathway to mitigate pathological cardiac hypertrophy. Exploring specific interactions and downstream effectors within this pathway could reveal novel targets for drug development or therapeutic interventions aimed at managing cardiac hypertrophy and associated complications. However, our study has several limitations. We employed global Faim2‐KO mice instead of cell type–specific KO, which limited the comprehensive investigation of FAIM2's cardiomyocyte‐specific effects in pathological cardiac hypertrophy in vivo. In addition, further exploration is required to investigate whether other signaling pathways are influenced by FAIM2. In conclusion, FAIM2 plays a pivotal role in modulating pressure overload–induced cardiac hypertrophy and fibrosis. Notably, FAIM2 acts as a negative regulator in the development of pathological cardiac hypertrophy by inhibiting the activation of the MAPK signaling cascade. These findings offer new insights into the mechanisms underlying pathological cardiac hypertrophy and may contribute to the exploration of effective therapeutic strategies for its treatment by targeting FAIM2. Sources of Funding This work was supported by the Key R&D Program of Jiangxi Province of China (20223BCG74003) and Educational Commission of Jiangxi Province of China (GJJ2201440). Ganzhou City “Science and Technology + Medical” union plan project (2023NS326667). Disclosures None. Supporting information Data S1 [153]JAH3-13-e034257-s001.pdf^ (905.9KB, pdf) This article was sent to Sakima A. Smith, MD, MPH, Associate Editor, for review by expert referees, editorial decision, and final disposition. Supplemental Material is available at [154]https://www.ahajournals.org/doi/suppl/10.1161/JAHA.124.034257 For Sources of Funding and Disclosures, see page 20. Contributor Information Wei Liao, Email: gzliaowei@163.com. Yijian Chen, Email: chenyj2005@163.com. REFERENCES