Abstract Background Pathological cardiac hypertrophy is regarded as a critical precursor and independent risk factor of heart failure, and its inhibition prevents the progression of heart failure. Switch‐associated protein 70 (SWAP70) is confirmed important in immunoregulation, cell maturation, and cell transformation. However, its role in pathological cardiac hypertrophy remains unclear. Methods and Results The effects of SWAP70 on pathological cardiac hypertrophy were investigated in Swap70 knockout mice and Swap70 overexpression/knockdown cardiomyocytes. Bioinformatic analysis combined with multiple molecular biological methodologies were adopted to elucidate the mechanisms underlying the effects of SWAP70 on pathological cardiac hypertrophy. Results showed that SWAP70 protein levels were significantly increased in failing human heart tissues, experimental transverse aortic constriction–induced mouse hypertrophic hearts, and phenylephrine‐stimulated isolated primary cardiomyocytes. Intriguingly, phenylephrine treatment decreased the lysosomal degradation of SWAP70 by disrupting the interaction of SWAP70 with granulin precursor. In vitro and in vivo experiments revealed that Swap70 knockdown/knockout accelerated the progression of pathological cardiac hypertrophy, while Swap70 overexpression restrained the cardiomyocyte hypertrophy. SWAP70 restrained the binding of transforming growth factor β‐activated kinase 1 (TAK1) and TAK1 binding protein 1, thus blocking the phosphorylation of TAK1 and downstream c‐Jun N‐terminal kinase/P38 signaling. TAK1 interacted with the N‐terminals (1–192) of SWAP70. Swap70 (193–585) overexpression failed to inhibit cardiac hypertrophy when the TAK1–SWAP70 interaction was disrupted. Either inhibiting the phosphorylation or suppressing the expression of TAK1 rescued the exaggerated cardiac hypertrophy induced by Swap70 knockdown. Conclusions SWAP70 suppressed the progression of cardiac hypertrophy, possibly by inhibiting the mitogen‐activated protein kinases signaling pathway in a TAK1‐dependent manner, and lysosomes are involved in the regulation of SWAP70 expression level. Keywords: cardiac hypertrophy, granulin precursor, heart failure, mitogen‐activated protein kinases, SWAP70 Subject Categories: Basic Science Research, Mechanisms, Hypertrophy __________________________________________________________________ Nonstandard Abbreviations and Acronyms GRN granulin precursor JNK c‐Jun N‐terminal kinase MAPK mitogen‐activated protein kinase MYH7/β‐MHC β‐myosin‐heavy‐chain NRVMs neonatal rat ventricular myocytes SWAP70 switch‐associated protein 70 TAB1 transforming growth factor β‐activated kinase 1 binding protein 1 TAC transverse aortic constriction TAK1 transforming growth factor β‐activated kinase 1 WT wild‐type Clinical Perspective. What Is New? * Generation of Swap70 knockout mice and Swap70 overexpression/knockdown primary cardiomyocytes revealed the protective effect of switch‐associated protein 70 (SWAP70) on myocardial hypertrophy. * Granulin precursor regulates the protein degradation of SWAP70 through lysosomes and reduces the degradation of SWAP70 by reducing the binding to SWAP70 under hypertrophic stimuli. SWAP70 inhibits cardiomyocyte hypertrophy by inhibiting transforming growth factor β‐activated kinase 1–transforming growth factor β‐activated kinase 1 binding protein 1 interaction, thus inhibiting the activation of the downstream transforming growth factor β‐activated kinase 1–JNK‐P38 pathway. What Are the Clinical Implications? * Our results indicated that SWAP70 may be a potential target for preventing cardiac hypertrophy, and the study on the upstream and downstream regulation of SWAP70 may provide ideas for future clinical studies. The adult mammalian heart contains ≈65% to 80% by volume and 30% to 40% by number of cardiomyocytes.[42] ^1 The size of the heart and cardiomyocyte after birth is closely related to the size or metabolic requirements of the human body. Diseases such as hypertension, coronary heart disease, and valvular disorders can lead to cardiomyocyte enlargement and further pathological cardiac hypertrophy, which is regarded as an adaptive myocardial response to pressure overload.[43] ^2 Constant stress damage to the heart eventually leads to myocardial systolic dysfunction, cytoskeleton remodeling, fibrosis, chamber dilation, increased risk of circulatory failure, and even death.[44] ^3 , [45]^4 Pathological cardiac hypertrophy is a critical precursor and independent risk factor for heart failure.[46] ^5 , [47]^6 Evidence indicates that inhibition of pathological cardiac hypertrophy can effectively prevent heart failure.[48] ^7 Through the exploration of related pathways and their corresponding mechanisms, great progress has been achieved in the control of hypertension and treatment of secondary cardiac hypertrophy attributable to other diseases. On the other hand, mechanisms, including cell metabolism, immune response, translational regulation, and posttranscriptional control neglected previously, have been demonstrated to negatively or positively regulate the progression of pathological cardiac hypertrophy.[49] ^7 , [50]^8 , [51]^9 Further studies about the corresponding cellular signaling pathways, such as phosphoinositide 3‐kinase or mitogen‐activated protein kinase (MAPK) pathways, explored their unique roles in the various forms of hypertrophy.[52] ^10 , [53]^11 , [54]^12 Today, although increasingly advanced medical treatments can improve the symptoms of heart failure, they cannot stop the progression of the disease or reverse the pathological progression.[55] ^13 , [56]^14 Exploration on the underlying mechanisms of pathological myocardial hypertrophy is essential to delay or even reverse the progression of cardiac hypertrophy. Switch‐associated protein 70 (SWAP70) is a rho guanine nucleotide exchange factor belonging to a small family of only 2 members, SWAP70 and FDCP 6 homolog (DEF6), which are originally discovered in B and T cells, respectively.[57] ^15 SWAP70, which has a unique protein structure and functional characteristics, plays important roles in normal cellular processes, including immunoregulation, cellular morphological changes, and cell maturation.[58] ^16 , [59]^17 , [60]^18 Although research on SWAP70 is mainly focused on immune cells (B cells, dendritic cells, and mast cells), studies on other nonimmune cells, such as liver cells,[61] ^19 fibroblasts,[62] ^20 blood cells,[63] ^21 and osteoclasts,[64] ^22 have also revealed a unique role of SWAP70 in disease progression. Deletion of Swap70 can lead to limited immune protection,[65] ^23 , [66]^24 , [67]^25 decreased cell invasion,[68] ^16 , [69]^26 and reduced cell differentiation and maturation.[70] ^18 , [71]^27 For example, Swap70 deficiency can lead to poor B cell migration.[72] ^25 Swap70 knockout mouse embryo fibroblasts (MEFs) grow more slowly and express less invasive phenotypes than wild‐type MEFs.[73] ^26 Swap70 deficiency in mice causes the accumulation of hematopoietic stem and precursor cells and reduces the differentiation of hematopoietic stem cells.[74] ^27 The viability and functional status of cardiomyocytes, as terminally differentiated cells, are critical to maintaining normal heart function. The MAPK pathway, which we find can be regulated by SWAP70 and inhibits the progression of nonalcoholic steatohepatitis,[75] ^19 appears closely related to pathological cardiac hypertrophy and plays a key role in its progression.[76] ^10 , [77]^28 Hyperactivation of the MAPK pathway can aggravate pathological cardiac hypertrophy, whereas inhibition of this pathway causes opposite results.[78] ^29 , [79]^30 However, the relationship between SWAP70 and pathological hypertrophy remains to be established. The present study aimed to examine the role of SWAP70 in pathological cardiac hypertrophy, with a focus on the MAPK pathway. The mechanisms underlying the regulation of SWAP70 protein expression were also investigated. This study may serve as a foundation for developing a strategy to prevent the progression of pathological cardiac hypertrophy. Methods The data that support the findings of this study are available from the corresponding author upon reasonable request. See Data [80]S1 and Tables [81]S1 through [82]S3 for detailed methods and materials used in the establishment of mouse models, detection of corresponding indicators, generation of Swap70 knockout mice, isolation of primary neonatal rat ventricular myocytes (NRVMs) and cell culture, histological staining, immunofluorescence staining, plasmid construction and adenovirus infection, immunoblot analysis, quantitative real‐time polymerase chain reaction, immunoprecipitation assay, and RNA sequencing. Human Heart Samples Left ventricular tissue specimens were procured from patients with end‐stage heart failure undergoing heart transplantation for hypertrophic cardiomyopathy or dilated cardiomyopathy, and normal left ventricular tissue specimens were collected from heart donors identified unsuitable for transplantation for noncardiac reasons. All heart tissue specimens were stored in liquid nitrogen or soaked in 4% paraformaldehyde. All human samples were obtained with written informed consent from the patients or their families and after authorization by the Hospital Committee for Investigation in Humans. All human sample collections were approved by the Human Research Ethics Committee of Zhongnan Hospital of Wuhan University (2022075K) and consistent with the principles outlined in the Declaration of Helsinki. Mice All animal experiments were approved by the Institutional Animal Care and Use Committee of the Zhongnan Hospital of Wuhan University (ZN2021201). The mice received humane care in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. C57BL/6 male mice (weight, 25.5–27 g; age, 9–11 weeks) were fed in a pathogen‐free environment (temperature, 22–24 °C; humidity, 40%–70%; and 12‐hour light cycle). The animals were in good health before transverse aortic constriction (TAC) or sham surgery and had access to food and water ad libitum. All data were obtained during this period, and operations and subsequent analyses were performed in a blinded manner. Statistical Analysis All data were analyzed using SPSS (IBM, Armonk, NY) and expressed as mean±SD. Data with normal distribution were subjected to Student's t‐test or 1‐way ANOVA to evaluate differences between 2 or multiple groups, respectively. The Bonferroni post hoc test (assuming homogeneity of variance) or Tamhane T2 post hoc test (assuming heterogeneity of variance) was applied for correction in 1‐way ANOVA. Data with nonnormal distribution were subjected to the Kruskal–Wallis nonparametric statistical test. Differences were considered statistically significant at P<0.05. Results SWAP70 Protein Level Is Increased in Response to Hypertrophic Stimuli To determine changes in SWAP70 expression in response to hypertrophic stimuli, we first measured SWAP70 expression in human failing heart tissues through immunohistochemical staining, Western blot, and quantitative real‐time polymerase chain reaction analyses. Western blot analysis revealed that SWAP70 protein expression was significantly higher in samples extracted from the above human failing heart tissues than that in normal tissues, and this result correlated well with the changes in heart failure markers (ANP [atrial natriuretic peptide], BNP [B‐type natriuretic peptide], and β‐myosin‐heavy‐chain [MYH7/β‐MHC]) (Figure [83]1A). However, the mRNA expression of SWAP70 showed no significant changes in these tissues (Figure [84]1B), suggesting that the increased protein expression of SWAP70 was posttranscriptionally regulated. Consistent with Western blot, immunohistochemical staining indicated higher protein levels of SWAP70 in failing heart tissues from patients with either hypertrophic cardiomyopathy or dilated cardiomyopathy than in normal tissues (Figure [85]1C). In addition, we detected the protein expression of SWAP70 in in vivo and in vitro cardiac hypertrophic models. The protein expression of SWAP70 was increased in cardiac tissues after 4 weeks of TAC surgery or in isolated primary NRVMs after 24 hours of phenylephrine treatment, and these changes were consistent with the changes in ANP, BNP, and MYH7 (Figure [86]1D and [87]1F). However, Swap70 mRNA levels showed minimal changes at indicated times during the in vitro and in vivo experiments (Figure [88]1E and [89]1G). These data revealed that SWAP70 protein expression was increased posttranscriptionally under hypertrophic stimuli. Figure 1. SWAP70 protein level is increased in response to hypertrophic stimuli. Figure 1 [90]Open in a new tab A, Immunoblotting and quantitation of SWAP70, ANP, BNP, MYH7/β‐MHC protein levels in human heart tissues from normal donors (n=5), HCM heart failure patients (n=5), and DCM heart failure patients (n=7). B, Relative mRNA levels of SWAP70 in human heart tissues from normal donors (n=5), HCM heart failure patients (n=5), and DCM heart failure patients (n=9) (NS: left, P=0.90; right, P=1.00). C, Left, expression of SWAP70 in human tissues from normal donors or heart failure patients arise from HCM or DCM. Right, quantitation results of immunohistochemical staining (n=4). D, Protein levels and quantitation results of SWAP70, ANP, BNP, MYH7 in mice hearts subjected to TAC surgery at the indicated time points (n=6). E, Relative mRNA levels of Swap70 in mice heart tissues after TAC operation at indicated time points (n=5) (NS: left, P=1.00; middle, P=0.11; right, P=0.81). F, Protein levels and quantitation results of SWAP70, ANP, BNP, MYH7 in NRVMs with ph PE (50 μmol/L) treatment at indicated time points (n=5 independent experiments). G, Relative mRNA levels of Swap70 in NRVMs with PE (50 μmol/L) treatment at indicated time points (n=5 independent experiments) (NS: left, P=1.00; middle, P=1.00; right, P=0.93). Data are shown as mean±SD. One‐way ANOVA with Bonferroni's post hoc analysis (A through C, E, G, MYH7 in F) or with Tamhane T2 post hoc test (MYH7 in A, D, F) was used. *P<0.05; **P<0.01 for indicated group vs control group. NS (no significance for indicated group vs control group. ANP indicates atrial natriuretic peptide; BNP, B‐type natriuretic peptide; DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; MYH7/β‐MHC, β‐myosin‐heavy‐chain; NRVMs, neonatal rat ventricular myocytes; NS, no significance; PE, phenylephrine; SWAP70, switch‐associated protein 70; and TAC, transverse aortic constriction. Hypertrophic Increase in SWAP70 Is Connected With the Inhibition of Lysosomal Degradation Regulated by Granulin Precursor To identify the source of increased SWAP70 protein in a hypertrophic environment, we first explored the half‐life of SWAP70 in NRVMs, and results showed that the half‐life of SWAP70 was ≈4 hours in the presence of protein translation inhibitor cycloheximide (Figure [91]2A). Further experiments in NRVMs showed that hypertrophic stimulation (phenylephrine treatment) significantly prolonged the half‐life of SWAP70, indicating that the high protein expression of SWAP70 may be ascribed to its reduced protein degradation under hypertrophic stimuli (Figure [92]2B). As shown in Figure [93]2C, SWAP70 protein expression was increased in the NRVMs treated with the lysosomal degradation inhibitor chloroquine but not obviously changed in the cells treated with the proteasome degradation inhibitor MG132, indicating that SWAP70 degradation was lysosome dependent under normal conditions. However, SWAP70 expression showed no obvious change after the treatment with chloroquine or MG132 in the presence of cycloheximide and phenylephrine compared with the control group (Figure [94]2D), suggesting that the increased SWAP70 protein expression after phenylephrine treatment can be ascribed to its reduced lysosomal degradation. To explore the possible upstream molecule(s) involved in the lysosomal regulation of SWAP70 protein expression, we performed protein mass spectrometry, intersected it with a subcellular localization database ([95]https://compartments.jensenlab.org/Search) by bioinformatics to find the molecules that can colocalize with lysosomes (9 molecules remained after this step), and finally used a protein database ([96]https://www.uniprot.org) to inquire about the function of these 9 molecules and gained 3 of them that were reported to involve in the regulation of molecular degradation[97] ^31 , [98]^32 , [99]^33 (Figure [100]2E). Further experiments in human embryonic kidney 293T cells transfected with the indicated molecules suggested that granulin precursor (GRN) regulated the lysosomal degradation of SWAP70 (Figure [101]2F). To confirm this finding, we infected NRVMs with Grn adenovirus at different multiplicities of infection. Results showed that SWAP70 protein expression decreased with the increased expression of Grn adenovirus (Figure [102]2G). However, the mRNA level of Swap70 did not change with GRN overexpression (Figure [103]S1A). Further coimmunoprecipitation assay in human embryonic kidney 293T cells and NRVMs revealed the interaction between SWAP70 and GRN (Figure [104]2H and Figure [105]S1B). As shown in Figure [106]2I and [107]2J, chloroquine or phenylephrine treatment can restrain the GRN‐regulated decrease in SWAP70 protein expression. Interestingly, analysis revealed that hypertrophic stimuli did not downregulate the protein expression of GRN or its 45‐kDa degradation product in NRVMs (Figure [108]2K). Both endogenous and exogenous upregulation of GRN protein could not lead to the downregulation of SWAP70 expression under stimulation conditions, suggesting that the degradation of SWAP70 mediated by GRN may be altered under hypertrophic stimuli. Considering that GRN itself has no enzymatic activity, we speculated that the interaction between SWAP70 and GRN changed under hypertrophic stimuli. Then, coimmunoprecipitation and immunofluorescence analyses revealed that GRN and SWAP70 showed decreased interaction and colocalization in the phenylephrine group (Figure [109]2L, [110]2M, and Figure [111]S1C). Moreover, immunofluorescence showed that GRN increased the colocalization of SWAP70 and lysosomal marker lysosomal associated membrane protein 1, which was suppressed by phenylephrine treatment (Figure [112]2N and Figure [113]S1D). Taken together, these results suggest that lysosomes contribute to the upregulation of SWAP70 protein expression under hypertrophic stimuli, and GRN participates in the lysosomal regulation of SWAP70. Figure 2. Hypertrophic increase in SWAP70 is connected with the inhibition of lysosomal degradation regulated by granulin precursor. Figure 2 [114]Open in a new tab A, Immunoblotting and quantitation of SWAP70 protein levels in NRVMs added with CHX (25 μmol/L) for indicated time points (n=5 independent experiments) (NS: left, P=0.54; right, P=0.21). B, Immunoblotting and quantitation of SWAP70 protein levels in NRVMs added with CHX (25 μmol/L) for indicated time points with or without the stimulation of PE (50 μmol/L) (n=5 independent experiments) (NS: left, P=0.09; right, P=0.08). C, The protein expression and quantitation results of SWAP70 in NRVMs treated with DMSO, CQ (50 μmol/L), or MG132 (50 μmol/L) in the presence of CHX (25 μmol/L) for 6 hours (n=5 independent experiments) (NS: P=0.15). D, The protein expression and quantitation results of SWAP70 in NRVMs treated with DMSO, CQ (50 μmol/L), or MG132 (50 μmol/L) in the presence of CHX (25 μmol/L) and PE (50 μmol/L) for 6 hours (n=5 independent experiments) (NS: left, P=1.00; right, P=0.60). E, Upper, mode pattern showed that NRVMs were infected with Ad‐GFP‐Flag‐Swap70 and treated with PE (50 μmol/L, 48 hours), and then incubated with anti‐IgG or Anti‐Flag for mass spectrometry. Below, mass spectrometry screened the lysosomal related molecules and chose 3 of which according to its cellular function and localization. F, Immunoblotting and quantitation of SWAP70 protein levels in human embryonic kidney 293T cells transfected with indicated molecules (n=5 independent experiments) (NS: left, P=0.24; right, P=0.28). G, Immunoblotting and quantitation of SWAP70 protein levels in NRVMs infected with Ad‐GFP‐Flag‐Grn adenovirus at different multiplicities of infection (n=5 independent experiments). H, CO‐IP assays displayed the interaction between exogenous SWAP70 and endogenous GRN in NRVMs. I, Immunoblotting and quantitation of SWAP70 protein levels in NRVMs infected with or without Ad‐GFP‐Flag‐Grn adenovirus in the presence or absence of CQ (25 μmol/L) for 12 hours (n=5 independent experiments) (NS: P=0.82). J, Immunoblotting and quantitation of SWAP70 protein levels in NRVMs accompanied with indicated adenovirus and drugs (n=5 independent experiments) (NS: Left, P=1.00; Right, P=1.00). K, Immunoblotting and quantitation of GRN protein levels in NRVMs with PE (50 μmol/L) treatment at indicated time points (n=5 independent experiments) (NS: P=0.50). L, CO‐IP assays performed the interaction between SWAP70 and GRN in NRVMs accompanied with indicated adenoviruses and treatments. M, Representative immunofluorescence images of the colocalization of endogenous SWAP70 and exogenous GRN in NRVMs added with CQ (25 μmol/L, 12 hours) and Ad‐Flag‐Grn adenoviruses in the presence or absence of PE (50 μmol/L, 24 hours). N, Representative immunofluorescence images of the colocalization of endogenous SWAP70 and lysosome marker LAMP1 in NRVMs accompanied with indicated adenoviruses and treatments. Scale bar, 25 μm. Data are shown as mean±SD. Student t‐test (B) and 1‐way ANOVA with Bonferroni's post hoc analysis (C, D, G, and J) or with Tamhane T2 post hoc test (A, F, I, K) were used. *P<0.05; **P<0.01 for indicated group vs control group. ^# P<0.05; ^## P<0.01 for PE group vs PBS group. NS for indicated group vs control group or PE group vs PBS group (B). CHX indicates cycloheximide; CO‐IP, coimmunoprecipitation; CQ, chloroquine; GRN, granulin precursor; LAMP1, lysosomal associated membrane protein 1; NRVMs, neonatal rat ventricular myocytes; NS, no significance; PE, phenylephrine; and SWAP70, switch‐associated protein 70. Swap70 Deficiency Accelerates Pathological Cardiac Hypertrophy Swap70 knockout mice were generated (Figure [115]S2A), and experimental pathological cardiac hypertrophy was induced by TAC surgery to evaluate the impact of SWAP70 on pathological cardiac hypertrophy. The efficiency of Swap70 deletion was verified using Western blot (Figure [116]3A). Before surgery, mice were measured and found no difference in weight, heart rate, blood pressure, and blood velocity between different genotypes (Figure [117]S2B through S2D). At 4 weeks after TAC surgery, the heart rate, blood velocity, and pressure gradient showed no difference between Swap70 knockout and wild‐type (WT) mice (Figure [118]S2E through S2G), while the Swap70 knockout mice displayed significantly increased heart size, heart weight, heart/body weight ratios, heart weight/tibia length ratios, and lung/body weight ratios compared with the WT mice (Figure [119]3B through [120]3F). Ultrasonic measurements of the left ventricular end‐systolic diameter and left ventricular end‐diastolic diameter showed similar trends (Figure [121]3G and [122]3H). Meanwhile, the ejection fraction and fractional shortening were lower in the Swap70 knockout mice than those in the WT mice after TAC surgery (Figure [123]3I). Hematoxylin–eosin staining showed that the cross‐sectional area of cardiomyocytes from the Swap70 knockout mice was obviously larger than that of cardiomyocytes from the WT mice under hypertrophic stimuli. Moreover, Swap70 deficiency aggravated the perivascular and interstitial fibrosis of the hypertrophic heart (Figure [124]3J). Corresponding to the hematoxylin–eosin and picrosirius red staining, hypertrophic and fibrosis phenotypes were accompanied by the upregulation of hypertrophic genes (Anp, Bnp, and Myh7) and fibrotic genes (collagen Iα1, collagen IIIα1, connective tissue growth factor [Ctgf], tissue inhibitor of metalloproteinase 1 [Timp1]) in the Swap70 knockout mice compared with the WT mice under hypertrophic stimuli (Figure [125]3K and [126]3L). However, inflammatory genes (insulin 6 [Il‐6], Il‐1b, tumor necrosis factor a [Tnfa]) showed no difference between Swap70 knockout mice and WT mice (Figure [127]S2H). Collectively, these results demonstrated that Swap70 deficiency accelerated pathological cardiac hypertrophy. Figure 3. Swap70 deficiency accelerates pathological cardiac hypertrophy. Figure 3 [128]Open in a new tab A, The protein expression of SWAP70 in mice heart tissues from WT or Swap70‐KO mice (n=4). B, Representative heart size of mice from indicated groups. Scale bar, 0.3 cm. C through F, Heart weight, heart/body weight ratios, heart weight/tibia length ratios, and lung/body weight ratios in WT and Swap70‐knockout mice at 4 weeks after sham or TAC surgery (n=10). G, Representative echocardiography images of indicated groups (n=10). H and I, Echocardiography assessed the parameters of LVEDd, LVESd, EF, and FS in WT or Swap70‐KO mice at 4 weeks after sham or TAC surgery (n=10). J, Representative H&E (upper left) and picrosirius red (lower left) staining in the heart tissues from WT or Swap70‐KO mice at 4 weeks after sham or TAC surgery. Scale bar, 50 μm. Quantitative results of the average cross‐sectional areas and left ventricular interstitial collagen volume is performed at the right (n=6). K and L, Relative mRNA levels of hypertrophy (Anp, Bnp, Myh7), and fibrosis (collagen Iα1, collagen IIIα1, Ctgf, Timp1) related genes in mice heart tissues from indicated groups (n=4). Data are shown as mean±SD. One‐way ANOVA with Bonferroni's post hoc analysis (C through F, H through L) or with Tamhane T2 post hoc test (left ventricular interstitial collagen volume in J, collagen Iα1 and Ctgf in L) were used. *P<0.05; **P<0.01 for WT TAC group vs WT sham group. ^## P<0.01 for Swap70‐KO TAC group vs WT TAC group. ANP indicates atrial natriuretic peptide; BNP, B‐type natriuretic peptide; Ctgf, connective tissue growth factor; EF, ejection fraction; FS, fractional shortening; H&E, hematoxylin–eosin; LVEDd, left ventricular end‐diastolic systolic diameter; LVESd, left ventricular end‐systolic diameter; MYH7, β‐myosin‐heavy‐chain; SWAP70, switch‐associated protein 70; Swap70‐KO, Swap70‐knockout; TAC, transverse aortic constriction; Timp1, tissue inhibitor of metalloproteinase 1; and WT, wild‐type. Swap70 Overexpression Suppresses the Progression of Cardiomyocyte Hypertrophy NRVMs were isolated and treated with phenylephrine to investigate the effect of SWAP70 on simulated cardiomyocyte hypertrophy. Knockdown or overexpression of Swap70 was induced using adenoviruses containing Swap70 short hairpin RNA or Swap70. Data showed that the NRVMs with Swap70 knockdown showed higher protein and mRNA expression levels of hypertrophic markers ANP, BNP, and MYH7 than the control group under hypertrophic stimuli (Figure [129]4A and [130]4B). Consistently, the NRVMs with Swap70 knockdown had larger cardiomyocyte areas than the control group under hypertrophic stimuli (Figure [131]4C). Meanwhile, the NRVMs with Swap70 overexpression showed lower expression of hypertrophic markers and smaller cardiomyocyte areas than the control group following phenylephrine treatment (Figure [132]4D through [133]4F). Taken together, these data indicated that Swap70 overexpression suppressed the progression of cardiomyocyte hypertrophy. Figure 4. Swap70 overexpression suppresses the progression of cardiomyocyte hypertrophy. Figure 4 [134]Open in a new tab A, Immunoblotting (left) and quantitation (right) of SWAP70, ANP, BNP, and MYH7 protein levels in NRVMs infected with Ad‐GFP‐sh‐Swap70 or Ad‐sh‐GFP in the presence or absence of PE (50 μmol/L) for 48 hours (n=5 independent experiments). B, Relative mRNA levels of Swap70 and hypertrophic marker genes (Anp, Bnp, and Myh7) in NRVMs from indicated groups (n=5 independent experiments). C, Representative immunofluorescence images (upper) of NRVMs infected with Ad‐GFP‐sh‐Swap70 or Ad‐sh‐GFP adenovirus in the presence or absence of PE (50 μmol/L) for 24 hours and stained with α‐actinin. Scale bar, 50 μm. Quantitative results (below) of the cardiomyocyte surface area from indicated groups (n=5 independent experiments). D, Immunoblotting (left) and quantitation (right) of SWAP70, ANP, BNP, MYH7 protein levels in NRVMs infected with Ad‐GFP‐Flag‐Swap70 or Ad‐GFP in the presence or absence of PE (50 μmol/L) for 48 hours (n=5 independent experiments). E, Relative mRNA levels of Swap70 and hypertrophic marker genes (Anp, Bnp, and Myh7) in NRVMs from indicated groups (n=5 independent experiments). F, Representative immunofluorescence images (upper) of NRVMs infected with Ad‐GFP‐Flag‐Swap70 or Ad‐GFP adenovirus in the presence or absence of PE (50 μmol/L) for 24 hours and stained with α‐actinin. Scale bar, 50 μm. Quantitative results (below) of the cell surface area from indicated groups (n=5 independent experiments). Data are shown as mean±SD. Student t‐test (A, D) and 1‐way ANOVA with Bonferroni's post hoc analysis (B, E, F) or with Tamhane T2 post hoc test (mRNA level of Myh7 in B, C) were used. *P<0.05; **P<0.01 for GFP PE group vs GFP PBS group. ^# P<0.05; ^## P<0.01 for Swap70 overexpression/knockdown PE group vs GFP PE group. ANP indicates atrial natriuretic peptide; BNP, B‐type natriuretic peptide; MYH7, β‐myosin‐heavy‐chain; NRVMs, neonatal rat ventricular myocytes; PE, phenylephrine; and SWAP70, switch‐associated protein 70. SWAP70 Alleviates the Activation of the Transforming Growth Factor β‐Activated Kinase 1—c‐Jun N‐Terminal Kinase 1/2‐P38 Pathway in Response to Hypertrophic Stimuli To investigate the possible downstream targets of SWAP70 in pathological cardiac hypertrophy, we utilized an RNA sequencing assay to compare the cardiac tissues from the Swap70 knockout mice and control mice under pressure overload. Bioinformatic analysis showed that differentially expressed genes were mainly related to fibrosis, heart function, and protein processing (Figure [135]5A through [136]5C). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the MAPK pathway was significantly changed by Swap70 deficiency (Figure [137]5D). Our previous research found that among some of the upstream molecules of the MAPK pathway, transforming growth factor β‐activated kinase 1 (TAK1) showed the strongest interaction with SWAP70.[138] ^19 We examined the activation of TAK1 and downstream c‐Jun N‐terminal kinase (JNK) 1/2‐P38 molecules in hypertrophic mice or NRVMs and found that Swap70 deficiency or knockdown aggravated the phosphorylation of these molecules and accentuated the hypertrophic phenotype of the myocardium in in vitro and in vivo experiments (Figure [139]5E and [140]5F). Nevertheless, these effects were suppressed in the NRVMs with Swap70 overexpression (Figure [141]5G). Moreover, we detected some landmark proteins in the phosphatidylinositide 3‐kinases–protein kinase B pathway found in sequencing analysis and in the nuclear factor kappa‐B pathway reported before that may be influenced by SWAP70.[142] ^18 Results showed that Swap70 knockdown/overexpression showed no influence on the phosphorylation of protein kinase B and Iκ‐β kinase (Figure [143]S3). These results indicated that Swap70 deficiency promotes the activation of MAPK pathway and the progression of pathological cardiac hypertrophy. Figure 5. SWAP70 alleviates the activation of the TAK1‐JNK1/2‐P38 pathway in response to hypertrophic stimuli. Figure 5 [144]Open in a new tab A, Hierarchical clustering analysis performed the global sample distribution profiles between WT mice and Swap70‐KO mice under TAC surgery based on the RNA sequencing (RNA‐seq, n=3). B, Gene set enrichment analysis of molecular events involved in fibrosis, heart function, and protein processing in RNA‐seq data. C, Heatmaps about the significantly altered genes concerned with cardiac hypertrophy. D, Kyoto Encyclopedia of Genes and Genomes pathway enrichment about the significantly altered pathways according to the RNA‐seq data. E, Immunoblotting analysis of phosphorylation and total TAK1, P38, JNK1/2 proteins in mice heart samples from indicated groups, hypertrophic marker proteins (ANP, BNP, and MYH7) were used to assess the severity of the hypertrophy (n=4). F, Immunoblotting analysis of phosphorylation and total TAK1, P38, JNK1/2 proteins in NRVMs infected with indicated adenovirus and stimulated with PE (50 μmol/L) for a time gradient, hypertrophic marker proteins (ANP, BNP, and MYH7) were used to assess the severity of the hypertrophy (n=4 independent experiments). G, Immunoblotting analysis of phosphorylation and total TAK1, P38, JNK1/2 proteins level in NRVMs infected with Ad‐GFP or Ad‐GFP‐Flag‐Swap70 and stimulated with PE (50 μmol/L) for a time gradient, hypertrophic marker proteins (ANP, BNP, and MYH7) were used to assess the severity of the hypertrophy (n=4 independent experiments). ANP indicates atrial natriuretic peptide; BNP, B‐type natriuretic peptide; JNK, c‐Jun N‐terminal kinase; MYH7, β‐myosin‐heavy‐chain; NRVMs, neonatal rat ventricular myocytes; PE, phenylephrine; Swap70‐KO, Swap70‐knockout; TAK1, transforming growth factor β‐activated kinase 1; and WT, wild‐type. SWAP70 Regulates Cardiomyocyte Hypertrophy by Suppressing the TAK1–Transforming Growth Factor β‐Activated Kinase 1 Binding Protein 1 Interaction To confirm whether SWAP70‐mediated cardioprotective effects are dependent on TAK1 under hypertrophic stimuli, we analyzed the relationship between SWAP70 and TAK1 and found that SWAP70 colocalized with TAK1 in the cytoplasm of NRVMs (Figure [145]6A). Further immunoprecipitation assays showed that SWAP70 interacted with endogenous TAK1 in the NRVMs under hypertrophic stimuli (Figure [146]6B). As in our previous report, TAK1 interacts with the N‐terminals (1–192) of SWAP70.[147] ^19 We established N‐terminal missing adenovirus (193–585) of Swap70 and found that SWAP70 (193–585) could not interact with endogenous TAK1 under hypertrophic stimuli (Figure [148]6C). Further experiment found that Swap70 (WT) instead of Swap70 (193–585) inhibited the phosphorylation of TAK1 and downstream JNK1/2‐P38 molecules, sequentially suppressing the progression of cardiomyocyte hypertrophy (Figure [149]6D). The expression of myocardial markers (ANP, BNP, and MYH7) showed that Swap70 (193–585) failed to inhibit cardiac injury in a hypertrophic environment (Figure [150]6D and [151]6E). The α‐actinin staining indicative of cardiomyocyte area showed similar results (Figure [152]6F). Moreover, we previously discovered that SWAP70 could restrain the activation of TAK1 by inhibiting the interaction of TAK1 and transforming growth factor β‐activated kinase 1 binding protein 1 (TAB1),[153] ^19 which is essential for the activation of TAK1.[154] ^34 We established Ad‐GFP‐Flag‐Tab1 adenovirus and found that SWAP70 could restrain the activation of TAK1‐JNK1/2‐P38 signaling caused by TAB1 overexpression, thus suppressing the expression of hypertrophic markers and inhibiting cardiomyocyte hypertrophy (Figure [155]S4). These data revealed that SWAP70 interacted with TAK1 and that SWAP70 overexpression mediated cardioprotective effects by suppressing the interaction between TAB1 and TAK1. Figure 6. SWAP70 regulates cardiomyocyte hypertrophy by interacting with TAK1. Figure 6 [156]Open in a new tab A, Representative immunofluorescence images of the colocalization of HA‐tagged SWAP70 and endogenous TAK1 in NRVMs infected with Ad‐HA‐Swap70 adenovirus in the presence of PE (50 μmol/L) for 24 hours. Scale bar, 25 μm. B, CO‐IP assays displayed the interaction between endogenous TAK1 and Flag‐tagged SWAP70 in NRVMs. C, CO‐IP assays displayed the interaction between endogenous TAK1 and Flag‐tagged SWAP70 (193–585) in NRVMs. D, Immunoblotting analysis of phosphorylation and total TAK1, P38, JNK1/2 proteins in NRVMs infected with indicated adenoviruses and stimulated with PE (50 μmol/L) for 48 hours, hypertrophic marker proteins (ANP, BNP, and MYH7) were used to assess the severity of the hypertrophy (n=5 independent experiments). E, Relative mRNA levels of hypertrophic marker genes (Anp, Bnp, and Myh7) in NRVMs from indicated groups (n=5 independent experiments) (NS: high, P=0.25; middle, P=0.08; low, P=0.65). F, Representative immunofluorescence images (left) of NRVMs infected with indicated adenovirus in the presence of PE (50 μmol/L) for 24 hours and stained with α‐actinin. Scale bar, 50 μm. Quantitative results (right) of the cell surface area from indicated groups (n=5 independent experiments) (NS: P=0.15). Data are shown as mean±SD. One‐way ANOVA with Bonferroni's post hoc analysis (E, F) or with Tamhane T2 post hoc test (mRNA levels of Myh7 in E) were used. ^## P<0.01 for indicated group vs control group. NS for indicated group vs control group. ANP indicates atrial natriuretic peptide; BNP, B‐type natriuretic peptide; CO‐IP, coimmunoprecipitation; JNK, c‐Jun N‐terminal kinase; MHY7, β‐myosin‐heavy‐chain; NRVMs, neonatal rat ventricular myocytes; NS, no significance; PE, phenylephrine; SWAP70, switch‐associated protein 70; and TAK1, transforming growth factor β‐activated kinase 1. Inhibiting the Activation of TAK1 Rescues the Aggravated Cardiomyocyte Hypertrophy Induced by Swap70 Knockdown Considering that TAK1 activation is essential in the progression of cardiomyocyte hypertrophy, we used the TAK1 inhibitor 5Z‐7‐oxozeaenol to explore the role of TAK1 activity in the progression of cardiomyocyte hypertrophy following Swap70 knockdown. Results showed that the phosphorylation of downstream JNK1/2‐P38 decreased with the suppression of TAK1 activity (Figure [157]7A). At this point, the protein and mRNA expression of myocardial markers (ANP, BNP, and MYH7) upregulated by Swap70 knockdown were countered by 5Z‐7‐oxozeaenol (Figure [158]7A and [159]7C). Immunofluorescence staining of α‐actinin revealed a similar trend in the cardiomyocyte surface area (Figure [160]7B). In addition, we established Ad‐GFP‐sh‐Tak1 adenovirus to further examine the functions of TAK1 in the progression of cardiomyocyte hypertrophy regulated by SWAP70 (Figure [161]7D and [162]7E). Similar to the results obtained with 5Z‐7‐oxozeaenol, Tak1 knockdown significantly decreased the activity of TAK1 and downstream phosphorylation of JNK1/2 and P38 molecules, thus reversing the exacerbation of cardiomyocyte hypertrophy induced by Swap70 knockdown following phenylephrine treatment (Figure [163]7F through [164]7H). These results demonstrated that SWAP70 protected against cardiomyocyte hypertrophy by inhibiting the activation of TAK1. Figure 7. Inhibiting the activation of TAK1 rescues the aggravated cardiomyocyte hypertrophy induced by Swap70 knockdown. Figure 7 [165]Open in a new tab A, Immunoblotting analysis of phosphorylation and total TAK1, P38, JNK1/2 proteins in NRVMs infected with indicated adenoviruses and treated with DMSO or 5Z‐7‐oxozeaenol (100 nmol/L, 12 hours) in the presence of PE (50 μmol/L, 48 hours), hypertrophic marker proteins (ANP], BNP, and MYH7) were used to assess the severity of the hypertrophy (n=5 independent experiments). B, Representative immunofluorescence images (left) of NRVMs infected with indicated adenovirus, treated with DMSO or 5Z‐7‐oxozeaenol (100 nmol/L, 12 hours) in the presence of PE (50 μmol/L, 24 hours) and stained with α‐actinin. Scale bar, 50 μm. Quantitative results (right) of the cell surface area from indicated groups (n=5 independent experiments). C, Relative mRNA levels of Swap70 and hypertrophic marker genes (Anp, Bnp, Myh7) in NRVMs from indicated groups (n=5 independent experiments). D, The protein expression of TAK1 in NRVMs after infection with Ad‐GFP‐sh‐Tak1 #1, #2, #3, or #4 adenovirus for 48 hours. E, Relative mRNA levels of Tak1 in NRVMs after infection with Ad‐GFP‐sh‐Tak1 #2 for 48 hours (n=5 independent experiments) F, Immunoblotting analysis of phosphorylation and total TAK1, P38, JNK1/2 proteins in NRVMs infected with indicated adenoviruses and stimulated with PE (50 μmol/L) for 48 hours, hypertrophic marker proteins (ANP, BNP, and MYH7) were used to assess the severity of the hypertrophy (n=5 independent experiments). G, Representative immunofluorescence images (upper) of NRVMs infected with indicated adenovirus, treated with PE (50 μmol/L) for 24 hours and stained with α‐actinin. Scale bar, 50 μm. Quantitative results (below) of the cell surface area from indicated groups (n=5 independent experiments). H, Relative mRNA levels of Swap70, Tak1, and hypertrophic marker genes (Anp, Bnp, and Myh7) in NRVMs from indicated groups (n=5 independent experiments). Data are shown as mean±SD. Student t‐test (E) and 1‐way ANOVA with Bonferroni's post hoc analysis (B, C, G, mRNA levels of Myh7 in H) or with Tamhane T2 post hoc test (H) were used. Kruskal‐Wallis nonparametric statistical test (mRNA levels of Tak1 in H) was used for nonnormal distribution data. *P<0.05; **P<0.01 for indicated group vs control group. ^## P<0.01 for indicated group vs sh‐Swap70 PE group. ANP indicates atrial natriuretic peptide; BNP, B‐type natriuretic peptide; JNK, c‐Jun N‐terminal kinase; MHY7, β‐myosin‐heavy‐chain; NRVMs, neonatal rat ventricular myocytes; PE, phenylephrine; and TAK1, transforming growth factor β‐activated kinase 1. Discussion The salient findings from our present study revealed that Swap70 deficiency could promote the progression of cardiac hypertrophy. Under hypertrophic stimuli, SWAP70 was less degraded by lysosome through an interaction change between GRN and SWAP70. Further gain‐ and loss‐of‐function approaches showed that Swap70 overexpression inhibited the progression of cardiomyocyte hypertrophy in a TAK1‐dependent manner by suppressing the TAK1‐TAB1 interaction and subsequent activation of MAPK pathway. Previous studies on SWAP70 mainly focused on immunity, tumor progression, and erythropoiesis.[166] ^35 , [167]^36 , [168]^37 , [169]^38 , [170]^39 , [171]^40 , [172]^41 SWAP70 interacts with F‐actin, interferon regulatory factor 4, and Rac1, thus participating in the membrane ruffling,[173] ^37 actin rearrangements,[174] ^38 phagocytosis,[175] ^39 micropinocytosis,[176] ^40 and differentiation[177] ^41 of cells. SWAP70 seems to allow cells to evolve into more dynamic forms and functions. However, its role in pathological cardiac hypertrophy remains unclear. In the present study, SWAP70 protein expression was significantly upregulated in hypertrophic cardiomyocytes, and Swap70 overexpression delayed the progression of cardiac hypertrophy. This result indicated that the hypertrophy‐induced upregulation of SWAP70 protein was a compensatory but insufficient response to cardiac injury. Gain‐ and loss‐of‐function studies revealed the protective effects of SWAP70 against the progression of pathological cardiac hypertrophy. Intriguingly, Swap70 deletion or overexpression alone did not exert significant effects on the mice or primary cardiomyocytes, indicating that SWAP70 mainly works under pathological conditions. On the other hand, the mRNA levels of Swap70 showed no significant change both in in vitro and in vivo experiments. Further exploration revealed that lysosomes may participate in the accumulation of SWAP70 in a hypertrophic environment. Through immunoprecipitation mass spectrometry, we identified an upstream regulatory molecule that mediates the lysosomal degradation of SWAP70, called GRN, which was reported to regulate lysosomal function by mediating the activity of lysosomal enzymes and trafficking of proteins to lysosomes.[178] ^42 , [179]^43 In the present study, GRN could downregulate the protein expression of SWAP70, while it was blocked under hypertrophic stimuli. However, similar to a previous report on age‐related cardiac hypertrophy,[180] ^44 the present study revealed that the protein expression of GRN showed no decrease in NRVMs, indicating the regulation form between SWAP70 and GRN may be altered under hypertrophic stimuli. A previous study found that SWAP70 interacts with Rac1 under stimulation but not under normal conditions.[181] ^39 Another study reported that a mutant of SWAP70 can exist at the plasma membrane without any stimulation while SWAP70 itself could not.[182] ^45 Similarly, our results showed an interaction change between SWAP70 and GRN in a hypertrophic environment. Thus, SWAP70 under normal or pathological conditions may have different configurations that influence the forms and functions of cells. In the present study, hypertrophic stimuli reduced the colocalization of SWAP70 with GRN or lysosomal marker lysosomal associated membrane protein 1. Hypertrophic stimuli possibly decreased the lysosomal degradation of SWAP70 by disrupting the interaction of SWAP70 with GRN. GRN at least partially participates in the maintenance of SWAP70 protein levels under normal conditions through the lysosomal degradation pathway. However, a more comprehensive study should be conducted to investigate the role of GRN in pathological cardiac hypertrophy with emphasis on SWAP70 regulation. Through RNA sequencing, we found that the MAPK pathway, which has been reported to effectively influence the progression of pathological cardiac hypertrophy,[183] ^10 , [184]^46 played an important role in the progression of pathological cardiac hypertrophy mediated by Swap70 deficiency. Swap70 overexpression suppressed the progression of cardiac hypertrophy possibly by inhibiting the interaction between TAK1 and TAB1, which is essential for the activation of TAK1,[185] ^34 and further restrained the phosphorylation of TAK1 and downstream JNK1/2‐P38 pathways. Similar to our previous research,[186] ^19 SWAP70 derived from different cell types may have multifunctional roles in different disease models. However, there still remain some elusive places in the relationship between TAK1 and pathological cardiac hypertrophy. Previous studies in mice found that TAK1 expression is higher in embryos or neonate mice and decreases gradually before adulthood. Widespread hyperactivation of TAK1 leads to the death of mice attributable to hypertrophic cardiomyopathy within 2 weeks of birth.[187] ^47 Similar to our results, upregulation of TAK1 phosphorylation has been observed in stress hypertrophy models.[188] ^48 Meanwhile, another study reported that Tak1 knockout mice under pressure overload show increased risk for cardiac hypertrophy and heart failure.[189] ^49 Therefore, the timing and extent of TAK1 activation are critical to its role in the heart. In the present study, restrained activation of TAK1 could inhibit the progression of cardiomyocyte hypertrophy caused by Swap70 knockdown under pressure overload. We speculate that a low level of TAK1 activation may be beneficial for the inhibition of pathological cardiac hypertrophy regulated by Swap70 knockdown. Moreover, it has been proved that TAK1 binds to the N‐terminals of SWAP70.[190] ^19 In the present study, N‐terminal deletion of Swap70 did not effectively inhibit the activation of TAK1, thus failing to protect against the progression of cardiac hypertrophy. This result indicates that Swap70 overexpression restrained the progression of cardiomyocyte hypertrophy in a TAK1‐dependent manner. There still exist some limitations in the present study. First, experiments with cardiac‐specific Swap70 overexpression or knockout mice could further examine the protective effects of SWAP70 against the progression of pathological cardiac hypertrophy. Second, a use of human‐induced pluripotent stem cells may help us better study the function of SWAP70 in hypertrophy and link the research to clinical applications. Third, if there exists a mutant isoform of the SWAP70 protein that can inhibit TAK1 activation and avoid lysosomal degradation simultaneously, it may be better for clinical application. We expect to further explore the lysosomal regulation mechanism of SWAP70 in future studies and try to find a mutant of SWAP70 with an activated conformation to obtain a functional and structurally stable SWAP70 protein for clinical application. Conclusions Our data showed that SWAP70 was upregulated in primary cardiomyocytes, mice, and human heart tissues under pressure overload. In a disease state, the increase in SWAP70 protein expression may partly result from the GRN‐mediated suppression of lysosomal degradation. Swap70 overexpression alleviated the progression of cardiomyocyte hypertrophy by inhibiting the activation of TAK1 and downstream JNK1/2‐P38 pathways. Reducing the activation of TAK1 could effectively alleviate cardiomyocyte hypertrophy aggravated by Swap70 knockdown. SWAP70 interacted with TAK1 and decreased the TAK1‐TAB1 interaction. Swap70 overexpression restrained the progression of cardiomyocyte hypertrophy in a TAK1‐dependent manner (Figure [191]8). This study revealed the regulation pattern of the expression and mechanism of SWAP70 under pathological conditions, which may provide a potential therapeutic target for the treatment of pathological cardiac hypertrophy. Figure 8. Schematic showing possible mechanisms underlying normal or hypertrophic environment associated with SWAP70. Figure 8 [192]Open in a new tab Under normal conditions, SWAP70 is degraded by lysosomes mediated by GRN to maintain the balance of SWAP70 protein level, while hypertrophic stress reduces the binding of SWAP70 to GRN and thus decreases the lysosomal degradation of SWAP70, leads to the increased SWAP70 protein level, decreases the interaction between TAK1 and TAB1, inhibits the phosphorylation of TAK1‐JNK 1/2‐P38 signaling pathways, and finally suppresses cardiac hypertrophy. The phosphorylation inhibition of TAK1 by SWAP70 is dependent on its N‐terminal interaction with TAK1. GRN indicates granulin precursor; JNK, c‐Jun N‐terminal kinase; SWAP70, switch‐associated protein 70; TAB1, transforming growth factor β‐activated kinase 1 binding protein 1; and TAK1, transforming growth factor β‐activated kinase 1. Sources of Funding This work was supported by National Science Foundation of China (82170505), Key Research and Development Project of Hubei Provincial Department of Science and Technology (2020BCB053), and Talent Project of Zhongnan Hospital of Hubei Province (20220501). Disclosures None. Supporting information Data S1 [193]Click here for additional data file.^ (3.5MB, zip) Acknowledgments