MED1 Deficiency in Macrophages Aggravates Isoproterenol-Induced Cardiac Fibrosis in Mice

Mediator 1 (MED1), a key subunit of the mediator complex, interacts with various nuclear receptors and functions in lipid metabolism and energy homeostasis. Dilated cardiomyopathy–related ventricular dilatation and heart failure have been reported in mice with cardiomyocyte-specific Med1 deficiency. However, the contribution of macrophage-specific MED1 in cardiac remodeling remains unclear. In this study, macrophage-specific Med1 knockout (Med1ΔMac) mice were generated and exposed to isoproterenol (ISO) to induce cardiac fibrosis; these mice showed aggravated cardiac fibrosis compared with Med1fl/fl mice. The levels of expression of marker genes for myofibroblast transdifferentiation [α-smooth muscle actin (SMA)] and of profibrotic genes, including Col1a1, Col3a1, Postn, Mmp2, Timp1, and Fn1, were significantly increased in the cardiac tissues of Med1ΔMac mice with ISO-induced myocardial fibrosis. In particular, the transforming growth factor (TGF)-β–Smad2/3 signaling pathway was activated. In bone marrow–derived and peritoneal macrophages, Med1 deficiency was also associated with elevated levels of expression of proinflammatory genes, including Il6, Tnfa, and Il1b. These findings indicate that macrophage-specific MED1 deficiency may aggravate ISO-induced cardiac fibrosis via the regulation of the TGF-β–SMAD2/3 pathway, and the underlying mechanism may involve MED1 deficiency triggering the activation of inflammatory cytokines in macrophages, which in turn may stimulate phenotypic switch of cardiac fibroblasts and accelerate cardiac fibrosis. Thus, MED1 is a potential therapeutic target for cardiac fibrosis. Mediator 1 (MED1), a key subunit of the mediator complex, interacts with various nuclear receptors and functions in lipid metabolism and energy homeostasis. Dilated cardiomyopathy–related ventricular dilatation and heart failure have been reported in mice with cardiomyocyte-specific Med1 deficiency. However, the contribution of macrophage-specific MED1 in cardiac remodeling remains unclear. In this study, macrophage-specific Med1 knockout (Med1ΔMac) mice were generated and exposed to isoproterenol (ISO) to induce cardiac fibrosis; these mice showed aggravated cardiac fibrosis compared with Med1fl/fl mice. The levels of expression of marker genes for myofibroblast transdifferentiation [α-smooth muscle actin (SMA)] and of profibrotic genes, including Col1a1, Col3a1, Postn, Mmp2, Timp1, and Fn1, were significantly increased in the cardiac tissues of Med1ΔMac mice with ISO-induced myocardial fibrosis. In particular, the transforming growth factor (TGF)-β–Smad2/3 signaling pathway was activated. In bone marrow–derived and peritoneal macrophages, Med1 deficiency was also associated with elevated levels of expression of proinflammatory genes, including Il6, Tnfa, and Il1b. These findings indicate that macrophage-specific MED1 deficiency may aggravate ISO-induced cardiac fibrosis via the regulation of the TGF-β–SMAD2/3 pathway, and the underlying mechanism may involve MED1 deficiency triggering the activation of inflammatory cytokines in macrophages, which in turn may stimulate phenotypic switch of cardiac fibroblasts and accelerate cardiac fibrosis. Thus, MED1 is a potential therapeutic target for cardiac fibrosis. Cardiac fibrosis is a major global health problem associated with nearly all forms of heart disease.1Geva T. Bucholz E.M. Is myocardial fibrosis the missing link between prematurity, cardiac remodeling, and long-term cardiovascular outcomes?.J Am Coll Cardiol. 2021; 78: 693-695Crossref PubMed Scopus (4) Google Scholar It can be characterized by excess extracellular matrix protein and the accumulation of collagen in myocardium, which leads to stiffening of the ventricles and heart failure.2Travers J.G. Kamal F.A. Robbins J. Yutzey K.E. Blaxall B.C. Cardiac fibrosis: the fibroblast awakens.Circ Res. 2016; 118: 1021-1040Crossref PubMed Scopus (920) Google Scholar Cardiac fibrosis occurs as a cardiac-remodeling process and can be provoked by various stresses, such as mechanical stress and sympathetic stress.3Xiao H. Li H. Wang J.J. Zhang J.S. Shen J. An X.B. Zhang C.C. Wu J.M. Song Y. Wang X.Y. Yu H.Y. Deng X.N. Li Z.J. Xu M. Lu Z.Z. Du J. Gao W. Zhang A.H. Feng Y. Zhang Y.Y. 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Mediator 1 (MED1), a major subunit of the mediator complex, influences transcription initiation and conveys regulatory information to the basal transcription machinery through its interactions with specific transcription factors, transcription coactivators, and proteins that induce epigenetic alterations and RNA polymerase II.7Allen B.L. Taatjes D.J. The Mediator complex: a central integrator of transcription.Nat Rev Mol Cell Biol. 2015; 16: 155-166Crossref PubMed Scopus (568) Google Scholar,8Tsai K.L. Yu X. Gopalan S. Chao T.C. Zhang Y. Florens L. Washburn M.P. Murakami K. Conaway R.C. Conaway J.W. Asturias F.J. Mediator structure and rearrangements required for holoenzyme formation.Nature. 2017; 544: 196-201Crossref PubMed Scopus (102) Google Scholar By interacting with various nuclear receptors and transcription factors, including peroxisome proliferator-activated receptors (PPARs), retinoic acid receptor (RAR), farnesoid X receptor (FXR), p53, GATA family members, p300, and CCAAT/enhancer-binding protein (C/EBP)-β, MED1 has key roles in lipid metabolism, energy homeostasis, and conditions such as cardiovascular disease.9Bai L. Jia Y. Viswakarma N. Huang J. Vluggens A. Wolins N.E. Jafari N. Rao M.S. Borensztajn J. Yang G. Reddy J.K. Transcription coactivator mediator subunit MED1 is required for the development of fatty liver in the mouse.Hepatology. 2011; 53: 1164-1174Crossref PubMed Scopus (51) Google Scholar, 10Bai L. Li Z. Li Q. Guan H. Zhao S. Liu R. Wang R. Zhang J. Jia Y. Fan J. Wang N. Reddy J.K. Shyy J.Y. Liu E. Mediator 1 is atherosclerosis protective by regulating macrophage polarization.Arterioscler Thromb Vasc Biol. 2017; 37: 1470-1481Crossref PubMed Scopus (32) Google Scholar, 11Zhu Y. Qi C. Jain S. Rao M.S. Reddy J.K. Isolation and characterization of PBP, a protein that interacts with peroxisome proliferator-activated receptor.J Biol Chem. 1997; 272: 25500-25506Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 12Chen W. Zhang X. Birsoy K. Roeder R.G. A muscle-specific knockout implicates nuclear receptor coactivator MED1 in the regulation of glucose and energy metabolism.Proc Natl Acad Sci U S A. 2010; 107: 10196-10201Crossref PubMed Scopus (62) Google Scholar, 13Jang Y. Park Y.K. Lee J.E. Wan D. Tran N. Gavrilova O. Ge K. MED1 is a lipogenesis coactivator required for postnatal adipose expansion.Genes Dev. 2021; 35: 713-728Crossref PubMed Google Scholar Germline deletion of Med1 is associated with mid-gestational embryo fatality, with developmental impairment of multiple organs, including the heart.14Zhu Y. Qi C. Jia Y. Nye J.S. Rao M.S. Reddy J.K. Deletion of PBP/PPARBP, the gene for nuclear receptor coactivator peroxisome proliferator-activated receptor-binding protein, results in embryonic lethality.J Biol Chem. 2000; 275: 14779-14782Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar,15Landles C. Chalk S. Steel J.H. Rosewell I. Spencer-Dene B. Lalani el N. Parker M.G. The thyroid hormone receptor-associated protein TRAP220 is required at distinct embryonic stages in placental, cardiac, and hepatic development.Mol Endocrinol. 2003; 17: 2418-2435Crossref PubMed Scopus (52) Google Scholar Furthermore, using a Cre-loxP system and tamoxifen-inducible Cre approach, Jia et al16Jia Y. Chang H.C. Schipma M.J. Liu J. Shete V. Liu N. Sato T. Thorp E.B. Barger P.M. Zhu Y.J. Viswakarma N. Kanwar Y.S. Ardehali H. Thimmapaya B. Reddy J.K. Cardiomyocyte-specific ablation of Med1 subunit of the mediator complex causes lethal dilated cardiomyopathy in mice.PLoS One. 2016; 11: e0160755PubMed Google Scholar and Spitler et al17Spitler K.M. Ponce J.M. Oudit G.Y. Hall D.D. Grueter C.E. Cardiac Med1 deletion promotes early lethality, cardiac remodeling, and transcriptional reprogramming.Am J Physiol Heart Circ Physiol. 2017; 312: H768-H780Crossref PubMed Scopus (22) Google Scholar reported that mice with cardiomyocyte-specific deficiency of Med1 (csMed1−/−) die within 10 days after weaning or 13 to 28 days post–tamoxifen administration due to ventricular dilatation and heart failure. Based on these findings, MED1 deficiency in the heart is speculated to lead to the down-regulation of multiple genes related to calcium signaling and PPAR-regulated energy metabolism, and to the up-regulation of genes linked to cardiac hypertrophy, cardiac fibrosis, and myocardial injury.16Jia Y. Chang H.C. Schipma M.J. Liu J. Shete V. Liu N. Sato T. Thorp E.B. Barger P.M. Zhu Y.J. Viswakarma N. Kanwar Y.S. Ardehali H. Thimmapaya B. Reddy J.K. Cardiomyocyte-specific ablation of Med1 subunit of the mediator complex causes lethal dilated cardiomyopathy in mice.PLoS One. 2016; 11: e0160755PubMed Google Scholar,17Spitler K.M. Ponce J.M. Oudit G.Y. Hall D.D. Grueter C.E. Cardiac Med1 deletion promotes early lethality, cardiac remodeling, and transcriptional reprogramming.Am J Physiol Heart Circ Physiol. 2017; 312: H768-H780Crossref PubMed Scopus (22) Google Scholar This suggests that MED1 is necessary for normal cardiac development and function. Given that those studies were performed in a mouse model with cardiomyocyte Med1 deficiency, it is not possible to delineate the role of macrophage MED1 in cardiac-specific functions. Recently, macrophage-specific Med1 knockout mice (Med1ΔMac) were generated by intercrossing of Med1fl/fl mice with Lyz2-Cre transgenic mice. Med1 deficiency in macrophages is associated with increased atherosclerosis in both apolipoprotein (Apo)-E−/− and low-density lipoprotein receptor (LDLR)−/− mice via regulation of macrophage polarization.10Bai L. Li Z. Li Q. Guan H. Zhao S. Liu R. Wang R. Zhang J. Jia Y. Fan J. Wang N. Reddy J.K. Shyy J.Y. Liu E. Mediator 1 is atherosclerosis protective by regulating macrophage polarization.Arterioscler Thromb Vasc Biol. 2017; 37: 1470-1481Crossref PubMed Scopus (32) Google Scholar MED1 is required for PPARγ-mediated M2 phenotype switch, with M2 marker genes induced but M1 marker genes suppressed.10Bai L. Li Z. Li Q. Guan H. Zhao S. Liu R. Wang R. Zhang J. Jia Y. Fan J. Wang N. Reddy J.K. Shyy J.Y. Liu E. Mediator 1 is atherosclerosis protective by regulating macrophage polarization.Arterioscler Thromb Vasc Biol. 2017; 37: 1470-1481Crossref PubMed Scopus (32) Google Scholar However, the specific pathways through which macrophage-specific MED1 is involved in cardiac remodeling are unclear. The purpose of this study was to investigate the role of macrophage-specific MED1 in cardiac fibrosis. The effects of macrophage deficiency of Med1 on ISO-induced cardiac fibrosis and the expression of fibrotic factors were investigated. The underlying mechanisms involved in the activation of the transforming growth factor (TGF)-β–SMAD2/3) signaling pathway and proinflammatory response in macrophages were studied in mice. Med1ΔMac (homozygous: Med1flox/flox-Lyz2-Cre) mice were generated and genotyped as described elsewhere.10Bai L. Li Z. Li Q. Guan H. Zhao S. Liu R. Wang R. Zhang J. Jia Y. Fan J. Wang N. Reddy J.K. Shyy J.Y. Liu E. Mediator 1 is atherosclerosis protective by regulating macrophage polarization.Arterioscler Thromb Vasc Biol. 2017; 37: 1470-1481Crossref PubMed Scopus (32) Google Scholar Mice with floxed Med1 (Med1fl/fl) were used as littermate controls.18Jia Y. Qi C. Kashireddi P. Surapureddi S. Zhu Y.J. Rao M.S. Le Roith D. Chambon P. Gonzalez F.J. Reddy J.K. Transcription coactivator PBP, the peroxisome proliferator-activated receptor (PPAR)-binding protein, is required for PPARalpha-regulated gene expression in liver.J Biol Chem. 2004; 279: 24427-24434Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar All mice were housed in a facility with a 12-hour light/dark cycle and fed a standard rodent chow and water ad libitum. Eight-week–old male Med1ΔMac and Med1fl/fl mice (n = 8 to 14 mice per group) were injected s.c. with isoflurane (ISO; 5 mg/kg; lot number WXBC2200V, Sigma-Aldrich, St. Louis, MO) or saline as vehicle control, daily for 3 or 7 days. Animal care and experimental procedures were preapproved by the Animal Care and Use Committee, Xi'an Jiaotong University (Xi'an, China; approval number XJTULAC2018-297). Hearts from Med1ΔMac and Med1fl/fl mice were fixed in 4% paraformaldehyde overnight and processed for embedding in paraffin. Sections, 5 μm thick, were cut and stained with hematoxylin and eosin (H&E) for measuring the cross-sectional area of cardiomyocytes. Picro Sirius Red and Masson's trichrome staining was performed in accordance with the manufacturer's instructions, and used for the detection of cardiac fibrosis. Immunohistochemistry (IHC) staining for α-smooth muscle actin (SMA; 1:400; catalog number 19245; Cell Signaling Technologies, Boston, MA), collagen α1(I) chain (Col1α1) (1:100; catalog number AF1840; Beyotime Biotechnology, Shanghai, China), and F4/80 (1:200, catalog number 70076, Cell Signaling Technologies) was performed to assess the level of collagen accumulation. Briefly, sections were blocked with 2% horse serum for 2 hours, and then incubated with primary antibodies overnight at 4°C, followed by a secondary antibody for 1 hour at room temperature. Sections were visualized using a diaminobenzidine (DAB) substrate kit (Origene, Shanghai, China). Histologic analysis and image processing were performed using a Leica DMRE microscope (Leica Microsystems, Wetzlar, Germany) equipped with digital image analysis software (Leica Application Suite V3.8) and camera (Spot Imaging, Sterling Heights, MI). The fibrosis was quantified by calculating the percentage of collagen staining with five randomly selected images in each section (n = 3 to 6 mice per saline group; n = 4 to 6 mice per ISO group) using software analysis with ImageJ software version 1.8.0 (NIH, Bethesda, MD; http://imagej.nih.gov.ij) bundled with Java 1.8.0_172. Bone marrow–derived macrophages (BMDMs) were isolated and differentiated with macrophage–colony stimulating factor (M-CSF) as described previously.19Trouplin V. Boucherit N. Gorvel L. Conti F. Mottola G. Ghigo E. Bone marrow-derived macrophage production.J Vis Exp. 2013; : e50966PubMed Google Scholar Briefly, bones from the hind leg of Med1fl/fl and Med1ΔMac mice (n = 3 mice per group) were collected and smashed in a mortar. Once the resident macrophages were removed from the bone marrow cell preparation, bone marrow cells were incubated with M-CSF (day 0). After 3 days, the cells, which were round and nonadherent before culture, started to differentiate into macrophages. After 7 days, the differentiated BMDMs were treated with ISO (1 μmol/L) for 24 hours. Pooled peritoneal macrophages (PMs) were collected from 8-week–old Med1fl/fl and Med1ΔMac mice (n = 6 mice per group) injected with 3% thioglycolate as previously described.10Bai L. Li Z. Li Q. Guan H. Zhao S. Liu R. Wang R. Zhang J. Jia Y. Fan J. Wang N. Reddy J.K. Shyy J.Y. Liu E. Mediator 1 is atherosclerosis protective by regulating macrophage polarization.Arterioscler Thromb Vasc Biol. 2017; 37: 1470-1481Crossref PubMed Scopus (32) Google Scholar Peritoneal cells were harvested at 3 days after i.p. injection, and macrophages were enriched by quick adhesion. Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and penicillin/streptomycin for 2 hours. After washing of nonadherent cells, adherent macrophages were treated with or without lipopolysaccharide (50 ng/mL; catalog number L2630; Sigma-Aldrich) for 6 hours. Total RNA was extracted from mouse heart and BMDMs by using Trizol reagent (Accurate Biotechnology, Hunan, China). The quality and concentration of total RNA were determined by NanoDrop 1100 (Thermo Fisher Scientific, Waltham, MA). Reverse transcription (RT) involved 1 μg of total RNA with the PrimeScript RT reagent kit and gDNA eraser (Takara Bio Inc., Da Lian, China). Quantitative real-time PCR (qPCR) was performed in triplicate for amplifying specific genes and normalized to β-actin level. Each PCR reaction involved 0.8 μL (10 μmol/L) of forward and reverse primers and 10 μL of 2× SYBR Green PCR Master Mix for a final volume of 20 μL. PCR reaction was performed with CFX Connect Real-Time System (Bio-Rad, Hercules, CA). The generation of specific PCR products was confirmed by melting curve analysis. Relative gene expression was measured by the comparative Ct method, X = 2−ΔΔCt. qPCR primers are listed in Table 1.Table 1Quantitative Real-Time PCR PrimersGenePrimerCol1a1F: 5′-GAGCGGAGAGTACTGGATCG-3′R: 5′-TACTCGAACGGGAATCCATC-3′Col3a1F: 5′-CCCAACCCAGAGATCCCATT-3′R: 5′-GAAGCACAGGAGCAGGTGTAGA-3′Mmp2F: 5′-ACCTGAACACTTTCTATGGCTG-3′R: 5′-CTTCCGCATGGTCTCGATG-3′Timp1F: 5′-CGAGACCACCTTATACCAGCG-3′R: 5′-ATGACTGGGGTGTAGGCGTA-3′TgfbF: 5′-ATCCTGTCCAAACTAAGGCTCG-3′R: 5′-ACCTCTTTAGCATAGTAGTCCGC-3′Fn1F: 5′-CCGGTGGCTGTCAGTCAGA-3′R: 5′-CCGTTCCCACTGCTGATTTATC-3′PostnF: 5′-TTTACAACGGGCAAATACTGGAAAC-3′R: 5′-GATGATCTCGCGGAATATGTGAA-3′TnfaF: 5′-ATGGCCTCCCTCTCATCAGT-3′R: 5′-CTTGGTGGTTTGCTACGACG-3′Il6F: 5′-CTTCCATCCAGTTGCCTTCTTG-3′R: 5′-AATTAAGCCTCCGACTTGTGAAG-3′Il1bF: 5′-CTTCCCCAGGGCATGTTAAG-3′R: 5′-ACCCTGAGCGACCTGTCTTG-3′Il18F: 5′-TCTTGGCCCAGGAACAATGG-3′R: 5′-CAGGCTGTCTTTTGTCAACGA-3′F4/80F: 5′-CTTTGGCTATGGGCTTCCAGTC-3′R: 5′-GCAAGGAGGACAGAGTTTATCGTG-3′ActbF: 5′-GATCTGGCACCACACCTTCT-3′R: 5′-GGGGTGTTGAAGGTCTCAAA-3′ Open table in a new tab Proteins were extracted from heart tissues of Med1fl/fl and Med1ΔMac mice (n = 3 to 4 mice per group) using the RIPA kit (Beyotime). Protein concentrations were measured with the bicinchoninic acid (BCA) kit (catalog number MK164230; Thermo Fisher Scientific). Protein samples (40 μg) underwent 10% SDS-PAGE, transferred to polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA), and immunoblotted with antibodies for α-SMA (catalog number 19245; Cell Signaling Technologies), matrix metalloproteinase (Mmp)-2 (catalog number AF1420; Beyotime), tissue inhibitor of metalloproteinase 1 (Timp1) (catalog number AF8163; Beyotime), F4/80 (catalog number 70076; Cell Signaling Technologies), Smad2 (catalog number 5339; Cell Signaling Technologies), p-Smad2 (catalog number 18338; Cell Signaling Technologies), Smad3 (catalog number AF1501; Beyotime), p-Smad3 (catalog number AF1759; Beyotime), TGF-β (catalog number AF0297; Beyotime). Antibodies for β-actin (catalog number sc-47778; Santa Cruz Biotechnology, Dallas, TX) or glyceraldehyde phosphate dehydrogenase (GAPDH; catalog number 2118S, Cell Signaling Technologies) were used as loading controls. The protein bands were visualized by enhanced chemiluminescence (ECL; EMD Millipore). Total RNA was extracted from pooled macrophages of Med1fl/fl and Med1ΔMac mice (n = 6 mice per group). The mRNA was purified by using poly-T oligo-attached magnetic beads. RNA libraries were generated with a NEBNext Ultra RNA Library Prep Kit (New England BioLabs, Ipswich, MA) in accordance with the manufacturer's recommendations. Library quality was assessed on an Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA), and then sequenced on an Illumina NextSeq 500 (Illumina, San Diego, CA). Heatmap was plotted using the Morpheus online tool (https://software.broadinstitute.org/morpheus; last accessed August 13, 2021). RNA-seq raw data have been deposited on Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; accession number GSE196680). To monitor cardiac function, echocardiography was performed using a Vevo 1100 imaging system (Fujifilm VisualSonics, Toronto, ON, Canada). Before the imaging studies, Med1fl/fl and Med1ΔMac mice treated with saline or ISO (n = 3 to 5 mice per group) were shaved from the mid-chest area and anesthetized by isoflurane. Parasternal long-axis view, short-axis view at the papillary muscle level and two-dimensional guided M-mode images were recorded. The following echocardiographic parameters, including systolic and diastolic interventricular septal thickness, systolic and diastolic left ventricular (LV) end-posterior wall dimension, systolic and diastolic LV internal dimension, ejection fraction, and fractional shortening, were recorded and calculated as described previously.20Gao S. Ho D. Vatner D.E. Vatner S.F. Echocardiography in mice.Curr Protoc Mouse Biol. 2011; 1: 71-83PubMed Google Scholar,21Chang S.C. Ren S. Rau C.D. Wang J.J. Isoproterenol-induced heart failure mouse model using osmotic pump implantation.Methods Mol Biol. 2018; 1816: 207-220Crossref PubMed Scopus (43) Google Scholar Data are expressed as means ± SEM. Two-tailed t-test or one-way analysis of variance (for comparison of three or more groups) followed by the Tukey post-hoc test was used for statistical analysis with the use of GraphPad Prism software version 8.0 (GraphPad Software Inc., San Diego, CA). P < 0.05 was considered statistically significant. Cardiac Med1 plays a crucial role in the maintenance of heart function by affecting multiple metabolic, compensatory, and reparative pathways.16Jia Y. Chang H.C. Schipma M.J. Liu J. Shete V. Liu N. Sato T. Thorp E.B. Barger P.M. Zhu Y.J. Viswakarma N. Kanwar Y.S. Ardehali H. Thimmapaya B. Reddy J.K. Cardiomyocyte-specific ablation of Med1 subunit of the mediator complex causes lethal dilated cardiomyopathy in mice.PLoS One. 2016; 11: e0160755PubMed Google Scholar,17Spitler K.M. Ponce J.M. Oudit G.Y. Hall D.D. Grueter C.E. Cardiac Med1 deletion promotes early lethality, cardiac remodeling, and transcriptional reprogramming.Am J Physiol Heart Circ Physiol. 2017; 312: H768-H780Crossref PubMed Scopus (22) Google Scholar The role of macrophage-specific Med1 in cardiac remodeling in mice was investigated in this study. Toward this end, macrophage-specific Med1-knockout mice were generated by crossbreeding Med1fl/fl mice with Lyz2-Cre mice.10Bai L. Li Z. Li Q. Guan H. Zhao S. Liu R. Wang R. Zhang J. Jia Y. Fan J. Wang N. Reddy J.K. Shyy J.Y. Liu E. Mediator 1 is atherosclerosis protective by regulating macrophage polarization.Arterioscler Thromb Vasc Biol. 2017; 37: 1470-1481Crossref PubMed Scopus (32) Google Scholar Med1fl/fl and Med1ΔMac mice were injected with ISO 5 mg/kg per day s.c. for 7 days. Heart sections were stained with H&E, Picro Sirius Red, and Masson's trichrome staining. Although there were no obvious morphology changes in cardiomyocytes, the cross-sectional area of cardiomyocytes showed an increased trend in Med1ΔMac mice with ISO treatment (Figure 1, A and B ). Compared with groups with saline treatment, perivascular and interstitial cardiac fibrosis were increased with ISO in both Med1fl/fl and Med1ΔMac mice (Figure 1, A and C–E). Med1ΔMac mice exhibited further increase in collagen content with a scar-like morphology when compared to that in Med1fl/fl mice (sirius red staining for perivascular fibrosis, 9.83% ± 0.32% versus 18.03 ± 1.05%, P < 0.01; sirius red staining for interstitial fibrosis, 17.02% ± 1.12% versus 21.69% ± 5.82%, P < 0.05; Masson's trichrome staining, 17.79% ± 0.79% versus 25.59% ± 3.10%, P < 0.05) (Figure 1, A and C–E). As an ever-present marker of cardiac fibrosis, the incorporation of α-SMA in myofibroblast is a key indicator of activated phenotype. Collagen type I α1 chain (Col1α1) is known for its contribution in the deposition of extracellular matrix. As shown by immunostaining, both α-SMA and Col1α1 were markedly increased in cardiac tissue from Med1ΔMac mice compared to Med1fl/fl littermates with ISO treatment (Figure 2A). On qPCR, mRNA levels of genes encoding fibrotic factors, including Col1a1, collagen type III α1 chain (Col3a1), periostin (Postn), matrix metallopeptidase 2 (Mmp2), tissue inhibitor of metalloproteinase 1 (Timp1), and fibronectin 1 (Fn1) were higher in cardiac tissue from the Med1ΔMac mice than the Med1fl/fl mice after ISO stimulation (Figure 2B). The increased protein levels of α-SMA, Mmp2, and Timp1 in cardiac tissue from Med1ΔMac mice were further confirmed by Western blot analysis (Figure 2, C and D). Together, the results in Figures 1 and 2 indicate that deficiency of Med1 in macrophages promotes the development of cardiac fibrosis in mice. TGF-β is a major cytokine that mediates tissue fibrosis, which in turn induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins.22Khalil H. Kanisicak O. Prasad V. Correll R.N. Fu X. Schips T. Vagnozzi R.J. Liu R. Huynh T. Lee S.J. Karch J. Molkentin J.D. Fibroblast-specific TGF-beta-Smad2/3 signaling underlies cardiac fibrosis.J Clin Invest. 2017; 127: 3770-3783Crossref PubMed Scopus (505) Google Scholar With ISO treatment, both mRNA and protein levels of TGF-β in cardiac tissues were significantly elevated in Med1ΔMac mice as compared with those in Med1fl/fl mice (Figure 3, A–C). Typically, TGF-β signaling mobilizes Smad2/3 transcription factors that control fibrosis by promoting the expression of myofibroblast-specific genes.23Derynck R. Zhang Y.E. Smad-dependent and Smad-independent pathways in TGF-beta family signalling.Nature. 2003; 425: 577-584Crossref PubMed Scopus (4396) Google Scholar On ISO treatment, the phosphorylation levels of Smad2/3 were significantly up-regulated in the cardiac tissue of the Med1ΔMac mice as compared with those in the Med1fl/fl mice (Figure 3, B, D, and E), suggesting a functional role of TGF-β–Smad2/3 signaling in profibrotic response in the Med1ΔMac mice. These findings suggest that Med1 deficiency in macrophages triggers the activation of the TGF-β–Smad2/3 signaling pathway, and thus aggravates cardiac fibrosis. To explore how macrophage-specific Med1 deficiency aggravates the ISO-induced fibrosis in the heart, ISO was administered to Med1fl/fl and Med1ΔMac mice for 3 days. As expected, Med1ΔMac hearts exhibited more collagen accumulation as compared with that in Med1fl/fl hearts after ISO treatment (Supplemental Figure S1). ISO treatment led to macrophage infiltration in cardiac tissue from Med1fl/fl mice. Med1 deficiency was more profound in macrophages, as evidenced by F4/80 immunostaining (Figure 4A). Additionally, mRNA and protein levels of F4/80 were significantly increased in the cardiac tissue from the Med1ΔMac mice with ISO treatment (Figure 4, B and C). These results indicate increased macrophage infiltration in Med1ΔMac mouse heart. IL18 plays a crucial role in initiating and maintaining the cardiac inflammatory cascade on β-adrenergic receptor (AR) insult.3Xiao H. Li H. Wang J.J. Zhang J.S. Shen J. An X.B. Zhang C.C. Wu J.M. Song Y. Wang X.Y. Yu H.Y. Deng X.N. Li Z.J. Xu M. Lu Z.Z. Du J. Gao W. Zhang A.H. Feng Y. Zhang Y.Y. IL-18 cleavage triggers cardiac inflammation and fibrosis upon beta-adrenergic insult.Eur Heart J. 2018; 39: 60-69Crossref PubMed Scopus (173) Google Scholar Thus, the expression of Il18 and related proinflammatory genes in cardiac tissue and macrophages in mice were examined. In the Med1fl/fl mice, cardiac expression of Il18 was markedly induced with ISO treatment. Med1ΔMac hearts exhibited further increases in Il18 as well as Il6 and Il1b (Figure 4D). The levels of these inflammatory genes, including Il6, Il1b, and Tnfa, were elevated in Med1−/− BMDMs, either at basal level or with ISO treatment (Figure 4, E and F). On RNA-seq analysis of PMs from Med1fl/fl and Med1ΔMac mice (http://www.ncbi.nlm.nih.gov/geo; accession number GSE196680), a heatmap revealed that the levels of a series of proinflammatory genes, such as Il18, Ccl2, Ccl9, and Il1a, were elevated in Med1−/− PMs compared with Med1+/+ PMs (Supplemental Figure S2). On lipopolysaccharide stimulation, Med1−/− PMs released more IL18, IL1α, IL6, CCL2, CCL9, CXCL1, CXCL2, and TNF in comparison with Med1fl/fl macrophages (Supplemental Figure S2). These results indicate that Med1 deficiency induces a more robust proinflammatory response, and this is tangentially associated with evidence for increased macrophage activation in the heart. To investigate the effects of macrophage-specific Med1 deficiency on cardiac structu