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Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling.

ABSTRACT: Cardiac fibrosis is characterized by excessive deposition of extracellular matrix proteins and myofibroblast differentiation. Our previous findings have implicated resistin in cardiac fibrosis; however, the molecular mechanisms underlying this process are still unclear. Here we investigated the role of resistin in fibroblast-to-myofibroblast differentiation and elucidated the pathways involved in this process. Fibroblast-to-myofibroblast transdifferentiation was induced with resistin or TGFβ1 in NIH-3T3 and adult cardiac fibroblasts. mRNA and protein expression of fibrotic markers were analyzed by qPCR and immunoblotting. Resistin-knockout mice, challenged with a high-fat diet (HFD) for 20 weeks to stimulate cardiac impairment, were analyzed for cardiac function and fibrosis using histologic and molecular methods. Cardiac fibroblasts stimulated with resistin displayed increased fibroblast-to-myofibroblast conversion, with increased levels of αSma, col1a1, Fn, Ccn2 and Mmp9, with remarkable differences in the actin network appearance. Mechanistically, resistin promotes fibroblast-to-myofibroblast transdifferentiation and fibrogenesis via JAK2/STAT3 and JNK/c-Jun signaling pathways, independent of TGFβ1. Resistin-null mice challenged with HFD showed an improvement in cardiac function and a decrease in tissue fibrosis and reduced mRNA levels of fibrogenic markers. These findings are the first to delineate the role of resistin in the process of cardiac fibroblast-to-myofibroblast differentiation via JAK/STAT3 and JNK/c-Jun pathways, potentially leading to stimulation of cardiac fibrosis.

SUBMITTER: Singh R

PROVIDER: S-EPMC8085100 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AimsMyocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process.Methods and resultsUsing Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardio-fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is mediated by TGF-β1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-β1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-β1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR-370 mediated Dlk1's regulation of cardiac fibroblast-myofibroblast differentiation by directly targeting TGFβ-R2/Smad-3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process.ConclusionThese findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differentiation by interfering with TGFβ/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFβ signalling leads to chronic fibrosis.

Project description:Direct cell reprogramming represents a promising new myocardial regeneration strategy involving in situ transdifferentiation of cardiac fibroblasts into induced cardiomyocytes. Adult human cells are relatively resistant to reprogramming, however, likely because of epigenetic restraints on reprogramming gene activation. We hypothesized that modulation of the epigenetic regulator gene p63 could improve the efficiency of human cell cardio-differentiation. qRT-PCR analysis demonstrated significantly increased expression of a panel of cardiomyocyte marker genes in neonatal rat and adult rat and human cardiac fibroblasts treated with p63 shRNA (shp63) and the cardio-differentiation factors Hand2/Myocardin (H/M) versus treatment with Gata4, Mef2c and Tbx5 (GMT) with or without shp63 (p < 0.001). FACS analysis demonstrated that shp63+ H/M treatment of human cardiac fibroblasts significantly increased the percentage of cells expressing the cardiomyocyte marker cTnT compared to GMT treatment with or without shp63 (14.8% ± 1.4% versus 4.3% ± 1.1% and 3.1% ± 0.98%, respectively; p < 0.001). We further demonstrated that overexpression of the p63-transactivation inhibitory domain (TID) interferes with the physical interaction of p63 with the epigenetic regulator HDAC1 and that human cardiac fibroblasts treated with p63-TID+ H/M demonstrate increased cardiomyocyte marker gene expression compared to cells treated with shp63+ H/M (p < 0.05). Whereas human cardiac fibroblasts treated with GMT alone failed to contract in co-culture experiments, human cardiac fibroblasts treated with shp63+ HM or p63-TID+ H/M demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes. These findings demonstrate that p63 silencing provides enhanced rat and human cardiac fibroblast transdifferentiation into induced cardiomyocytes compared to a standard reprogramming strategy. p63-TID overexpression may be a useful reprogramming strategy for overcoming epigenetic barriers to human fibroblast cardio-differentiation.

Project description:Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and ?-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-?-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.

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Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling.

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