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Cardiac Fibroblast Foxm1 Deficiency Prevents Pressure Overload-Induced Cardiac Remodeling via the Usp10/p38γ MAPK Axis

ABSTRACT: Aims: Cardiac fibroblasts (CFs) play a crucial role in cardiac remodelling, which is a common cause of heart failure (HF). However, the molecular mechanisms underlying the fibroblast-to-myofibroblast transition remain largely unknown. Foxm1 is well known in various cardiopulmonary pathologies. However, Foxm1-driven CF activation in the progression of cardiac remodelling to HF remains to be investigated. Methods: Changes in Foxm1 expression were assessed in samples from patients with HF and mice with transverse aortic constriction (TAC)-induced cardiac remodelling. Pharmacologic antagonist FDI-6 was used to explore the effects of Foxm1 inhibition on post-TAC outcomes. Tcf21-Cre and PostnMCM were used to evaluate Foxm1 loss- and gain-of-function in CFs and myofibroblasts, respectively. Cardiac function and remodelling were examined by echocardiography and histological analysis. Foxm1 downstream target genes were identified by mass spectrometry (MS) and transcriptomic analysis. Post-translational regulation was evaluated by in vitro chromatin immunoprecipitation, co-immunoprecipitation, and ubiquitination assays. Pharmacological inhibition of Usp10 or knockout of p38γ in vivo verified the signalling pathway by which Foxm1 regulated cardiac remodelling. Results: Foxm1 was upregulated in human HF samples as well as in the mouse cardiac remodelling model. CFs were the primary cell type responsible for Foxm1 upregulation. Foxm1 pharmacological inhibition or genetic knockout in CFs or myofibroblasts significantly attenuated TAC-induced cardiac remodelling and HF. Conversely, conditional overexpression of Foxm1 in CFs or myofibroblasts resulted in more severe pathological cardiac remodelling and dysfunction. Combined RNA-sequencing and MS analysis revealed that Foxm1 promoted Usp10 expression by binding to its promoter. Usp10 interacted with p38γ, resulting in p38γ deubiquitination and thus inﬂuencing the downstream p38 mitogen-activated protein kinase (MAPK) signalling pathway. Pharmacological inhibition of Usp10 or genetic knockout of p38γ ameliorated the exacerbated TAC-induced cardiac remodelling in mice with myofibroblast-specific Foxm1 overexpression. Conclusion: Our findings reveal an essential role of Foxm1 in CF activation during cardiac remodelling. These results suggest that targeting the Foxm1/Usp10/p38γ MAPK axis may represent a new potential therapeutic strategy against pathological cardiac remodelling and HF.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: fengze cai

LAB HEAD: shuai song

PROVIDER: PXD050281 | Pride | 2024-03-03

REPOSITORIES: Pride

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Project description:Aims: Cardiac fibroblasts (CFs) play a crucial role in cardiac remodelling, which is a common cause of heart failure (HF). However, the molecular mechanisms underlying the fibroblast-to-myofibroblast transition remain largely unknown. Foxm1 is well known in various cardiopulmonary pathologies. However, Foxm1-driven CF activation in the progression of cardiac remodelling to HF remains to be investigated. Methods: Changes in Foxm1 expression were assessed in samples from patients with HF and mice with transverse aortic constriction (TAC)-induced cardiac remodelling. Pharmacologic antagonist FDI-6 was used to explore the effects of Foxm1 inhibition on post-TAC outcomes. Tcf21-Cre and PostnMCM were used to evaluate Foxm1 loss- and gain-of-function in CFs and myofibroblasts, respectively. Cardiac function and remodelling were examined by echocardiography and histological analysis. Foxm1 downstream target genes were identified by mass spectrometry (MS) and transcriptomic analysis. Post-translational regulation was evaluated by in vitro chromatin immunoprecipitation, co-immunoprecipitation, and ubiquitination assays. Pharmacological inhibition of Usp10 or knockout of p38γ in vivo verified the signalling pathway by which Foxm1 regulated cardiac remodelling. Results: Foxm1 was upregulated in human HF samples as well as in the mouse cardiac remodelling model. CFs were the primary cell type responsible for Foxm1 upregulation. Foxm1 pharmacological inhibition or genetic knockout in CFs or myofibroblasts significantly attenuated TAC-induced cardiac remodelling and HF. Conversely, conditional overexpression of Foxm1 in CFs or myofibroblasts resulted in more severe pathological cardiac remodelling and dysfunction. Combined RNA-sequencing and MS analysis revealed that Foxm1 promoted Usp10 expression by binding to its promoter. Usp10 interacted with p38γ, resulting in p38γ deubiquitination and thus inﬂuencing the downstream p38 mitogen-activated protein kinase (MAPK) signalling pathway. Pharmacological inhibition of Usp10 or genetic knockout of p38γ ameliorated the exacerbated TAC-induced cardiac remodelling in mice with myofibroblast-specific Foxm1 overexpression. Conclusion: Our findings reveal an essential role of Foxm1 in CF activation during cardiac remodelling. These results suggest that targeting the Foxm1/Usp10/p38γ MAPK axis may represent a new potential therapeutic strategy against pathological cardiac remodelling and HF.

Project description:Purpose: Cardiac fibroblasts (CFs) contribute to pathological remodeling by proliferating and secreting extracellular matrix. In contrast, physiological remodeling is an adaptive response to excessive demands of exercise that results in increased heart size without accompany fibrosis or deterioration of cardiac function. We hypothesize that these divergent outcomes are partially a function of cell-autonomous and paracrine cardioprotection afforded by CFs specifically during physiological remodeling. Our objective is to define the gene expression program (GEP) of CFs isolated from mouse models of physiological (swim) and pathological (thoracic aortic constriction (TAC) or myocardial infarction (MI)) remodeling to identify novel mechanisms underlying the development of pathological cardiac fibrosis and the cardioprotective benefits of exercise. Methods: Cardiomyocytes (CMs) were isolated by gravity sedimentation and CFs were isolated by differential plating for 2 hours from single-cell suspensions of 10-14 week old male C57BL/6 hearts subjected to the following 7 conditions: sedentary CFs (n=3), sham surgery CFs (n=2), 3 day TAC CFs (n=3), 10 day TAC CFs (n=3), 4 day swim CFs (n=3), 10 day swim CFs (n=3), and 5 day MI CFs (n=2). CMs were isolated from sedentary 10-14 week old male C57BL/6 hearts (n=3). Total RNA was isolated using the Qiagen RNeasy Plus Kit and genomic DNA removed using Qiagen gDNA columns. RNA quality was assessed with the Agilent Bioanalyzer and only samples with RIN >8.5 were used for sequencing. Library construction was performed using the TruSeq Stranded mRNA Sample Preparation Kit from 100ng poly-A mRNA. An Illumina HiSeq 2500 was used to generate 20-25 million single end reads of 100nt for each sample. The sequence reads that passed quality filters (Trimmomatic-0.32) were analyzed at the transcript isoform level using STAR_2.4.2a for SHRiMP_2_2_3 mapping to mouse genome assembly GRCm38.p4 version mm10 and processed with Cufflinks-2.0.2. Results: We determined that CFs display distinct GEPs that are inversely regulated in pathological and physiological remodeling. Spearman correlation and Principal Component Analysis (PCA) of genes with FPKM >1 and q >0.05 revealed consistency between biological replicates and highlighted active changes in GEPs during physiological and pathological remodeling both of which diverged from control samples in principal component (PC) 1 and PC2. CFs isolated from mice 5 days after initiation of MI also diverged from controls, TAC, and swim samples, demonstrating a different fibroblast GEP in ischemic and pressure overload induced remodeling. This analysis also revealed that our control models (sham surgery and sedentary) were indistinguishable from each other, therefore we typically treated sham and sedentary mice as a single control group in further studies. These initial results reveal that CFs differentially respond to pathological and physiological cues in as few as 3 days by enacting dynamic transcriptional changes in vivo. Consistent with previous reports of myofibroblast activation, Ingenuity Pathway Analysis (IPA) revealed that the most significantly enriched GO terms in pathological fibroblasts included stimulation of Rho-dependent contractile pathways and integrin-linked kinase (ILK) signaling, the latter of which was downregulated in a comparison between 10-day swim vs. control CFs. IPA transcription factor analysis comparing 10-day swim and 10-day TAC CFs further identified upregulation of detoxification and inflammatory transcription factors and downregulation of the serum response factor (SRF) after exercise. Furthermore, independent motif analysis using HOMER 2.0 identified the CArG box (SRF binding site) as the most enriched transcription factor binding site within 5kb of genes that are upregulated in 10-day TAC vs sham CFs. Conclusions: We have defined the GEPs of CFs that underlie physiological and pathological cardiac remodeling. Not only is the GEP of pathological CFs enriched in Rho-dependent pathways and SRF target genes, but the GEP of physiological CFs is enriched in detoxification and inflammatory target genes. We also demonstrate that the CFs change their transcriptional signature consistently and rapidly. Within 3-4 days of initiating extracellular stimuli, distinct profiles are observed in CFs isolated from swim-trained, pressure overload, or ischemic models of cardiac remodeling.

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Cardiac Fibroblast Foxm1 Deficiency Prevents Pressure Overload-Induced Cardiac Remodeling via the Usp10/p38γ MAPK Axis

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