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Transcriptome and proteome profiling of activated cardiac fibroblasts supports target prioritization in cardiac fibrosis

ABSTRACT: Activated cardiac fibroblasts (CF) play a central role in cardiac fibrosis, a condition associated with most cardiovascular diseases. Conversion of quiescent into activated CF sustains heart integrity upon injury. However, permanence of CF in active state inflicts deleterious heart function effects. Mechanisms underlying this cell state conversion are still not fully disclosed, contributing to a limited target space and lack of effective anti-fibrotic therapies. To prioritize targets for drug development, we studied CF remodeling upon activation at transcriptomic and proteomic levels, using three different cell sources: primary adult CF (aHCF), primary fetal CF (fHCF), and induced pluripotent stem cells derived CF (hiPSC-CF). All cell sources showed a convergent response upon activation, with clear morphological and molecular remodeling associated with cell-cell and cell-matrix interactions. Quantitative proteomic analysis identified known cardiac fibrosis markers, such as FN1, CCN2 and Serpine1, but also revealed targets not previously associated with this condition, including MRC2, IGFBP7 and NT5DC2. Exploring such targets to modulate CF phenotype represents a valuable opportunity for development of anti-fibrotic therapies. Also, we demonstrate that hiPSC-CF is a suitable cell source for preclinical research, displaying significantly lower basal activation level relative to primary cells, while being able to elicit a convergent response upon stimuli.

ORGANISM(S): Homo sapiens

PROVIDER: GSE202714 | GEO | 2022/12/01

REPOSITORIES: GEO

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Project description:In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) automatically triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These dramatic phenotypic switches confound interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we utilized poly (ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic cardiac ECM, respectively. As hypothesized, low hydrogel stiffness converted activated CFs into a quiescent state with reduced abundance of a-smooth muscle actin (a-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated b-galactosidase (SA-b-Gal) activity and increased expression of p16 and p21, which are cyclin-dependent kinase (CDK) inhibitors and markers of senescence. Using dynamically stiffening hydrogels with phototunable crosslinking capabilities, we demonstrate that substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low stiffness hydrogels, with a dramatic reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-kB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings further demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment to which they are exposed.

Project description:Cardiac hypertrophy is an important and independent risk factor for the development of cardiac myopathy that may lead to heart failure. Cardiac hypertrophy manifests as an enlargement of the individual cardiomyocytes, which impairs the function of the heart. The only way to cure end-stage cardiac myopathy is by heart transplantation, a possibility limited due to lack of donor hearts. Therefore, early diagnosis of cardiac hypertrophy is needed in order to be able to initiate interventions that may prevent further progression of the disease. The mechanisms underlying the development of cardiac hypertrophy are yet not well understood. To increase the knowledge about mechanisms and regulatory pathways involved in the progression of cardiac hypertrophy, we have developed a human induced pluripotent stem cell (hiPSC)-based in vitro model of cardiac hypertrophy and performed extensive characterization of the model using multi-omics analyses. In a series of experiments, hiPSC-derived cardiomyocytes were stimulated with Endothelin-1 for 8, 24, 48 and 72 hours and their transcriptome and secreted proteome were analyzed thoroughly. The transcriptomic data show many enriched canonical pathways related to cardiac hypertrophy already at the earliest time point, e.g., cardiac hypertrophy signaling, actin cytoskeleton signaling and PI3K/AKT signaling. Cluster analysis of the differentially expressed genes showed that there are numerous clusters of genes that are dysregulated over the time period of 8 to 72h. An integrated transcriptome-secretome analysis enabled the identification of multimodal biomarkers of high relevance for monitoring early cardiac hypertrophy progression. Taken together, the results from this study demonstrate that our in vitro model displays a hypertrophic response on transcriptomic- and secreted proteomic level. The results also provide novel insight into the underlying mechanisms of cardiac hypertrophy and novel putative early cardiac hypertrophy biomarkers have been identified that will be further validated to assess their clinical relevance.

Project description:Conversion of cardiospheres derived primitive cardiac stromal cells into myofibroblasts is typically associated with hypoxia conditions, metabolic insults, and/or inflammation, all of which are predisposing factors to cardiac fibrosis and heart failure. We hypothesized that this conversion could be also mediated by response of these cells to mechanical cues through activation of the Hippo transcriptional pathway. The objective of the present study was to assess the role of cellular/nuclear straining forces acting in myofibroblast differentiation of cardiospheres derived primitive cardiac stromal cells under the control of YAP transcription factor and to validate this finding using a pharmacological agent that interferes with the interactions of the YAP/TAZ complex with their cognate transcription factors TEADs, under high-strain and pro-fibrotic stimulation. We employed high content imaging, 2D/3D culture, atomic force microscopy mapping and molecular methods to prove the role of cell/nuclear straining in YAP-dependent fibrotic programming in a mouse model of ischemia-dependent cardiac fibrosis and in human-derived primitive cardiospheres derived primitive cardiac stromal cells . We also tested treatment of cells with Verteporfin, a drug known to prevent the association of the YAP/TAZ complex with their cognate transcription factors TEADs. Results suggested that pharmacologically targeting the YAP-dependent pathway overrides the pro-fibrotic activation of cardiospheres derived primitive cardiac stromal cells by mechanical cues in vitro, and that this occurs even in the presence of pro-fibrotic signaling mediated by TGF-β1. In vivo administration of Verteporfin in mice with permanent cardiac ischemia reduced significantly fibrosis and morphometric remodeling but did not improve cardiac performance. Our study indicates that preventing molecular translation of mechanical cues in cardiospheres derived primitive cardiac stromal cells reduces the impact of cardiac maladaptive remodeling with a positive effect on fibrosis.

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Transcriptome and proteome profiling of activated cardiac fibroblasts supports target prioritization in cardiac fibrosis

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