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Effect of the cardiac long non-coding RNA Charme depletion on the maturation and paracrine signaling of resident cardiac fibroblasts [RNA-Seq 1]

ABSTRACT: Charme is a murine long non-coding RNA necessary for the embryonic development of the heart. In vivo Charme knock-out causes prominent alterations of tissue structure, due to cardiac hyperplasia, and leads to the development of cardiac dysfunctions. Although its role in cardiomyocytes has been deeply investigated, no evidence has been collected about its function in other cardiac cell types. Cardiac fibroblasts (CFs) play pivotal roles in the development and homeostatic maintenance of the cardiac tissue. This study investigated the phenotype and function of resident CFs isolated from Charme knockout (CharmeKO) mice, and revealed impaired maturation and functionality. CharmeKO hearts show decreased levels of collagen I content in the extracellular matrix, associated with reduced extracellular matrix-related gene expression and matrix remodelling ability of the CFs. CharmeKO CFs show impaired phenotypic conversion into myofibroblasts and reduced responsiveness to activation stimuli, accompanied by the retention of features proper of unactivated mesenchymal cells. Charme depletion also affects CF paracrine function, determining an impoverishment in cardioprotective cytokines in their secretome, with consequent reduced ability to mediate PI3K/Akt pathway activation in cardiomyocytes and to induce the angiogenic process in endothelial cells. CharmeKO CFs also proved to be less supportive to cardiomyocyte maturation in an in vitro model of cardiac differentiation, thus indicating a potential contribution to the in vivo impairment of cardiomyocyte maturation. Overall, the evidence collected suggests that Charme depletion in the heart impacts CF maturation capacity, leading to their impaired function, which can potentially contribute to the alterations observed in the CharmeKO mice. These findings pave the way to deeper investigations of the intercellular signalling occurring in the heart upon Charme ablation with the aim of identifying how microenvironment homeostasis contributes to cardiomyocyte maturation, and is potentially involved in cardiac diseases.

ORGANISM(S): Mus musculus

PROVIDER: GSE296379 | GEO | 2026/06/03

REPOSITORIES: GEO

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Project description:Charme is a murine long non-coding RNA necessary for the embryonic development of the heart. In vivo Charme knock-out causes prominent alterations of tissue structure, due to cardiac hyperplasia, and leads to the development of cardiac dysfunctions. Although its role in cardiomyocytes has been deeply investigated, no evidence has been collected about its function in other cardiac cell types. Cardiac fibroblasts (CFs) play pivotal roles in the development and homeostatic maintenance of the cardiac tissue. This study investigated the phenotype and function of resident CFs isolated from Charme knockout (CharmeKO) mice, and revealed impaired maturation and functionality. CharmeKO hearts show decreased levels of collagen I content in the extracellular matrix, associated with reduced extracellular matrix-related gene expression and matrix remodelling ability of the CFs. CharmeKO CFs show impaired phenotypic conversion into myofibroblasts and reduced responsiveness to activation stimuli, accompanied by the retention of features proper of unactivated mesenchymal cells. Charme depletion also affects CF paracrine function, determining an impoverishment in cardioprotective cytokines in their secretome, with consequent reduced ability to mediate PI3K/Akt pathway activation in cardiomyocytes and to induce the angiogenic process in endothelial cells. CharmeKO CFs also proved to be less supportive to cardiomyocyte maturation in an in vitro model of cardiac differentiation, thus indicating a potential contribution to the in vivo impairment of cardiomyocyte maturation. Overall, the evidence collected suggests that Charme depletion in the heart impacts CF maturation capacity, leading to their impaired function, which can potentially contribute to the alterations observed in the CharmeKO mice. These findings pave the way to deeper investigations of the intercellular signalling occurring in the heart upon Charme ablation with the aim of identifying how microenvironment homeostasis contributes to cardiomyocyte maturation, and is potentially involved in cardiac diseases.

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.

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Effect of the cardiac long non-coding RNA Charme depletion on the maturation and paracrine signaling of resident cardiac fibroblasts [RNA-Seq 1]

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