Background: Charme is a murine long non-coding RNA with specific expression in striated muscles, presenting a human orthologue with 45% identity. Its depletion leads to incomplete muscle differentiation. Charme-/- mice display an altered cardiac phenotype with increased wall thickness and reduced chamber dimensions due to myocyte hyperplasia. Left ventricular dilatation and reduced fractional shortening are detected in aged Charme-/- mice. Embryonic myocardial architecture reveals aberrant compaction and ventricular hypotrabeculation, resembling some congenital cardiomyopathies. Cardiac mesenchymal stromal cells (CMSCs) play key roles in tissue homeostasis and pathophysiology, by maintaining the extra-cellular matrix and by paracrine action on all myocardial cells. Purpose: To elucidate the phenotype, paracrine function, and myofibroblast differentiation capacity of resident CMSCs in the Charme-/- mouse model. Methods: CMSCs were isolated from 5-week-old wild-type (WT) and Charme-/- mice by explant culture, pooling at least 4 different hearts per culture. Results: Charme-/- CMSCs showed increased features of primitive phenotypes, such as enhanced spontaneous spheroid growth (123,6±11,9 vs 51,0±6,4 spheroids/well; N=8, p<0,0001) and clonogenesis efficiency (2,7±0,5-fold; N=7, p<0,05) compared to WT cells. Charme-/- CMSCs also showed increased migration ability (Charme-/-: 68,5±2,7% vs WT: 80,2±0,7% wound area; N=2, p<0,005). Flow cytometry identified a reduced proportion of lin-/Sca1+/CD90+ primitive mesenchymal cells in Charme-/- versus WT CMSCs (7,4±1,6% vs 56,9±6,5%; N=6, p<0,05). Western blot analysis on Charme-/- whole cardiac tissue showed reduced collagen I and collagen I/III protein ratio compared to WT hearts (0,17±0,1 vs 0,85±0,1 normalized OD N=4, p<0,005). Stimulation with TGFb-1 resulted in lower expression of fibroblast activation markers compared to WT CMSCs, as assessed by immunofluorescence staining for aSMA (0,5±0,1 vs 0,8±0,1 mean fluorescence intensity; N=6-12, p<0,05), and collagen I/III mRNA expression ratio (1,3±0,1 vs 2,2±0,4 2^-DCt; N=3, p<0,01). The secretome of Charme-/- CMSCs revealed a general depletion in many cardioprotective cytokines, included in the KEGG term "PI3K-Akt signalling pathway" (FDR<0,0005), and classified in Gene Ontology categories of myoblast differentiation and fusion (GO:1901739, GO:1901741, GO:0045663, GO:0045661; FDR<0,05). In line with this, the secretome of TGFb1-stimulated Charme-/- CMSCs was less effective in sustaining cardiomyocyte survival versus the WT CMSC secretome (1,7±0,1 vs 1,9±0,1 normalized OD at 48h; N=5, p<0,005). Conclusions: Charme-/- CMSCs show a less mature phenotype associated to reduced collagen I presence in situ, reduced activation and differentiation upon stimulation, and impaired cardioprotective paracrine functions, suggesting a key role of Charme in the physiology of the cardiac stroma, possibly mediating in part the altered cardiac phenotype of Charme-/- mice.
The role of Charme in the phenotype and function of resident cardiac mesenchymal stromal cells
Francesca Pagano
2023
Abstract
Background: Charme is a murine long non-coding RNA with specific expression in striated muscles, presenting a human orthologue with 45% identity. Its depletion leads to incomplete muscle differentiation. Charme-/- mice display an altered cardiac phenotype with increased wall thickness and reduced chamber dimensions due to myocyte hyperplasia. Left ventricular dilatation and reduced fractional shortening are detected in aged Charme-/- mice. Embryonic myocardial architecture reveals aberrant compaction and ventricular hypotrabeculation, resembling some congenital cardiomyopathies. Cardiac mesenchymal stromal cells (CMSCs) play key roles in tissue homeostasis and pathophysiology, by maintaining the extra-cellular matrix and by paracrine action on all myocardial cells. Purpose: To elucidate the phenotype, paracrine function, and myofibroblast differentiation capacity of resident CMSCs in the Charme-/- mouse model. Methods: CMSCs were isolated from 5-week-old wild-type (WT) and Charme-/- mice by explant culture, pooling at least 4 different hearts per culture. Results: Charme-/- CMSCs showed increased features of primitive phenotypes, such as enhanced spontaneous spheroid growth (123,6±11,9 vs 51,0±6,4 spheroids/well; N=8, p<0,0001) and clonogenesis efficiency (2,7±0,5-fold; N=7, p<0,05) compared to WT cells. Charme-/- CMSCs also showed increased migration ability (Charme-/-: 68,5±2,7% vs WT: 80,2±0,7% wound area; N=2, p<0,005). Flow cytometry identified a reduced proportion of lin-/Sca1+/CD90+ primitive mesenchymal cells in Charme-/- versus WT CMSCs (7,4±1,6% vs 56,9±6,5%; N=6, p<0,05). Western blot analysis on Charme-/- whole cardiac tissue showed reduced collagen I and collagen I/III protein ratio compared to WT hearts (0,17±0,1 vs 0,85±0,1 normalized OD N=4, p<0,005). Stimulation with TGFb-1 resulted in lower expression of fibroblast activation markers compared to WT CMSCs, as assessed by immunofluorescence staining for aSMA (0,5±0,1 vs 0,8±0,1 mean fluorescence intensity; N=6-12, p<0,05), and collagen I/III mRNA expression ratio (1,3±0,1 vs 2,2±0,4 2^-DCt; N=3, p<0,01). The secretome of Charme-/- CMSCs revealed a general depletion in many cardioprotective cytokines, included in the KEGG term "PI3K-Akt signalling pathway" (FDR<0,0005), and classified in Gene Ontology categories of myoblast differentiation and fusion (GO:1901739, GO:1901741, GO:0045663, GO:0045661; FDR<0,05). In line with this, the secretome of TGFb1-stimulated Charme-/- CMSCs was less effective in sustaining cardiomyocyte survival versus the WT CMSC secretome (1,7±0,1 vs 1,9±0,1 normalized OD at 48h; N=5, p<0,005). Conclusions: Charme-/- CMSCs show a less mature phenotype associated to reduced collagen I presence in situ, reduced activation and differentiation upon stimulation, and impaired cardioprotective paracrine functions, suggesting a key role of Charme in the physiology of the cardiac stroma, possibly mediating in part the altered cardiac phenotype of Charme-/- mice.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
