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| Author(s): |
Agatha Ribeiro da Silva
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD) |
| Defense date: | 2019-09-27 |
| Examining board members: |
Jose Eduardo Krieger;
Leonardo Pinto de Carvalho;
Edilamar Menezes de Oliveira;
Luciana Venturini Rossoni
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| Advisor: | Jose Eduardo Krieger |
| Abstract | |
Ventricular conduction system (VCS) abnormalities have been associated with arrhythmias and many of the mechanisms for determining VCS remain unknown. We hypothesized that fibroblasts adjacent to the VCS influence cardiomyocyte maturation to fast-conducting fibers and could be associated with arrhythmias following stem cell transplantation in myocardial infarction models. We used ventricular cardiomyocyte cocultures of neonatal rats and cardiac fibroblasts to test this hypothesis in vitro. Additionally, we produced the deletion of the candidate gene IRX3 in iPS stem cells to test the hypothesis that IRX3 influences cardiomyocyte differentiation in specialized cardiac conduction cells. Co-culture of cardiac fibroblasts increased the expression of the VCS marker, Nav1.5, and altered intracellular calcium transients (CaTs) consistently with the rapidly conducting cell profile. Interestingly, fibroblastconditioned media was sufficient to recapitulate the increased gene and protein expression of VCS markers (Gja5 / Connexin 40, Scn5a / Nav1.5 and Irx3) and also inhibit the ventricular working cardiomyocyte phenotype (producing reduced cell area and decreased connexin 43 expression). We have also shown that induction of the fastconducting phenotype, illustrated by the increased protein expression of Connexin 40, requires juxtacrine signaling mediated by Notch1 activation in cardiomyocytes and fibroblasts, as demonstrated by pharmacological inhibition of the pathway in both cell types. Finally, hiPSC IRX3 KO resulted in cardiomyocytes with gene expression profile and CaTs consistent with ventricular working cardiomyocytes (higher expression of troponin I and connexin 43; lower expression of SCN5A; faster CaTs) in vitro. Together, we provide evidence that cardiac fibroblasts secrete factors that induce cardiomyocyte maturation to fast-conducting cells dependent on Notch1 signaling and that differentiation to conduction-specialized cells depends on the IRX3 gene, as its deletion favored the onset of ventricular working cardiomyocytes, which may be important for the development of more efficient strategies for cardiac regeneration (AU) | |
| FAPESP's process: | 17/05829-4 - Evaluation of the influence of fibroblasts on maturation and induction of cardiomyocytes with arrhythmogenic potential |
| Grantee: | Agatha Ribeiro da Silva |
| Support Opportunities: | Scholarships in Brazil - Master |