Profiling cardiac fibroblasts in regenerative hearts after myocardial infarction
Authors: Gomes R , Manuel F , Pereira C , Nascimento DS 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fundação para a Ciência e Tecnologia (FCT)

Introduction
Mouse neonates at postnatal day 1 (P1) are able to regenerate their hearts after myocardial infarction (MI) by reactivating cardiomyocyte proliferation and neovascularization, with little to no fibrosis. However, this process is transient and 7 day-old animals develop a reparative response as observed in adults [1]. After MI, these animals undergo permanent loss of myocardial tissue that is replaced by a rigid fibrotic scar to avoid organ rupture, having devastating consequences for heart function. Although the role of cardiac fibroblasts (CF) - the main orchestrators of fibrosis – in adults is well-documented, no study has unveiled the role of CF during neonatal regeneration. Recent work from our group showed that at least a subset of neonatal CF is able to provide pro-regenerative cues to cardiomyocytes, pointing to a beneficial role of CF in MI resolution.

Purpose
The ultimate goal of our research is to unravel CF-mediated mechanisms that confer regenerative potential to the neonate and re-activate these processes in the adult.

Methods
Mouse ventricles from E16, P1, P3 and P7 mice were subjected to targeted RNA-sequencing. To unveil non-myocyte cell dynamics in the first week after injury in regenerative (P1) hearts, myocardial infarction (MI) was induced by permanent ligation of the left descending coronary artery. To specifically assess the impact of CF in heart regeneration, a Tcf21iCre knock-in mouse line, carrying the diphtheria toxin receptor, was generated, rendering Tcf21+ CF - the majority of CF in the heart - susceptible to diphtheria toxin.

Results
Transcriptional profiling around birth highlighted severe extracellular matrix (ECM) changes from regenerative (P1) to reparative stages (P7). Coherently, from P1 to P7 CF were found to populate the myocardium and undergo a phenotypic shift that explained the transcriptional alterations observed for ECM-encoding genes, indicating a role of CF in the regeneration to repair transition. After MI at P1, CF were found to be readily and transiently recruited to the ischemic site, peaking at day 5 post-MI and returning to basal levels at day 7, a period in which cardiomyocyte proliferation and neovascularization were up-regulated. Of note, no evidence was found of fibrotic tissue or myofibroblasts from 7 days post-MI onwards. Contrarily, MI at P7 resulted permanent loss of cardiomyocytes, impaired neovascularization and formation of aberrant fibrosis as a result from exuberant and persistent fibroblast recruitment and activation. To evaluate the functional impact of fibroblast ablation during the regenerative response, ablation of Tcf21+ CF was performed after MI at P1. CF removal resulted impaired cardiac cell proliferation, indicating that CF recruited after MI are essential for effective neonatal regeneration.

Conclusion
After birth, CF undergo a switch from a regenerative to reparative phenotype that contributes to the loss of regenerative capacity after birth.