A sinalização colinérgica–Gi como eixo modulador da proteção e da maturação cardíaca

Abstract

Introduction: G protein-coupled receptors (GPCRs) play a central role in the regulation of cardiac function and heart development by modulating processes such as heart rate, contractility, cardiac remodeling, and cardiomyocyte (CM) maturation. While hyperactivation of Gs and Gq signaling pathways is associated with the development of cardiac pathologies and accelerated maturation, activation of the Gi pathway (classically stimulated by acetylcholine (ACh) via the M2 muscarinic receptor) has been associated with cardioprotection and modulation of cellular growth. However, despite these advances, the mechanisms underlying these effects, as well as the cellular bases involved, remain poorly explored. In this context, we investigated the role of cholinergic/Gi signaling both in the response of the adult heart to adrenergic stress and in the regulation of postnatal cardiac maturation. For this purpose, this thesis was divided into two chapters. Chapter I: To evaluate the therapeutic role of the Gi pathway in adult cardiomyocytes, we used a murine model with selective Gi activation in CMs through the DREADD system (Myh6-hM4Di), pharmacologically activated by clozapine-N-oxide (CNO). In in vitro and in vivo models of adrenergic overload induced by isoproterenol (ISO), we demonstrated that Gi activation in CMs reduces cellular contractility and prevents pathological cardiac remodeling, including cardiac and cellular hypertrophy, cardiomyocyte death, inflammatory infiltration, fibroblast activation, and increased collagen expression. Moreover, late Gi activation was able to reverse already established cardiac injury, highlighting an active role of this pathway in orchestrating cardiac repair, possibly through communication between cardiomyocytes, inflammatory cells, and fibroblasts. Chapter II: To investigate the role of cholinergic activity in postnatal cardiac maturation, we used different murine models with either increased or reduced cholinergic activity: VAChT-KD mice, Chat-ChR2 mice, and DREADD-based activation of Gi signaling between postnatal days 1 and 21 (P1–P21), the period that encompasses the maturation window. We also used neonatal CM cultures. Increased systemic ACh availability resulted in delayed maturation, characterized by increased CM proliferation, reduced cellular growth, and a higher proportion of mononucleated cardiomyocytes. In contrast, global reduction of ACh accelerated maturation, promoting increased cell size and higher multinucleation. Selective activation of the Gi pathway in neonatal cardiomyocytes reproduced the delayed maturation phenotype, indicating that ACh effects are predominantly mediated through the ACh–M2–Gi axis. In vitro studies further demonstrated that cardiomyocyte-derived ACh exerts an autocrine/paracrine role in controlling cellular growth, as inhibition of its degradation reduced CM area, whereas blockade of its release or muscarinic signaling promoted cellular hypertrophy. Conclusion: Collectively, our data demonstrate that the cholinergic/Gi pathway exerts a dual role in the heart, acting as a protective mechanism against adrenergic insults in the adult heart and as a negative modulator of structural and functional cardiomyocyte maturation during the postnatal period. Thus, the dynamic balance between adrenergic and cholinergic signaling emerges as a key determinant of both cardiac adaptation to stress and cardiac development, positioning Gi signaling as a potential therapeutic target across different physiological and pathological cardiac contexts: Keywords: Cardiomyocyte; Cardiac remodeling; GPCRs; Gi pathway; cardiac maturation.