Gi activation in cardiomyocytes reverses cardiac damage induced by adrenergic overload

G-protein coupled receptors (GPCRs) are key modulators of cardiac function. Hyperactive Gs and Gq pathways are linked to cardiac disease, while Gi activation via ACh binding to muscarinic type 2 receptors promotes cardioprotection. We aimed to selectively activate Gi signaling in cardiomyocytes (CMs) to assess its therapeutic potential in a adrenergic overload-induced cardiac damage model. To selectively activate Gi in CMs, we crossed the R26-LSL-Gi-hMD4i model with Myh6-CRE, creating a Myh6-Cre/hM4Di mice, in which upon N-Oxide-Clozapine (CNO) stimulation activates Gi selectively in cardiomyocytes (CMs). Male and female mice (8 weeks old) were subjected to: Protocol 1: CMs were isolated, electrically stimulated to contract, and treated with Isoproterenol (ISO 50nmol/L), ACh, or CNO (100nmol/L). Protocol 2: Myh6-Cre/hM4Di mice were treated in vivo with ISO (25 mg/kg/day) with or without CNO (0.5 mg/kg/day) for 7 days. Mice were sacrificed on day 8. The data was derived from at least three independent experiments. Normality was assessed using the Shapiro-Wilk test. For normal data, an unpaired Student's t-test or one-way ANOVA with Tukey's post hoc test was used. For non-normal data, the Mann-Whitney U or Kruskal-Wallis test was applied, followed by the Games-Howell post hoc test when appropriate. A significance threshold of p < 0.05 was applied. To confirm the activation of the Gi pathway in CMs the cells were exposed to CNO or ACh (positive control for Gi), and contraction was recorded. Myh6-Cre/hM4Di cells exhibited reduced shortening area after CNO stimulation, while no effect was observed in cells from Myh6-CRE or hM4Di littermate mice. Next, we investigated whether Gi activation in CMs could attenuate the ISO-induced tissue injury in vivo (protocol 2). CNO treatment of Myh6-Cre/hM4Di mice prevented ISO-induced pathological cardiac remodeling, including hypertrophy, inflammation and fibrosis. To explore whether Gi activation could reverse pre-existing cardiac injury, we started CNO injection on day 4 of a 7-day ISO treatment, at a stage when cell death and inflammation were already occurring. We found that late CNO treatment reversed ISO-induced cardiac and CM hypertrophy. Flow cytometry analyses showed a reduction in inflammatory cells, which directly impacted the fibrotic and functional cardiac outcomes. This was evidenced by fewer myofibroblasts, reduced collagen I and III transcripts, and improved echocardiographic parameters, confirming the efficacy of late CNO treatment in cardiac recovery. These results demonstrate, for the first time that targeted Gi activation in CMs protects against adrenergic overload-induced cardiac injury, highlighting CM-Gi's role in cardiac repair. CNPq, CAPES e FAPEMIG This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.