Abstract Body: Acetylcholine (ACh) is a neurotransmitter involved in regulating cardiac function, and its production is significantly reduced following myocardial damage. Previous studies demonstrated that administration of ACh reduced infarct size in myocardial infarction (MI) and ischemic-reperfusion (I/R) injury in in vivo animal models. As well, ACh activated anti-inflammatory pathways, and promoted cell survival under hypoxic conditions. However, the broad activity of ACh leads to side effects that hinder the development of new therapeutic approaches for preventing myocardial damage. Moreover, previously used models have not fully replicated the intricate cardiac microenvironment, limiting the understanding of its clinical potential. In this context, our laboratory has recently developed in vitro cardiac spheroids (CSs) comprised of stem cell-derived cardiomyocytes, fibroblasts and endothelial cells to better mimic the molecular, cellular, and extracellular features typical of the human cardiac microenvironment. This study delves into evaluating the cardioprotective effects of ACh using three different delivery methods: i) freely-dissolved 100µM ACh; ii) ACh-producing cholinergic nerves (CNs); and iii) ACh-loaded nanoparticles (ACh-NPs). Myocardial damage in CSs was achieved by exposing them to either I/R-like conditions through oxygen level changes or by exposing them to doxorubicin (DOX), a well-known cardiotoxic drug. Our analyses of cell viability and death, contractile function, and gene expression profiles through qPCR in in vitro I/R- and DOX-induced CSs revealed that increased ACh levels protect against the reduction in cell viability, fractional shortening % (FS%), as well as mitigate changes in gene associated with myocardial damage. Furthermore, the cardioprotective effects of ACh-NPs were evaluated in an in vivo MI mouse model. Our ultrasound imaging, histology and bulk RNAseq analyses showed that injecting ACh-NPs in the myocardium improved the ejection fraction % (EF%) by 20.24% +/- 2.925% in MI animals, prevented cardiac fibrosis and activated signalling pathways regulating cell survival and proliferation. Altogether, our findings support the cardioprotective role of ACh against I/R and DOX-induced myocardial damage, underscoring the potential use of ACh-NPs as a novel therapeutic approach.