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Introduction: The production of acetylcholine (ACh), a neurotransmitter regulating cardiac function, is significantly reduced following myocardial damage in vivo. Previous studies demonstrated that ACh administration can be used to reduce infarct size following myocardial infarction (MI) and ischemic-reperfusion (I/R) injury in in vivo animal models. This has been associated with ACh-mediated activation of anti-inflammatory pathways, as well as improved cell survival under hypoxic conditions. Nevertheless, in order to avoid unspecific effects due to ACh’s broad activity on a number of receptors throughout the body, its use in the clinic has been limited, preventing the development of new therapeutic approaches for preventing myocardial damage. Therefore, this project aimed at evaluating the potential protective effects of ACh by combining advanced in vitro models of myocardial damage using vascularised cardiac spheroids. In an effort to translate in vitro findings, we tested the protective effects in an in vivo models of myocardial damage in mice.
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, fibroblast and endothelial cells to better mimic the molecular, cellular, and extracellular features typical of the human cardiac microenvironment.
Methods: We evaluated 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) (Figure 1). These were tested in vitro by comparing the three delivery methods in cardiac spheroids that modelled I/R conditions. These were achieved by exposing cardiac spheroids to changes in oxygen levels from normoxia (5% oxygen), hypoxia (0% oxygen) and normoxia (5% oxygen). Additionally, myocardial damage in cardiac spheroids was also achieved by exposing them to doxorubicin (DOX), a well-known cardiotoxic drug. Control and injured (I/R or DOX) cardiac spheroids were tested for changes in viability/toxicity by calcein-AM and ethidium homodimer staining, respectively, as well as for changes in contractile activity by measuring contraction frequency and fractional shortening %. In vivomyocardial damage was achieved by ligating the LAD in a mouse model established in our laboratory. ACh-NPs were directly injected into the muscle wall of infarcted animals right before ligating the LAD. Mice were imaged using ultrasound techniques to measure changes in ejection fraction %. Hearts were also isolated at 28 days to isolate tissue for histological, immunofluorescence and transcriptomics analyses.
Results: Our analyses revealed that increased ACh levels protect against the reduction in cell viability, fractional shortening % (FS%), as well as mitigate changes in genes associated with myocardial damage in cardiac spheroids. 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.
Discussion and conclusion: 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.
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