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Introduction Cardiac pericytes (contractile cells enwrapping capillaries) are vital for heart microvasculature, contributing to vascular stability and coronary blood flow regulation. They play a key role in angiogenesis, vessel maturation, and repair following myocardial infarction (MI). While their functions and contractility in response to vasoactive agents like endothelin-1 (ET1) and noradrenaline (NA) in vivo are well understood, research on their functional properties under neural stimulation and in the context of regenerative therapy remains limited. This study examines whether human cardiac pericytes retain their functional phenotype after expansion in vitro, explores their response to neural stimulation, and provides insight into their potential use for vascular regeneration after MI.Methods Cardiac pericytes were obtained from cardiac surgery waste tissue and cultured. Cells were then treated for 24, 48 or 72 h with either cholinergic agonist carbachol (0.1µM) or adrenergic agonist isoprenaline (0.1µM) to assess effects of ex vivo expansion and neural stimulation on their functional phenotype. Cells were then loaded with Ca 2+ indicator Cal-520 and pericyte Ca2+ responses to ET1 (10nM) and NA (10µM) were recorded from single cells. ET1 and NA were applied alternately, with 5 min washout period between applications.Results None of the treatments had adverse effects on the functional capacity of pericytes, as evidenced by robust Ca 2+ responses to ET-1 and NE after treatment with carbachol or isoprenaline. At baseline, cells stimulated with carbachol or isoprenaline exhibited greater fluorescence than control cells. Both vasoconstrictors increased intracellular Ca2 + levels, with ET-1 eliciting a stronger response than NA. Treated cells generated a larger response to vasoconstrictors compared to controls. Peak fluorescence was higher in cells exposed to ET-1 than in those exposed to NA (figure 1A). Response frequency was similar across all groups. Upon a second application of a vasoconstrictor, cells exhibited a comparable response to both ET-1 and NA (figure 1B).Conclusion Human cardiac pericytes cultured and stimulated in vitro for 24–72 h with synthetic cholinergic and beta-adrenergic agonists exhibit repeatable changes in intracellular Ca 2+ concentration when alpha-1 and ETA/ETB receptors are activated. This response aligns with the in vivo evidence showing that ET and NA elicit strong vasoconstricting effects on pericytes and suggests that pericytes expanded in vitro retain functional capacity. Further research is needed to assess the mechanism of action of Ca2+ flux.Abstract BS31 Figure 1Amplitude of Ca2+ responses to endothelin-1 and noradrenaline in control cells: A) after first ET1/NA application; B) after second ET1/NA application