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Acute heat stress (AHS) causes massive death of fish and other animals during the periods of abnormal temperature increase or in cases of hot water draining into the natural reservoirs. Since AHS affects functioning of different organism systems including cardiovascular, ectothermic animals living at low temperatures are very vulnerable. The aim of the present study was to elucidate the effects of acute heat stress on cardiac function of European sculpin (Myoxocephalus scorpio) in vivo and in vitro. ECG registration with two stainless steel electrodes implanted into pericardial cavity was used to investigate AHS effects on heart function in vivo in non-narcotized free-swimming sculpin. Before the surgery sculpins were anesthetized (tricaine 0.17g/L). The experiments were performed in a 20L tank with running seawater after a 24hr recovery period. During the experiments the temperature was rising with speed of 2°C/hr from 3°C up to 26°C. In a series of experiments atropine (2mg/kg) and propranolol (2mg/kg) were injected into pericardial cavity. The effects of AHS on ionic currents and action potentials (AP) configuration in isolated cardiomyocytes were studied in vitro using whole-cell patch clamp. All experiments were approved by local bioethical committee. AHS caused significant increase of heart rate (HR) and decrease of PR-, QRS- and QT-intervals till the breakpoint temperature (21°C); further heating caused opposite changes. Autonomic blockade led to a decrease in breakpoint temperature and maximum HR. At 26°C single P-waves and QRS-complexes were observed. AHS changed AP configuration in isolated cardiomyocytes and caused a decrease in AP duration at levels of repolarization 50% and 90%. The maximum rising slope was enhanced by AHS till the breakpoint (26°C), the further warming caused significant decrease. AHS enhanced the repolarizing potassium currents IK1 and IKr in isolated cardiomyocytes. Sodium current INa and calcium L-type current ICaL increased with heating till the breakpoint; further temperature rise was followed by a dramatic decrease. AHS has profound effect on the processes of impulse generation and conduction in fish heart due to the changes in ionic currents and AP configuration in cardiomyocytes. Presumably, AHS can cause arrhythmic events leading to animals’ death. Autonomic system plays important role in the adaptation to AHS.