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Autonomic regulation plays a central role in cardiac contractility and excitability in numerous vertebrate species. However, the role of autonomic regulation is less understood in fish physiology. Here, we used Goldfish as a model to explore the role of autonomic regulation. A transmural electrocardiogram recording showed perfusion of the Goldfish heart with isoproterenol increased the spontaneous heart rate, while perfusion with carbamylcholine decreased the spontaneous heart rate. Cardiac action potentials obtained via sharp microelectrodes exhibited the same modifications of the spontaneous heart rate in response to isoproterenol and carbamylcholine. Interestingly, the duration of the cardiac action potentials lengthened in the presence of both isoproterenol and carbamylcholine. To evaluate cardiac contractility, the Goldfish heart was perfused with the Ca 2+ indicator Rhod-2 and ventricular epicardial Ca 2+ transients were measured using Pulsed Local Field Fluorescence Microscopy. Following isoproterenol perfusion, the amplitude of the Ca 2+ transient significantly increased, the half duration of the Ca 2+ transient shortened, and there was an observable increase in the velocity of the rise time and fall time of the Ca 2+ transient, all of which are compatible with the shortening of the action potential induced by isoproterenol perfusion. On the other hand, carbamylcholine perfusion significantly reduced the amplitude of the Ca 2+ transient and increased the half duration of the Ca 2+ transient. These results are interesting because the effect of carbamylcholine is opposite to what happens in classically used models, such as mouse hearts, and the autonomic regulation of the Goldfish heart is strikingly similar to what has been observed in larger mammalian models resembling humans., Competing Interests: The authors declare the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bazmi and Escobar.)