Our animal facilities are Association for Assessment and Accreditation of Laboratory Animal Care accredited and Office of Laboratory Animal Welfare certified and fully comply with all regulations, policies, and standards that protect animal welfare. Animal use in our studies were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, Revised 1996) and the Institutional Animal Care and Use Committee guidelines of the University of California Merced (Protocol # 2008–201). The animals were bought from Toledo Goldfish, United States.

Adult Goldfish were anesthetized by immersion in ice-cold water containing 0.16 mg ml^–1 tricaine methanesulfonate for 2–5 min. To assure the Goldfish were completely anesthetized prior to decapitation, the tail was held with a small, curved tweezer, and gently moved. Once the Goldfish were completely anesthetized, they were decapitated, and the intact heart was removed from the chest cavity.

Goldfish hearts were dissected, and the bulbous arteriosus was cannulated onto a gauge 27 needle and perfused in a Langendorff system at a rate of 60 μl/min driven by gravity. Multiple solutions were perfused through the bulbus arteriosus with the aid of a self-designed μ-manifold. Goldfish hearts were perfused with a fish ringer solution containing: NaCl 137 mm, KCl 5.4 mm, CaCl₂ 1.8 mm, MgCl₂ 0.5 mm, HEPES 10 mm, and glucose 5.5 mm. The Ca²⁺ dye Rhod-2 AM was perfused into the heart with a Harvard pump for 30–45 min. The temperature of the bath containing the heart was set to 28°C. The temperature was controlled with the aid of a Peltier unit positioned at the bottom of the recording chamber and measured with a linearized semiconductor temperature sensor.

The Goldfish heart was perfused with fish ringer solution containing 4 mm blebbistatin, prior to obtaining any electrophysiological recordings to suppress cardiac motion. In order to elicit a sympathetic response, the Goldfish heart was perfused with fish ringer solution containing 100 nm isoproterenol for 10 min before the start of any AP and Ca²⁺ transient recordings. To determine if the sympathetic response to isoproterenol could be reversed, the Goldfish heart was perfused with fish ringer solution for a prolonged amount of time. Indeed, the effects of isoproterenol could be completely reversed if the Goldfish heart were continuously perfused with fish ringer solution for 20 min. In contrast, to elicit a parasympathetic response the Goldfish heart was perfused with fish ringer solution containing 5 μm carbamylcholine for 10 min prior to the start of any AP and Ca²⁺ transient recording. The effects of carbamylcholine could be completely reversed after continuously perfusing the heart with fish ringer solution for 60 min. Recordings obtained prior to perfusion with isoproterenol or carbamylcholine were considered as control, and recordings obtained following isoproterenol or carbamylcholine perfusion were considered as experimental.

In whole heart experiments, there are two main causes of variance. First, no two animals have entirely identical hearts, regardless of the species. Second, although we are measuring Ca²⁺ transients and APs in the same region (the mid-region of the left ventricle) of the heart, it is impossible to perform the recordings in the same precise location between different hearts. Thus, the data are presented as the measured times with their standard error (SEM). To assess electrical changes, AP traces were first normalized then evaluated at their respective repolarization times. Specifically, the time it takes for the AP to reach 30, 50%, or 90% repolarization, referred to here as APD30, APD50, or APD90, respectively.

Each wave of the electrocardiogram (QRS complex, T wave, and J wave) was measured using its corresponding half duration. The recorded Ca²⁺ transients were normalized between zero (minimum fluorescence) and one (maximum fluorescence) in order to evaluate the kinetics of the recorded Ca²⁺ transients. The kinetic parameters of the Ca²⁺ transients evaluated were the rise time (time for the Ca²⁺ transient to rise from, 10 to 90% of its maximum amplitude), half duration (duration of the Ca²⁺ transient at 50% of the maximum amplitude), and fall time (time for the Ca²⁺ transient to fall from 90 to 10% of the maximum amplitude). The AP parameters and Ca²⁺ transient kinetics obtained for the control and the experimental groups were evaluated and normalized to their respective control values for each heart used. After this normalization, data were compiled, and statistical analysis was performed.

The data are presented as multiple measurements (n; dot cloud) recorded for different measurements (n) on different hearts (N) with the mean ± SEM (solid lines). To determine if the agonists produced a significant effect, the distribution of the data before and after administration was compared using a two-sample Kolmogorov–Smirnov test (OriginPro, 2019). The difference was significant if the value of p < 0.01.

In order to elicit a sympathetic response and assess the 𝛽-adrenergic regulation of the Goldfish heart, we first perfused the heart with 100 nm isoproterenol. Goldfish ventricular chronotropic properties were examined via AP recordings and spontaneous heart rate recordings (Figure 1). Perfusion of the Goldfish intact heart with 100 nm isoproterenol altered the AP morphology (Figure 1A) and had a positive chronotropic effect, significantly increasing the heart rate by 46% (Figures 1B,C; from 0.87 ± 0.01 Hz to 1.27 ± 0.02 Hz). Interestingly, all kinetic parameters of the AP significantly changed following isoproterenol perfusion; APD30 increased from 228.10 ± 14.40 ms to 237.90 ± 11.80 ms (Figure 2A), APD50 decreased from 353.90 ± 30.40 ms to 300.30 ± 20.00 ms (Figure 2B), and APD90 increased from 455.40 ± 20.10 ms to 468.70 ± 27.00 ms (Figure 2C).

The effects of catecholamines on whole heart electrical activity were assessed through transmural electrocardiogram recordings (Figure 3). The 3 main components of the Goldfish electrocardiogram are presented in Figure 3A and consist of the QRS complex (ventricular depolarization), J wave (likely due to a voltage gradient due to the presence of a prominent AP notch in the epicardium but not the endocardium), ending with T wave (ventricular repolarization). Application of isoproterenol altered the morphology of the Goldfish electrocardiogram (Figure 3A) and increased the heart rate (Figures 3B,C; from 1.10 ± 0.40 Hz to 3.10 ± 0.70 Hz). Furthermore, application of isoproterenol also significantly altered the duration of the QRS wave, T wave, and J wave (Figure 4). The QRS complex significantly decreased from 22.70 ± 1.30 ms to 17.30 ± 2.80 ms (Figure 4A), the T wave significantly increased from 164.70 ± 53.20 ms to 292.10 ± 58.10 ms (Figure 4B), the J wave significantly increased from 126.10 ± 42.30 ms to 333.10 ± 105.30 ms (Figure 4C).

In many vertebrate species, stimulation of either autonomic nervous system branch will not only alter cardiac excitability, but also cardiac contractility. In order to assess if eliciting a sympathetic response altered the inotropic and/or the lusitropic properties of the Goldfish ventricle, experiments were performed in which the amplitude and kinetics of the Ca²⁺ transient were examined in the presence and absence of 100 nm isoproterenol (Figure 5). Stimulation of 𝛽-adrenergic receptors altered the morphology of the Ca²⁺ transient (Figure 5A) and significantly increased the normalized amplitude of the Ca²⁺ transient (Figure 5B; from 1.00 ± 0.07 to 1.10 ± 0.04). To detect if isoproterenol significantly altered the kinetics of the Ca²⁺ transient, the three following parameters of the Ca²⁺ transient were assessed (Figure 6): rise time (RT), fall time (FT), and half duration (HD). A significant change in any aforementioned kinetical parameter is a reflection of a significant corresponding change in myocardial Ca²⁺ handling dynamics. Perfusion of isoproterenol significantly increased the velocity of every Ca²⁺ transient kinetic parameter in the Goldfish heart (Figures 6A–C; RT: from 27.98 ± 4.60 ms to 22.47 ± 3.50 ms, FT: from 150.08 ± 22.80 ms to 135.88 ± 20.30 ms, and HD: from 148.60 ± 8.10 ms to 134.87 ± 5.20 ms), implying perfusion with isoproterenol increased the rate of relaxation of the Goldfish myocardium during diastole, resulting in a positive lusitropic effect (Figure 6B). The presence of a 𝛽-adrenergic drive suggests the presence of a parasympathetic one, as they are the two antagonistic branches of the autonomic nervous system.

The parasympathetic nervous system, on the other hand, is thought to be the dominant branch of the autonomic nervous system. To elicit a parasympathetic response and induce a cholinergic response, 5 μm carbamylcholine was administered to the Goldfish intact heart. As before, the chronotropic properties of the heart were assessed via AP and spontaneous heart rate recordings. Not surprisingly, the administration of carbamylcholine altered the AP morphology (Figure 7A) and had a negative chronotropic effect, reducing the heart rate by 92.2% (Figures 7B,C; from 0.98 ± 0.05 Hz to 0.13 ± 0.08 Hz). Not surprisingly, carbamylcholine administration significantly prolonged all three kinetic parameters of the AP (Figures 8A–C; APD30 increased from 235.90 ± 12.10 ms to 295.30 ± 11.50 ms, APD50 increased from 388.10 ± 23.90 ms to 651.40 ± 49.50 ms, and APD90 increased from 446.70 ± 14.60 ms to 833.60 ± 30.00 ms, respectively).

To further assess how cholinergic stimulation altered whole heart electrical activity in the Goldfish model, transmural electrocardiograms were recorded in the presence and absence of 5 μm carbamylcholine (Figure 9). The morphology of the Goldfish electrocardiogram changed dramatically in response to cholinergic stimulation (Figure 9A) and significantly decreased the spontaneous heart rate (Figures 9B,C; from 1.00 ± 0.04 Hz to 0.15 ± 0.09 Hz).

Cholinergic stimulation significantly altered the duration of the QRS complex, the T wave, and the J wave (Figure 10). The QRS complex significantly decreased from: 33.20 ± 2.40 ms to 31.90 ± 1.80 ms (Figure 10A), the T wave significantly increased from 370.20 ± 3.70 ms to 379.70 ± 14.40 ms (Figure 10B), and the J wave significantly increased from 169.20 ± 30.20 ms to 326.60 ± 23.30 ms (Figure 10C). The increased J wave duration is likely reflective of the decreased heart rate observed in Figures 7B, 9C.

In many vertebrate species, stimulation of either autonomic nervous system branch will not only alter cardiac excitability, but also cardiac contractility. To assess if stimulation of either autonomic nervous system branch altered the inotropic and/or the lusitropic properties of the Goldfish ventricle, experiments were performed in which the amplitude and kinetics of the Ca²⁺ transient were examined in the presence and absence of a cholinergic agonist.

To assess cholinergic regulation of contractility specifically, Ca²⁺ transients were recorded from the epicardial wall of the Goldfish ventricle in the presence and absence of 5 μm carbamylcholine (Figure 11). Administration of carbamylcholine had a negative inotropic effect, as the amplitude of the Ca²⁺ transient (Figure 11A) decreased in the presence of carbamylcholine. This negative inotropic effect is also presented in Figure 11B, where the normalized amplitude of the Ca²⁺ transient significantly decreased from 1.00 ± 0.10 to 0.49 ± 0.03 following cholinergic stimulation.

The three kinetical properties of the Ca²⁺ transient, including the rise time (RT), fall time (FT), and half duration (HD) were also evaluated to better understand how stimulation of the cholinergic pathway affected Ca²⁺ handling kinetics in the Goldfish myocardium (Figure 12). Although administration of 5 μm carbamylcholine did not significantly increase the rise time of the Ca²⁺ transient (Figure 12A; 30.20 ± 5.40 ms to 31.20 ± 3.20 ms), it did significantly increase the half duration of the Ca²⁺ transient (Figure 12C; from 151.80 ± 2.30 ms to 160.30 ± 4.00 ms). This effect can be due to the longer APs induced via cholinergic stimulation. Interestingly we were unable to observe a significant difference in the relaxation time (Figure 12B; from 161.50 ± 15.10 ms to 150.70 ± 12.10 ms) of the Ca²⁺ transient. This suggests carbamylcholine application did not significantly modify the lusitropic property of the Goldfish myocardium in these experiments; however, a significant change in the half duration of the Ca²⁺ transient does indicate the presence of an intrinsic parasympathetic tone in the Goldfish isolated heart, capable of modifying Ca²⁺ transient kinetics.

It is well-established stimulation of 𝛽-adrenergic receptors will have a positive chronotropic, dromotropic, inotropic, and lusitropic effect in any vertebrate species exhibiting full autonomic regulation. In the fish model, the autonomous rhythm of the heart is determined by the pacemaker region located near the atrial chamber, identified over 100 years ago (Keith and Mackenzie, 1910). Pacemaker APs are categorized by a gradual and slow diastolic depolarization (Phase 4), toward the threshold voltage of the AP upstroke (Phase 0; Saito, 1973; Harper et al., 1995; Haverinen and Vornanen, 2007; Tessadori et al., 2012). There are three main mechanisms by which an organism can modulate its heart rate, all of which end with an altered slope of the diastolic depolarization during diastole. This slope, set by the sinoatrial node, can be modified positively (by the sympathetic nervous system) or negatively (by the parasympathetic nervous system) by shifting the maximum diastolic potential, or decreasing the rate of depolarization, or (positively or negatively) shifting the membrane potential threshold; all of which could either increase or decrease the time required for the membrane potential to reach the threshold and fire an AP. In the Goldfish model, administration of isoproterenol altered ventricular AP morphology and had a positive chronotropic effect (Figures 1A–C). Remarkably, previous studies have observed isoproterenol to induce strikingly similar AP morphological changes in canine endocardial myocytes and guinea pig cardiomyocytes (O’Hara and Rudy, 2012; Szentandrássy et al., 2012; Sala et al., 2018).

The changes in the AP morphology can be better observed in Figure 2, where the kinetics of the ventricular AP are presented following adrenergic stimulation with isoproterenol. Perfusion with isoproterenol lead to a significant increase in APD30 and APD90 (Figures 2A,C, respectively) and a significant decrease in APD50 (Figure 2B). Interestingly, canine endocardial myocytes treated with isoproterenol exhibit lengthening of APD90 (Szentandrássy et al., 2012; Sala et al., 2018), similar to what we observed for the Goldfish. Unlike mammals, many fish species lack the slow component of the delay rectifier current (IKs), the main current system mediating repolarization effects of adrenergic stimulation on cardiac AP duration (Vornanen, 2017). The absence of this repolarizing current could explain the counterintuitive prolongation of APD30 and APD90 in the presence of an adrenergic stimulus. However, the role of I_Ks in response to adrenergic stimulation is not yet elucidated in the Goldfish heart, and further studies are necessary to corroborate this hypothesis.

Transmural electrocardiograms were recorded in the presence and absence of isoproterenol (Figure 3) to examine its effect on whole heart electrical activity. Indeed, isoproterenol perfusion not only altered the morphology of the electrocardiogram (Figure 3A) but also reaffirmed the positive chronotropic effect (Figures 3B,C) of isoproterenol presented in Figure 1. The positive chronotropic effect could be due to an increased slope of diastolic depolarization, as many other mammals exhibit the same pattern in response to adrenergic stimulation (Randall et al., 2020).

Isoproterenol’s significant effect on whole heart excitability (Figure 4) not only reaffirms the presence of a positive chronotropic effect, but also suggests the instigation of a positive dromotropic effect. Because the QRS complex represents ventricular depolarization, its duration indirectly measures intraventricular impulse conduction. Thus, the positive dromotropic effect induced by isoproterenol can best be observed in Figure 4A, where administration of the catecholamine significantly reduced the duration of the QRS complex and increased the rate of intraventricular impulse conduction. Furthermore, isoproterenol significantly increased the duration of the T wave, and significantly prolonged the duration of the J wave (Figures 4B,C). An increased J wave duration is consistent with the prolongation of the APD30 in the presence of 100 nm of isoproterenol (Figure 2A). These modifications observed in the electrocardiogram further solidify the hypothesis that Goldfish exhibit sympathetic regulation, as perfusion with a catecholamine significantly modified cardiac excitability. The cardiac AP alters the mechanical function of vertebrate hearts by increasing intracellular free Ca²⁺ concentration, ultimately inducing cardiac contractions (Coraboeuf, 1978; Randall et al., 2020). In the Goldfish heart, Ca²⁺ influx through the LTCCs is the most likely trigger of Ca²⁺ release from the sarcoplasmic reticulum (Bazmi and Escobar, 2020), which ultimately augments cardiac contractile properties. Therefore, modifications in Ca²⁺ handling dynamics are essential for understanding how cardiac excitability alters cardiac contractility.

Epicardial Ca²⁺transient recordings from the Goldfish ventricle show administration of isoproterenol altered the morphology of the Ca²⁺ transient and significantly increased the normalized amplitude of the Ca²⁺ transient (Figures 5A,B), a trend also observed in guinea pigs (Katra et al., 2004). The positive chronotropic effect (Figures 1C, 3C) in response to adrenergic stimulation could be explained by the alterations present in the Ca²⁺ transient dynamics followed by isoproterenol perfusion. A significant increase in the Ca²⁺ transient amplitude following adrenergic stimulation (Figure 5B) suggests isoproterenol increased the Ca²⁺ current, likely through the LTCC (Bazmi and Escobar, 2020). As previously discussed, adrenergic stimulation activates a cascade of events that phosphorylate numerous Ca²⁺ handling proteins, including PLN on serine 16 and threonine 17. Phosphorylation of PLN removes its inhibitory effect on the cardiac sarcoplasmic endoplasmic reticulum ATPase, thus increasing Ca²⁺ load into the sarcoplasmic reticulum. An increased Ca²⁺ transient amplitude increases the influx of positive charges into the myocardium and reduces the AP threshold; both of which increase the conduction velocity of the AP, resulting in a positive dromotropic effect. Furthermore, an increased Ca²⁺ current will increase the amount of Ca²⁺ in the sarcoplasmic reticulum and ultimately increase Ca²⁺ induced Ca²⁺ release. This would then increase the strength of contraction, resulting in the positive inotropic effect observed in Figure 5. To our knowledge, a positive inotropic effect in response to adrenergic stimulation has yet to be observed in the ventricle of any other fish species (Vornanen and Tuomennoro, 1999; Molina et al., 2007; Vornanen et al., 2010; Abramochkin and Vornanen, 2017). However, it is likely previous studies did not observe positive dromotropic effects considering many of them performed similar experiments on isolated cardiomyocytes and not at the intact heart level. Looking at changes in the kinetic properties of the Goldfish heart, it is likely isoproterenol also had a positive lusitropic effect. Although all three kinetical parameters of the Goldfish Ca²⁺ transient decreased following isoproterenol perfusion (Figures 6A–C), the lusitropic effect can be best observed in Figure 6B, as the fall time of the Ca²⁺ transient significantly decreased, suggesting isoproterenol increased the rate of myocardial relaxation during diastole.

As previously mentioned, an organism with an adrenergic drive could potentially also have a cholinergic drive, as they are the two antagonistic branches of the autonomic nervous system. Cholinergic control, however, is stronger than adrenergic control and has a negative chronotropic, dromotropic, inotropic, and lusitropic effect (Randall et al., 1968; Urbá-Holmgren et al., 1977; Laurent et al., 1983; Farrell, 1984; Axelsson et al., 1987). In the Goldfish model, perfusing the heart with 5 μm carbamylcholine prolonged the AP (Figure 7A) and had a negative chronotropic effect (Figures 7B,C). The strong negative chronotropic effect induced by cholinergic stimulation could be mediated by an ACh activated potassium current (IK_Ach); a major current found in fish atrial myocytes responsible for the repolarization of the membrane potential (Molina et al., 2007; Vornanen et al., 2010; Abramochkin and Vornanen, 2017). Furthermore, the activation of a muscarinic receptor will produce inhibition of the adenylyl cyclase reducing the levels of cAMP, preventing PKA-mediated phosphorylation. It is important to note, however, the levels of cAMP are finely regulated by PDEs, which contribute to the lowered cAMP concentrations. Nevertheless, lowered PKA levels result in a reduction in key phosphorylation sites, which ultimately decrease the slope of the diastolic depolarization and decrease heart rate. Furthermore, administration of carbamylcholine significantly prolonged all three kinetical parameters of the AP duration (Figures 8A–C). Considering previous research has shown the presence of Ca²⁺ dependent inactivation of the LTCC in Goldfish ventricular myocytes (Bazmi and Escobar, 2020), it is likely a decreased sarcolemmal Ca²⁺ influx mediated by carbamylcholine (Figure 11B) decelerated inactivation and prolonged the duration of the action potential.

In order to determine how the stimulation of cholinergic response modulated whole heart electrical activity, electrocardiograms were recorded in the presence and absence of carbamylcholine (Figure 9). Perfusion with carbamylcholine altered electrocardiogram morphology (Figure 9A) and reaffirmed the negative chronotropic effect (Figures 9B,C) presented in Figure 7B. Modification of whole heart excitability in response to muscarinic stimulation is presented in Figure 10. Interestingly, the duration of the QRS complex decreased in response to carbamylcholine administration, suggesting a slight positive dromotropic response, something typically observed in tachycardia. Currently, little is known about the depolarizing ventricular currents in the Goldfish which could provide further insight as to why cholinergic stimulation would reduce the duration of the QRS complex. Carbamylcholine perfusion also significantly increased the T and J wave durations (Figures 10A–C); however, the increased J wave was expected as there was a corresponding increase in APD30 (Figure 8A).

The negative chronotropic effect induced by stimulation of the muscarinic receptors could also be explained by modifications of the Ca²⁺ transient. Administration of 5 μM carbamylcholine modified Ca²⁺ transient morphology and significantly decreased the amplitude of the Ca²⁺ transient, suggesting stimulation of muscarinic receptors may have had a negative inotropic effect (Figures 11A,B). This is particularly interesting because previous studies conducted in isolated cardiac myocytes suggest muscarinic stimulation produced minor changes in cardiac chronotropic and inotropic properties in the fish heart (Laurent et al., 1983; Fritsche and Nilsson, 1990; Steele et al., 2009). This discrepancy, however, could be explained by the fact that other experiments were conducted in isolated myocytes, while our experiments were performed in the intact heart. As the heart is an electrically coupled organ, isolation of cardiac myocytes disrupts this electrical coupling, which may alter cardiac contractile properties.

A reduction in the Ca²⁺ current amplitude (Figure 11B) is likely to have reduced the slope of the diastolic depolarization and, as such, induced a negative chronotropic effect. As mentioned before, there are numerous mechanisms by which this slope may change. During cholinergic stimulation, PDEs and inhibition of adenylyl cyclase reduce cAMP levels which not only lower the activation of PKA, but also reduce stimulation of HCN channels. The current produced by these channels, I_f, typically increases the slope of the diastolic depolarization. However, in the presence of a cholinergic agonist, stimulation of If decreases, thus reducing the slope of the diastolic depolarization and ultimately reducing heart rate. Interestingly, HCN4 pacemaker channels have only been identified in the pacemaker region of the Goldfish and Zebrafish (Tessadori et al., 2012; Newton et al., 2014). Another possible mechanism by which muscarinic receptor stimulation induced a negative chronotropic response could be activation (IK_Ach), although the contribution of this current is still poorly elucidated in fish ventricular myocytes (Molina et al., 2007; Vornanen et al., 2010; Abramochkin and Vornanen, 2017). Activation of IK_ACh would induce hyperpolarization of the maximum diastolic potential, decreasing the heart rate. A decreased Ca²⁺ current and activation of IK_ACh also lead to a negative dromotropic effect as a decreased Ca²⁺ current will decrease the influx of positive charges, increase the threshold of the AP, and decrease the mean diastolic potential; all of which reduce AP conduction velocity and induce a negative dromotropic effect.

Modifications presented in the kinetic properties of the Goldfish heart following carbamylcholine perfusion (Figure 12) suggest stimulation of the muscarinic receptor induced a minor negative lusitropic effect. While the half duration of the Ca²⁺ transient significantly increased in response to carbamylcholine perfusion (Figure 12C), the rise time and fall time were not significantly altered (Figures 12A,B). These results are interesting because the effect of carbamylcholine is in the opposite direction of what happens in mouse hearts and is very similar to larger mammals (Aguilar-Sanchez et al., 2019).