INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

GENETIC SCREENS IN ZEBRAFISH provide a means to scan the genome for genes important to embryonic physiological function (35, 36). However, many important homeostatic mechanisms are not fully developed until well after embryogenesis, and it is not known if the functions of a gene in the embryo predict those in the adult. The regulation of heart rate, for example, differs dramatically from embryonic to adult animals (4, 5, 18, 30). Neuronal and humoral control and developmentally regulated and chamber-specific participation of voltage-gated currents are all core determinants of adult cardiac pacing. Additional factors that change with growth and that may affect heart rate include oxygen delivery to the thickening myocardium, calcium storage and release mechanisms, sarcomeric kinetics, the rate of force development in cardiomyocytes, and properties of the pacemaker potential (19).

How these many heart rate regulators are orchestrated to provide carefully controlled sequential beating of the chambers is not clear. Many heart cells appear to have pacemaking properties when isolated in culture. The "pacemaker current" (I_h) is one current shared among cardiac myocytes, both in the sinus node and throughout atria and ventricles. There are several lines of evidence that I_h is among the most important currents for pacemaking in the heart (for a review, see Ref. 11). It is an unusual current in that it provides inward current upon hyperpolarization of the cell membrane. The inward current helps lead to sufficient depolarization to initiate an action potential. I_h is a presumptive target for autonomic nervous system regulation of heart rate. Adrenergic stimulation increases I_h activity and thereby speeds heart rate. Cholinergic stimulation decreases I_h activity and thereby slows heart rate (9). I_h properties differ between the embryonic and adult heart and between the atrium and ventricle. As the heart matures, I_h conductance in the atrium and the developing conduction system increases, whereas the current density of I_h in the ventricle drops dramatically (6). In addition, the threshold for activation of ventricular I_h is shifted to an even more hyperpolarized voltage (27, 29), thus reducing its effect on pacemaking.

The genetic evidence that I_h does regulate cardiac pacemaking in the intact animal comes from the mutation slow mo. We discovered this mutation as part of a large-scale zebrafish genetic screen. The homozygous slow mo fish has a slow heart rate (sinus bradycardia) evident from the first heartbeats (3). This is the only noticeable effect of the mutation. At present, the mutation resides in an unknown gene. In a prior study, we developed the means to culture and voltage-clamp cells from embryonic zebrafish hearts and found these cells manifested a variety of currents, including I_h. In the embryonic zebrafish heart, I_h appears to have two kinetically distinct components, as has also been described in cardiomyocytes from the rabbit heart (10, 20, 24) and in neurons from the rat (31). Myocardial cells of slow mo mutant embryos have a markedly reduced I_h, with a near ablation of the fast component (3).

Most of the 150 cardiac mutations discovered in zebrafish genetic screens are lethal (2, 7, 32, 36). Although slow mo mutant embryos do have reduced viability, we have been able to carry several through to adulthood. In the adult fish, we found that the slow mo mutation causes a chronic bradycardia even though the heart is fully innervated. Here, we report maturational changes in the properties of I_h that have a chamber-specific profile. The adult slow mo mutant fish continues to manifest bradycardia. This bradycardia is present in the setting of a dramatic reduction in I_h, especially in atrial cells. This demonstrates that I_h continues to play a central role in pacemaking in the adult heart.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Fish care. Zebrafish were raised and maintained as described in (37). Fish were classified as adults after achievement of sexual maturity at ~3 mo of age. Homozygote genotypes for slow mo mutant adults were confirmed before analysis.

Heart rate counts. Adult fish (4-18 mo old) were transferred to an aqueous solution of propofol at a final concentration of 5 µg/ml. Fish were anesthetized for 5 min and then transferred to the counting chamber. The chamber consisted of a cuvette immersed in propofol at the same concentration used in the anesthetizing container. The chamber made it possible to turn the fish ventral side up to directly observe the heart beat. The heart rate was counted for a full 60 s under direct vision using a dissecting microscope. Once the counting was finished, the fish was transferred to normal fish water without propofol. All fish survived the counting experiments. Temperature of all solutions used for the propofol counting experiments fell within the range of 20.0-23.0°C. A second set of counting experiments was conducted with the fish anesthetic tricaine (MS-222, Sigma) at a final concentration of 0.16 mg/ml (37), and the temperature was maintained at 26°C throughout. Heart rates were always determined blind to the genotype of the fish.

Cardiomyocyte culture. Cells were prepared from adult zebrafish (4-18 mo old). Fish were anesthetized by allowing the fish to swim in an aqueous solution of tricaine for 3-5 min. Each fish was in turn placed in L15 media (GIBCO-BRL) and pinned ventral side up, and the entire heart was dissected out and placed in a solution of L15 supplemented with penicillin G (200 IU/ml) and streptomycin (200 µg/ml). The heart could reliably be removed within 30 s. Once three hearts had been placed in the antibiotic supplemented L15, the atrium and ventricle were separated under direct vision. The chambers of interest (either atria or ventricles) were then opened widely and transferred to 750 µl of solution A [containing (in mM) 135 NaCl, 5.4 KCl, 1 MgCl₂, and 10 HEPES (pH 7.20 with NaOH)]. The solution was supplemented with penicillin G-streptomycin (26). The chambers were allowed to stir for 10 min at room temperature in a small cuvette. While the hearts continued to stir, 750 µl of solution B (solution A supplemented with 2 mg/ml collagenase I, 0.28 mg/ml protease XIV, 200 IU/ml penicillin G, and 200 µg/ml streptomycin) was added. The hearts were then allowed to stir for 60 min at room temperature to digest the tissue. The digestion solution was then transferred to an Eppendorf tube, and the cells were pelleted by spinning at 2,000 rpm for 2 min. The supernatant was removed, and 1 ml of L15 supplemented with penicillin G-streptomycin was added. The cells were spun as before, and the supernatent was again removed. The cells were then resuspended in 400 µl of L15 supplemented with penicillin G-streptomycin. The cells were rested for 10 min at room temperature, and single cells were then obtained by gentle trituration. Cells were placed on plain glass coverslips. The cells were used for recording on the following day. The cells were prepared blind to the genotype of the fish.

Electrophysiology. Standard whole cell recording techniques were applied to single cells. All data were acquired and analyzed blind to genotype. For recording I_h from both atria and ventricles, the bath solution contained (in mM) 40 NaCl, 40 KCl, 2 CaCl₂, 1 MgCl₂, 40 N-methyl-D-glucamine, 10 tetraethylammonium chloride, 10 HEPES, and 10 glucose (pH 7.4 with NaOH). The corresponding pipette solution contained (in mM) 70 KF, 70 KCl, 1 MgCl₂, 10 EGTA, and 10 HEPES (pH 7.4 with NaOH). For recording sodium current (I_Na) from both atria and ventricles, the bath solution contained (in mM) 30 NaCl, 110 choline chloride, 2 KCl, 1 MgCl₂, 2 CaCl₂, 2 BaCl₂, 10 HEPES, and 10 glucose (pH 7.4 with NaOH). The corresponding pipette solution contained (in mM) 70 CsCl, 70 CsF, 0.5 MgCl₂, 10 EGTA, and 10 HEPES (pH 7.4 with CsOH). Pipettes were pulled using borosilicate glass and polished to resistances of 1.6-3.2 M. All I_Na recordings had series resistance compensation employed at 90%. I_h were acquired without such compensation. Data acquisition and analysis were carried out using pCLAMP 6 software (Axon Instruments; Foster City, CA). All recordings were at room temperature (20.5-23.0°C). The amplitude of I_h was determined from the amplitude of the Boltzmann fit to the steady-state activation curve determined at a test voltage of 130 mV. The amplitude of I_Na was determined from the amplitude of the Boltzmann fit to the steady-state inactivation curve determined at a test voltage of 30 mV. The leak current and nonlinear open channel properties were minimized by taking all of the current amplitudes at a constant test voltage. The kinetics of activation for I_h were determined at 130 mV. Data traces were filtered at 1 kHz and fitted with either a single- or double-exponential curve. All traces were initially fit with a single exponential. The need for a second component was determined by a trial fit with two components. If the fitting algorithm converged and gave a nontrivial second component (i.e., time constant and amplitude > 0), then two components were assumed. The fitting and number of components was determined blind to the genotype. In the majority of cases, the number of components was easily confirmed by visual inspection. Cell capacitance was determined for all cells, and all currents are reported as current densities. Cell capacitance was determined by nulling all capacitative charge in the on-cell configuration, converting to the whole cell configuration, and then immediately determining the cell capacitance by providing the cell with a voltage ramp and measuring the resulting current. As a check, we integrated the amount of current passed during a small voltage step (10 mV). The cell capacitance determined using these two separate methods gave results within 5% of each other.

Statistics. All comparisons were made using robust statistics (15). The robust median and the 99% confidence interval about the robust median were determined for the parameters of interest. The confidence interval is based on the biweight locator and estimate of scale.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Adult slow mo zebrafish have a reduced heart rate. Of the nearly 150 published mutations affecting the heart in zebrafish, only slow mo homozygotes are viable past embryonic development. Slow mo fish grow at the same rate as wild-type siblings but do appear to have a slightly reduced viability. As shown in Fig. 1, slow mo mutant adults hearts are histologically indistinguishable from wild-type hearts. Overall morphology and size are not different between wild-type fish and slow mo mutants (data not shown).

View larger version (129K): [in this window] [in a new window]   Fig. 1.   Heart size and morphology are not altered in slow mo (smo) mutant adults. Histological cross sections through adult (9 mo old) fish at the level of the heart preserve orientation and shape and demonstrate that slow mo mutant hearts (B) are not grossly different from wild-type (WT) adult fish hearts (A). Bar, 250 µm.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Heart rate is reduced in adult slow mo zebrafish. Adult zebrafish were anesthetized with propofol, and the heart rate was counted under direct vision. Heart rate counts were binned at increments of 5 beats/min (bpm), and the resulting histogram for each genotype is shown; n = 81 WT fish (A) and 75 slow mo fish (B) for the generation of the histograms. The 99% confidence interval is shown by the dark gray bars in each histogram. The solid line at the center of the bar indicates the robust median. The slow mo population had a slower heart rate than the WT population.

In wild-type adult cardiomyocytes, ventricular I_h is smaller than atrial I_h. In other species, it has been noted that there are chamber-specific properties to I_h, with a difference in current density and voltage dependence between the atrium and ventricle that presumably would make the ventricle less likely to activate I_h, thereby favoring atrial pacemaking. Our prior work using embryonic zebrafish revealed a reduced I_h in embryonic slow mo cardiomyocytes. In our earlier work (3), it was not technically feasible to separately culture atrial and ventricular myocytes from the embryonic heart, so we did not distinguish whether chamber-specific differences were present in the embryo. During those prior studies, we did observe a range of current densities and activation voltages for I_h in the mixed population of cells from the embryonic heart, so it is possible that embryonic cells do have chamber-specific properties. It is important to note that the differences between slow mo and wild-type I_h are dramatic throughout the entire voltage range of activation.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Pacemaker current (Ih) is reduced in both the atrium and ventricle of adult slow mo zebrafish. A: representative Ih recorded from individual atrial and ventricular cells demonstrate two findings. First, slow mo Ih are reduced in size compared with WT Ih in both the atrium and ventricle. Second, ventricular currents are smaller than atrial currents for both WT and slow mo. Currents were elicited by hyperpolarizing the cells from a holding voltage of 40 mV to more negative voltages (130 to 50 mV in 10-mV increments). After the 8-s hyperpolarization, the voltage was stepped to 130 mV for 1 s and then returned to 40 mV. Currents were elicited every 15 s. The data are shown without any leak subtraction, and the calibration bars pertain to each current family. B: histograms and 99% robust confidence intervals for the current density of Ih show that the population of slow mo cells (b) from atria (n = 39) and ventricles (n = 36) have less current density than the population of WT cells (a) from atria (n = 36) and ventricles (n = 30). Current densities of Ih were determined at 130 mV as previously described (3). For atrial cells, current densities were binned every 5 pA/pF. The 99% robust confidence interval is shown by the dark gray bars in each histogram. The solid line at the center of the bar indicates the robust median. In atrial cells, slow mo current density of Ih was lower than that in WT. Note the excess number of slow mo atrial cells having little or no current density. For ventricular cells, current densities were binned every 1.5 pA/pF. In ventricular cells, the slow mo current density of Ih was lower than that in WT. Note the excess of slow mo ventricular cells having little or no current density. Note the difference in the y-axis between the atrial and ventricular histograms. Atrial current density was larger than ventricular current density for both genotypes.

I_h is reduced in adult slow mo atrial cardiomyocytes. As shown in Fig. 3A, I_h in adult slow mo atrial cells is smaller than in wild-type atrial cells. To quantitate the difference in I_h between wild-type and slow mo cells, we compared the current density between a group of wild-type cells (n = 36) and a group of slow mo cells (n = 39). The current density was significantly smaller in slow mo atrial cells (wild-type: robust median, 33.3 pA/pF; 99% robust confidence interval, 25.2-41.4 pA/pF; slow mo: robust median, 9.7 pA/pF; 99% robust confidence interval, 4.8-14.5 pA/pF). A histogram of our data along with the 99% confidence interval on the robust median is presented in Fig. 3B. The histograms demonstrate that the slow mo atrial cells have a lower current density of I_h and that many slow mo cells have no discernable I_h.

I_h is reduced in adult slow mo ventricular cardiomyocytes. Even though wild-type ventricular I_h is small compared with atrial I_h, we could still observe a diminution of ventricular I_h in the slow mo mutant (Fig. 3A). The current density of I_h was smaller in slow mo ventricular cells compared with wild-type cells (wild-type: n = 30; robust median, 7.6 pA/pF; 99% robust confidence interval, 4.9-10.2 pA/pF; slow mo: n = 36; robust median, 2.0 pA/pF; 99% robust confidence interval, 1.1-2.9 pA/pF). A histogram of these data along with the 99% confidence interval on the robust median is shown in Fig. 3B. The histograms demonstrate that slow mo ventricular cells have a lower current density of I_h and that many slow mo ventricular cells have no discernable I_h. Because of the small size of ventricular I_h, we were not able to confidently perform a kinetic analysis of I_h activation, but, as shown in Table 1, we found no significant difference between wild-type and slow mo I_h in terms of V_1/2 or V_s (Table 1). We also tested both wild-type and slow mo ventricular cardiomyocytes for their responsiveness to internal cAMP. We included 100 µM cAMP in the patch pipette and found that V_1/2 markedly shifted in the depolarized direction (wild-type: n = 8; robust median, 75.3 mV; 99% robust confidence interval, 88.1 to 62.5 mV; slow mo: n = 6; robust median, 80.5 mV; 95% robust confidence interval, 92.7 to 68.2 mV).

The slow mo mutation has minimal effects on I_Na in adult zebrafish. As a control for the specificity of the effects of the slow mo mutation on ion currents other than I_h, we examined the inward I_Na, found in nearly all of the cells of both slow mo and wild-type hearts. Typical recordings from these cardiomyocytes are shown in Fig. 4A. In our wild-type chamber-specific analysis, we found some distinctions between ventricular and atrial I_Na in zebrafish hearts. Atrial I_Na has a steady-state inactivation voltage that is more negative than ventricular I_Na, and there is a reduced voltage sensitivity to steady-state inactivation with atrial I_Na compared with ventricular I_Na (Table 1). However, these features were not different between wild-type and slow mo mutant cells. In addition, we found no significant differences in the current density at 30 mV of atrial I_Na between wild-type (n = 43) and slow mo cells (n = 36) or of ventricular I_Na between wild-type (n = 44) and slow mo cells (n = 46) (Table 1 and Fig. 4B).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Sodium current (INa) is not affected in either the atrium or ventricle by the slow mo mutation. A: representative INa recorded from individual atrial and ventricular cells demonstrate two findings. First, slow mo INa are the same size compared with WT in both the atrium and ventricle. Second, the ventricular currents have slower kinetics than atrial currents for both WT and slow mo. All currents were elicited by stepping the voltage to 30 mV after a 1-s prepulse to voltages ranging from 120 to 10 mV in 10-mV increments. The prepulse results in steady-state inactivation, which is then measured by a test pulse to 30 mV. Currents were elicited every 3 s. The data are shown without any leak subtraction, and the calibration bars pertain to each current family. B: histograms and 99% robust confidence intervals for current density of INa are shown for each chamber and genotype. a: WT cells; b: slow mo cells. INa was determined by plotting the peak inward current at 30 mV as a function of prepulse voltage and fitting the resulting curve with a Boltzmann function. The current density was then obtained by dividing the current amplitude at 30 mV (as determined from the fit) by the cell capacitance. Because the current amplitude was determined at a fixed voltage (30 mV), leak current and nonlinear current-voltage issues were avoided. For all cells, current densities were binned every 25 pA/pF. The 99% robust confidence interval is shown by the dark gray bars in each histogram. The solid line at the center of the bar indicates the robust median. In both atrial and ventricular cells, slow mo current density of INa was not statistically different than that in WT.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study provides genetic evidence that I_h is an essential component of cardiac pacemaking in the adult zebrafish. A defect in the slow mo gene causes a reduced heart rate in adult zebrafish and, at the cellular level, a reduction in the activity of I_h in both chambers of the heart.

The electrophysiological phenotype of slow mo differs slightly between the adult and embryonic heart. We (3) previously reported that the slow mo mutation affects embryonic heart rate, providing evidence that I_h is essential for pacing in the embryo. Our study of cardiomyocytes derived from embryonic hearts revealed that the slow mo mutation appears to perturb predominantly the fast component of I_h, leaving the slow component unaltered (3). In the adult zebrafish atria, however, where both the fast and slow components could be distinguished, slow mo reduces the amplitude of both components of I_h. The time constants for each component are unchanged, but the amplitude of the currents contributed by the fast as well as the slow components are reduced (Table 1). A comparison of the kinetics of embryonic wild-type I_h with adult atrial I_h shows a slowing of both fast and slow components in the adult compared with the embryo. An alteration in our recording solutions and the inclusion of tetraethylammonium in the bath solution for recording the adult I_h may have played at least some part in slowing adult I_h kinetics. A more likely contributing factor is the fact that the voltage range over which the adult cells activate is nearly 13 mV more negative than that in embryonic cells. This would tend to slow the adult kinetics for any given voltage step. A similar slowing has been noted during development of rat ventricular I_h (27). Despite the reduced amplitude of slow mo I_h, I_h still responds to internal cAMP by shifting the voltage at which it activates into a more depolarized region. In this regard, slow mo I_h behaves like the wild-type I_h in both the atrium and ventricle.

Genes encoding I_h channels have recently been cloned. These genes were termed hyperpolarization-activated cyclic nucleotide-gated (HCN) genes for the properties of the channels they encode (8, 13, 21, 28). HCN channels are members of the voltage-gated K⁺ channel family and possess a COOH-terminal cyclic nucleotide-binding domain. Expression studies of HCN family members have revealed that HCN isoforms have different activation kinetics, suggesting that the different kinetic components of I_h measured in cardiomyocytes reflect individual contributions from separate HCN isoforms (22). Perhaps the slow mo mutation affects gene products in a developmentally differential pattern and the embryonic I_h profile is differentially affected compared with the adult. A change of isoform profiles in development has been documented for I_h in the rat ventricle (29).

We found that atrial cardiomyocytes in the zebrafish heart have a greater current density of I_h than ventricular cardiomyocytes, as is true for other vertebrates. This has been teleologically ascribed to the beneficial effect of restricting pacemaking to pacemaking tissues, thereby avoiding ectopic pacemaking. The specific strategy for I_h distribution patterning appears to differ from species to species. Human, mouse, rabbit, and rat cardiomyocytes manifest a negative shift of the voltage dependence of activation of I_h in tissues where pacemaking is usually inappropriate (12, 27, 29). This is not true for zebrafish cardiomyocytes, in which the atrial and ventricular cells differ not in the activation voltage for I_h but rather in the current density over the entire activation range. This has also been reported in the rat, in which ventricular current density of I_h is reduced compared with atrial current density, without a shift in activation voltage (6).

How the slow mo gene normally affects I_h must await its cloning. Although it is formally possible that slow mo is an I_h gene, our preliminary linkage studies do not suggest this to be the case. Alternatively, it might be a modifier of the function of the channel. Most known modifiers of I_h increase or decrease I_h activity via a shift in the voltage dependence of activation. This is true, for example, of adrenergic and cholinergic agonists (11), calcium (23), substance P via the NK₁ receptor (17), and opioids (16, 33). Modifications of native I_h that alter the size of I_h without changing the activation voltage or kinetics, as is true of the slow mo mutation, include inhibition of phosphatase activity (1) and the activation of epidermal growth factor receptor tyrosine kinase (38). Perhaps the slow mo mutation affects the phosphorylation state of an HCN channel isoform or the phosphorylation state of a modifier of the I_h channel. The phosphorylation state of modifier proteins has recently been shown to affect channel activity of the skeletal ryanodine (RYR) channel [calsequestrin (34)] and the cardiac RYR2 receptor [FKBP12.6 (25)].