Defective "pacemaker" current (Ih) in a zebrafish mutant with a slow heart rate

Abstract

At a cellular level, cardiac pacemaking, which sets the rate and rhythm of the heartbeat, is produced by the slow membrane depolarization that occurs between action potentials. Several ionic currents could account for this pacemaker potential, but their relative prominence is controversial, and it is not known which ones actually play a pacemaking role in vivo. To correlate currents in individual heart cells with the rhythmic properties of the intact heart, we have examined slow mo (smo), a recessive mutation we discovered in the zebrafish Danio rerio. This mutation causes a reduced heart rate in the embryo, a property we can quantitate because the embryo is transparent. We developed methods for culture of cardiocytes from zebrafish embryos and found that, even in culture, cells from smo continue to beat relatively slowly. By patch-clamp analysis, we discovered that a large repertoire of cardiac currents noted in other species are present in these cultured cells, including sodium, T-type, and L-type calcium and several potassium currents, all of which appear normal in the mutant. The only abnormality appears to be in a hyperpolarization-activated inward current with the properties of Ih, a current described previously in the nervous system, pacemaker, and other cardiac tissue. smo cardiomyocytes have a reduction in Ih that appears to result from severe diminution of one kinetic component of the Ih current. This provides strong evidence that Ih is an important contributor to the pacemaking behavior of the intact heart.

Figure 1

The heart rate in smo homozygotes is slow throughout development and at different temperatures. Heart rates were counted upon visual inspection of unanesthetized embryos developing at room temperature (25°C), our standard aquaculture temperature (28.5°C), or at an elevated temperature (32°C). At least 40 animals were examined for each time point at each temperature. The heart rates of both the wild type siblings (A) and smo mutants (B) rose with developmental stage at all temperatures examined. Smo heart rates were consistently lower than wild type. Additionally, as shown in A, wild type hearts were very responsive to shifts in temperature. The mutant hearts (B), however, had an abnormal decrease in heart rate when raised at 32°C. hpf, hours postfertilization.

Figure 2

A hyperpolarization-activated I_h current is present in zebrafish cardiac myocytes. (A) Activation of I_h current by hyperpolarization. An individual dissociated cardiomyocyte was voltage-clamped from a holding potential of −40 mV to various hyperpolarizing voltages (−70 to −130 mV in 10-mV increments) once every 15 s. Each hyperpolarization was followed by a step to −130 mV to determine the current at a constant voltage. The current amplitude immediately after the voltage step to −130 mV was then plotted as a function of the activation voltage. The data were fitted with a Boltzmann function. The amplitude of the I_h current was given by the net amplitude of the Boltzmann function. The leak current was determined by the baseline of the fit and was not part of the I_h determination. Current traces were filtered at 1 kHz. (B) Tail current analysis of the I_h reversal potential. After I_h was activated for 1 s by a step from 0 mV to −130 mV, another step was applied to a new voltage (from −50 to +30 mV in 10-mV increments). The tail current amplitude immediately after the voltage clamp settled is plotted as a function of the tail current voltage. The tail currents reversed at −14 mV, as determined by the line fitted to the data points. A perfectly potassium-selective channel would have a reversal potential of −49 mV in the solutions used. The dihydropyridine niguldipine (1 μM) was included to block the calcium currents present in these cells. If the cell was held at 0 mV and not hyperpolarized to activate the I_h current, no tail currents were observed (data not shown). Current traces were filtered at 2 kHz. The voltage protocol was delivered once every 4 s. (C) Inhibition of I_h by cesium. I_h currents with and without 2 mM CsCl applied extracellularly, in response to a step from −40 mV to −130 mV. Current traces were filtered at 1 kHz.

Figure 3

I_h is reduced in smo cardiac myocytes. (A) Representative I_h, I_Kr, and I_Ca,T currents from wild-type and smo cells. I_h was elicited from a holding voltage of −50 mV by steps to various voltages (−65 to −130 mV in 5-mV increments). A test voltage of −130 mV was applied at the end of each hyperpolarizing step. I_Kr was activated by a ≈1-s prepulse to various voltages (+60 to −70 mV in 10-mV increments); the inwardly rectifying current was then measured at a constant test voltage of −120 mV. I_Ca,T was elicited from a holding potential of −100 mV to voltages ranging from −90 to +50 mV once every 2 s. Current traces were filtered at 1 kHz (I_h) or 5 kHz (I_Kr and I_Ca,T). (B) Amplitude of I_h, I_Kr, and I_{Ca, T} currents from many wild-type and smo cells. The data are displayed as box plots. The box delimits the central 50% of the data values. The median is shown as a solid line tagged with a solid box, and the mean is shown as a solid circle. The 99% confidence interval for a robust median is shown by the gray shaded zone. The confidence interval is based on the biweight locator and estimate of scale (25). Boxplots of the I_h current amplitudes are shown for wild-type (n = 47) and smo (n = 48) cells. Boxplots of the I_Kr current amplitudes also are shown for wild-type (n = 31) and smo (n = 23) cells and for I_Ca,T, wild-type (n = 20), and smo (n = 20) cells. The size of whole cell I_h currents in smo cells was very small (average 12.0 pA), making it difficult to fit some of the data to a Boltzmann function. The amplitude comparison between wild-type and smo cells was thus repeated but with recording conditions that were chosen to enhance the amplitude of the I_h currents. The bath solution was as described in Materials and Methods except that KCl was increased to 140 mM, 2 mM BaCl₂ was added, and NaCl was omitted. The pipette solution was supplemented with 100 μM cAMP. The I_h current amplitude was indeed enhanced 2.5-fold by the changes in recording solutions, and the effects of the smo mutation on the I_h current were the same. For the I_h current, the confidence interval was 110–260 pA in wild-type cells (n = 19) compared with 5–44 pA in smo cells (n = 20). I_Kr current was again unchanged by the mutation: 99% confidence interval was 67–358 pA for wild-type (n = 20) and 109–261 pA for smo cells (n = 19). The reduction in I_h current in smo cells was not due to a difference in cell size between wild-type and smo because the cell capacitances were the same for the two genotypes; the 99% confidence interval on the robust median for wild-type (n = 34) was 2.3–3.2 pF and for smo (n = 28) was 2.4–3.1 pF. Data were obtained for both I_h and I_Kr from each cell whenever possible. The amplitude of the I_h current was determined as shown in Fig. 2A from the amplitude of the Boltzmann fit, and the amplitude of the I_Kr current was determined in an analogous fashion. The current amplitude for I_Ca,T was determined at −20 mV after leak subtraction. The leak current and nonlinear open channel properties were minimized for both the I_h and I_Kr records by taking all of the current amplitudes at a constant voltage. For each value in the legend, we report a range of numbers giving the 99% confidence interval for the robust median. WT, wild-type.

Figure 4

The reduced I_h current in smo cells is due to the reduction of a fast component normally seen in wild type. (A) Representative I_h currents from a wild type and a smo cell in response to a step from −40 to −130 mV. I_h currents were recorded using the solutions noted in the Fig. 3 legend. The wild type current trace was fit with a sum of two exponentials; the individual exponential components are shown in the dashed lines, and their sum lies on top of the current trace. The smo current was well described by a single exponential. (B) Amplitude and kinetic analysis of wild type and smo I_h currents. Wild type I_h currents (from 68 cells) were fitted with two exponentials (Fast and Slow); the amplitude and time constant for each component are displayed separately. smo I_h currents (from 65 cells) were fitted with either one or two exponentials (Fast and Slow); the amplitude and time constant for each component are displayed separately. The amplitude of the fast component is markedly reduced in smo cells. The kinetics of both the fast and slow components are similar. The shaded area in each box shows the 99% confidence interval about the robust median. WT, wild type.