Altered electrophysiological properties in transgenic cardiomyocytes

Action potentials, stimulated at a frequency of 1 Hz, recorded from cells isolated from the hearts of WT mice displayed a very rapid repolarization typical of cells with a prominent transient outward current (Powell et al. 1980; Fiset et al. 1997), whereas those recorded from cells isolated from the hearts of NGF mice showed a much slower repolarization and, consequently, a longer action potential duration. Typical action potentials are shown in Fig. 1, and the mean data illustrate that the action potential duration at 50 and 90 % repolarization (APD₅₀ and APD₉₀) was significantly prolonged in NGF compared with WT cells. In WT cells the mean APD₅₀ was 4·69 ± 0·49 ms (n = 9) compared with 9·42 ± 1·55 ms (P < 0·01; n = 8) in NGF cells, a prolongation of 101 %, and the APD₉₀ in WT cells was 20·77 ± 5·11 ms (n = 9) compared with 50·34 ± 10·54 ms in NGF cells (P < 0·05; n = 8), a prolongation of 142 %. The resting membrane potentials of WT and NGF cells were not significantly different (WT, -67·4 ± 3 mV, n = 9; NGF, -75·2 ± 3 mV, n = 8). The prolonged action potential in the NGF cardiomyoctyes might result from an alteration in the properties of a variety of ionic currents including outward K⁺ currents and inward Ca²⁺ currents, and these possibilities were investigated.

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Figure 1. Comparison of action potentials and mean action potential duration data from WT and NGF myocytes Action potentials (1 Hz stimulation) recorded from WT (A) and NGF (B) myocytes. Arrows indicate 0 mV. C, mean APD50 and APD90 data in WT (, n = 9) and NGF (, n = 8) myocytes. * P < 0·05, ** P < 0·01.

A prominent transient outward K⁺ current (I_to) was the dominant outward current recorded in both WT and NGF myocytes, as has been reported previously in mice (Benndorf & Nilius, 1988). In other species, such as dog, the transient outward K⁺ currents consist of two components: a Ca²⁺-independent 4-AP-sensitive current and a Ca²⁺-dependent current. We focused on the former, which has previously been shown to be decreased in a variety of models of heart failure (Benitah et al. 1993; Beuckelmann et al. 1993; Cerbai et al. 1994; Tomita et al. 1994; Potreau et al. 1995; Kääb et al. 1996), and suppressed the Ca²⁺-dependent current by the inclusion of 11 mM EGTA in the patch pipette. Experiments described below demonstrate that all the I_to recorded under these conditions was sensitive to block by 4-AP, consistent with the presence of only the Ca²⁺-independent I_to.

Typical records of I_to recorded from a WT and a NGF myocyte are shown in Fig. 2A and B, respectively, and demonstrate the rapid inactivation of I_to superimposed on a steady-state current. I_to recorded from NGF cells was markedly reduced in amplitude compared with I_to in WT cells. The current was measured as the difference between the peak and end-pulse current and normalized to cell capacitance. Figure 2C shows the current-voltage relation of I_to in WT and NGF cells and clearly shows the reduction in the current density in NGF myocytes compared with WT myocytes: the amplitude of I_to activated by a step depolarization to +60 mV was 20·3 ± 4·23 pA pF^-1 in WT cells (n = 28) compared with 9·76 ± 1·02 pA pF^-1 in NGF cells (n = 23; P < 0·05). The steady-state current, estimated by measuring the current at the end of the pulse relative to zero current and normalized to cell capacitance, was smaller in NGF cells, but this difference was not statistically significant (WT, 16·6 ± 3·42 pA pF^-1, n = 28; NGF, 10·2 ± 0·72 pA pF^-1, n = 23; P > 0·05).

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Figure 2. A comparison of the properties of Ito in WT and NGF myocytes Typical Ito recorded from WT (A) and NGF (B) myocytes from a holding potential of -60 mV, activated by 500 ms step depolarizations to potentials between -40 and +60 mV. C, mean transient outward current density in WT () and NGF myocytes (). * P < 0·05. D, voltage-dependent activation (WT, , n = 28; NGF, , n = 23) and steady-state inactivation (WT, , n = 9; NGF, , n = 14) of Ito. Dotted and continuous lines are fits to the NGF and WT data, respectively.

The reduction in the amplitude of I_to in NGF cells might reflect a change in the voltage range over which I_to activates or inactivates. Therefore, the voltage dependence of activation and the steady-state inactivation of I_to were investigated. Examination of the activation of I_to at different voltages fitted with a Boltzmann function (Fig. 2D) revealed no difference between the voltage dependence of activation of I_to in WT and NGF myocytes. The V_½ was 18·0 ± 1·59 mV in WT cells (n = 28) and 20·1 ± 1·59 mV in NGF cells (n = 23), and the slope was 16·7 ± 0·4 mV in WT cells and 15·8 ± 0·81 mV in NGF cells (Fig. 2D). Similarly, the steady-state inactivation of I_to in WT and NGF cells fitted with a Boltzmann function was not significantly different; the V_½ of inactivation for WT cells was -27·9 ± 2·7 mV (n = 9) compared with -25·6 ± 2 mV (n = 14) for NGF cells, and the slope was 8·3 ± 0·6 mV for WT cells and 9·6 ± 0·3 mV in NGF cells (Fig. 2D).

The rate of inactivation of I_to was quantified by fitting curves to the current inactivating during a step depolarization to +60 mV of 1 s duration. The inactivation of I_to recorded from all WT myocytes (n = 16) could consistently be fitted by two exponential functions with a fast and a slow time constant. In twenty out of twenty-four cells, the inactivation of I_to from NGF myocytes was also best fitted by fast and slow time constants. Figure 3A and B shows two typical records of I_to from a WT and a NGF myocyte activated by a 500 ms step depolarization to +60 mV and fitted with an exponential function (dotted line). Mean data for those cells fitted with a double-exponential function showed no significant difference between WT and NGF fast and slow time constants (Fig. 3C). The amplitude of the fast time constant (amplitude 1) was significantly smaller in NGF cells compared with WT (Fig. 3D). In WT cells, amplitude 1 was 8·6 ± 1·8 pA pF^-1 (n = 16) and in NGF mice, amplitude 1 was 4·5 ± 0·6 pA pF (n = 20, P < 0·05). There was no difference in the amplitude of the slow time constant (amplitude 2) in NGF and WT cells (WT cells, 7·2 ± 0·9; NGF cells, 6·0 ± 0·8; P > 0·05). In four of the NGF cells, I_to was fitted by only a single exponential with a relatively slow time constant of 115·2 ± 9 ms and an amplitude of 11·9 ± 3 pA pF^-1 (n = 4; data not shown).

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Figure 3. Analysis of the kinetics of inactivation of Ito in WT and NGF myocytes A and B, Ito activated by a 500 ms step depolarization to +60 mV from -60 mV holding potential and fitted with two exponential time constants of decay (dotted line) in WT (A) and NGF (B) myocytes. C and D, mean fast (1) and slow (2) time constant and amplitude data from the decay of Ito in WT (, n = 16) and NGF (, n = 20) myocytes. * P < 0·05.

The difference in the cardiac I_to in WT and NGF heart described above might result from a change in the expression of the type of channel or channel combination that forms I_to in the mice overexpressing NGF. A great many K⁺ channels have been cloned from the heart that might contribute to the total cardiac I_to, and they have different sensitivities to block by agents such as TEA and 4-AP. Therefore, the pharmacology of the adult mouse I_to was compared in WT and NGF myocytes. Figure 4 shows records of I_to recorded from a WT and a NGF myocyte before (control) and after exposure to 5 mM TEA. In both the WT (upper panel) and NGF (lower panel) myocytes, TEA had no effect on I_to, and this result was observed in an additional four WT and four NGF myocytes. Figure 5 shows the effect of 5 mM 4-AP on I_to recorded from WT and NGF myocytes, and in this case the currents from both cells were completely blocked. Therefore, there was no difference between the sensitivity of I_to in WT and NGF cells to TEA and 4-AP. 4-AP will also block another K⁺ current, the sustained or ultra-rapid outward K⁺ current I_K(ur) (Wang et al. 1993). To study this current in more detail, we used a lower concentration of 4-AP (50 µM), which selectively blocks I_K(ur) (Wang et al. 1993). We found that the current sensitive to 50 µM 4-AP in WT and NGF myocytes was significantly smaller in amplitude in NGF myocytes than in WT myocytes following a depolarizing step to +60 mV (WT current, 5·62 ± 0·79 pA pF^-1, n = 11; NGF current, 2·75 ± 0·75 pA pF^-1, n = 6; P < 0·05; Fig. 6A). Typical currents recorded from a WT and a NGF cell are shown in Fig. 6B for control (a), after exposure to 50 µM 4-AP (b) and the 4-AP-sensitive current, I_K(ur) (c).

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Figure 4. Comparison of the effect of 5 mM TEA on Ito in WT and NGF myocytes Typical records of Ito activated by 500 ms step depolarizations from -60 to +60 mV from WT (upper traces) and NGF (lower traces) myocytes before (Control) and after exposure to TEA. Arrows indicate zero current level.

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Figure 5. Comparison of the effect of 5 mM 4-AP on Ito in WT and NGF myocytes Typical records of Ito activated by 500 ms step depolarizations from -60 to +60 mV from WT (upper traces) and NGF (lower traces) myocytes, before (Control) and after exposure to 4-AP. Arrows indicate zero current level.

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Figure 6. Comparison of IK(ur) (the current sensitive to 50 µM 4-AP) in WT and NGF myocytes A, mean current sensitive to 50 µM 4-AP in WT (, n = 11) and NGF (, n = 6) myocytes. * P < 0·05. B, typical records of Ito from WT and NGF myocytes for control (a), in the presence of 4-AP (b), and the 4-AP-sensitive current obtained by subtraction (c). Currents were activated by 500 ms step depolarizations from a holding potential of -60 mV.

The prolongation of APD observed in the NGF myocytes might also be due to a reduction in inward Ca²⁺ channel current, I_Ca. Therefore, the amplitude of the I_Ca in WT and NGF myocytes was compared and the current-voltage relationship determined (Fig. 7). The amplitude of peak I_Ca was not different in WT and NGF cells; in WT cells, peak I_Ca was 5·91 ± 0·8 pA pF^-1 (n = 17), and in NGF myocytes, 5·96 ± 1 pA pF^-1 (n = 21). There was also no significant difference in the characteristics of the current-voltage relationship of I_Ca in WT and NGF myocytes.

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Figure 7. Comparison of the voltage-dependent activation of ICa in WT and NGF myocytes Mean voltage-dependent activation of the L-type Ca2+ channel current in WT and NGF myocytes. Currents were activated by 40 ms step depolarizations from a holding potential of -40 mV.

To test whether the observed reductions in I_to and I_K(ur) in the NGF myocytes could account for the prolongation of APD, the change in APD in a WT cell was measured while I_to was partially blocked, such that it was of a similar amplitude as I_to recorded in the NGF cells. This was achieved by applying 1 mM 4-AP, which at this concentration also blocked I_K(ur). As shown in Fig. 8A, the action potential recorded from a WT cell in normal Tyrode solution was more than doubled in duration after exposure to 1 mM 4-AP. In this cell, APD₉₀ was increased by 157 %, a similar increase to that observed in cells isolated from the hearts of NGF mice. The shape of the action potential in the presence of 1 mM 4-AP was similar, although not identical, to the action potentials recorded from cells from the hearts of the NGF mice (see Fig. 1). In this experiment the 4-AP was washed out, allowing the action potential to return to almost the same duration as in control, and the cell was voltage clamped to a -60 mV holding potential. I_to currents were then recorded and 4-AP was applied again. 4-AP (1 mM) reduced I_to current at +60 mV by 61 % (Fig. 8B), comparable to the difference in the amplitude of I_to in the WT and NGF cells (see Fig. 2C).

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Figure 8. Effect of 1 mM 4-AP on APD and Ito in a WT myocyte A, action potentials recorded from a single WT myocyte in control, in the presence of 1 mM 4-AP and after washout of 4-AP. Action potentials were stimulated at 1 Hz. B, Ito recorded from the same cell after washout of 4-AP (a) and after re-exposure to 1 mM 4-AP (b).

Changes in -adrenergic responses and -AR density

Since the overexpression of NGF increases the sympathetic innervation of the heart and raises plasma catecholamine levels, it was of interest to study the response of the -adrenergic signalling cascade. Therefore, since I_Ca is known to be regulated by this system, we studied the effect of -AR stimulation by isoprenaline on peak I_Ca. Trains of pulses were applied to the potential at which peak I_Ca was activated in the absence and presence of 1 µM isoprenaline. We found that the response to isoprenaline was smaller in cells isolated from the NGF mice; after isoprenaline, I_Ca was increased in WT cells by 75·1 ± 11·5 % (n = 8) while in NGF cells, the increase with isoprenaline was significantly less at 44·4 ± 7·7 % (n = 8, P < 0·05; Fig. 9).

To investigate the basis for differences in -AR agonist responses between NGF and WT cardiomyocytes, the density of -ARs was measured. With ICYP, a selective ligand for myocardial -ARs, binding was monophasic and revealed that total -AR density (B_max) was increased by 55 % in NGF tissue compared with WT, whilst the dissociation constants for total -ARs did not differ between the WT and NGF mice (Table 2). Further analysis of -AR subtypes was carried out using the highly selective ₁-AR antagonist CGP 20712A and showed that the increase in -AR density detected in NGF tissue was due entirely to an increase in ₂-ARs in NGF tissue compared with WT tissue, with no change in the level of ₁-ARs. The dissociation constants for the -AR subtypes did not differ between the WT and NGF mice. These data are summarized in Table 2.

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Figure 9. Effect of isoprenaline on peak ICa in WT and NGF myocytes Trains of pulses (40 ms duration) to activate peak ICa were applied at 5 s intervals from a holding potential of -40 mV and peak current was measured. Cells from WT (, n = 8) and NGF (, n = 8) hearts were exposed to 1 µM isoprenaline and the percentage increase in peak current is shown. * P < 0·05.

Table 2. Myocardial -AR density and subtypes in wild-type and NGF transgenic mice

	-AR density			Dissociation constants
	Total (fmol (mg protein)-1)	1-AR subtype (fmol (mg protein)-1)	2-AR subtype (fmol (mg protein)-1)	KD (pM)	KD1 (nM)	KD2 (µM)
Wild-type	35 ± 5	23 ± 3	12 ± 2	51 ± 9	10 ± 3	3·5 ± 1·2
NGF transgenic	55 ± 5 *	27 ± 3	28 ± 3 **	59 ± 12	8 ± 3	3·8 ± 1·2

S.E.M., n = 9-11 in each group. * P < 0·05, ** P < 0·001 compared with wild-type by Student's t test. K_D, dissociation constant for total -AR population; K_D1 and K_D2, dissociation constants for ₁- and ₂-ARs, respectively.

Table 3. Basal and stimulated adenylyl cyclase activity in wild-type and NGF transgenic mice assayed by measuring cAMP levels

		Stimulated activity
	Basal activity (pmol (mg protein)-1 min-1)	Isoprenaline (pmol (mg protein)-1 min-1)	Gpp(NH)p (pmol (mg protein)-1 min-1)	Forskolin (pmol (mg protein)-1 min-1)	n
Wild-type	2·1 ± 0·2	4·6 ± 0·8	13·2 ± 0·9	71·9 ± 5·3	11
NGF transgenic	2·7 ± 0·3	1·6 ± 0·2 *	14·3 ± 1·5	61·2 ± 5·5	11

Values are means ± S.E.M. * P < 0·01 by Student's t test. Basal adenylyl cyclase activity was obtained in the presence of GTP. Stimulated adenylyl cylcase levels were the net increases above the basal levels under each assay condition.

Since the response of the Ca²⁺ channel current to isoprenaline, a non-selective -AR agonist, was depressed in NGF cells, but the density of ₂-ARs was increased, it was thought possible that the chronic increase in noradrenaline levels in the NGF myocardium might have uncoupled intracellular signalling pathways. To address this issue, the responses of the -AR-coupled adenylyl cyclase system to isoprenaline, Gpp(NH)p and forskolin were studied by measuring cAMP production. In NGF tissue, isoprenaline elicited a significantly smaller response than in tissue from WT mice (Table 3). This diminished response was not due to reduced adenylyl cyclase activity as the responses of NGF tissue to Gpp(NH)p and forskolin were not signicantly different in NGF tissue compared with WT (Table 3).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Overexpression of NGF in the heart results in chronic heart failure characterized by cardiac hypertrophy secondary to an elevation of catecholamines caused by sympathetic hyperinnervation of the heart (Hassankhani et al. 1995). The elevated catecholamine levels are thought to be confined exclusively to the heart, and therefore this transgenic mouse provides a novel model in which to study the pathological influence of the increased sympathetic activity on the heart. Cardiac hypertrophy, occurring in many common cardiovascular diseases or induced in experimental models by chronic pressure or volume overload, is generally characterized by prolonged APD (see, for example, Aronson, 1980; Scamps et al. 1985; Kleiman & Houser, 1988; Nordin et al. 1989; Bouron et al. 1992; Ryder et al. 1993; Ten Eick & Basset, 1997). Accordingly, in this transgenic mouse with cardiac hypertrophy, the action potential was more than doubled in duration compared with WT mice, with no change in the resting membrane potential. Alterations in the duration of the action potential may predispose the heart cells to dispersion of repolarization, leading to re-entrant arrhythmias (Di Diego & Antzelevitch, 1994). In particular, prolonged action potentials favour the development of after-depolarizations, which in turn can induce triggered arrhythmias, and therefore these changes are potentially pathological.

The significant decrease in the current density of I_to and I_K(ur) in cells isolated from the NGF mouse observed in this study is likely to have contributed to the prolonged APD. A great many K⁺ channels have been described in the heart (Barry & Nerbonne, 1996) and I_to and I_K(ur) are two K⁺ currents which play a particularly important role during the action potential. They contribute to shaping the early phase of the cardiac action potential and the refractoriness of myocardial tissue (Wang et al. 1993; Campbell et al. 1997). In the mouse heart, block of the K⁺ current sensitive to 50 µM 4-AP (namely I_K(ur)) caused significant changes in APD and, as a result, in force of contraction (Fiset et al. 1997). A reduction in I_to has been reported before in a variety of models of cardiac failure including pressure overload-induced cardiac hypertrophy (Benitah et al. 1993; Tomita et al. 1994; Potreau et al. 1995), hypertrophy secondary to hypertension (Cerbai et al. 1994), and pacing-induced heart failure (Kääb et al. 1996), and in cells isolated from patients with terminal heart failure (Beuckelmann et al. 1993), and is thought to contribute to long-QT and possibly to the generation of arrhythmias in heart failure.

The molecular nature of I_to and I_K(ur) in the mouse is not completely clear, but the Kv1.5 channel is thought to underlie I_K(ur) (Fiset et al. 1997) and has been cloned from mouse (Attali et al. 1993). Channels from the Kv4 family are thought most likely to underlie cardiac I_to in canine and rat, and both Kv4.2 and Kv4.3 mRNAs have been found in the mouse ventricle and atrium (Fiset et al. 1998). These channels share a similar sensitivity to 4-AP and insensitivity to TEA as I_to observed in the mouse heart (Dixon et al. 1996; Yeola & Snyders, 1997). Therefore it seems likely that genes from the Kv4 family also code for I_to in the mouse heart.

The mechanism by which the current density of I_to is reduced in the failing heart is not known. One hypothesis is that the current density decreases in hypertrophy because of an increase in the size of the cell membrane which is not accompanied by a corresponding increase in the expression of the K⁺ channels. However, the increase in capacitance in the NGF cells was only 12 %, whereas the density of I_to and I_K(ur) in NGF myocytes was only 40-50 % of those currents measured in WT cells. Given these data, it seems more likely that there was a downregulation in the number of channels or of a channel subunit expressed during the development of heart failure in the NGF mouse. It is possible that gene expression may be altered during the development and progression of heart failure. Indeed, the expression of K⁺ channel genes has previously been shown to change during heart failure: a downregulation of Kv4.2 and Kv4.3 was reported in the hearts of rats with renovascular hypertension which caused an increase in afterload on the heart (Takimoto et al. 1997), and in a rat model of heart failure in which cardiac hypertrophy was induced by myocardial infarction, the levels of Kv2.1 and Kv4.2 mRNA and gene product were reduced whereas no reduction was seen in the mRNA or protein levels of Kv1.5, the channel that has been shown to underlie I_K(ur) (Feng et al. 1997; Fiset et al. 1998). However, in two other studies, one on hypertrophied rat ventricle and the second on human atrial cells from patients with chronic atrial fibrillation, significant reductions in Kv1.5 mRNA levels were found (Matsubara et al. 1993; Van Wagoner et al. 1997). Therefore, it seems likely that K⁺ channel expression can be altered in hypertrophy despite some variations between the different models studied.

While these studies indicate that K⁺ channel proteins are downregulated in hypertrophy, it is also possible that in the NGF mouse heart there was a change in the assembly of channel subunits or regulatory subunits, such as -subunits, which modify channel properties. However, since we did not see any change in the electrophysiological or pharmacological properties of the K⁺ currents which were reduced in density in the NGF mouse heart, there is little evidence to suggest changes in such proteins occurred in this model.

Another possible explanation for the observed decrease in I_to in the NGF mouse heart is an increase in ₁-adrenergic stimulation as a result of the elevated catecholamine levels in the hearts of the NGF mice. Unlike -ARs (see later), it is thought that the absolute number of ₁-adrenergic receptors remains the same in heart failure (Bristow et al. 1988). ₁-Adrenergic agonist-induced inhibition of I_to has been observed in rabbit atrial (Fedida et al. 1990) and rat ventricular cells (for example, see Ertl et al. 1991) through activation of protein kinase C, and although some of these reductions in I_to are relatively small they may at least contribute to the observed reduction in I_to in NGF cells.

The prolongation of APD in the NGF mice was probably not due to an increase in Ca²⁺ current. No difference in the Ca²⁺ current density in WT and NGF myocytes was observed. Conflicting results have previously been reported concerning changes in Ca²⁺ current density and characteristics in various models of hypertrophy. The current density was reported to be unchanged (Scamps et al. 1985; Kleiman & Houser, 1988; Cerbai et al. 1994), increased (Keung, 1989; Ryder et al. 1993) and decreased (Nuss & Houser, 1991; Rossner, 1991; Bouron et al. 1992) in different models of heart failure. It seems likely that variations between species and the degree of heart failure might influence the changes in the Ca²⁺ current in these different models (see Lee et al. 1997).

The effect of 1 mM 4-AP on the action potential duration and the outward K⁺ currents provides further support for the hypothesis that the reduction in I_to and I_K(ur) in cells from NGF hearts was sufficient to account for the action potential prolongation in the NGF mice. This concentration of 4-AP caused a 61 % reduction in the I_to current and reduced the end-pulse current (reflecting block of I_K(ur)) by more than half, similar to the amplitude of I_to and I_K(ur) observed in myocytes isolated from the NGF mice. Any differences between the shape of the action potentials recorded in the presence of 1 mM 4-AP and of those recorded from cells isolated from the hearts of NGF mice may reflect slight differences in the amount of each type of K⁺ current either blocked by 4-AP or reduced following overexpression of NGF. However, in the presence of 4-AP, the action potential was more than doubled in duration. This was an increase similar to that recorded from the NGF mouse cells and therefore consistent with the prolonged action potential in cells from NGF mice resulting from a decrease in these currents.

Both decreases (Bristow et al. 1988) and increases (Vatner et al. 1985) in -AR density in heart failure have been reported previously, and in this study we found an increase in -AR density in NGF transgenic mouse cardiac tissue. Further analysis of -AR subtypes revealed the novel finding that only ₂-ARs were increased in density. However, despite this increase, the response of I_Ca to isoprenaline was reduced in NGF myoyctes. This was not due to a decrease in receptor affinity as the dissociation constants were not changed. Instead, we found that isoprenaline-stimulated adenylyl cyclase activity was depressed in NGF compared with WT cardiac tissue, although there was no change in the response of adenylyl cyclase to Gpp(NH)p or forskolin, findings which are consistent with an uncoupling of the -ARs from the adenylyl cyclase signalling pathway and which explain the diminished response of I_Ca to -AR stimulation.

It is possible that the increase in ₂-AR density occurred as a compensatory mechanism in response to the uncoupling of the receptors, and these changes occurred as a result of the exposure of the heart to the excess levels of catecholamines in the NGF mice. Thus the chronic catecholamine excess influences both receptor expression in the heart and the coupling to the G-protein-linked signal transduction pathways, pathophysiological processes observed previously in the failing human heart (Bristow et al. 1982).

In conclusion, the overexpression of NGF in the mouse heart results in a chronic catecholamine excess and heart failure characterized by a prolongation of the cardiac action potential. This change is due, at least in part, to a reduction in the density of two outward K⁺ currents, I_to and I_K(ur), which are important in cardiac repolarization. In addition, there was a reduction in -AR function; despite an increase in the density of ₂-ARs in the NGF myocardium, the functional coupling of the receptors to the adenylyl cyclase signalling pathway was reduced such that the response of the I_Ca to isoprenaline was markedly diminished. These potentially pathological changes may be involved in the occurrence of ventricular arrhythmias in hypertrophied hearts, and this mouse provides a novel system in which to study such changes.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

Aronson, R. S. (1980). Characteristics of action potentials of hypertrophied myocardium from rats with renal hypertension. Circulation Research 47, 443-454	[Medline]
Attali, B., Lesage, F., Ziliani, P., Guillemare, E., Honore, E. X., Waldmann, R., Hugnot, J. P., Mattei, M. G., Lazdunski, M. & Barhanin, J. (1993). Multiple mRNA isoforms encoding the mouse cardiac Kv1.5 delayed rectifier K+ channel. Journal of Biological Chemistry 268, 24283-24289	[Abstract]
Barry, D. M. & Nerbonne, J. M. (1996). Myocardial potassium channels: electrophysiological and molecular diversity. Annual Review of Physiology 58, 363-394	[Abstract]
Benitah, J. P., Gomez, A. M., Bailly, P., Da Ponte, J. P., Berson, G. X., Delgado, C. & Lorente, P. (1993). Heterogeneity of the early outward current in ventricular cells isolated from normal and hypertrophied rat hearts. The Journal of Physiology 469, 111-138	[Abstract]
Benndorf, K. & Nilius, B. (1988). Properties of an early outward current in single cells of the mouse ventricle. General Physiology and Biophysics 7, 449-466	[Medline]
Beuckelmann, D. J., Nabauer, M. & Erdmann, E. (1993). Alterations of K+ currents in isolated human ventricular myocytes from patients with terminal heart failure. Circulation Research 73, 379-385	[Abstract]
Bouron, A., Potreau, D. & Raymond, G. (1992). The L-type calcium current in single hypertrophied cardiomyocytes isolated from the right ventricle of ferret heart. Cardiovascular Research 26, 662-670	[Medline]
Bristow, M. R., Ginsburg, R., Minobe, W., Cubicciotti, R. S. X., Sageman, W. S., Lurie, K., Billingham, M. E., Harrison, D. C. & Stinson, E. B. (1982). Decreased catecholamine sensitivity and beta-adrenergic-receptor density in failing human hearts. New England Journal of Medicine 307, 205-211	[Abstract]
Bristow, M. R., Minobe, W., Rasmussen, R., Hershberger, R. E. X. & Hoffman, B. B. (1988). Alpha-1 adrenergic receptors in the nonfailing and failing human heart. Journal of Pharmacology and Experimental Therapeutics 247, 1039-1045	[Abstract]
Campbell, D., Rasmusson, R., Corner, M. & Strauss, H. (1997). The cardiac calcium-independent outward potassium current: kinetics, molecular properties and role in ventricular repolarization. In Cardiac Electrophysiology: From Cell to Bedside, 2nd edn, ed. Zipes, D. & Jaliffe, J., pp. 83-96. W. B. Saunders and Co., Philadelphia.
Cerbai, E., Barbieri, M., Li, Q. & Mugelli, A. (1994). Ionic basis of action potential prolongation of hypertrophied cardiac myocytes isolated from hypertensive rats of different ages. Cardiovascular Research 28, 1180-1187	[Medline]
Di Diego, J. M. & Antzelevitch, C. (1994). High [Ca2+]o-induced electrical heterogeneity and extra-systolic activity in isolated canine ventricular epicardium. Circulation 89, 1839-1850	[Abstract]
Dixon, J. E., Shi, W., Wang, H.-S., McDonald, C., Yu, H., Wymore, R. S., Cohen, I. S. & McKinnon, D. (1996). Role of the Kv4.3 K+ channel in ventricular muscle. A molecular correlate for the transient outward current. Circulation Research 79, 659-668	[Abstract/Full Text]
Dooley, D. J., Bittinger, H. & Reymann, N. C. (1986). CGP 20712A: A useful tool for quantitating 1- and 2- adrenoceptors. European Journal of Pharmacology 130, 137-139	[Medline]
Ertl, R., Jahnel, U., Nawrath, H., Carmeliet, E. & Vereecke, J. (1991). Differential electrophysiologic and inotropic effects of phenylephrine in atrial and ventricular heart muscle preparations from rats. Naunyn-Schmiedeberg's Archives of Pharmacology 344, 574-581.	[Medline]
Fan, T. H., Liang, C. S., Kawashima, S. & Banerjee, S. P. (1987). Alterations in cardiac beta-adrenoceptor responsiveness and adenylate cyclase system by congestive heart failure in dogs. European Journal of Pharmacology 140, 123-132	[Medline]
Fedida, D., Shimoni, Y. & Giles, W. R. (1990). Alpha-adrenergic modulation of the transient outward current in rabbit atrial myocytes. The Journal of Physiology 423, 257-277	[Abstract]
Feng, J., Wible, B., Li, G. R., Wang, Z. & Nattel, S. (1997). Antisense oligodeoxynucleotides directed against Kv1.5 mRNA specifically inhibit ultrarapid delayed rectifier K+ current in cultured adult human atrial myocytes. Circulation Research 80, 572-579	[Abstract/Full Text]
Fiset, C., Clark, R. B., Janzen, K. M., Winkfein, R. & Giles, W. R. (1998). Molecular identity of K+ currents in adult mouse atrium and ventricle. Biophysical Journal 74, A207.
Fiset, C., Clark, R. B., Larsen, T. S. & Giles, W. R. (1997). A rapidly activating sustained K+ current modulates repolarization and excitation-contraction coupling in adult mouse ventricle. The Journal of Physiology 504, 557-563	[Abstract]
Francis, G. S., Cohn, J. N., Johnson, G., Rector, T. S., Goldman, S. & Simon, A. (1993). Plasma norepinephrine, plasma renin activity, and congestive heart failure. Relations to survival and the effects of therapy in V-HeFT II. The V-HeFT VA cooperative studies group. Circulation 87, VI 40-48	[Medline]
Hart, G. (1994). Cellular electrophysiology in cardiac hypertrophy and failure. Cardiovascular Research 28, 933-946	[Medline]
Hassankhani, A., Steinhelper, M. E., Soonpaa, M. H., Katz, E. B. X., Taylor, D. A., Andrade-Rozental, A., Factor, S. M., Steinberg, J. J., Field, L. J. X. & Federoff, H. J. (1995). Overexpression of NGF within the heart of transgenic mice causes hyperinnervation, cardiac enlargement, and hyperplasia of ectopic cells. Developmental Biology 169, 309-321.	[Medline]
Kääb, S., Nuss, H. B., Chiamvimonvat, N., O'Rourke, B., Pak, P. H. X., Kass, D. A., Marban, E. & Tomaselli, G. F. (1996). Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circulation Research 78, 262-273	[Abstract/Full Text]
Keung, E. C. (1989). Calcium current is increased in isolated adult myocytes from hypertrophied rat myocardium. Circulation Research 64, 753-763	[Abstract]
Kleiman, R. B. & Houser, S. R. (1988). Calcium currents in normal and hypertrophied isolated feline ventricular myocytes. American Journal of Physiology 255, H1434-1442	[Medline]
Kottkamp, H., Budde, T., Lamp, B., Haverkamp, W., Borggrefe, M. X. & Breithardt, G. (1994). Clinical significance and management of ventricular arrhythmias in heart failure. European Heart Journal 15, suppl. D, 155-163.
Lee, J. K., Kodama, I., Honjo, H., Anno, I., Kamiua, K. & Toyama, J. (1997). State-dependent changes in membrane currents in rats with monocrotaline-induced right ventricular hypertrophy. American Journal of Physiology 272, H2833-2842	[Medline]
McPherson, G. A. (1985). Analysis of radioligand binding experiments. A collection of computer programs for the IBM PC. Journal of Pharmacological Methods 14, 213-228	[Medline]
Matsubara, H., Suzuki, J. & Inada, M. (1993). Shaker-related potassium channel, Kv1.4, mRNA regulation in cultured rat heart myocytes and differential expression of Kv1.4 and Kv1.5 genes in myocardial development and hypertrophy. Journal of Clinical Investigation 92, 1659-1666	[Medline]
Nordin, C., Siri, F. & Aronson, R. S. (1989). Electrophysiologic characteristics of single myocytes isolated from hypertrophied guinea-pig hearts. Journal of Molecular and Cellular Cardiology 21, 729-739	[Medline]
Nuss, H. B. & Houser, S. R. (1991). Voltage dependence of contraction and calcium current in severely hypertrophied feline ventricular myocytes. Journal of Molecular and Cellular Cardiology 23, 717-726	[Medline]
Potreau, D., Gomez, J. P. & Fares, N. (1995). Depressed transient outward current in single hypertrophied cardiomyocytes isolated from the right ventricle of ferret heart. Cardiovascular Research 30, 440-448	[Medline]
Powell, T., Terrar, D. A. & Twist, V. W. (1980). Electrical properties of individual cells isolated from adult rat ventricular myocardium. The Journal of Physiology 302, 131-153	[Abstract]
Rossner, K. L. (1991). Calcium current in congestive heart failure of hamster cardiomyopathy. American Journal of Physiology 260, H1179-1186	[Medline]
Ryder, K. O., Bryant, S. M. & Hart, G. (1993). Membrane current changes in left ventricular myocytes isolated from guinea pigs after abdominal aortic coarctation. Cardiovascular Research 27, 1278-1287	[Medline]
Scamps, F., Mayoux, E., Charlemagne, D. & Vassort, G. (1985). Calcium current in single cells isolated from normal and hypertrophied rat heart. Effects of beta-adrenergic stimulation. Circulation Research 67, 199-208.	[Abstract]
Schwartz, P. J., Vanoli, E., Zaza, A. & Zuanetti, G. (1985). The effect of antiarrhythmic drugs on life-threatening arrhythmias induced by the interaction between acute myocardial ischemia and sympathetic hyperactivity. American Heart Journal 109, 937-948	[Medline]
Takimoto, K., Li, D. Q., Hershman, K. M., Li, P., Jackson, E. K. & Levitan, E. S. (1997). Decreased expression of of Kv4.2 and novel Kv4.3 K+ channel subunit mRNAs in ventricles of renovascular hypertensive rats. Circulation Research 81, 533-539	[Abstract/Full Text]
Ten Eick, R. E. & Basset, A. L. (1997). Cardiac hypertrophy and altered cellular electrical activity of the myocardium: possible electrophysiological basis for myocardial contractility changes. In Physiology and Pathophysiology of the Heart, ed. Sperelakis, N., pp. 521-542. Martinus Nihoff, The Netherlands.
Tomita, F., Bassett, A. L., Myerburg, R. J. & Kimura, S. (1994). Diminished transient outward currents in rat hypertrophied ventricular myocytes. Circulation Research 75, 296-303	[Abstract]
Van Wagoner, D. R., Pond, A. L., McCarthy, P. M., Trimmer, J. S. & Nerbonne, J. M. (1997). Outward K+ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation. Circulation Research 80, 772-781	[Abstract/Full Text]
Vatner, D. E., Vatner, S. F., Fujii, A. M. & Homey, C. J. (1985). Loss of high affinity cardiac -adrenergic receptors in dogs withheart failure. Journal of Clinical Investigation 76, 2259-2264	[Medline]
Wang, Z., Fermini, B. & Nattel, S. (1993). Sustained depolarization-induced outward current in human atrial myocytes. Evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents. Circulation Research 73, 1061-1076	[Abstract]
Yeola, S. W. & Snyders, D. J. (1997). Electrophysiological and pharmacological correspondence between Kv4.2 current and rat cardiac transient outward current. Cardiovascular Research 33, 540-547	[Medline]

Corresponding author

R. S. Kass: College of Physicians and Surgeons of Columbia University, Department of Pharmacology, 630 West 168th Street, New York, NY 10032, USA.

Email: rsk20{at}colombia.edu

Authors' present addresses

B. M. Heath: University Department of Pharmacology, Oxford, OX1 3QT, UK.

Email: bronagh.heath{at}pharm.ox.ac.uk

H. J. Federoff, E. Dong * and S. Liang *: Departments of Neurology, Microbiology, Immunology and * Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.

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