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. 2000 Aug 1;526(Pt 3):561–569. doi: 10.1111/j.1469-7793.2000.t01-1-00561.x

Sympathetic innervation alters activation of pacemaker current (If) in rat ventricle

Jihong Qu *, Ira S Cohen †, Richard B Robinson *
PMCID: PMC2270045  PMID: 10922008

Abstract

  1. Pacemaker current (If) exists in both neonatal and adult ventricles, but activates at more negative voltages in the adult. This study uses whole-cell patch clamp to investigate the factors that may contribute to the maturational shift of If, comparing neonatal rat ventricular myocytes that were cultured for 4-6 days either alone, in co-culture with sympathetic nerves, or with neurotransmitters chronically present in culture.

  2. If recorded from nerve-muscle co-cultures had a significantly more negative and shallower activation-voltage relation than that from control muscle cultures, which was reflected in the midpoint potential (V50) and slope factor (K) of activation. This effect of innervation was prevented by the sustained presence in the culture of the α1-adrenergic antagonist prazosin (Pz) at 10−7 M.

  3. In parallel experiments, myocytes treated with noradrenaline (NA) at 10−7 M or neuropeptide Y (NPY) at 10−7 M during culture had the same If activation as control cells, but cells treated with NA and NPY together had a significantly more negative and shallower activation curve. Maximum conductance and reversal potential were unchanged.

  4. The effect of chronic exposure to NA + NPY was prevented by the sustained presence of either Pz or the NPY Y2 selective antagonist T4-[NPY(33-36)]4 (3.5 × 10−7 M) in the culture, indicating a requirement for both α1-adrenergic and NPY Y2 activation.

  5. Substituting NA with the α1A-adrenergic selective agonist A61603 (5-10 × 10−9 M), in the presence of NPY, did not alter If, suggesting the involvement of α1B- rather than α1A-adrenoceptors. Further, sequential exposure to NPY followed by NA was effective in reproducing the action of chronic simultaneous exposure to these agonists, but sequential exposure to NA followed by NPY was ineffective.

  6. The results are consistent with past studies indicating that NPY affects the functional expression of the α1B-adrenergic cascade and suggest that sympathetic innervation induces a negative shift of If in ventricle via a combined action at α1B-adrenergic and NPY Y2 receptors. This effect of innervation probably contributes to the developmental maturation of If activation.

The pacemaker current, If, is widely distributed through the mammalian heart, but with markedly different activation voltages depending on region, age and disease status. In the primary pacemakers of the adult sinoatrial node the current is activated at voltages negative to -40 mV (DiFrancesco, 1991), while in Purkinje fibres the activation threshold is reported to be close to -90 mV (Yu et al. 1993). In the newborn rat ventricle, the current is observed at less negative voltages, with its threshold at approximately -70 mV (Robinson et al. 1997; Cerbai et al. 1999), and this is consistent with other studies in embryonic chick ventricle (Satoh & Sperelakis, 1991; Brochu et al. 1992). A large If activating at physiological voltages is also observed in ventricular cells from aged spontaneously hypertensive rats (Cerbai et al. 1994) and failing human heart (Cerbai et al. 1997). In contrast, in the normal adult ventricle If activates at much more negative voltages that are outside the physiological range. For example, threshold values negative to -120 mV are reported for both dog and guinea-pig ventricle cells (Yu et al. 1993; Ranjan et al. 1998). In the adult rat ventricle, Robinson et al. (1997) and Fares et al. (1998) observed thresholds of -113 and -108 mV, respectively, while Ranjan et al. (1998) reported one of -80 mV. Expression data are consistent with the persistence of pacemaker current in the adult ventricle, with message for several isoforms of the pacemaker channel being observed in the ventricles of both newborn and adult rat (Shi et al. 1999).

Thus, it appears that as the ventricle matures into its adult role as a non-pacing region of the heart, the contribution of the pacemaker current is reduced by shifting its activation voltage to more negative levels rather than by turning off gene expression. During disease, the activation voltage can apparently shift in a positive direction, leading, potentially, to restoration of physiological activity that could contribute to ventricular arrhythmogenesis. The mechanism(s) controlling this developmental and pathological regulation are presently unknown. However, it has previously been demonstrated that the ontogeny of sympathetic innervation can contribute to the developmental maturation of other cardiac ionic currents, including the Na+ current (Zhang et al. 1992) and the L-type Ca2+ current (Ogawa et al. 1992; Protas & Robinson, 1999). We therefore investigated the possibility that sympathetic innervation contributes to the age-dependent negative shift of If activation, and if so what neural factors are responsible for this action.

METHODS

Preparation of myocytes

One- to two-day-old Wistar rats were killed by decapitation in accordance with protocols approved by the Institutional Animal Care and Use Committee of Columbia University. Ventricular myocytes were isolated from the hearts by a previously described trypsin dispersion procedure (Protas & Robinson, 1999). After removal of the hearts, the paravertebral sympathetic chains were removed, dissected free from associated tissue, and dissociated using a trypsin treatment (Drugge et al. 1985). Nerve cells were suspended in minimal essential medium (MEM) with 10 % fetal calf serum and 20 ng ml−1 nerve growth factor, and plated at a density of approximately 600 cells mm−2 into Petri dishes previously coated with fibronectin. Freshly dissociated muscle cells were added 2 h later to create nerve-muscle co-cultures. On days 1 (24 h post-culture) and 4, fresh serum-free medium (SFM, Rybin & Steinberg, 1996) was added. In some experiments, neurotransmitters or receptor agonists and/or antagonists were added to the cultures at the same time as fresh SFM was added or replaced. As indicated in Results, these substances included noradrenaline (NA, 10−7 M), neuropeptide Y (NPY, 10−7 M), prazosin (Pz, 10−7 M), T4-[NPY(33-36)]4 (3.5 × 10−7 M), or A61603 (5-10 × 10−9 M). NA, NPY and Pz were ordered from Sigma (USA); T4-[NPY(33-36)]4 was ordered from the Foundation for Cardiovascular Research and Hypertension (Lausanne, Switzerland); A61603 was provided to our research group by Dr Gregg Rokosh. On the day of the experiment, the monolayer culture was resuspended by a brief (2-3 min) exposure to 0.25 % trypsin and replated in agonist- and antagonist-free SFM onto fibronectin-coated 9 mm × 22 mm glass coverslips. The myocytes were studied 2-8 h after resuspension.

Recording and analysis of If

In this study, the whole-cell patch clamp technique was employed on 4- to 6-day-old cultures and co-cultures in which myocytes were resuspended and replated as single cells to permit recording of If. Experiments were carried out on cells superfused at 35-36°C. Extracellular solution contained (mM): NaCl, 140; MgCl2, 1; KCl, 10; CaCl2, 1.8; Hepes, 5; glucose, 10; pH 7.4. MnCl2 (2 mM) and BaCl2 (3 mM) were added to the superfusate to eliminate Ca2+ and K+ currents, which can obscure If. The patch pipette solution included (mM): aspartic acid, 130; KOH, 146; NaCl, 10; CaCl2, 2; EGTA-KOH, 5; Mg-ATP, 2; Hepes-KOH, 10; pH 7.2. Pipette resistance was typically 2-4 MΩ. The administration and washout of 4 mM CsCl were used to confirm that the time-dependent current recorded was If. During measurements, If was defined as the time-dependent (i.e. leak-subtracted) component taken at the end of a 3 s voltage step from a holding potential of -35 mV to test voltages between -55 and -125 mV with increments of 5 mV. A 3 s duration was set to achieve near steady state at most test voltages. This isochronal I-V relation, divided by the driving force, was converted to an activation curve after the reversal potential was determined (see below), and then fitted by a Boltzmann function (y = 1/(1 + exp ((V – V50)/K))) to give the midpointpotential (V50) and slope factor (K) of activation. The maximum conductance (gmax) and reversal potential (Erev) were determined by linear regression (y = gmax(V – Erev)) against a fully activated I-V relation for If (Accili et al. 1997). The fully activated I-V relation was determined by running a protocol which consisted of first holding the membrane potential at -35 mV and then applying steps to a series of test voltages after a full activation at -125 mV or no activation by depolarizing to -15 mV. The amplitude of the resulting difference tail current was measured as the fully activated current for that test voltage. An Axopatch-200B amplifier and pCLAMP 7.0 software (Axon Instruments, Inc.) were used for data acquisition and analysis in this study. All the If data reported here are given as means ±s.e.m. Statistical significance was examined using ANOVA for multiple comparisons or Student's t test for paired comparisons, and accepted at P < 0.05. Throughout the paper, n refers to the number of cells studied.

RESULTS

Effect of in vitro innervation on If activation

Comparison of the fully activated I-V relation of If in muscle cells grown in culture in the absence or presence of sympathetic neurons indicated no difference in either maximum conductance (34 ± 5 and 37 ± 5 pS pF−1 for non-innervated and innervated cultures, respectively) or reversal potential (-36.1 ± 1.5 and -40.4± 1.3 mV). However, as illustrated in Fig. 1, innervation had a significant effect on the shape of the activation relation. If activation relations were obtained from the groups of non-innervated myocytes (muscle (M) cultures), innervated myocytes (nerve-muscle (NM) co-cultures), and innervated myocytes with the α1-adrenergic antagonist prazosin (Pz) present in culture. With Boltzmann fitting, the mean V50 values in the non-innervated (M) and innervated (NM) myocytes were -77 ± 1 and -88 ± 5 mV, respectively. This difference is consistent with that seen between neonatal and adult ventricles reported by Robinson et al. (1997). The mean K values were 6.7 ± 0.6 and 10.3 ± 1.4 mV for M and NM cultures, respectively, representing a shallower activation-voltage relation in NM cultures than M cultures. The differences in both the V50 and K of activation between NM and M cultures were statistically significant, but no differences were observed when prazosin was constantly present in the innervated muscle cultures. The mean V50 and K values for the NM + Pz group were -75± 1 mV and 7.3 ± 0.7 mV.

Figure 1. Effect of in vitro innervation on If activation in neonatal rat ventricular myocytes.

Figure 1

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Average If activation curves from the non-innervated muscle (M) cultures (•, n = 9), innervated muscle (NM) cultures (□, n = 5), and innervated muscles with prazosin (10−7 M) chronically present in culture (NM + Pz, ○, n = 6). The dashed lines are the Boltzmann fit to the experimental data. V50 values were -77 ± 1, -88 ± 5 and -75 ± 1 mV for M, NM and NM + Pz cultures, respectively; corresponding K values were 6.7 ± 0.6, 10.31 ± 1.4, and 7.3 ± 0.7 mV. The differences in V50 and K between NM and M or NM + Pz cultures are statistically significant (ANOVA).

Effect of chronic conditioning with neurotransmitters

Since our initial observations indicated that the effect of innervation on If activation was prevented by the α1-adrenergic antagonist prazosin, we investigated whether the neurotransmitter NA could reproduce the negative shift in If activation when cultures were grown in the sustained presence of this compound for 4-6 days. Figure 2 illustrates families of current traces and corresponding I-V relations and activation curves for a control muscle cell and one grown in the sustained presence of NA. There was no difference in either V50 or K between the control myocyte and the myocyte treated with NA. This suggested that α1-adrenergic activation might be necessary but not sufficient for the negative shift in the If activation observed in the innervated culture. Since we have previously reported that NPY, which is known to be present in and released from sympathetic neurons, contributes (at 10−7 M) to the innervation-dependent maturation of other ionic currents (Protas & Robinson, 1999) and α1-adrenergic signalling cascades (Sun et al. 1991), we also tested the effect of chronic NPY on If. As seen in Fig. 2, chronic NPY was not effective in shifting If activation in the negative direction.

Figure 2. Action of neurotransmitters on If in neonatal rat ventricular myocytes.

Figure 2

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Top panels, families of current traces from a myocyte (M), a myocyte cultured in the sustained presence of noradrenaline (NA, 10−7 M) and one cultured in the sustained presence of neuropeptide Y (NPY, 10−7 M). The voltages for the selected current traces were -55, -70, -85, -100 and -110 mV in each panel. Middle panels, the isochronal I-V relations for If from the same cells as in the top row. Bottom panels, the activation-voltage relations of If, converted by fitting the I-V relations in the middle row with a Boltzmann function (dotted lines). The calculated parameters were also used to generate the dotted lines in the middle panels.

However, when cultures were grown in the combined presence of both NA and NPY, the activation was substantially more negative and shallower, as illustrated in the example shown in Fig. 3. The average activation curves are shown and compared in Fig. 4. V50 was -94 ± 6 mV in NA + NPY-treated cultures, in comparison to -77 ± 1 mV in control cultures (M), -82 ± 3 mV in NA-treated cultures and -81 ± 3 mV in NPY-treated cultures. Only the V50 value for the NA + NPY group differed significantly from the M group. In addition, K also differed significantly between M cultures and NA + NPY-treated cultures (6.7 ± 0.6 vs. 10.8 ± 1.2 mV), consistent with the shallower activation curve for the NA + NPY-treated cultures than for the M or NA-treated cultures (7.4 ± 1.2 mV) or for the cultures treated with NPY (7.3 ± 1.1 mV) alone. Moreover, the effect on If activation achieved by sustained NA + NPY treatment is comparable to that resulting from sympathetic innervation of the myocytes in culture (V50= -94 ± 6 vs. -88 ± 5 mV and K = 10.8± 1.2 vs. 10.3 ± 1.4 mV for NA + NPY vs. NM).

Figure 3. Action of combined neurotransmitters on If in neonatal rat ventricular myocytes.

Figure 3

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Top panels, the families of current traces from a control myocyte (M, reproduced from Fig. 2) and a myocyte cultured in the sustained combined presence of NA and NPY (NA + NPY), both at 10−7 M. The voltages for the selected current traces were -55, -70, -85, -100 and -110 mV for the M myocyte, and -90, -105, -120, -135 and -145 mV for the NA + NPY-treated myocyte. Middle panels, the isochronal I-V relations for If from the same cells. Bottom panels, the activation-voltage relations of If, converted with the Boltzmann fit (dotted lines) from the I-V relations in the middle row. The calculated parameters were also used to generate the dotted lines in the middle panels. Note the different voltage scale for the NA + NPY-treated myocyte in the middle and bottom rows, compared to the M myocyte.

Figure 4. Effect of neurotransmitters on If activation.

Figure 4

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Average If activation curves for control cultures (M, ○, n = 9) and cultures with NA (▵, n = 7), NPY (▿, n = 4) and NA + NPY (□, n = 6). The dashed lines are the Boltzmann fit to the experimental data. V50 values were -77 ± 1, -82 ± 3, -81 ± 3 and -94± 6 mV for M cultures and NA-, NPY- and NA + NPY-treated cultures, respectively; corresponding K values were 6.7 ± 0.6, 7.4 ± 1.2, 7.3 ± 1.1 and 10.8 ± 1.2 mV. The differences in V50 and K between M and NA + NPY-treated cultures are statistically significant (ANOVA) while those between M and NA-treated cultures or between M and NPY-treated cultures are not. Note that the more negative and shallower activation curve for cultures grown in the sustained presence of NA + NPY is comparable to the effect of innervation seen in Fig. 1. Data for M cultures were taken from Fig. 1.

Acute exposure to NA + NPY during electrophysiological recording, both applied at the same concentration of 10−7 M as was used chronically in this study, did not alter the activation of If compared to control cultures (n = 3, data not shown). Taken together, these data indicate that the effect of innervation requires sustained activation of both the α1-adrenergic and NPY signalling cascades and that preventing activation of one of these cascades (e.g. by prazosin in the co-cultures) inhibits the action of innervation.

As stated earlier, in vitro innervation altered the activation relation for If without affecting maximal conductance or reversal potential. Similar results were obtained following chronic exposure of myocytes to a combination of NA and NPY in culture. A linear regression, y =gmax(V – Erev), was performed against the fully activated I-V data to obtain the maximum conductance, gmax, and reversal potential, Erev. Figure 5 shows I-V relations for the fully activated If under various culture conditions. The slopes of the linear regression to the experimental data, representing gmax, were 34 ± 5, 33 ± 4, 31 ± 3 and 31 ± 7 pS pF−1 for the control muscle cultures, and the myocytes cultured with NA, NPY and NA + NPY, respectively. Corresponding Erev values, as reflected as the slope of the linear regression, were -36.1 ± 1.5, -39.8 ± 1.1, -336.7 ± 0.8 and -39.6 ± 1.9 mV. There was no evident difference either in conductance or reversal potential throughout these conditions.

Figure 5. Fully activated I-V relations of If in control muscle (M) cultures and myocytes cultured with NA, NPY or NA + NPY.

Figure 5

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The straight lines are the linear regressions (y =gmax(V – Erev)) against the experimental data. The panel of voltage pulses shown above the plots is the protocol for recording a fully activated I-V relation (see Methods). The prepulses to -15 and -125 mV are 2.8 s long and test pulses are 1 s. There was no difference in gmax or Erev among these preparations (ANOVA); n = 4-6.

Nature of the action of NPY and NA

The previously reported chronic effects of NPY to promote maturation of other ionic currents or signalling cascades have been ascribed to the Y2 subtype of NPY receptor (Sun et al. 1998; Protas & Robinson, 1999). To determine if the effect on If involved the same receptor subtype, additional experiments were conducted in which ventricular myocytes cultured with NA + NPY were chronically exposed to the Y2-selective antagonist T4-[NPY(33-36)]4 (at 3.5 × 10−7 M, the approximate Kd at the Y2 receptor; Grouzmann et al. 1998). As revealed in Fig. 6, the effect on If activation exerted by the sustained presence of NA + NPY was abolished by T4-[NPY(33-36)]4, indicating that the action of NPY is via the neuropeptide Y Y2 receptor subtype. V50 was -77 ± 2 mV for cultures treated with NA + NPY combined with T4-[NPY(33-36)]4 in comparison to -77 ± 1 mV for M cultures and -94 ± 6 mV for NA + NPY-treated cultures. Corresponding K values were 6.4 ± 0.8, 6.7 ± 0.6 and 10.8 ± 1.2 mV.

Figure 6. Subtype selectivity of the NA and NPY signalling pathways affecting If.

Figure 6

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A, the midpoint potentials (V50) of If activation for control cultures (M, n = 9) and cultures treated with NA + NPY (n = 6), NA + NPY plus the Y2-selective NPY antagonist T4-[NPY(33-36)]4 at 3.5 × 10−7 M (n = 5), NA + NPY plus the α1-adrenergic antagonist prazosin (Pz) at 10−7 M (n = 6), and NPY plus the α1A-adrenergic selective agonist A61603 at 5-10 × 10−9 M (n = 4). B, the slope factors (K) under the same conditions as in A. *P < 0.05 (ANOVA). Data for M and NA + NPY groups were taken from Fig. 4.

Additional experiments were performed to begin to elucidate the α1-adrenergic cascade involved. As with sympathetic innervation, NA and NPY together were ineffective when prazosin (10−7 M) was present in the culture. The mean V50 and K were -75± 1 mV and 6.5 ± 1.0 mV, for myocytes chronically exposed to NA + NPY and Pz (Fig. 6). These data confirm that the combined effect of NA and NPY also requires the α1-adrenergic pathway. In additional experiments, NA was replaced with the α1A-adrenergic selective agonist A61603 (EC50= 3.6 × 10−9 M) at 5-10 × 10−9 M. This concentration has been found to be effective in activating the α1A-adrenergic cascade in these cultures of newborn rat ventricular myocytes (Autelitano & Woodcock, 1998; Steinberg et al. 1999). After treatment with A61603 and NPY, If was found to be equivalent to that under control conditions (V50=-76± 1 mV; K = 6.0± 0.3 mV). Therefore A61603 was ineffective as a substitute for NA (Fig. 6), suggesting that the α1A-adrenergic receptor subtype is not involved and raising the possibility that the relevant subtype might be the α1B-adrenergic receptor.

Previous studies have demonstrated that the α1B-adrenergic cascade is only functionally available in the rat ventricle after sympathetic innervation (Drugge et al. 1985; Steinberg et al. 1985), that this effect of innervation is dependent on NPY (Sun et al. 1991) and that, based on the profile of activity of NPY peptide analogues, the relevant NPY receptor subtype may be Y2 (Sun et al. 1998). It therefore seemed possible that the dependence of the effect on If of both NPY and NA occurs because of a sequential action of NPY on the availability of the α1B-adrenergic cascade and the subsequent chronic activation of that cascade by NA, and that it is in fact the activation of the α1B-adrenergic cascade that modifies If. In this case, sequential exposure to NPY followed by NA should cause a similar change in the voltage dependence of If to that induced by simultaneous exposure to both agonists, while sequential exposure to NA followed by NPY should be ineffective. Figure 7 illustrates that this was the case. Cultures were exposed to either NPY or NA alone from day 1 to day 4. The culture medium was then changed to one containing the alternate agonist and the cells were maintained for another 2 days prior to the patch clamp experiment. Cells studied on day 6 that had been sequentially exposed to NPY followed by NA had a shallow activation curve (K = 13.0± 1.4 mV) with a midpoint potential (V50=-91± 4 mV) that was negative to that of control cells. In contrast, cells exposed first to NA followed by NPY had activation curves that did not differ from control (K = 5.9± 0.8 mV; V50=-75± 1 mV).

Figure 7. Simultaneous and sequential activation of the signalling pathways affecting If.

Figure 7

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Average If activation curves for the conditions of control (M) (○, n = 9), cultures exposed first to NA and then to NPY (▪, n = 4) and cultures exposed first to NPY and then NA (•, n = 9). The dashed lines are the Boltzmann fit to the experimental data. V50 values were -77 ± 1, -75 ± 1 and -91 ± 4 mV for M, NA → NPY and NPY → NA cultures, respectively; corresponding K values were 6.7 ± 0.6, 5.9 ± 0.8 and 13.0 ± 1.4 mV. The differences in V50 and between M and NPY → NA cultures are statistically significant (ANOVA) while those between M and NA → NPY cultures are not. Concentrations of NA and NPY were both 10−7 M. Data for M taken from Fig. 1.

Taken together, these data suggest that the hyperpolarizing shift in If activation seen with sympathetic innervation requires activation of an α1-adrenergic receptor distinct from the α1A-subtype by neurally released NA as well as the activation of the NPY Y2 receptor by neurally released NPY. The data are consistent with a mechanism of action involving a Y2-dependent effect on the availability of the α1B-adrenergic cascade and a subsequent activation of that cascade.

DISCUSSION

This work demonstrates that the activation of pacemaker channels (If) is altered in cultured ventricular myocytes from newborn rats by the sustained presence of sympathetic neurons or neurotransmitters. The two in vitro conditions caused equivalent changes in the activation curve. The decreased steepness and corresponding negative shift in the midpoint of the activation voltage are in a direction that mimics that observed during normal development, although we did not observe a marked negative shift in the threshold of activation, as has been reported in developmental studies (Robinson et al. 1997). The effect on If was evident in the voltage dependence of its activation, but not in its maximum conductance or reversal potential. The absence of a decrease in conductance is consistent with data from our past studies of pacemaker current in adult versus newborn rat ventricle (Robinson et al. 1997) and the persistent expression of pacemaker channel (HCN) message in the developing rat ventricle (Shi et al. 1999). A recent developmental study in rat ventricle reported that If current density decreased with age without a shift in activation voltage (Cerbai et al. 1999). While the reason for the discrepancy between that study and the adult ventricle data of Robinson et al. (1997) and Fares et al. (1998) is not clear, it is intriguing that the largest change in current density reported in the study of Cerbai et al. occurred around days 5-10 after birth, coinciding with the onset of sympathetic innervation of the rat ventricle (Robinson, 1996).

The neurotransmitters that were applied in this study and induced the alterations in If activation were NA and NPY, both applied at 10−7 M, a concentration proven to have effects on other ion channels during culture (Protas & Robinson, 1999). The data show that NA and NPY produce a statistically significant negative shift in the midpoint potential and a shallower slope of activation only when present together; neither NA nor NPY alone altered If under chronic conditioning. These changes in If activation were reproduced in the ventricular myocytes innervated in vitro by sympathetic neurons. The decreased slope of the activation-voltage relation upon innervation or sustained exposure to NA and NPY is distinct from the typical effect on pacemaker current of acute exposure to catecholamines or other substances that alter the intracellular cAMP level, which cause a parallel shift in the activation-voltage relation and a corresponding change in both the midpoint potential and the threshold (DiFrancesco, 1996). It thus seems unlikely that innervation or long term NA/NPY exposure are acting strictly via a sustained reduction in the intracellular cAMP level.

The shallow activation curves following in vitro innervation or culture with NA + NPY (slope factors of 10.3 and 10.8 mV, respectively) are comparable to that reported by Fares et al. (1998) in freshly isolated adult rat ventricle cells (slope factor of 10.3 mV). However, the magnitude of the negative shift of activation of If by in vitro innervation or neurally released substances seen in this work is approximately half of that observed in developmental studies (Robinson et al. 1997). One contribution to this difference in magnitude may be the different measurements employed. The previous work used threshold measurements, while full activation relations were examined in the present study. However, as stated above, we did not observe a marked effect of these in vitro treatments on the threshold of activation. The difference in magnitude of the shift may also reflect inadequate exposure to nerves or neural factors during our 4-6 day culture period in comparison to the normal developmental process. Alternatively, it is possible that not all factors and/or regulatory cascades that contribute to normal maturation have been identified and tested in this in vitro study of If regulation. In this regard, it is interesting that two If isoforms, HCN2 and HCN4, are reported to be present at the message level in both newborn and adult rat ventricle, and the relative abundance changes with development (Shi et al. 1999). As measured by RNAse protection assay, the ratio of HCN2 to HCN4 is 5:1 in the neonatal and 13:1 in the adult ventricle. It is not known at this time if this change in isoform expression is an absolute requirement for the negative shift of If activation with age, and in fact existing data on expression of HCN2 and HCN4 in heterologous expression systems give conflicting results on the midpoints of activation of these isoforms (Ludwig et al. 1999; Ishii et al. 1999; Moroni et al. 2000). However, a recent study reported sensitivity of cardiac HCN2 message to in vivo thyroid hormone levels (Pachucki et al. 1999), suggesting that factors other than innervation may well contribute to expression of specific HCN isoforms and perhaps also impact on the functional characteristics of the corresponding current. Whether innervation also modulates isoform expression is unknown, but one could hypothesize that innervation preferentially alters the activation voltage and possibly expression of one HCN isoform. As a result, the slope of If activation would be shallower in innervated and mature cells. This is indeed what was seen for If activation in the myocytes treated with NA + NPY or innervated with neurons in this study. Alternatively, the negative shift and/or shallow slope may reflect the relative presence or absence of a subsidiary subunit, or post-translational modification of the channel protein. While direct testing of the effect of in vivo or in vitro innervation on isoform expression could prove problematic (due to variable contamination by neural elements, among other difficulties), in vitro chemical conditioning of myocyte cultures by NA + NPY, thyroid hormone, or other potential developmental factors is likely to provide future insight into the mechanism of regulation of the voltage dependence of If.

While the signalling cascades by which sympathetic innervation modulates If have not been fully elucidated, the fact that the effect of sympathetic innervation can be inhibited by prazosin demonstrates that an α1-adrenergic cascade must be involved in the action. The observation that NA alone does not mimic the action of innervation suggests that the α1-adrenergic cascade may be necessary but not sufficient. In fact, the combined presence of NA and NPY was required to cause a negative shift in If activation. It is known that α1-adrenoceptors are heterogeneous. The α1A- and/or α1D-subtypes function in newborn and adult ventricles while the α1B-subtype appears functional only in adult or innervated cells, although the receptor is present in neonates (Rybin et al. 1996). The experiments with the α1A-subtype selective agonist A61603 combined with NPY indicate that the α1A-adrenergic receptor is not involved and suggest a possible role for the α1B-receptor subtype. The experiments with the Y2-selective NPY antagonist T4-[NPY(33-36)]4 suggest that the Y2 NPY receptor is also involved in the effect on If.

In this respect it is relevant that the α1B-adrenergic pathway itself requires chronic exposure to neurally released NPY acting at Y2 receptors to become functionally available. Neonatal rat ventricle exhibits a positive chronotropic response to α1-adrenergic activation, while the adult ventricle exhibits a negative chronotropic response (Drugge et al. 1985). This maturational change is reproduced in culture when neonatal rat ventricle myocytes are co-cultured with sympathetic neurons (Drugge et al. 1985), and the maturational change is accelerated or delayed in vivo when neonatal animals are treated with nerve growth factor or its antibody to accelerate or delay sympathetic innervation, respectively (Malfatto et al. 1990). Evidence that the effect of innervation on expression of the negative chronotropic α1-adrenergic response is mediated by NPY comes from studies showing that the effect of in vitro innervation is prevented by chronic exposure of co-cultures to an NPY antagonist and mimicked by chronic exposure of non-innervated myocytes to NPY in culture (Sun et al. 1991). The negative chronotropic α1-adrenergic response is via the α1B-adrenergic receptor subtype (del Balzo et al. 1990; Sun et al. 1998), and the effect of NPY on maturation of this cascade appears to be via the Y2 receptor subtype (Sun et al. 1998). Thus, one interpretation of the present data is that sustained exposure to neurally released NA modulates If through an α1B-adrenergic cascade, but that this cascade is not functionally available until the myocytes have been exposed to sustained levels of neurally released NPY acting through a Y2 receptor. In this scenario, the actions of NPY and NA are actually sequential. This interpretation is supported by the experiments in which myocytes were exposed sequentially to NPY and NA. When the NPY exposure preceded the NA exposure, the voltage dependence of If was altered. However, when cells were first exposed to NA and only subsequently to NPY, the If of the treated cells did not differ from that of control myocytes.

Acknowledgments

The authors wish to thank Ms Ema Stasko for technical assistance and Dr Lev Protas for helpful discussions. This work was supported by NIH PPG HL-28958, and was carried out during the tenure of a post-doctoral fellowship from the American Heart Association, Heritage Affiliate, Inc., awarded to Dr Jihong Qu.

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