Nitric Oxide Can Increase Heart Rate by Stimulating the Hyperpolarization-Activated Inward Current, If
Abstract We investigated the chronotropic effect of increasing concentrations of sodium nitroprusside (SNP, n=8) or 3-morpholinosydnonimine (SIN-1, n=6) in isolated guinea pig spontaneously beating sinoatrial node/atrial preparations. Low concentrations of NO donors (nanomolar to micromolar) gradually increased the beating rate, whereas high (millimolar) concentrations decreased it. The increase in rate was (1) enhanced by superoxide dismutase (50 to 100 U/mL, n=6), (2) prevented by the guanylyl cyclase inhibitors 6-anilino-5,8-quinolinedione (5 μmol/L, n=6) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (10 μmol/L, n=6), and (3) mimicked by 8-bromo-cGMP (n=6) with no additional positive chronotropic effect of SIN-1 (n=5). The response to 10 μmol/L SNP (n=28) or 50 μmol/L SIN-1 (n=16) was unaffected by ICa-L antagonism with nifedipine (0.2 μmol/L) but was abolished after blockade of the hyperpolarization-activated inward current (If) by Cs+ (2 mmol/L) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride (1 μmol/L). The effect on If was further evaluated in rabbit isolated patch-clamped sinoatrial node cells (n=21), where we found that 5 μmol/L SNP or SIN-1 caused a reversible Cs+-sensitive increase in this current (+130% at −70 mV and +250% at −100 mV). In conclusion, NO donors can affect pacemaker activity in a concentration-dependent biphasic fashion. Our results indicate that the increase in beating rate is due to stimulation of If via the NO-cGMP pathway. This may contribute to the sinus tachycardia in pathological conditions associated with an increase in myocardial production of NO.
Although the role of NO in modulating myocardial contractility has been extensively investigated, the effects of NO on heart rate have received comparatively little attention.1 2 It is well known that systemic administration of NO donors (eg, SNP) is associated with an increase in heart rate, and this is thought to be due to a neurally mediated reflex response to the fall in arterial blood pressure.3 However, SNP can also increase heart rate in heart transplant recipients4 before sympathetic reinnervation can occur,5 suggesting that NO donors might stimulate SAN activity independent of the arterial baroreflex. The evidence supporting this hypothesis is, however, inconclusive. In isolated right atria, low concentrations of the NO donor SIN-1 had no effect on beating rate, whereas very high concentrations had a negative chronotropic effect.6 Conversely, in a study aimed to assess the role of NO in modulating arrhythmias in isolated perfused hearts, Pabla and Curtis7 noted an increase in beating rate in response to a low concentration of SNP and a negative chronotropic effect after blockade of the NO synthase with NG-nitro-l-arginine methyl ester. This suggests that NO might independently stimulate pacemaker activity.
To test this hypothesis, we investigated whether exogenous NO could affect the spontaneous beating rate of an isolated guinea pig SAN/atrial preparation. We found that SNP and SIN-1 caused a biphasic, concentration-dependent, chronotropic response. The increase in beating rate was prevented by guanylyl cyclase inhibitors and could be mimicked by 8-Br-cGMP. Furthermore, this positive chronotropic effect was not affected by ICa-L antagonism but was abolished by blockers of If. Finally, in rabbit isolated SAN cells, we showed a marked Cs+-sensitive increase in If with both SNP and SIN-1. When taken together, these results indicate that the increase in rate with NO donors is due to stimulation of If via the NO-cGMP pathway.
Materials and Methods
Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and the Animals (Scientific Procedures) Act 1986 (UK).
Guinea Pig SAN/Atrial Preparation
Guinea pigs (400 to 450 g) of either sex were killed by cervical dislocation and exsanguinated. The heart was rapidly removed and placed in a dissecting dish with Tyrode’s solution aerated with 95% O2/5% CO2 at 35°C to 37°C. Heparinized Tyrode’s solution (1000 U/mL) was immediately perfused through the aorta, and the ventricles were carefully dissected and removed. Sutures (Ethicon 6/0 silk) were placed at the lateral edges of the two atria. The preparation was then transferred to a preheated (37±0.1°C), continuously oxygenated, water-jacketed bath containing 60 mL of Tyrode’s solution. The atria were mounted vertically with the suture in the right atrium attached to a stainless steel hook, and the left atrium was attached to an isometric force transducer (HSE F30), which was connected to a laboratory-built amplifier. Data were acquired on a Power Macintosh 8500 computer using a Biopac MP100 data acquisition system and AcqKnowledge 3.5 software. Beating rate was triggered from contraction, and the signals were displayed in real time. Data were stored on an optical disk for off-line analysis.
Solution and Drugs
The Tyrode’s solution contained (mmol/L) NaCl 120, KCl 4, MgCl2 2, NaHCO3 25, CaCl2 1.8, NaH2PO4 0.1, and glucose 11. The solution was aerated with 95% O2/5% CO2 (pH 7.4), and its temperature was continuously monitored (Digitron 1408-K gauge) and kept at 37±0.1°C.
Two different NO donors, SNP (Sigma) and SIN-1 (Sigma Chemical Co),8 were used. In addition, SNAP (Affiniti Ltd) was tested as an NO donor with S-nitrosylating properties.9 CsCl (2 mmol/L, Sigma) and ZD7288 (1 μmol/L, Zeneca Pharmaceuticals) were used as selective blockers of If,10 11 12 and NIF (0.2 μmol/L, Sigma) was used to antagonize ICa-L.13 NIF (0.2 μmol/L) was used, because in a preliminary set of experiments, this concentration was the highest that elicited a stable bradycardia without arresting the preparation. SOD (Sigma), an enzyme known to enhance NO-dependent effects through scavenging the superoxide anion,14 inhibitors of guanylyl cyclase LY8358315 (Calbiochem) and ODQ16 17 (Tocris Cookson UK), and the membrane-permeable cGMP analogue 8-Br-cGMP18 (Sigma) were used to evaluate the mechanism of the chronotropic effect of NO donors.
CsCl, ZD7288, and NIF were added from stock solutions of 1 mol/L, 1 mmol/L, and 0.1 mmol/L, respectively. Solutions of SNP or SNAP (in water of pH 7.4) and SIN-1 (in water of pH 5.4 to 5.8) were prepared immediately before application.8 9 All water used was of reagent grade from an Elga water purification system. Exchange of the solution during experiments (see “Protocols”) was achieved from a jacketed reservoir kept at 37°C.
Before starting each protocol, we kept the mounted atria in Tyrode’s solution for 120 to 200 minutes (the medium was changed every 20 minutes), until their beating rate stabilized (within 5 bpm for 40 minutes). Since SNP, SIN-1,8 8-Br-cGMP, and NIF are very light-sensitive, all experiments were carried out in a darkened room.
Chronotropic Response to Incremental Concentrations of NO Donors
SNP (n=6) or SIN-1 (n=8) was applied cumulatively to the tissue bath in half-logarithmic increments (the next dose added after a stable response to the previous concentration was reached) to achieve a range of concentrations from 5×10−8 to 10−2 mol/L for SNP and from 5×10−8 to 10−3 mol/L for SIN-1. The concentration-response relation to SIN-1 was also determined (n=6) in the presence of SOD (50 to 100 U/mL) to minimize the possible role of superoxide (an agent generated in addition to NO during SIN-1 breakdown) or peroxynitrite (a product of NO and superoxide)9 19 in eliciting the chronotropic effect.
It is known that under physiological conditions NO can react with thiol groups in proteins to form S-nitrosothiols, which may serve as biologically active intermediates of NO.20 Furthermore, S-nitrosylation (NO+ transfer) can account for both cGMP-dependent20 21 and cGMP-independent19 effects of NO. For these reasons, we also tested the chronotropic effect of increasing concentrations of the S-nitrosothiol SNAP9 (n=7 plus n=3 control preparations for the effect of the carrier, N-acetyl-d,l-penicillamine; concentration range, from 5×10−8 to 10−3 mol/L).
Role of cGMP in the Positive Chronotropic Response to Exogenous NO
Modulatory effects of NO donors on membrane channels can occur both via indirect (cGMP-dependent) and direct (redox-modulation) mechanisms.19 22 We investigated the role played by the cGMP-dependent pathway in the positive chronotropic effect of NO donors by evaluating (1) the chronotropic effect of increasing concentrations of a membrane-permeable analogue of cGMP, 8-Br-cGMP (10−6 to 10−3 mol/L, n=6), and (2) the concentration-response relation to SIN-1 in the presence of 8-Br-cGMP (1 mmol/L, 20-minute preincubation, n=5) or in the presence of a guanylyl cyclase inhibitor, LY83583 (5 μmol/L, 40-minute preincubation, n=6)1 15 18 or ODQ (10 μmol/L, 40-minute preincubation, n=6).16 17
Chronotropic Effect of SNP in the Presence of NIF
Each experiment was preceded by a control response to SNP (10 μmol/L, the concentration causing submaximal positive chronotropic effect; see Fig 1⇓) and a washout. Subsequently, NIF (0.2 μmol/L, n=10) was added, and when a stable beating rate was reached, the same dose of SNP was reapplied. The time course of the experiment was as follows: SNP (10 minutes)→washout (20 minutes)→NIF (20 minutes)→SNP (10 minutes).
Chronotropic Effect of SNP in the Presence of If Blockers
Experiments were preceded by a control response to SNP (10 μmol/L) and a washout. An If antagonist, either 2 mmol/L CsCl (n=10) or 1 μmol/L ZD7288 (n=8), was then added, and when a stable beating rate was reached, the same dose of SNP was reapplied. The time course of the experiment was as follows: SNP (10 minutes)→washout (20 minutes)→Cs+ (15 minutes) or ZD7288 (45 minutes)→SNP (10 minutes).
Chronotropic Response to SIN-1 in the Presence of NIF or If Blockade
To evaluate whether some nonspecific properties of SNP8 might affect the chronotropic response during If or ICa-L blockade, the effect of 50 μmol/L SIN-1 (concentration causing submaximal effect; see Fig 1⇑) on the beating rate was tested before and after treatment with NIF (0.2 μmol/L) or CsCl (2 mmol/L) as described above (n=8 in each series).
Isolated Rabbit SAN Cells
Cell Isolation and Solutions
Pacemaker cells were isolated from the SAN of New Zea-land White rabbits (700 to 900 g) killed by cervical dislocation. The isolation protocol and composition of external solution have been described in detail previously.23 In brief, thin strips of SAN tissue (≈0.5×3 mm) were placed in Ca2+-free Tyrode’s solution for 5 minutes and subsequently incubated at 37°C for 30 to 40 minutes in the presence of collagenase (Sigma, 230 U/mL) and elastase (Sigma, 15 U/mL). After the strips were maintained in Krebs’ buffer at 4°C for at least 1 hour, single cells were released from the tissue by glass pipette suction.
The whole-cell patch-clamp mode (amphotericin-permeabilized patches; internal solution containing [mmol/L] KCl 140, HEPES 5, EGTA 1, and MgSO4 1.8, titrated to pH 7.4 with KOH, and amphotericin, 200 μg/mL) was used for electrical recordings from single SAN cells.
A temperature of 36±0.5°C was maintained throughout each experiment. For details regarding recording methods and data acquisition, see Reference 2323 .
In 21 cells, after successful seal formation and amphotericin permeabilization, a two-pulse voltage-clamp protocol was used to test for If from the holding potential of −40 to −70 mV (1 second) and then from −40 to −100 mV (1 second).
Effect of SNP on the Amplitude of If (n=11)
After a control recording, the solution was changed for the one containing 5 μmol/L SNP (prepared immediately before application), and subsequent recordings were made at 3, 5, and 10 minutes. Washout of SNP was attempted in six cells.
Effect of Cs+ on If in the Presence of SNP (n=6)
The same two-pulse protocol was used (see above) to evaluate whether CsCl (2 mmol/L) inhibits the effect of SNP (5 μmol/L) on If. The time course of recordings was as follows: control→SNP (5 and 10 minutes after application)→SNP plus Cs+ (5 and 10 minutes)→SNP only (5 and 10 minutes).
Control Experiments With SIN-1 (n=4)
The If protocol (as above) was used to test whether SIN-1 modulates If in a similar manner to that of SNP. In addition, in the same cells we evaluated the effect of SIN-1 on ICa-L. In all experiments, exposure to SNP or SIN-1 was performed in a darkened room.
Data are presented as mean±SEM. For experiments on SAN/atrial preparations, one-way repeated measures ANOVA followed by Scheffé’s post hoc test was used to evaluate the effect of increasing NO donor or 8-Br-cGMP concentrations on beating rate and to assess the effect of antagonists of pacemaker currents within the same group of experiments. One-way factorial ANOVA (followed by Scheffé’s post hoc test) was used to compare the chronotropic effect of SIN-1 alone versus SIN-1 in the presence of SOD and the effect NO donors after the application of NIF versus If blockers. Student’s t test was used to compare changes in the magnitude of If during exposure to SNP in isolated pacemaker cells and to evaluate the effect of Cs+. Statistical significance was accepted at P<.05.
Immediately after the SAN/atrial preparations were placed in the experimental chamber, the mean spontaneous beating rate was 254±3 bpm. During the period of stabilization (120 to 200 minutes), the beating rate decreased in an exponential fashion until it reached a stable value, which averaged 179±3 bpm (n=97). Six SAN/atrial preparations were discarded, since their beating rates did not remain stable.
Chronotropic Response to NO Donors
Fig 1⇑ shows the chronotropic effect of increasing concentrations of SNP and SIN-1 on spontaneously beating SAN/atria. SNP caused a progressive increase in beating rate, which became significantly different from baseline at concentrations from 5 to 50 μmol/L. The peak positive chronotropic response to SNP was reached at 50 μmol/L (rate increase of 77±7 bpm, P<.05). Further increments in SNP concentration resulted in a stepwise decrease in the beating rate. At the highest concentration of SNP used (10 mmol/L), the beating rate was lowered by 83±9 bpm (P<.05) compared with the average maximal positive chronotropic effect of this agent (Fig 1⇑).
The concentration-response curve to SIN-1 was similar to that for SNP (Fig 1⇑). However, the peak increase in the beating rate with SIN-1 (+36±4 bpm, P<.05) was significantly lower than that with SNP and occurred at 100 μmol/L. The highest concentration of SIN-1 (1 mmol/L) caused a decrease in spontaneous rate by 32±5 bpm compared with the peak rate achieved in response to this drug.
In the presence of SOD (Fig 1⇑), the positive chronotropic effect of SIN-1 was significantly enhanced, with the peak increase in beating rate averaging 51±5 bpm (P<.05 versus the effect of SIN-1 alone).
In summary, both SNP and SIN-1 caused a biphasic concentration-dependent chronotropic response, with a gradual increase in beating rate for low concentrations and a decrease in beating rate for high concentrations of either NO donor. The response to SIN-1 was enhanced in the presence of SOD.
Chronotropic Response to SNAP
Incremental concentrations of SNAP, an S-nitrosylating compound and NO donor,9 caused a progressive increase in beating rate, which became statistically significant for concentrations of ≥5 μmol/L and peaked at 0.5 and 1 mmol/L (increase of 37±5 and 37±6 bpm, P<.05, Fig 2⇓). Conversely, N-acetyl-d,l-penicillamine, used in the same range of concentrations as SNAP, had no effect on the beating rate. At concentrations of 0.5 and 1 mmol/L, the positive chronotropic effect of SNAP was often preceded by a short-lived (1- to 2-minute) decrease in the beating rate.
Role of cGMP-Dependent Pathway
Application of increasing concentrations of 8-Br-cGMP resulted in an progressive increase in beating rate (Fig 3⇓). The peak effect was observed at the highest concentration of 8-Br-cGMP (increase of 62±6 bpm, P<.05). In the presence of 8-Br-cGMP, the positive chronotropic effect of low concentrations of SIN-1 (≤0.1 mmol/L) was abolished while the decrease in rate in response to higher concentrations was still present (Fig 4A⇓). LY8358315 caused a nonsignificant decrease in the spontaneous rate (−8.6%) and prevented the positive (but not the negative) chronotropic effect of SIN-1 (Fig 4B⇓). However, LY83583, in addition to inhibiting the guanylyl cyclase,15 appears to have other biological actions that can affect NO-dependent pathway(s), eg, generation of oxygen-derived free radicals24 and direct inactivation of NO.15 25 For that reason, we also evaluated the concentration-response relation to SIN-1 in the presence of ODQ, a novel specific inhibitor of guanylyl cyclase.16 17 In the presence of ODQ, low concentrations of SIN-1 (≤0.1 mmol/L) did not alter the beating rate, whereas higher concentrations decreased it (Fig 4C⇓).
In summary, the positive chronotropic response to NO donors (1) was prevented by LY83583 or ODQ and (2) was mimicked by a membrane-permeable analogue of cGMP with no additional effect of the NO donor in its presence.
Effect of Antagonizing ICa-L on the Positive Chronotropic Response to SNP
Fig 5A⇓ (trace a) shows representative raw data of the effect of SNP (10 μmol/L) on the beating rate before and after ICa-L was antagonized with NIF (0.2 μmol/L). The control response to SNP resulted in an average increase in beating rate of 49±9 bpm (P<.05), which was fully reversed after washout of SNP. NIF decreased the beating rate from 184±7 to 131±8 bpm (−29%, P<.05). When SNP was reapplied in the presence of NIF, it still caused an increase in the beating rate of 61±14 bpm (P<.05, Fig 5A⇓, trace a, and Fig 5B⇓).
In summary, the positive chronotropic response to the NO donor SNP was maintained when the L-type Ca2+ current was antagonized by NIF.
Effect of Blocking If on the Positive Chronotropic Response to SNP
We tested whether applying 2 mmol/L CsCl or 1 μmol/L ZD7288 to block If would attenuate the positive chronotropic response to SNP. Fig 5A⇑ (trace b and trace c) shows examples of raw data from these experiments (mean values are shown in Fig 5B⇑). SNP (10 μmol/L) caused a comparable increase in beating rate in both groups (by 45±7 bpm in the group in which Cs+ was subsequently applied and by 46±8 bpm in the ZD7288 group, P<.05 for either group), which was fully reversed after washout. Cs+ (2 mmol/L) and ZD7288 (1 μmol/L) decreased the spontaneous rate by 60±3 bpm (−32%, P<.05) and 106±7 bpm (−58%, P<.05), respectively. When applied in the presence of either If blocker, SNP no longer had a significant positive chronotropic effect (5±2 bpm, P=NS).
In summary, the positive chronotropic response to SNP was virtually abolished in the presence of If blockade with either Cs+ or ZD7288.
Effect of NIF Versus Cs+ on the Positive Chronotropic Response to SIN-1
To test whether the chronotropic effect of SNP could be attributed to some nonspecific properties of this agent,8 we repeated our experiments (n=8 for NIF and n=8 for Cs+) using the NO donor SIN-1 (50 μmol/L).
In Fig 6A⇓, two original rate traces are shown, one from the NIF group (trace a) and one from the Cs+ group (trace b); data for all experiments are summarized in Fig 6B⇓. SIN-1 increased the beating rate by 30±7 bpm in the NIF group and 33±5 bpm in the Cs+ group (P<.05 for either group), and this was fully reversed after washout. Application of NIF (0.2 μmol/L) or Cs+ (2 mmol/L) caused a comparable significant decrease in rate by 58±10 bpm (−32%) and 62±7 bpm (−33%), respectively (P=NS for differences between the two groups). In the presence of NIF, SIN-1 increased the beating rate by 51±12 bpm (P<.05). After the application of Cs+, however, the positive chronotropic effect of SIN-1 was abolished (+1±1 bpm, P=NS).
In summary, the increase in beating rate in response to SIN-1 was maintained in the presence of NIF but was completely prevented by blocking If with Cs+.
Effect of SNP and SIN-1 on If in Single SAN Pacemaker Cells
Consistent with previous reports,26 the control amplitude of If varied in different cells from −5 to −113 pA for the first hyperpolarizing voltage-clamp pulse (−40 to −70 mV) and from −10 to −242 pA for the second pulse (−40 to −100 mV).
Effect of SNP on the Amplitude of If
After exposure to SNP, the amplitude of If activated by the first pulse increased in all but one cell, whereas all cells showed an increase in If in response to the second pulse (in one cell, the patch was lost before the recording at 10 minutes). With the first pulse, the average increase in If with SNP was 48±21% at 3 minutes, 85±20% at 5 minutes, and 134±19% at 10 minutes (P<.05, Fig 7B⇓). The corresponding values with the second pulse were 193±38%, 213±33%, and 254±38% (P<.05, Fig 7B⇓).
Washout of the NO donor was attempted in six cells: in three cells, a full reversal of the amplitude of If was observed; in two, the patch was lost after the solution was changed; and in one, the magnitude of the current was not back to the control value after 25 minutes.
Effect of Cs+ on If in the Presence of SNP
The stimulation of If by 5 μmol/L SNP was suppressed 5 minutes after the application of 2 mmol/L CsCl in the presence of SNP (from 234±19% to 30±12% of the control value for the first pulse and from 354±38% to 78±36% of the control value for the second pulse, P<.05 for both pulses) (Fig 7A⇑ and Fig 7B⇑). After 10 minutes of exposure to Cs+ in the presence of SNP, If could not be elicited by the first pulse in 50% of the cells (average amplitude, 24±16% of the control value; P<.05; Fig 7B⇑), whereas the mean amplitude of If with the second pulse was 27±8% of the control value (P<.05, Fig 7B⇑). Ten minutes after Cs+ washout, the amplitude of If was 242±24% of the control value during the first pulse and 247±29% of the control value during the second pulse.
Effect of SIN-1 on If and ICa-L
SIN-1 (n=4) consistently increased the amplitude of If in all studied cells (If amplitude averaged 278±115% of the control value at 3 minutes and 336±194% after 5 minutes at −100 mV), but it did not stimulate ICa-L (ICa-L amplitude was 97±10% of the control value at 3 minutes and 94±12% after 5 minutes of exposure to SIN-1).
In summary, in isolated SAN cells the amplitude of the pacemaker current, If, was increased by SNP or SIN-1 (5 μmol/L). This effect was markedly and reversibly suppressed by 2 mmol/L Cs+. In contrast, the amplitude of ICa-L was not increased.
The new findings from this study are as follows: (1) NO donors modulate mammalian heart rate in a concentration-dependent biphasic fashion, with a gradual increase in beating rate for low concentrations and a decrease in beating rate for high concentrations. (2) The positive chronotropic effect appears to be NO-mediated and cGMP dependent. (3) The increase in beating rate with NO donors is maintained in the presence of the L-type Ca2+ channel antagonist NIF, but it is virtually abolished after If blockade. (4) Direct recordings in rabbit isolated SAN cells showed a marked, reversible, and Cs+-sensitive increase in If with SNP or SIN-1, whereas ICa-L was not increased.
Chronotropic Effect of NO Donors
Previous studies of the effect of exogenous NO on the beating rate of mammalian heart in vitro produced inconsistent results. In the isolated rat right atrium, Kennedy et al6 showed that concentrations of SIN-1 from 0.01 μmol/L to 0.3 mmol/L did not significantly affect the beating rate, whereas higher concentrations decreased it. Conversely, Pabla and Curtis7 noted an increase in the beating rate of 20% in Langendorff-perfused rat hearts in response to 10 μmol/L SNP. Furthermore, in this preparation, pharmacological blockade of endogenous NO synthase was associated with a reduction in beating rate by 15%.7 This indicates that endogenously released NO might exert a tonic positive chronotropic effect that can be mimicked by NO donors.
The greater magnitude of the positive chronotropic response to SNP compared with SIN-1 (Fig 1⇑) may be consistent with the different mechanisms by which these donors release NO. SNP has been reported to generate NO intracellularly,27 whereas SIN-1 releases NO in aqueous solution, and this is rapidly scavenged in oxygenated buffer.8 Since NO+ can serve as a biologically relevant intermediate of NO21 and since the modulation of membrane channels by NO+ can differ from that by the free radical NO,19 it was important to evaluate the chronotropic effect of an NO+-donating compound. Interestingly, we found that incremental concentrations of the S-nitrosothiol SNAP9 can elicit a progressive increase in beating rate similar to that produced by the NO donors SNP or SIN-1.8 9 However, unlike SNP or SIN-1, SNAP did not produce a persistent negative chronotropic effect in high concentrations (Fig 3⇑). Thus, an increase in beating rate could be elicited by both an S-nitrosothiol and NO donors (at least in nanomolar to micromolar concentrations). This is consistent with data showing that S-nitrosothiols can serve as guanylyl cyclase–stimulating intermediates of NO and NO donors.20 21
Increase in Beating Rate Is Due to NO and Occurs via a cGMP-Dependent Mechanism
In many tissues, NO is known to exert its effects through the stimulation of guanylyl cyclase and the increase in cGMP.1 2 Our findings provide evidence for the involvement of NO-cGMP pathways in the positive chronotropic effect of NO donors. In particular, the enhancement of the chronotropic response to SIN-1 in the presence of SOD (Fig 1⇑) is consistent with the primary involvement of NO. Furthermore, we show that inhibition of endogenous guanylyl cyclase by LY83583 or ODQ prevents the increase in beating rate with SIN-1 (Fig 4B⇑ and 4C⇑), whereas the membrane-permeable analogue of cGMP, 8-Br-cGMP, can mimic it (Fig 3⇑). Finally, in the presence of 8-Br-cGMP, SIN-1 did not produce an additional positive chronotropic effect (Fig 4A⇑). These data are consistent with the involvement of cGMP in eliciting the positive chronotropic response to NO donors.
Functional Evidence That If Mediates the Positive Chronotropic Response to NO
Positive Chronotropic Effect of NO Donors Is Abolished in the Presence of Cs+ or ZD7288
If is a highly modulated current that plays an important role in maintaining pacemaker activity10 26 28 and in mediating the chronotropic response to autonomic agonists.26 28 Consistent with other reports,11 29 we found that the reduction in spontaneous beating rate of SAN/atrial preparations was greater with ZD7288 (1 μmol/L) than with Cs+ (2 mmol/L). Both of these blockers of If, however, were equally effective in preventing the positive chronotropic effect of the NO donor SNP (Fig 5⇑). Likewise, the increase in beating rate in response to SIN-1 could not be elicited in the presence of Cs+ (Fig 6⇑).
The ability of If blockers to prevent the increase in the beating rates of SAN/atrial preparations in response to SNP and SIN-1 indicates that (1) the positive chronotropic effect of NO results from the modulation of If in cardiac pacemaker cells and (2) the mechanism underlying the positive chronotropic effect is common for both NO donors.
Positive Chronotropic Effect of NO Donors Is Intact in the Presence of NIF
ICa-L is essential for myocardial contraction and for pacemaking in the SAN.26 In isolated pacemaker cells, this current is selectively blocked by NIF.13 The lack of attenuation of the NO-induced increase in the beating rate after pretreatment with NIF (Figs 5⇑ and 6⇑) indicates that stimulation of ICa-L in the cardiac pacemaker cells is unlikely to play a major role in the positive chronotropic effect of exogenous NO.
Stimulation of If in Isolated Pacemaker Cells
In isolated pacemaker cells, SNP or SIN-1 (5 μmol/L) caused a time-dependent increase in If (Fig 7⇑), which was suppressed by 2 mmol/L CsCl. This is consistent with a recent finding by Janigro et al,30 who showed that the If-like current in endothelial cells of the blood-brain barrier was markedly increased by low concentrations of SNP (from 1 to 10 μmol/L) and by 1 μmol/L SIN-1. Our results indicate that the increase in the beating rate in response to exogenous NO is primarily mediated by stimulation of If and not ICa-L. This is in keeping with data from other groups showing that NO donors have no effect on basal ICa-L in isolated cells from the SAN18 or atrioventricular node.31
We have shown that the positive chronotropic effect of NO donors can be mimicked by increasing concentrations of a membrane-permeable analogue of cGMP. Interestingly, DiFrancesco28 has demonstrated that If can be stimulated by cGMP in a concentration-dependent fashion. These data are consistent with our hypothesis that activation of the NO-cGMP-If pathway is responsible for the chronotropic effect of NO donors.
Our data provide evidence for stimulation of If by exogenous NO in rabbit isolated pacemaker cells; the work by Han et al18 showed that NO participates in the cholinergic inhibition of isoproterenol-stimulated ICa-L in the same preparation. This suggests that NO can play an important role in promoting both the positive chronotropic effects32 and the heart rate deceleration associated with vagal reflexes.33 34
NO donors are widely used in cardiology, and our results suggest that they can have a biphasic concentration-dependent effect on pacemaking in the heart. Several in vivo observations support our findings on the SAN/atrial preparation. For instance, low doses of molsidomine (the prodrug of SIN-1) can increase heart rate without significantly affecting arterial blood pressure.35 Likewise, the intracoronary injection of a low dose of SNP has been shown to increase the rate of canine hearts in situ in the absence of changes in arterial pressure.36 Conversely, a slight reduction in heart rate was observed when 50-fold-higher doses of SNP were used in a similar experiment in humans.37
From our results using exogenous NO, it could be extrapolated that stimulation of If by endogenous NO might play a part in the sinus tachycardia that accompanies pathological conditions associated with an increase in both sympathetic activity and myocardial production of NO (eg, septic shock and heart failure).38 39 Moreover, If has been recently found in ventricular myocytes from diseased human hearts,40 suggesting that our findings might have wider implications for the role of NO in the performance of the failing heart.
Selected Abbreviations and Acronyms
|8-Br-cGMP||=||8-bromoguanosine 3′:5′-cyclic monophosphate|
|ICa-L||=||L-type Ca2+ current|
|If||=||hyperpolarization-activated inward current|
|NO||=||nitric oxide (and its congeners)|
We gratefully acknowledge the support of the British Heart Foundation and the Garfield Weston Trust. Dr Musialek is a recipient of an Overseas Research Award (UK) and is supported by the Wellcome Trust and by Hoechst Pharmaceuticals, Germany. Dr Lei is supported by the Medical Research Council, UK.
- Received November 22, 1996.
- Accepted April 15, 1997.
- © 1997 American Heart Association, Inc.
Kelly RA, Balligand J-L, Smith TW. Nitric oxide and cardiac function. Circ Res. 1996;79:363-380.
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