Positive Chronotropic Actions of Parathyroid Hormone and Parathyroid Hormone–Related Peptide Are Associated With Increases in the Current, If, and the Slope of the Pacemaker Potential
Background The classic calciotropic hormone parathyroid hormone (PTH) and its paracrine factor parathyroid hormone–related protein (PTHrP) both increase heart rate.
Methods and Results We used standard electrophysiological techniques to study the effects of PTH and PTHrP on isolated rabbit sinus node, isolated canine Purkinje fibers, and disaggregated rabbit sinus node myocytes. Sinus node maximum diastolic potential, activation voltage, and amplitude were unchanged by PTH or PTHrP (P>.05). However, the slope of phase 4 and the automatic rate were increased at PTH and PTHrP ≥10 nmol/L (P<.05). Comparable results were seen in canine Purkinje fibers. We then used the perforated-patch technique to study the If pacemaker current in sinus node. PTH 12.5 nmol/L and PTHrP 12.5 to 18 nmol/L increased If at −65 mV by 68±41% (n=5) and 69±50% (n=5), respectively. Actions of both agents were reversible. The increase in If appeared to result from a change in maximal conductance and not a shift in the voltage dependence of activation.
Conclusions These observations provide, for the first time, direct electrophysiological support for the chronotropic actions of PTH and PTHrP. They suggest that classic hormones and paracrine factors can have multiple functions and that in the case of PTH and PTHrP, a newly recognized action is to alter automaticity directly.
Parathyroid hormone is best known as a regulator of extracellular calcium homeostasis, but it now appears that this product of the parathyroid glands is a multipurpose hormone with diverse effects on many cellular functions, including those of the heart (eg, see References 11 -3). Its genetic cousin, PTHrP, was discovered in the search for the cause of hypercalcemia of cancer,4–8 but it too is now recognized to have other physiological properties.9 In contrast to PTH, PTHrP does not circulate in the blood under normal circumstances, but it is present in virtually all cells. This observation has given rise to the concept that PTHrP is an autocrine/paracrine substance released and acting locally.10 Both PTH and PTHrP have potent actions on the cardiovascular system.1–3,9 The heart is of particular interest because PTHrP is a product of the cardiac myocyte and its release is stimulated by mechanical forces like stretch.9
PTH and PTHrP increase heart rate independently of autonomic reflexes.1,2,11–14 They also are positive inotropic agents.12 In a recent study, the inotropic effects of PTH and PTHrP at very low concentrations (0.1 to 10 nmol/L) were attributable to their direct effects to increase coronary blood flow and heart rate.10 Enhanced contractility appears to result primarily from these two actions and not from any direct effects on contractile force.
The mechanisms responsible for the actions of PTH and PTHrP on cardiac automaticity are not known. The purpose of this study was to test the hypothesis that PTH and PTHrP influence pacemaker properties in cardiac cells. We therefore investigated the actions of PTH and PTHrP on transmembrane potentials and automaticity of isolated canine Purkinje fibers and rabbit sinus node and on the If pacemaker current of disaggregated rabbit sinus node cells.
Male New Zealand White rabbits (1.3 to 1.8 kg) were injected with pentobarbital 30 mg/kg IV. For studies of isolated sinus node preparations, the right atrium was removed and pinned into a dissecting bath to facilitate identification and excision of the sinus node, as previously described.15 The sinus node preparations were then placed into the tissue bath and superfused with Tyrode’s solution containing (in mmol/L) NaCl 131, NaHCO3 18, KCl 4, CaCl2 2.7, MgCl2 0.5, NaH2PO4 1.8, and dextrose 5.5. The Tyrode’s solution was gassed with 95% O2/5% CO2, warmed to 37°C (pH 7.3±0.05, mean±SEM), and pumped through the tissue bath at a flow rate of 12 mL/min, with chamber content changed three times a minute. The bath was connected to ground with a 3 mol/L KCl/Ag/AgCl junction.
All preparations were impaled with 3 mol/L KCl-filled glass capillary microelectrodes with tip resistances of 10 to 30 MΩ. The electrodes were coupled by a Ag/AgCl junction to an amplifier with high-input impedance and input capacity neutralization (model Duo 773, World Precision Instruments). Transmembrane action potentials and V̇max were displayed on a digital storage oscilloscope (model 4074, Gould) or chart recorder (model 2400, Gould). The system was calibrated as previously described.16,17 Approximately 60 minutes of equilibration of the spontaneously beating sinus node preparations was permitted, and only those whose rates varied by ≤±5% were studied further. These were superfused with graded concentrations of PTH or PTHrP, and effects on spontaneous rate, MDP, activation voltage (the inflection point for the action potential upstroke) amplitude, and slope of phase 4 depolarization were determined.
For voltage-clamp experiments, sinus node myocytes were prepared as previously described.18 We used the permeabilized patch for clamping of isolated sinus node myocytes to reduce or eliminate rundown of ion currents. The method was originally described by Horn and Marty19 with nystatin and then modified by Rae et al20 to use amphotericin B. The initial 100 μm of the pipette tip was filled by suction with the pipette solution, and the rest of the pipette was backfilled with pipette solution containing amphotericin B (240 μg/ml) as reported by Gao et al.21
If is an inward current activated by hyperpolarization. It gives rise to a time-dependent increase in membrane slope conductance when activated. The most likely contaminating current on hyperpolarization is due to the deactivation of the delayed rectifier IK. This “tail current” should give rise to a decreasing slope conductance. If is blocked by 1 to 4 mmol/L cesium, whereas the tail current of IK is not.22 In a series of control experiments originating at a holding potential of −35 mV, confirmation that the current was, in fact, If was provided by the following: (1) there was an increase in slope conductance during the activation of the current and (2) the increasing inward current and the increase in slope conductance were eliminated by addition of 2 mmol/L cesium.
We used an Axopatch IB amplifier at 1 kHz filtering and the pClamp program for collection and analysis of data, respectively. The data were filtered in pClamp at 20 Hz. The pipette solution contained (in mmol/L) potassium aspartate 130, MgCl2 2, EGTA 11, NaHEPES 10, CaCl2 5, Na2ATP 2, and Na2GTP 0.1. The solution was titrated to pH 7.2 with the addition of 40 mmol/L KOH. The external solution contained (in mmol/L) NaCl 40, KCl 5.4, CaCl2 1.8, MgCl2 1, HEPES 5, and glucose 10, titrated to pH 7.4 with 2.0 mmol/L NaOH. All recordings were made at 34±0.5°C during any given experiment.
In a subset of experiments, mongrel dogs weighing 10 to 20 kg were anesthetized with sodium pentobarbital (30 mg/kg IV). Their hearts were removed through a left lateral thoracotomy and immersed in cold Tyrode’s solution. Free-running Purkinje fibers were dissected from the right and left ventricles and placed in a tissue bath superfused with Tyrode’s solution. For stimulated preparations, standard techniques were used to deliver 1.0-ms square-wave pulses at 2 times threshold through bipolar Teflon-coated silver electrodes.16,17
Action potentials were studied in Purkinje fibers driven at a cycle length of 2000 ms. After a 60-minute stabilization period in control Tyrode’s solution containing [K+]o=2.7 mmol/L, baseline data were recorded. The fibers then were superfused with Tyrode’s solution containing graded concentrations of PTH or PTHrP. The transmembrane potential characteristics recorded were the MDP, action potential amplitude, V̇max,16 APD50 and APD90, and the average rate of depolarization during phase 4 (phase 4 slope).
To study the effects of PTH or PTHrP on automaticity, Purkinje fibers superfused with Tyrode’s solution containing [K+]o=2.7 mmol/L were allowed to beat spontaneously. Only those whose rate varied by ±5% or less were used in experiments. After equilibration, fibers were exposed to PTH or PTHrP in graded concentrations.
Statistical analysis of action potential data were performed by ANOVA, and if the F value indicated P<.05, Bonferroni’s test was performed.23 Statistical analysis of If data was performed on the ratios of If amplitudes in PTH or PTHrP compared with that in control. The ratios were log transformed to more closely approximate a normal distribution. Either t tests or ANOVA was then used. Data are reported as mean±SEM. A value of P<.05 was considered significant.
Effects of PTH and PTHrP on the Sinus Node Action Potential and Automaticity
We first studied the effects of PTH on sinus node action potentials and automaticity. PTH had no effect on MDP or activation voltage, but it significantly increased the slope of phase 4 depolarization and spontaneous rate (Table 1⇓). A representative experiment is presented in Fig 1⇓. A comparable result was found for PTHrP (n=4). Here, the control slope of phase 4 was 66±6 mV/s, and rate was 120±6 bpm. At 10 and 100 nmol/L PTHrP, the slope of phase 4 increased to 82±7 and 92±9 mV/s, respectively, and the rate was 146±6 and 157±7 bpm (all values P<.05 versus control).
We then asked whether interventions known to modulate If or calcium current predictably altered automaticity and the slope of phase 4 in sinus node. Hence, we superfused four rabbit sinus node preparations with cesium 2 mmol/L and four others with verapamil 100 nmol/L. After equilibration with cesium or verapamil, respectively, the preparations were superfused with PTH 1 to 100 nmol/L in the continued presence of cesium or verapamil. As shown in Table 2⇓, verapamil reduced action potential amplitude, consistent with calcium channel blockade. A representative experiment (see Fig 2⇓) demonstrates the marked effect of cesium to suppress the action of PTH; in contrast, PTH retains its positive chronotropic effect in the presence of verapamil. Summary data are provided in Fig 3⇓. Whereas verapamil does not significantly alter the effect of PTH on phase 4 slope or rate, cesium significantly reduces the PTH-induced increases in both variables.
Effects of PTH and PTHrP on the If Current
We used the perforated-patch technique to study If, the inward current activated by hyperpolarization, which contributes to diastolic depolarization.24 Typical results with PTH 12.5 nmol/L are provided in Fig 4⇓. Panel A shows control currents activated on hyperpolarization from a holding potential of −35 mV. Even at −45 mV, there is a small, time-dependent increase in inward current. The amplitude of this current increases and the kinetics become more rapid as the hyperpolarization is increased to a maximum of −85 mV.
When PTH is applied, there is an increase in time-dependent current at all potentials (panel B) that is entirely reversible on washout (panel C). The amplitude of the time-dependent current for panels A through C is plotted against test potential in panel D for the 1.5-second voltage-clamp pulses of this experiment. These results indicate a clear, reversible increase in the amplitude of the time-dependent current at each potential tested. In a total of four experiments, PTH at 12.5 nmol/L increased the inward current activated by hyperpolarization at −65 mV by 68±41% (range, 13% to 130% increase). To examine the voltage dependence of PTH action on the time-dependent current, we plotted the ratio of the amplitude of the time-dependent current If in PTH to that in control (IPTH/ICon) against the test potential for our four experiments. No clear voltage dependence was observed (panel E), and statistical analysis with ANOVA indicated that the effect of voltage was not significant. However, when we t-tested the pooled PTH data against the pooled control values at all voltages, the difference was significant (P<.05).
We performed a similar set of experiments with PTHrP, one example of which is provided in Fig 5⇓. Again, panel A represents the control traces obtained with 2-second hyperpolarizing voltage-clamp steps from a holding potential of −35 mV. Panels B and C show that PTHrP 18 nmol/L reversibly increased the time-dependent current elicited with voltage-clamp hyperpolarization. The average increase in five experiments with PTHrP concentrations of 12.5 to 18 nmol/L at −65 mV was 69±50% (range, 12% to 221% increase). The isochronal current-voltage relation for panels A through C of this experiment is plotted in panel D. The percentage increase in time-dependent current was roughly equal at all potentials. When we plotted the ratio IPTHrP/ICon against the test potential for our five experiments, no clear voltage dependence was observed (panel E). We performed the same statistical tests as for the PTH data, above. ANOVA indicated no significant effect of voltage. A t test of the pooled PTHrP data against pooled controls at all voltages demonstrated that the effect of PTHrP was significant (P<.05). This suggests that both PTH and PTHrP may cause an increase in conductance rather than a voltage shift in activation. This was further examined by a two-pulse voltage-clamp protocol in which the first step is to the middle of the activation curve for If and the second step is near the top of the curve.24 If the increase in If is due to a positive shift of the If activation curve and not to an increase in maximal If conductance, then in the presence of PTHrP, there should be a larger time-dependent current in response to the first step and a smaller time-dependent current in response to the second step. In this experiment, we used three different voltages for the first step (−65, −75, or −85 mV). The second voltage step was to −110 mV. The data provided in Fig 6⇓ clearly indicate that the time-dependent current is larger in the presence of PTHrP in response to both voltage steps. This indicates a change in the maximal If current, ¯If. We obtained the same results in each of seven experiments (five with PTHrP and two with PTH) in which this two-pulse protocol was executed. The increases we observed were 1.36±0.12, 1.37±0.08, and 1.47±0.10 at −65, −75, and −85 mV, respectively, for the first pulse (all P<.05 versus control). The increases at −110 mV were 1.54±0.12, 1.49±0.11, and 1.57±0.12 for first pulses of −65, −75, and −85 mV, respectively (all P<.05 versus control). The effect of voltage was tested with ANOVA and found not to be significant.
Effects of PTH and PTHrP on Purkinje Fiber Automaticity
The fact that an agent alters impulse initiation or action potential characteristics in one tissue is not necessarily generalizable to other tissues. For this reason, we studied the effects of PTH and PTHrP on the action potential and automaticity of canine Purkinje fibers. PTH had no effect on maximum diastolic potential (control=−96±1.9 mV), activation voltage (control=−91±2.0 mV), overshoot (control=36±1.9 mV), V̇max (control=566±52 V/s), or repolarization (control APD50=242±8 ms, APD90=347±14 ms) of Purkinje fibers driven at a basic cycle length of 2000 ms. However, it increased the slope of phase 4 depolarization from a control of 4.4±0.5 mV/s to 5.6±0.6 mV/s at PTH 10 nmol/L (P<.05). This increase in phase 4 slope would be predicted to increase automaticity in Purkinje fibers as it did in sinus node. A comparable result was found for PTHrP; no effect was seen on any variable except for the slope of phase 4, which was 4.1±0.7 mV/s in control and 5.3±0.9 mV/s at PTHrP 10 nmol/L (n=6, P<.05). In spontaneously beating Purkinje fibers, both automatic rate and the slope of phase 4 were increased by both agonists. For both groups (n=6 for PTH and n=4 for PTHrP), no change was seen in MDP (control=−92 mV for both) or activation voltage (control =−82 mV for both). PTH 10 nmol/L increased the slope of phase 4 from a control of 3.4±0.7 to 4.6±0.7 mV/s and increased the rate from 10.1±2.1 to 17.8±3.0 bpm (both P<.05). Similarly, PTHrP 10 nmol/L increased phase 4 slope from 1.9±0.1 to 2.4±0.2 mV/s (P<.05) and rate from 10.0±1.5 to 13.2±0.6 bpm (P<.05).
The purpose of the present study was to investigate mechanisms that contribute to the increase in heart rate that occurs on exposure to PTH or PTHrP. The previously demonstrated positive chronotropic action of PTH10 could have been based on increases in vascular dilation, on autonomic interactions, and/or on a primary action on sinus node. The design of our experiments, using intact sinus node, isolated sinus node myocytes, and intact Purkinje fiber bundles obviates any possible effects on neuronal or vascular elements.
The studies with intact sinus node and Purkinje fibers show unequivocally that both PTH and PTHrP increase the slope of phase 4 depolarization while manifesting no other effect on the transmembrane action potential. This action would be consistent with an increase in automaticity, which in fact is demonstrated in Table 1⇑ and Fig 1⇑: PTH and PTHrP in the 10 nmol/L concentration range both increase beating rate. Multiple ionic currents contribute to the pacemaker potential. If, whose role in sinus node has been particularly emphasized,24 is blocked by cesium. Calcium current, too, contributes to impulse initiation, and parathyroid hormone is known to increase calcium current.25,26 Hence, the experiments reviewed in Fig 2⇑ were of particular importance to us in that they emphasize the relatively greater effect of cesium than of verapamil in blocking the positive chronotropic actions of PTH. Moreover, in one additional experiment (not shown here), we found that even 2 μmol/L verapamil could not block the positive chronotropic action of PTH. Experiments in intact preparations can, of course, only permit one to surmise ionic mechanisms. However, the voltage-clamp experiments, while not ruling out an effect of PTH and PTHrP on calcium current (previously reported by others25,26), indicate a clear effect on If.
If current, which activates on hyperpolarization in both Purkinje fibers and sinus node,24 is a major determinant of automaticity in cardiac pacemaker fibers.27 Our voltage-clamp experiments using the permeabilized patch technique point to a plausible explanation for the positive chronotropic effect of PTH and PTHrP. Both agents reversibly increase the magnitude of the inward current activated by hyperpolarization. Such increases in this current as with catecholamines are known to increase heart rate.28 Our results suggest an increase in maximal conductance rather than a voltage shift in activation. This result would suggest that its signaling pathway may differ from those of the β-agonists, in which a voltage shift occurs.29 As stated above, our observed increases in If current should increase automaticity but do not rule out the additional effects of PTH and PTHrP on other ion currents, such as ICa,L.25,26 It is for this reason that our experiments with cesium and verapamil on isolated sinus node assume added importance.
In conclusion, the electrophysiological observations in this study indicate possible mechanisms by which PTH and PTHrP lead to enhanced automaticity and increased heart rate. As such, our experiments add to the cumulative evidence pointing clearly to important cardiovascular actions of PTH and PTHrP. It is apparent that these calciotropic hormones have the capability to interact with cells in the heart, which are not classically recognized as targets. It is most likely that in such a situation, the active agent would be PTHrP, produced locally in the myocytes, rather than circulating PTH, which is produced only in the parathyroid glands.30 The significant effects of PTHrP call attention to potential roles this peptide may play in modulating cardiac function under conditions of stress.
Selected Abbreviations and Acronyms
|APD50||=||action potential duration to 50% repolarization|
|APD90||=||action potential duration to 90% repolarization|
|MDP||=||maximum diastolic potential|
|PTHrP||=||parathyroid hormone–related protein|
This study was supported by US Public Health Service–NHLBI grants HL-28958, HL-20558, and DK-32333. The authors express their gratitude to Dr Natalia Egorova for her assistance with some of the experiments and to Eileen Franey for her careful attention to the preparation of the manuscript.
- Received March 6, 1997.
- Revision received July 11, 1997.
- Accepted August 1, 1997.
- Copyright © 1997 by American Heart Association
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