Essential role of diastolic oscillatory potentials in adrenergic control of guinea pig sino-atrial node discharge

Abstract

Background: The diastolic oscillatory after-potential Vos and pre-potential ThVos play an essential role in the pacemaker mechanism of sino-atrial node (SAN). The aim of this study was to investigate whether these oscillatory potentials are also involved in adrenergic control of SAN discharge.

Methods: Vos and ThVos were visualized by superfusing guinea pig SAN in high [K+]o. The actions of adrenergic agonists on oscillatory potentials were studied by means of a microelectrode technique. Statistical significance was determined by means of Student's paired t-test.

Results: In non-spontaneous SAN, norepinephrine (NE) decreased the resting potential into a voltage range ("oscillatory zone") where increasingly larger ThVos appeared and initiated spontaneous discharge. In slowly discharging SAN, NE gradually increased the rate by increasing the amplitude and slope of earlier-occurring ThVos and of Vos until these oscillations fused with initial diastolic depolarization (DD1). In the presence of NE, sudden fast rhythms were initiated by large Vos that entered a more negative oscillatory zone and initiated a large ThVos. Recovery from NE exposure involved the converse changes. The beta-adrenergic agonist isoproterenol had similar actions. Increasing calcium load by decreasing high [K+]o, by fast drive or by recovery in Tyrode solution led to growth of Vos and ThVos which abruptly fused when a fast sudden rhythm was induced. Low [Ca2+]o antagonized the adrenergic actions. Cesium (a blocker of If) induced spontaneous discharge in quiescent SAN through ThVos. In spontaneous SAN, Cs+increased Vos and ThVos, thereby increasing the rate. Cs+ did not hinder the positive chronotropic action of NE. Barium increased the rate, as Cs+ did.

Conclusion: Adrenergic agonists: (i) initiate SAN discharge by decreasing the resting potential and inducing ThVos; (ii) gradually accelerate SAN rate by predominantly increasing size and slope of earlier and more negative ThVos; (iii) can induce sudden fast rhythms through the abrupt fusion of large Vos with large ThVos; (iv) increase Vos and ThVosby increasing cellular calcium; and (v) do not modify the oscillatory potentials by means of the hyperpolarization-activated current If. The results provide evidence for novel mechanisms by which the SAN dominant pacemaker activity is initiated and enhanced by adrenergic agonists.

Figure 1

Norepinephrine induces SAN discharge through ThV_os. In 13.6 mM [K⁺]_o, NE was administered during the recording of panels b-h at normal and at higher gain. In panel b, the rightward oblique arrows indicate ThV_os and the leftward oblique arrow V_os. The dash lines emphasize the change in slope of DD₁ past the peak of V_os, the peak being indicated by the short bars next to DD₁. The traces labeled with an empty and a filled star were superimposed in inset 1. In the higher gain c-f traces, the shaded areas emphasize the progressive growth of V_os. The c-g traces have been superimposed in inset 2, the arrow pointing to the shortening of the AP. The events during the recovery from NE exposure are illustrated in inset 3. The top trace was recorded also at higher gain (see rightward oblique arrow) and the ThV_os marked by an asterisk has been superimposed upon the previous AP (horizontal arrow). In panel 4, means and standard error of the mean are shown in control (empty columns) and in the presence of NE (filled columns). The parameters shown are the rate in min^-1, the action potential amplitude in mV (APa), the diastolic depolarization amplitude in mV (DDa), the amplitude of V_os in mV, the depolarizing slope of V_os(V_osds) and of ThV_os (ThV_osds) in mV s^-1, and force in mg. Asterisks indicate statistical difference with respect to control (P < 0.05).

Figure 2

Norepinephrine increases SAN discharge by increasing ThV_os and V_os. The SAN was spontaneously active in 13 mM [K⁺]_o. In panel a, the shaded areas emphasize V_os and ThV_os, as labeled. The dash lines extrapolate the depolarizing slope of ThV_os. NE was administered between the vertical arrows (b-g panels). Inset 1 shows the gradual increase of ThV_os (dots) and V_os (see rectangle) by NE. In inset 2, means and SEM in control and in the presence of NE are shown. Other explanations are as in the legend of Fig.1.

Figure 3

Sudden onset of fast discharge in the presence of isoproterenol. In panel a, the non-spontaneous SAN was driven at 6 min^-1 in 11.2 mM [K⁺]_o. Isoproterenol was administered between the downward arrows. In panel b, the first 3 APs were driven, but once the fast sudden rhythm started, the drive was discontinued. The sudden cessation of the fast rhythm is shown in panel d. In panel e, the same SAN was driven at 6 min^-1, but the drive was discontinued prior to isoproterenol administration. In panel f, the arrow points to the ThV_os that initiated the sudden fast rhythm. The fast rhythm ceased during recovery and a driven AP is shown in panel h. The means and SEM are shown in inset 1. Other explanations are as in the legend of Fig. 1.

Figure 4

Induction of sudden fast rhythm by V_os and ThV_os on lowering high [K⁺]_o. In panels A and B, [K⁺]_o was decreased while the same SAN was driven. The bars mark the peak of V_os. The downward arrows mark the ThV_os that initiated an AP followed by the fast rhythm. The horizontal arrow points to the stimulus artifact of the last driven AP. The traces labeled with a dot and a rhombus are shown at higher gain in inset 1. In inset 2, the circle labels a ThV_os that missed the threshold during the subsiding of the fast rhythm. In panel C, the upward oblique arrows indicate driven APs that initiated runs of fast discharge. The downward oblique arrows indicate ThV_os that initiated an AP followed by runs of fast discharge.

Figure 5

V_os, ThV_os and sudden fast discharge during recovery from high [K⁺]_o to Tyrode solution. V_os, ThV_os and sudden fast discharge during recovery from high [K⁺]_o to Tyrode solution. The SAN was quiescent in 19.2 mM [K⁺]_o and recovery in the Tyrode solution started at the downward arrow and continued for the rest of the figure. In panel A, subthreshold stimuli (15 V, 3 ms; leftward oblique arrows) were applied at 6 min^-1. Subthreshold ThV_os is shown at higher gain in inset 1. In panel B, the star indicates ThV_os leading to spontaneous discharge and the rightward oblique arrow points to the depolarising slope of a ThV_os that initiated an AP and fast rhythm. In the inset 2, the arrow points to the peak of V_os. The trace indicated by a horizontal line ending with two dots is shown at higher gain in inst 3, where the dots and the shaded areas emphasize the growth of V_os. In the inset 4, the subthreshold stimuli initiated ThV_os at the resting potential (upward arrow), but not during early DD₁ (leftward oblique arrow). The dots show ThV_os that began prior to the electrical stimulus.

Figure 6

Growth of V_os and ThV_os leading to sudden fast rhythm. The first part of A and B panels was recorded in 19.2 mM [K⁺]_o at normal and higher gain, respectively. Recovery in Tyrode solution began at the downward arrow and continued for the rest of the figure. The section of panel B between the two arrows is shown at greater time base in panel C. The dots emphasize the growth of V_os and the circles that of ThV_os.

Figure 7

Induction of fast rhythm by overdrive. A spontaneously discharging SAN was overdriven at 60 min^-1 for 30 s in panel A. In inset 1, the traces labeled with a star and a rhombus are shown superimposed in a, and the traces labeled with a rhombus and a dot are superimposed in b. In panel B, the SAN was overdriven at 120 min^-1 for 30 s. In inset 2, the traces labeled with a star and a rhombus were superimposed in a, and those with a rhombus and a dot in b.

Figure 8

Low [Ca²⁺]_o antagonizes the effects of NE. In panel a, the arrow points to the superimposition of the small deflection on the previous AP. NE was administered between the downward arrows. In panel e, the control trace labeled with a square was superimposed on the trace marked by a star. [Ca²⁺]_o was decreased as indicated (panes f-h). In panel g, the ThV_os that missed the threshold is labeled with a rhombus and the following AP has been superimposed on it (arrow) in the bottom trace recorded at higher gain. In panel h, the trace labeled by a star is the same trace labeled with the same symbol in panel e. Panel i was recorded in the presence of NE during recovery from low [Ca²⁺]_o.

Figure 9

Cesium does not suppress either V_os and ThV_os or SAN discharge. The APs recorded at normal and higher gain are shown in panel A (Control). Cs⁺ was administered as indicated above panel A. In inset 1, the traces labeled with a square and rhombus are superimposed, with the MDP cut off. Recovery is illustrated in panel B. Means and SEM are shown in the inset 3. Other explanations are in the text and as in legend of Fig. 1.

Figure 10

Norepinephrine increases the rate of discharge in the presence of Cs⁺. In high [K⁺]_o, Cs⁺ administration was initiated at the downward arrow and continued for the rest of the figure. NE was added to the Cs⁺ solution as indicated above traces b, c and d. Recovery in Cs⁺ solution (no NE) is shown in trace e. Means and SEM are shown in the inset 2 (Control, +cesium, +NE) and 3 (Control, +NE, +cesium). Other explanations are in the text and as in legend of Fig. 1.

Figure 11

Barium increases V_os and ThV_os slope and amplitude, and rate of discharge. In panel A, APs in high [K⁺]_o are shown at normal and higher gain. Ba²⁺ was administered as indicated above the top trace. The dots label ThV_os occurring gradually sooner during DD₂. In panel B, the recovery from Ba²⁺ exposure is shown. Inset 1 shows the changes in slope and amplitude of ThV_os during (top trace) and after barium exposure (bottom trace). In inset 2 and 3, the arrows indicate the direction of change during and after barium exposure, respectively. Means and SEM are shown in inset 4. Other explanations are in the text and as in legend of Fig. 1.