Intracellular calcium dynamics and acceleration of sinus rhythm by beta-adrenergic stimulation

Abstract

Background: Recent evidence indicates that membrane voltage and Ca2+ clocks jointly regulate sinoatrial node (SAN) automaticity. Here we test the hypothesis that sinus rate acceleration by beta-adrenergic stimulation involves synergistic interactions between these clock mechanisms.

Methods and results: We simultaneously mapped intracellular calcium (Ca(i)) and membrane potential in 25 isolated canine right atrium, using previously described criteria of the timing of late diastolic Ca(i) elevation (LDCAE) relative to the action potential upstroke to detect the Ca2+ clock. Before isoproterenol, the earliest pacemaking site occurred in the inferior SAN, and LDCAE was observed in only 4 of 25 preparations. Isoproterenol infusion (1 micromol/L) increased sinus rate and shifted pacemaking site to superior SAN, concomitant with the appearance of LDCAE preceding the action potential upstroke by 98+/-31 ms. Caffeine had similar effects, whereas sarcoplasmic reticulum Ca2+ depletion with ryanodine and thapsigargin prevented isoproterenol-induced LDCAE and blunted sinus rate acceleration. Ca(i) transient relaxation time during isoproterenol was shorter in superior SAN (124+/-34 ms) than inferior SAN (138+/-24 ms; P=0.01) or right atrium (164+/-33 ms; P=0.001) and was associated with a lower sarcoplasmic reticulum Ca2+ ATPase pump to phospholamban protein ratio in SAN than in right atrium. Hyperpolarization-activated pacemaker current (I(f)) blockade with ZD 7288 modestly blunted but did not prevent LDCAE or sinus rate acceleration by isoproterenol.

Conclusions: Acceleration of the Ca2+ clock in the superior SAN plays an important role in sinus acceleration during beta-adrenergic stimulation, interacting synergistically with the voltage clock to increase sinus rate.

Figure 1

Identification of SAN. A, Intact canine RA preparation. The white shaded area is the SAN. The numbers show the V_m recording sites in B. B, V_m (blue) recording of a spontaneous sinus beat. V_m was recorded in SAN (sites 1-4), inferior (site 5), posterior (sites 6-10), and anterior (sites 11-15) to SAN. V_m of SAN only shows typical phase 4 DD (arrows). C, Masson's trichrome staining of SAN and adjacent RA. The general outline of the SAN is indicated with the black arrows. RAA, RA appendage; IVC, inferior vena cava; SVC, superior vena cava; ST, sulcus terminalis; A, anterior; P, posterior; S, superior; I, inferior.

Figure 2

Activation pattern of SAN and surrounding RA during baseline spontaneous sinus beat. A, Isochronal map. The number on each isochronal line indicates the time of activation (ms), with the earliest activation as time zero. The white shaded area is the SAN. B, The V_m (blue) and Ca_i (red) recordings from the superior (a), middle (b), inferior (c) SAN and RA (d) presented in A. Arrows point to LDCAE. C, Magnified view of Ca_i and V_m tracings of inferior SAN. Note that LDCAE (arrow) occurred prior to phase 0 of AP (0 ms), and much earlier than the p wave on ECG. D, Ca_i and V_m ratio maps at times from −40 ms before to 20 ms after phase 0 AP of C.

Figure 3

Activation pattern of SAN and surrounding RA during ISO infusion of 0.3 μmol/L. A, Isochronal map of V_m. The number on the each isochronal line indicates time (ms). White shaded area is the SAN. B, The V_m (blue) and Ca_i (red) recordings from the superior (a), middle (b), inferior (c) SA nodes and RA (d) presented in A. C, Magnified view of Ca_i and V_m tracings of superior SAN. Note the robust LDCAE (solid arrow) before phase 0 of AP (0 ms), which in turn was much earlier than onset of p wave on ECG. D, The V_m and Ca_i ratio maps at times from −60 ms before to 180 ms after phase 0 AP of C. The LDCAE (broken arrows in frame −40 and −20 ms) was followed by the Ca_i sinkhole during early diastole (solid arrow in frame 180 ms). E, (a) Ca_i and dCa_i/dt. (b) V_m and dV_m/dt. The onset of LDCAE and DD were marked with arrows.

Figure 4

The spatial changes of Ca_i and V_m around the leading pacemaker site (*) in SAN. A, The Ca_i and V_m ratio maps showing recording site. B, The changes of Ca_i and V_m tracings along the anterior (A)-posterior (P) direction. C, The changes of Ca_i and V_m tracings along the superior (S)-inferior (I) direction. D, The spatial change of LDCAE and DD slopes.

Figure 5

Co-localization of LDCAE and the leading pacemaker site. A, Upward shift of the leading pacemaker site with LDCAE during ISO infusion. (a) Ca_i ratio maps of SAN at each sinus rate. (b) Corresponding Ca_i tracings from superior (1, 2), middle (3, 4) and inferior (5, 6) SAN. At 95 bpm, the sites 4 and 5 had most prominent LDCAEs (asterisks). As sinus rate gradually increased, the sites of Ca_i elevation progressively moved upward. At the maximum sinus rate of 173 bpm, the site 2 had the most apparent LDCAE. B, Differential responses of different SAN sites to ISO. (a) The Ca_i and V_m tracings from inferior, middle, and superior SAN sites at different sinus rates. (b) The LDCAE and DD slopes of superior SAN at different sinus rates.

Figure 6

The differences of Ca_i physiology between SAN and RA. A, (a) Ca_i tracing from superior (1), middle (2) and inferior (3) SAN, and from anterior (4) and posterior RA (5). (b) The Ca_i tracing during baseline (left panels) and ISO infusion of 1 μmol/L (right panels). The earliest activation sites were inferior (3) and superior SAN (1) during baseline and ISO infusion, respectively. Arrow points to a rapid reduction of Ca_i at the superior SAN, which was followed by rapid onset of LDCAE (asterisk). B, The comparison of 90% Ca_i relaxation time among superior, inferior SAN and RA during baseline and ISO infusion. C, The Western analyses of SERCA2a (SER) and phospholamban (PLB) at superior SAN (SN-S), inferior SAN (SN-I) and RA. D, The SERCA2a/PLB ratio of all preparations analyzed.

Figure 7

The effect of ryanodine and ZD 7288 on intact canine SAN. A, Dose dependent decrease of heart rate with ryanodine infusion. B, The impaired ISO dose-dependent increase of heart rate by ryanodine infusion of 3 μmol/L. C, The Ca_i and V_m tracings during 3 μmol/L ryanodine alone infusion (left panels), concomitant 3 μmol/L ryanodine and 1 μmol/L ISO infusion (middle panels), and 10 μmol/L ryanodine, 200 nmol/L thapsigargin and 1 μmol/L ISO infusion (right panels). D, The Ca_i and V_m tracings during 3 μmol/L ZD 7288 infusion (left panels). ISO infusion of 1 μmol/L produced LDCAE (arrows) at the superior SAN in the presence of ZD 7288 (right panels). The Ca_i and V_m tracings were recorded from superior (1), middle (2) and inferior (3) SANs. Ryd, ryanodine; Thap, thapsigargin.

Figure 8

Spontaneous heart rate of canine intact SAN depends upon both Ca²⁺-related mechanisms and I_f current. Bars show a change in heart rate (% from baseline) induced by different pharmacological interventions. The grey bars show the changes during 3 μmol/L ZD 7288, 3 μmol/L ryanodine, and 10 μmol/L ryanodine plus 200 nmol/L thapsigargin infusion without ISO, while the black bars show the changes during 1 μmol/L ISO infusion.