Intracellular Ca2+ release contributes to automaticity in cat atrial pacemaker cells

Abstract

1. The cellular mechanisms governing cardiac atrial pacemaker activity are not clear. In the present study we used perforated patch voltage clamp and confocal fluorescence microscopy to study the contribution of intracellular Ca2+ release to automaticity of pacemaker cells isolated from cat right atrium. 2. In spontaneously beating pacemaker cells, an increase in subsarcolemmal intracellular Ca2+ concentration occurred concomitantly with the last third of diastolic depolarization due to local release of Ca2+ from the sarcoplasmic reticulum (SR), i.e. Ca2+ sparks. Nickel (Ni2+; 25-50 microM), a blocker of low voltage-activated T-type Ca2+ current ((ICa,T), decreased diastolic depolarization, prolonged pacemaker cycle length and suppressed diastolic Ca2+ release. 3. Voltage clamp analysis indicated that the diastolic Ca2+ release was voltage dependent and triggered at about -60 mV. Ni2+ suppressed low voltage-activated Ca2+ release. Moreover, low voltage-activated Ca2+ release was paralleled by a slow inward current presumably due to stimulation of Na+-Ca2+ exchange (INa-Ca). Low voltage-activated Ca2+ release was found in both sino-atrial node and latent atrial pacemaker cells but not in working atrial myocytes. 4. These findings suggest that low voltage-activated ICa,T triggers subsarcolemmal Ca2+ sparks, which in turn stimulate INa-Ca to depolarize the pacemaker potential to threshold. This novel mechanism indicates a pivotal role for ICa,T and subsarcolemmal intracellular Ca2+ release in normal atrial pacemaker activity and may contribute to the development of ectopic atrial arrhythmias.

Figure 1. Ca²⁺ sparks precede the action potential in spontaneously beating latent atrial pacemaker cells

Simultaneous recordings of membrane voltage (V_m; A) and subsarcolemmal Ca²⁺ concentration ([Ca²⁺]_ss; B). The lower traces in each panel show amplified recordings of diastolic voltage (A) and [Ca²⁺]_ss (B). The amplified traces in B show an increase in [Ca²⁺]_ss during the late pacemaker depolarization preceding the action potential (‘Ca²⁺ pedestal’). [Ca²⁺]_ss started to increase at threshold potentials of about -58 mV. C, local [Ca²⁺] transients recorded by averaging over a short distance (1 μm) of the scanned line intersecting with a site displaying localized Ca²⁺ release (arrow in D). The arrows mark individual Ca²⁺ sparks. D, surface plot representation of the linescan image illustrating changes in [Ca²⁺]_ss during the diastolic depolarization preceding the first action potential (indicated by the horizontal bar in C). The inset in D indicates the positioning of the line.

Figure 2. Pacemaker Ca²⁺ sparks are triggered by a voltage-dependent mechanism

Changes in [Ca²⁺]_ss (line plot presentation, A; linescan image, C) and membrane currents (B) in a latent atrial pacemaker cell in response to the voltage clamp protocol shown below the current trace. A depolarizing voltage clamp ramp from -70 to -45 mV (400 ms) prior to the voltage step to +10 mV (150 ms) elicited LVCR which reproduced the diastolic Ca²⁺ pedestal seen in spontaneous pacemaker cells (left panels). In the same cell, when the voltage ramp was omitted (right panels), no sparks were detected prior to the [Ca²⁺]_ss transient elicited by the step depolarization. Prolongation of the interval between beats also did not result in detection of spontaneous Ca²⁺ sparks (not shown).

Figure 3. Nickel inhibits diastolic Ca²⁺ release in spontaneously active and voltage clamped pacemaker cells

A, Ni²⁺ (50 μM) reversibly slowed the rate of a spontaneously beating latent atrial pacemaker cell by selectively decreasing the slope of the late phase of diastolic depolarization. B, Ni²⁺ (50 μM) inhibited the diastolic Ca²⁺ pedestal (ii, and surface plots in iii and iv) without a significant reduction of the [Ca²⁺]_ss transient triggered by the action potential (i). Spontaneous [Ca²⁺]_ss transients were aligned to the point of maximum rate of rise. ctrl, control. C, in another voltage clamped pacemaker cell, Ni²⁺ (25 μM) inhibited LVCR initiated at -59 mV. Peak I_Ca,L elicited by the step depolarization was unaffected by Ni²⁺. D, [Ca²⁺]_ss and membrane current in response to a ramp-step depolarization in a third latent atrial pacemaker cell. During the ramp depolarization the current deviated from the expected linear change in background current (dashed line). The onset of inward current coincided with the rise in [Ca²⁺]_ss indicative of I_Na-Ca stimulation.

Figure 4. LVCR is present in primary pacemakers from the SA node, but absent in non-pacemaker atrial muscle cells

Response of membrane current (i) and [Ca²⁺]_ss (ii) to a ramp-step depolarization (left panels) or a continuous ramp (right panels) in a non-pacemaker atrial muscle cell (A and B) and a SA node pacemaker cell (C and D). In the regular atrial muscle cell, Ca²⁺ release was triggered at -36 mV. In the SA node pacemaker cell, two components of Ca²⁺ release were elicited at -52 and -36 mV.