Changes in intra-luminal calcium during spontaneous calcium waves following sensitization of ryanodine receptor channels

Abstract

Cardiac contraction during systole is dependent on action potential-triggered Ca(2+) release from the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) channels. SR Ca(2+) release can also occur spontaneously during diastole, which causes a decrease in Ca(2+) content within the SR and contributes to arrhythmogenesis. Here, we use measurements of cytosolic Ca(2+) and intra-SR Ca(2+) ([Ca(2+)](SR)) to examine how RyR sensitization alters spontaneous SR Ca(2+) release events in rabbit ventricular myocytes. RyR sensitization with caffeine (250 microM) increased the open probability of single RyR channels, increased the initial frequency and amplitude of local SR Ca(2+) release events (Ca(2+) sparks), and decreased the [Ca(2+)](SR) level where Ca(2+) sparks terminated. In intact myocytes, caffeine applied during rest after steady-state electrical stimulation increased the frequency of spontaneous Ca(2+) waves and decreased the [Ca(2+)](SR) level where waves terminated. These effects caused a marked loss of SR Ca(2+) content. Therefore, increasing RyR activity has complex effects on cardiac function. Increased RyR activity during systole is beneficial as it increases SR Ca(2+) release and contractile strength. However, increased RyR activity during diastole produces spontaneous, arrhythmogenic Ca(2+) release events that lower SR Ca(2+) content and subsequently decrease contractility.

Figure 1

The Ca²⁺ release unit of ventricular myocytes. (A) Illustration of a Ca²⁺ release unit highlighting the close proximity of L-type Ca²⁺ channels (LTCC) in the t-tubule membrane and RyR clusters in the junctional SR membrane. A spontaneous Ca²⁺ spark is observed using the high-affinity Ca²⁺ indicator rhod-2 as an increase in cytosolic fluorescence (red), or with the low-affinity Ca²⁺ indicator fluo-5N as an intra-SR Ca²⁺ depletion (Ca²⁺ blink, green). (B) 3-dimensional (time, width, amplitude) fluorescence profiles of a Ca²⁺ spark (red) and a Ca²⁺ blink (green). Profiles correspond to the average of 40 simultaneously recorded Ca²⁺ spark/Ca²⁺ blink pairs from permeabilized ventricular myocytes.

Figure 2

RyR sensitization increases single channel activity and Ca²⁺ spark amplitude, and decreases the [Ca²⁺]_SR termination level of Ca²⁺ sparks. (A) Representative traces of a single RyR channel incorporated into a planar lipid bilayer under control conditions (left) and in the presence of 250 μM caffeine (right). C represents the closed state and O represents the open state of the channel. (B) Representative confocal line scan images of a cytosolic Ca²⁺ spark (red image) and its corresponding intra-SR Ca²⁺ blink (green image) recorded in a permeabilized ventricular myocyte in the absence (left) and presence (right) of 250 μM caffeine. Presented between images are averaged (n = 10) fluorescence (F/F₀) profiles of Ca²⁺ sparks (red) and Ca²⁺ blinks (green) in the presence and absence of 250 μM caffeine.

Figure 3

Simultaneous cytosolic and intra-SR Ca2+ measurements during spontaneous Ca2+ waves. (A) 2-Dimensional images of [Ca2+]i (rhod-2, upper, red) and [Ca2+]SR (fluo-5N, lower, green) at rest (t = 7.8 s) and during propagation of a spontaneous Ca2+ wave in an intact ventricular myocytes in the presence of 250 μM caffeine (shown at 8.3, 8.5 and 8.7 s). (B) Cytosolic (red traces) and intra-SR (green traces) fluorescence profiles of 2 Ca2+ transients during ECC (arrows denote electrical stimulation), followed by rest from pacing. During rest, 250 μM caffeine was applied to sensitize RyRs, and a spontaneous Ca2+ wave propagated through the cell (this wave is shown in (A)). Profiles in (B) correspond to fluorescence from 5 μm-wide regions of interest (gray boxes 1, 2, 3 in illustration to left of profiles). Each region of interest was separated by 20 μm. Circles in (B) represent time where images in (A) were acquired.

Figure 4

RyR sensitization decreases the minimum [Ca2+]SR level during a Ca2+ wave. Example line-scan images and fluorescence profiles of 2 Ca2+ transients during ECC (arrows denote electrical stimulation) followed by an intra-SR Ca2+ depletion wave in an intact ventricular myocyte in the absence (A) and presence (B) of 250 μM caffeine. Profiles represent spatially averaged fluorescence over the width (y-axis) of the line-scan images. For analysis of wave kinetics and wave nadir, fluorescence was averaged along a single line positioned parallel to the wavefront, as indicated by break marks. (C) Summary data showing [Ca2+]SR prior to waves and the minimum [Ca2+]SR level (wave nadir) during waves in the absence and presence of 250 μM caffeine. All experiments shown were performed in the presence of 25 nM isoproterenol to increase [Ca2+]SR and induce Ca2+ waves. F/F0 of 1 corresponds to diastolic [Ca2+]SR during electrical stimulation. *p < 0.05 versus control, n = 6.