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

Abstract

Cardiac contraction during systole is dependenton action potential-triggered Ca²⁺ release from the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) channels. SR Ca²⁺ release can also occur spontaneously during diastole, which causes a decrease in Ca²⁺ content within the SR and contributes to arrhythmogenesis. Here, we use measurements of cytosolic Ca²⁺ and intra-SR Ca²⁺ ([Ca²⁺]_SR) to examine how RyR sensitization alters spontaneous SR Ca²⁺ release events in rabbit ventricular myocytes. RyR sensitization with caffeine (250 μM) increased the open probability of single RyR channels, increased the initial frequency and amplitude of local SR Ca²⁺ release events (Ca²⁺ sparks), and decreased the [Ca²⁺]_SR level where Ca²⁺ sparks terminated. In intact myocytes, caffeine applied during rest after steady-state electrical stimulation increased the frequency of spontaneous Ca²⁺ waves and decreased the [Ca²⁺]_SR level where waves terminated. These effects caused a marked loss of SR Ca²⁺ content. Therefore, increasing RyR activity has complex effects on cardiac function. Increased RyR activity during systole is beneficial as it increases SR Ca²⁺ release and contractile strength. However, increased RyR activity during diastole produces spontaneous, arrhythmogenic Ca²⁺ release events that lower SR Ca²⁺ content and subsequently decrease contractility.

Introduction

Cardiac excitation-contraction coupling (ECC) is triggered by the Ca²⁺-induced Ca²⁺ release (CICR) mechanism.¹ During ECC, depolarization of the sarcolemma membrane opens L-type Ca²⁺ channels (LTCCs), and the subsequent Ca²⁺ influx activates sarcoplasmic reticulum (SR) Ca²⁺ release channels, ryanodine receptors (RyRs), and causes a massive release of Ca²⁺ into the cytosol. The global cytosolic Ca²⁺ transient that triggers contraction is composed of the temporal and spatial summation of thousands of individual CICR events that occur in a signaling microdomain called the dyadic cleft. In this region, the LTCCs in the t-tubule membrane (an invagination of the surface sarcolemma) come in close proximity to RyR clusters in the junctional SR, and form a specialized signaling complex termed the Ca²⁺ release unit (Fig. 1A). The concerted opening of a cluster of RyRs generates the fundamental CICR event of ECC, the Ca²⁺ “spark”,² which is visualized using confocal fluorescence microscopy and high-affinity Ca²⁺ indicators (e.g., rhod-2) as a localized increase in cytosolic fluorescence (Fig. 1A and B upper). Alternatively, the Ca²⁺ released from the SR during a Ca²⁺ spark can be measured directly using low-affinity Ca²⁺ indicators (e.g., fluo-5N) entrapped within the SR; this reciprocal intra-SR Ca²⁺ depletion is termed a Ca²⁺ “blink”^3,4 (Fig. 1A and B lower).

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.

The intrinsic sensitivity of the RyR to cytosolic Ca²⁺ underlies the process of CICR and therefore cardiac contraction. However, recent evidence shows that channel gating is also regulated by the Ca²⁺ concentration within the lumen of the SR ([Ca²⁺]_SR). In planar lipid bilayer experiments channel open probability is markedly enhanced by increasing Ca²⁺ on the luminal side of the channel,⁵ an effect that is removed following enzymatic digestion of the luminal side of the channel protein.⁶ In the cellular environment this property of the channel manifests as an increase in Ca²⁺ spark frequency and an increase in the number of Ca²⁺ release units that respond to an action potential as Ca²⁺ content within the SR rises (e.g., during adrenergic stimulation). However, elevated SR Ca²⁺ content also leads to spontaneous Ca²⁺ release events during diastole, including arrhythmogenic Ca²⁺ waves. Ca²⁺ waves occur when the Ca²⁺ released during a spontaneous Ca²⁺ spark diffuses to neighboring release junctions, triggers CICR, and forms a regenerative wave of CICR that travels from release junction to release junction.⁷ This form of diastolic Ca²⁺ release is highly arrhythmogenic as the Ca²⁺ released during the wave is extruded by the electrogenic Na⁺-Ca²⁺ exchanger (NCX), which depolarizes the sarcolemma and leads to spontaneous electrical activity such as delayed afterdepolarizations.^8,9 Furthermore, diastolic Ca²⁺ release depresses cardiac contractility by impairing relaxation, as well as by decreasing SR Ca²⁺ content and Ca²⁺ release during subsequent systole.

In a recent study,¹⁰ we investigated how sensitization of RyR channels alters Ca²⁺ sparks and SR Ca²⁺ release in rabbit ventricular myocytes. Using a low dose of the RyR agonist caffeine (250 μM), we sensitized RyRs and monitored SR Ca²⁺ release using simultaneous measurements of cytosolic Ca²⁺ ([Ca²⁺]_i) and [Ca²⁺]_SR. These experiments showed that RyR sensitization alters both activation and termination of Ca²⁺ sparks, and both of these mechanisms contribute to an initial increase in global Ca²⁺ transient amplitude during ECC. However, during steady-state pacing, the initial increase in systolic SR Ca²⁺ release (positive inotropic effect) as well as the increased frequency and amplitude of diastolic Ca²⁺ sparks led to drastically reduced SR Ca²⁺ content and a subsequent decrease in systolic SR Ca²⁺ release (negative inotropic effect). In the follow-up study presented here, we tested whether RyR sensitization alters the properties of an additional form of diastolic SR Ca²⁺ release: spontaneous, arrhythmogenic Ca²⁺ waves. We find that RyR sensitization increases the occurrence of Ca²⁺ waves, lowers the [Ca²⁺]_SR level where release terminates during a wave, and leads to a substantial loss of Ca²⁺ from the SR during diastole.

Results and Discussion

In this investigation we sensitized the cardiac RyR using 250 μM caffeine, and examined spontaneous Ca²⁺ release using measurements of [Ca²⁺]_i and [Ca²⁺]_SR. As shown in Figure 2A, application of 250 μM caffeine to a single cardiac RyR channel incorporated into a planar lipid bilayer increased the open probability of the channel without altering single channel conductance. This concentration of caffeine increased the open probability of RyR channels (127% increase from 0.026 ± 0.009 to 0.059 ± 0.013, p < 0.05, n = 5) by reducing the mean closed time (49% decrease from 128.7 ± 25.3 ms to 65.6 ± 46.4 ms, p < 0.05, n = 5) without altering the mean open time (3.6 ± 1.1 ms to 3.9 ± 1.5 ms). In the cellular environment, where RyRs function cooperatively as release clusters, this increase in channel sensitivity in the presence of 250 μM caffeine is observed as an initial increase in Ca²⁺ spark frequency^10–12 and amplitude (Fig. 2B, red traces), and a decrease in the [Ca²⁺]_SR level where Ca²⁺ sparks terminate (Fig. 2B and green traces). We next examined the effects of RyR sensitization on spontaneous Ca²⁺ waves in intact rabbit ventricular myocytes. As shown in the sequence of two-dimensional confocal images in Figure 3A, a Ca²⁺ wave is observed as a propagating increase in [Ca²⁺]_i (red images) or as the reciprocal propagating [Ca²⁺]_SR depletion (green images). Figure 3B presents the [Ca²⁺]_i and [Ca²⁺]_SR fluorescence profiles from 3 regions of the cell shown in Figure 3A (each separated by 20 μm) during electrical stimulation (1 Hz) as well as during rest after stimulation. Following cessation of electrical stimulation the cell was exposed to 250 μm caffeine and a spontaneous Ca²⁺ wave was observed (Fig. 3A). The fluorescence profiles from different areas of the cell superimpose during electrical stimulation, due to the synchronous nature of ECC in ventricular myocytes. As expected, this effect is evident in both the [Ca²⁺]_i and [Ca²⁺]_SR fluorescence profiles. In contrast, during the spontaneous Ca²⁺ wave the fluorescence profiles from the different regions no longer superimpose, and are temporally dissociated as the wave propagates through successive regions of the cell. This example also illustrates the marked loss of Ca²⁺ from the SR following a Ca²⁺ wave, which can be seen by comparing the initial [Ca²⁺]_SR prior to the Ca²⁺ wave with the [Ca²⁺]_SR remaining after the wave (Fig. 3B). Under control conditions, Ca²⁺ waves were not typically observed after rest from steady state pacing (2 waves in 24 cells). However, in the presence of 250 μM caffeine Ca²⁺ waves were more frequent (16 waves in 24 cells), which caused a substantial decrease in total SR Ca²⁺ content after 10 s of rest from pacing (48 ± 9% decrease in [Ca²⁺]_SR in the presence of 250 μM caffeine versus 15 ± 4% under control conditions, n = 5, p < 0.05). Similar to the effect of increased Ca²⁺ spark frequency in the presence of caffeine, the increased propensity for spontaneous Ca²⁺ waves reflects RyRs which are sensitized to release activation.¹³ To examine if release termination is also altered during spontaneous Ca²⁺ waves, we examined the minimum [Ca²⁺]_SR level observed during Ca²⁺ waves. As rabbit ventricular myocytes did not typically exhibit Ca²⁺ waves under control conditions, we added 25 nM of the β-adrenoreceptor agonist isoproterenol to increase activity of the sarcoplasmic-endoplasmic reticulum Ca²⁺ ATPase, elevate SR Ca²⁺ content, and increase the probability of spontaneous Ca²⁺ waves. In an individual cell in the presence of 25 nM isoproterenol, the [Ca²⁺]_SR level where Ca²⁺ waves terminated was variable, independent of initial SR Ca²⁺ content (data not shown). This is in contrast to spontaneous Ca²⁺ sparks and Ca²⁺ release during ECC, where Ca²⁺ release terminates at a set [Ca²⁺]_SR irrespective of initial [Ca²⁺]_SR, ^4,10 and suggests that Ca²⁺ wave termination is a more complex phenomenon. As shown in the line-scan image and [Ca²⁺]_SR profile of a spontaneous Ca²⁺ wave in Figure 4A, the Ca²⁺ wave terminated with a substantial amount of Ca²⁺ remaining in the SR. Similar results were reported in atrial myocytes,¹⁴ where 45% of the original [Ca²⁺]_SR remained in the SR immediately following a Ca²⁺ wave. This observation contrasts with experimental evidence in permeabilized rabbit ventricular myocytes¹⁵ which showed nearly complete depletion of [Ca²⁺]_SR during spontaneous Ca²⁺ waves. However, our results are from intact cells, while the investigation of MacQuaide et al.¹⁵ used permeabilized conditions with elevated cytosolic Ca²⁺ (600 nM), which may promote RyR opening and more substantially deplete [Ca²⁺]_SR. ¹⁶ Furthermore, similar to our previous results on Ca²⁺ sparks and Ca²⁺ transients, RyR sensitization with 250 μM caffeine decreased the [Ca²⁺]_SR where release terminated during a Ca²⁺ wave (Fig. 4B and C). This is an important observation, because decreasing the [Ca²⁺]_SR level of release termination during a Ca²⁺ wave will increase cytosolic Ca²⁺ wave amplitude, increase Ca²⁺ extrusion via NCX, and exacerbate loss of Ca²⁺ from the SR. Furthermore, as the NCX is an electrogenic transporter (3 Na⁺ for 1 Ca²⁺), increased Ca²⁺ extrusion via NCX will depolarize the sarcolemmal membrane and increase the propensity for spontaneous electrical activity.

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.

In summary, our previous study¹⁰ and this work show that RyR sensitization has effects on both release activation and release termination. During systole in response to the action potential, RyR sensitization increases release junction recruitment and makes SR Ca²⁺ release highly synchronized. In addition, at each activated release junction during ECC, the amount of Ca²⁺ release increases due to a decrease in the [Ca²⁺]_SR level where release terminates. The effects of RyR sensitization during systole are therefore beneficial, and cause an increase in SR Ca²⁺ release and contractility. On the contrary, the effects of RyR sensitization during diastole have detrimental effects on ECC and cardiac function. Increasing sensitivity to release activation causes an increase in the frequency of diastolic Ca²⁺ release events such as spontaneous Ca²⁺ sparks and waves. The increased frequency of diastolic release events, combined with the increased amount of Ca²⁺ released during each release event (via the altered release termination level), causes substantial loss of [Ca²⁺]_SR during diastole. Therefore, when [Ca²⁺]_SR reaches a new steady-state following RyR sensitization, Ca²⁺ release is depressed even though RyRs are sensitized.¹⁰ Furthermore, the increased frequency and amplitude of diastolic release events increases electrogenic Ca²⁺ extrusion via NCX and increases the likelihood of arrhythmogenic delayed afterdepolarizations. As increasing evidence points to “sensitized” RyRs following activation of neurohormonal signaling pathways and during the progression of disease,^17–21 these results have important implications for the altered cellular Ca²⁺ homeostasis associated with these conditions.

Methods

Myocyte isolation

Rabbit ventricular myocytes were isolated from male New Zealand White Rabbits (2.5 kg) using retrograde Langendorff perfusion with Liberase Blendzyme 4 solution as previously described.¹⁰ All protocols were approved by the Institutional Animal Care and Use Committee.

Confocal microscopy

Ventricular myocytes were incubated with 5 μM fluo-5N/AM (Molecular Probes/Invitrogen, Carlsbad, CA) with 0.25% Pluronic F-127 for 2.5 hours at 37°C to load the SR with fluo+5N. For Ca²⁺ spark and blink measurements, myocytes were permeabilized with 0.005% saponin for 30 s, followed by application of the internal solution containing rhod-2.⁴ The internal cell solution contained (in mM): 100 K⁺ aspartate, 15 KCl, 5 KH₂PO₄, 5 MgATP, 0.35 EGTA, 0.12 CaCl₂, 0.75 MgCl₂, 10 phosphocreatine, 5 U/ml creatine phosphokinase, 8% dextran (M_r: 40,000), 10 HEPES, 0.04 rhod-2 tripotassium salt (Molecular Probes/Invitrogen); pH 7.2 with KOH. Free [Ca²⁺] and [Mg²⁺] of this solution were 150 nM and 1 mM, respectively.

Intact (fluo-5N loaded) myocytes were incubated with 5 μM rhod-2/AM (Molecular Probes/Invitrogen) for 10 minutes, followed by a 10 minute wash. The external solution contained (in mM): 135 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 D-Glucose,10 HEPES; pH 7.4 with NaOH. For Ca²⁺ wave measurements, intact myocytes were electrically stimulated at 1 Hz for 20 s, followed by 10 s of rest. During the rest period, cytosolic and/or intra-SR Ca²⁺ dynamics during spontaneous Ca²⁺ waves were measured in the presence or absence of 250 μM caffeine. In some experiments, 25 nM isoproterenol was added to increase [Ca²⁺]_SR and induce waves. Fluo-5N signals were corrected for the Ca²⁺-insensitive component of the fluorescence (F_Min) obtained after complete SR Ca²⁺ depletion with 10 mM caffeine, and are presented as F/F₀ (F − F_Min/F₀ − F_Min), where F₀ is resting or diastolic (1 Hz) fluorescence.

Laser scanning confocal microscopy was used to monitor changes in [Ca²⁺]_i (rhod-2) and [Ca²⁺]_SR (fluo-5N) in either frame-scan mode (60 fps at 512 × 128, A1R, Nikon, Japan) or line-scan mode (3 ms/line, Radiance 2000/MP, Biorad, UK). Fluo-5N was excited at 488 nm with emission collected at 515 ± 15 nm, while rhod-2 was excited at 543 nm with emission collected at >600 nm. All experiments were performed at room temperature (20–23°C).

Ryanodine receptor single channel recordings

Single RyR channel activity was measured using SR vesicles isolated from ventricular tissue, which were incorporated into planar lipid bilayers.²² Cs⁺ was used as the charge carrier for recordings, with the cis (cytosolic) and trans (luminal) chambers containing (in mM): 400 CsCH₃SO₃, 0.1 CaCl₂, 20 HEPES; pH 7.3. Free [Ca²⁺] in the cis-chamber was adjusted to 3 μM with EGTA. Single channel currents were sampled at 5 kHz using an Axopatch 200B amplifier (Axon Instruments/Molecular Devices Corporation) at a holding potential of −20 mV, and filtered at 1 kHz.

Statistics

Data are presented as individual observations or as mean ± SEM of n measurements. Statistical comparisons between groups were performed with the Student’s t test. Differences were considered statistically significant at p < 0.05.

Abbreviations

[Ca²⁺]_i

cytosolic calcium concentration

[Ca²⁺]_SR

intra-sarcoplasmic reticulum free calcium concentration

CICR

calcium-induced calcium release

ECC

excitation-contraction coupling

LTCC

L-type Ca²⁺ channel

NCX

Na⁺/Ca²⁺ exchanger

RyR

ryanodine receptor

SR

sarcoplasmic reticulum