Redox modification of ryanodine receptors underlies calcium alternans in a canine model of sudden cardiac death

Abstract

Aims

Although cardiac alternans is a known predictor of lethal arrhythmias, its underlying causes remain largely undefined in disease settings. The potential role of, and mechanisms responsible for, beat-to-beat alternations in the amplitude of systolic Ca²⁺ transients (Ca²⁺ alternans) was investigated in a canine post-myocardial infarction (MI) model of sudden cardiac death (SCD).

Methods and results

Post-MI dogs had preserved left ventricular (LV) function and susceptibility to ventricular fibrillation (VF) during exercise. LV wedge preparations from VF dogs were more susceptible to action potential (AP) alternans and the frequency-dependence of Ca²⁺ alternans was shifted towards slower rates in myocytes isolated from VF dogs relative to controls. In both groups of cells, cytosolic Ca²⁺ transients ([Ca²⁺]_c) alternated in phase with changes in diastolic Ca²⁺ in sarcoplasmic reticulum ([Ca²⁺]_SR), but the dependence of [Ca²⁺]_c amplitude on [Ca²⁺]_SR was steeper in VF cells. Abnormal ryanodine receptor (RyR) function in VF cells was indicated by increased fractional Ca²⁺ release for a given amplitude of Ca²⁺ current and elevated diastolic RyR-mediated SR Ca²⁺ leak. SR Ca²⁺ uptake activity did not differ between VF and control cells. VF myocytes had an increased rate of reactive oxygen species production and increased RyR oxidation. Treatment of VF myocytes with reducing agents normalized parameters of Ca²⁺ handling and shifted the threshold of Ca²⁺ alternans to higher frequencies.

Conclusion

Redox modulation of RyRs promotes generation of Ca²⁺ alternans by enhancing the steepness of the Ca²⁺ release–load relationship and thereby providing a substrate for post-MI arrhythmias.

1. Introduction

Sudden cardiac death (SCD) due to sustained ventricular arrhythmias [ventricular tachycardia or ventricular fibrillation (VF)] is a major cause of mortality in patients following myocardial infarction (MI).^1–3 In these patients, ventricular ectopy appears to be part of the pathogenic mechanism of arrhythmias, whereas electrical heterogeneity and dispersion of repolarization are thought to be essential for the arrhythmogenic substrate.⁴ One important source of repolarization dispersion is cardiac alternans, a beat-to-beat alternation in action potential duration (APD) and contractile force or intracellular Ca²⁺ transient.^5,6 Ca²⁺ dynamics, and in particular regulation of Ca²⁺ release from the sarcoplasmic reticulum (SR), has been recognized as a key factor in the genesis of electromechanical alternans.^6–9

Several factors have been implicated in the disturbances of Ca²⁺ signalling that result in alternans. These include slowed SR Ca²⁺ uptake,^10,11 incomplete recovery of ryanodine receptor (RyR) from inactivation,^12,13 and increased steepness of the Ca²⁺ release–SR Ca²⁺ content relationship.^11,14 However, the relative importance of these factors in the genesis of alternans remains a subject of debate. Moreover, most studies of alternans have been performed in myocytes from healthy hearts using various experimental interventions or computer simulations using mathematical models of Ca²⁺ cycling. Thus, the mechanisms of alternans in clinically relevant disease models remain largely unexplored.

Altered redox balance and redox-mediated changes in Ca²⁺ handling are increasingly recognized as important pathogenic factors in different cardiac diseases, including both ischaemic and non-ischaemic disease processes.¹⁵ Reactive oxygen species (ROS) have the capability to affect Ca²⁺ handling via redox modification of components of the excitation–contraction (EC) coupling machinery, including RyRs.¹⁶ However, the potential role of redox-mediated alterations of Ca²⁺ signalling in the genesis of alternans and arrhythmias in general remains to be investigated.

In the present study, the potential role of, and mechanisms responsible for, Ca²⁺ alternans was investigated using a canine post-MI model of SCD. This well-characterized model is known to recapitulate many aspects of humans with healed MIs and a residual risk of SCD (recently reviewed in Billman¹⁷) including altered repolarization, altered autonomic balance, and responses to pharmacological interventions. The present study reports susceptibility to Ca²⁺ alternans at the tissue and myocyte levels in post-MI animals with a demonstrated vulnerability to sustained ventricular tachycardia and VF. Ca²⁺ alternans resulted from the increased steepness of the SR Ca²⁺ release–content relationship and is attributable to enhanced RyR activity secondary to redox modification of the channel protein.

2. Methods

Expanded Methods section is given in Supplementary material online.

2.1. Canine model of sustained ventricular tachyarrhythmia

All in vivo procedures^18,19 were approved by the Ohio State University Institutional Animal Care and Use Committee and conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). An anterior wall MI was induced by ligation of the left anterior descending coronary artery, and a vascular occluder was placed on the left circumflex coronary artery at the time of surgery. A minimum of 4 weeks after surgery and the MI, the dogs were risk-stratified for arrhythmia susceptibility by an exercise plus ischaemia test as described previously.¹⁷ This test reliably induced ventricular flutter that deteriorated into VF. A minimum of 7 days elapsed between the exercise plus ischaemia test and tissue or myocyte isolation to avoid any acute effects of recent ischaemia. Only dogs with VF in response to the exercise plus ischaemia test were included in the present study (VF animals, n = 13 female, age 2–3 years, weight 18–26 kg); a group of 20 normal dogs (male/female, age 1–4 years, weight 15–25 kg) served as controls. Left ventricle (LV) function was assessed in a subset of control and VF animals using echocardiography. For the dogs used in the present study, LV fractional shortening was 41 ± 2 and 41 ± 1% in control (n = 10) and VF (n = 5) animals, respectively.²⁰

2.2. Optical AP mapping in the canine wedge preparation

Canine wedge preparations were isolated from normal (n = 4) and VF (excluding both infracted and border zone tissue, n = 4) dogs and placed in an imaging chamber for optical mapping while being perfused with Tyrode solution. Wedges were stained with the voltage-sensitive dye, di-4-ANEPPS (15 µM, Molecular Probes, OR, USA) and then administered cytochalasin D (6 µM) to ensure that motion artefact was prevented. Optical APs were recorded with high spatial (0.9 mm), temporal (1.0 ms), and voltage (0.5 mV) resolutions from cells spanning the entire LV wall. AP alternans was induced by decreasing pacing cycle lengths until 1-to-1 capture was lost.

2.3. Ca²⁺ imaging in myocytes

Myocytes were isolated from LV mid-myocardial wall by using standard techniques as described previously.²⁰ In the VF group, cells were isolated from region located at least 6–8 cm from the centre of the infarct to exclude necrotic and border zone area. Whole-cell patch-clamp recordings of AP and Ca²⁺ currents (I_Ca) were performed with an Axopatch 200B amplifier (Axon Instruments, CA, USA). Intracellular Ca²⁺ imaging was performed using an Olympus Fluoview 1000 confocal microscope in line-scan or XY mode. Intra-SR Ca²⁺ levels were studied by loading myocytes with 10 µM Fluo-5N AM (Molecular Probes) for 3–4 h at 37°C. Rhod-2 or Fluo-3 Ca²⁺ indicators were used to monitor cytosolic Ca²⁺. To facilitate [Ca²⁺]_SR measurements, voltage-clamp experiments involving [Ca²⁺]_SR recordings were performed in the presence of 100 nM isoproterenol (Iso, Calbiochem, CA, USA), a β-adrenergic agonist, which enhances SR Ca²⁺ release by increasing I_Ca and stimulating SERCA activity in both control and VF myocytes (see Supplementary material online, Figure S1).

2.4. Measurements of ROS production and RyR free thiol content

Changes in ROS production were measured with the fluorescent indicator 5-(and-6) chloromethyl-2′,7′-dichlorodihydrofluoroscein diacetate (DCFDA, 10 µM, Molecular Probes).²¹ After subtraction of background fluorescence, the signal (ΔF) was normalized to maximum fluorescence attained by application of 10 mM H₂O₂ (F_MAX).

The content of free thiols in RyRs was determined with monobromobimane (mBB, Calbiochem) fluorescence method.²² Heavy SR vesicles were prepared from fresh tissue samples from control and VF hearts under non-reducing conditions.²³ Samples were incubated with 400 µM mBB for 1 h in the dark at room temperature; subsequently, proteins were acetone precipitated and subjected to SDS–PAGE (4–20% gradient gel, Bio-Rad, CA, USA). mBB fluorescence was normalized to RyR amount determined using Coomassie Blue staining of the gels run in parallel.

2.5. Data analysis

Results are presented as mean ± SE. Statistical significance was evaluated either by the appropriate Student's t-test or by one-way ANOVA where appropriate. A P-value of <0.05 was considered significant.

3. Results

3.1. AP alternans in wedge preparations

In order to examine potential arrhythmogenic mechanisms in the post-infarction hearts of dogs susceptible to VF, optical APs were recorded from the transmural wall of LV wedge preparations while alternans was induced. The rate-dependence of APD alternans was significantly shifted towards slower heart rates, and the alternans magnitude was increased in wedges from VF animals compared with controls, indicating greater susceptibility to APD alternans in these preparations (Figure 1A–C). Furthermore, spatially discordant alternans occurred in all VF wedge preparations but in only 50% of the control. The presence of spatially discordant alternans significantly increased maximum repolarization gradients in VF wedge preparations from 4.3 ± 2.0 to 13.5 ± 3.3 mm/ms (P < 0.03). In addition, rapid pacing-induced VF was only observed in the VF preparations, never in control (see Supplementary material online, Figure S2). Baseline APD was similar in wedges from hearts of both control (254 ± 15 ms) and VF (262 ± 14 ms, P= NS) animals. Conduction velocity in control wedges (0.43 ± 0.06 m/s) was also similar to that in VF wedges (0.41 ± 0.06 m/s, P= NS). These results suggest that cardiac alternans is a potential mechanism for arrhythmogenesis in the myocardium of VF animals.

Figure 1.

Increased susceptibility to AP and Ca²⁺ alternans in VF hearts. (A) Representative mid-myocardial APs recorded at a stimulation rate of 230 bpm from a control and VF heart. (B) Dependence of the amplitude of APD alternans on stimulation rate recorded in VF and control wedge preparations. (C) APD alternans threshold in VF hearts (203 ± 23 bpm) is significantly lower than the threshold recorded in control (261 ± 12 bpm; *P < 0.05 vs. control). (D) Representative recordings of membrane potential with corresponding line-scan images and temporal profiles of Fluo-3 fluorescence in control and VF myocytes. (E and F) Average amplitudes of [Ca²⁺]_c and duration of corresponding APs were measured with consecutive stimuli in control and VF cells paced with 0.5 and 1 Hz, respectively. A1 and A2 are [Ca²⁺]_c amplitudes of two consecutive stimuli, as shown in (D). Average amplitudes of [Ca²⁺]_c (G) and APD90 (H) alternans in control and VF myocytes were measured at indicated stimulation frequencies. *P < 0.05, vs. control.

3.2. AP and Ca²⁺ alternans in isolated cardiomyocytes myocytes

Experiments performed on isolated myocytes demonstrated that APD alternans was paralleled by, and occurred in phase with, alternations in the amplitude of Ca²⁺ transients, i.e. Ca²⁺ alternans (Figure 1D–H). Consistent with the results in wedge preparations, myocytes from VF hearts showed AP alternans at slower stimulation frequencies than controls.

To explore the mechanisms of Ca²⁺ alternans, the next series of experiments were performed under voltage-clamp conditions (Figure 2). Additionally, in these experiments, the role of changes in intra-SR [Ca²⁺] in the generation of alternans was examined by measuring [Ca²⁺]_SR simultaneously with cytosolic [Ca²⁺] using SR-entrapped Fluo-5N and cytosolic Rhod-2. Representative cytosolic Ca²⁺ and [Ca²⁺]_SR measurements in a control and VF myocyte paced at 1 Hz are shown in Figure 2A. Cytosolic Ca²⁺ alternans in both control and VF cells occurred in phase with changes in diastolic [Ca²⁺]_SR. Alternans was more pronounced and occurred at substantially lower [Ca²⁺]_SR in VF cells than in controls. These results obtained at a fixed membrane potential showed that changes in Ca²⁺ handling alone could produce Ca²⁺ and, therefore, APD alternans. These data also point to a role for [Ca²⁺]_SR fluctuations in the genesis of Ca²⁺ alternans.

Figure 2.

Properties of SR Ca²⁺ release in control and VF myocytes displaying Ca²⁺ alternans during 1 Hz stimulation. (A) Representative I_Ca traces and corresponding line-scan images and temporal profiles of Rhod-2 and Fluo-5N fluorescence recorded in voltage-clamped control and VF cells. Upper traces show voltage protocol used. (B) Dependence of the SR Ca²⁺ release gain function ([Ca²⁺]_c amplitude/density of peak I_Ca) on [Ca²⁺]_SR measured with consecutive stimuli in control and VF cells paced at 1 Hz. (C) The slope of gain–[Ca²⁺]_SR function, calculated from the data presented in (B), was 1.0 ± 0.4 (n = 9) in control and 3.9 ± 0.8 (n = 10) in VF myocytes, respectively. *P < 0.05 vs. control.

Increased steepness of the Ca²⁺ release–load relationship is thought to be a key factor contributing to the development of Ca²⁺ alternans,^11,14 although the contribution of this mechanism has been recently challenged.¹² We measured directly the steepness of release–load relationships in control vs. VF myocytes exhibiting Ca²⁺ alternans. EC coupling gain, defined as the Ca²⁺ transient amplitude normalized to the peak of corresponding I_Ca density, was plotted as a function of the end-diastolic [Ca²⁺]_SR of the preceding Ca²⁺ release cycle for the large and small releases in control and VF myocytes (Figure 2). As shown in Figure 2B and C, the steepness of Ca²⁺ release–load relationship was markedly increased in VF cells compared with control cells.

3.3. Ca²⁺ release during EC coupling

To better understand the mechanism(s) responsible for altered cellular Ca²⁺ dynamics in myocytes from the hearts of animals susceptible to VF, SR Ca²⁺ release in control vs. VF myocytes was characterized in more detail. Figure 3A shows representative recordings of changes in cytosolic and SR Ca²⁺ concentrations along with I_Ca during steps to varying membrane potentials in a control and a VF myocyte. The results are summarized in Figure 3B and C that plot the voltage dependencies of the peak amplitude of cytosolic Ca²⁺ transients, the nadir of the luminal Ca²⁺ depletion signals, and I_Ca amplitude for the control and VF groups. Peak and decay of I_Ca measured at 0 mV were similar between the two groups (see Supplementary material online, Table S1). The amplitude of maximum cytosolic Ca²⁺ transients (at 0 mV) was also preserved in VF myocytes, although diastolic [Ca²⁺]_SR was markedly reduced from 1.1 ± 0.3 (n = 7) observed in control to 0.4 ± 0.1 mM (n = 8, P < 0.05). Notably, the magnitudes of both the cytosolic and luminal Ca²⁺ signals increased with small depolarizations, resulting in flattened and broadened voltage-dependence curves. Figure 3D shows that the ratio of gain of Ca²⁺-induced Ca²⁺ release to [Ca²⁺]_SR is significantly increased in VF myocytes, suggesting that RyR functional activity was increased in VF myocytes during EC coupling.

Figure 3.

Voltage-dependent characteristics of Ca²⁺-induced Ca²⁺ release in control and VF myocytes. (A) Representative traces of I_Ca, line-scan images and temporal profiles of Rhod-2 and Fluo-5N fluorescence recorded in control and VF cells. I_Ca and corresponding [Ca²⁺]_c were evoked by depolarizing steps from a holding potential of −50 mV to the indicated potentials. (B) Voltage-dependence of the amplitude of [Ca²⁺]_c and nadir of SR Ca²⁺ depletion in control and VF cells.*P < 0.05 vs. control. Voltage-dependence of the peak I_Ca (C) and of the gain/diastolic [Ca²⁺]_SR ratio (D). *P < 0.05 vs. control.

3.4. Ca²⁺ sparks and SR Ca²⁺ leak

In order to examine further the relationship between susceptibility to VF and SR Ca²⁺ release, Ca²⁺ sparks were measured in permeabilized myocytes at a constant baseline cytosolic [Ca²⁺] (see Supplementary material online, Figure S3). Ca²⁺ spark frequency was significantly increased, whereas the amplitude of the Ca²⁺ sparks was significantly decreased in VF myocytes compared with controls. These effects were associated with decreased SR Ca²⁺ content, as determined by applications of caffeine (see Supplementary material online, Figure S3D and E). Increased Ca²⁺ spark frequency observed at reduced SR Ca²⁺ content suggested that RyR functional activity was enhanced, resulting in leaky SR Ca²⁺ stores in VF myocytes.

3.5. SERCA-mediated SR Ca²⁺ uptake and Na⁺/Ca²⁺ exchanger activity

In addition to elevated SR Ca²⁺ leak, the reduced SR Ca²⁺ content in VF myocytes could result from the inhibition of SR Ca²⁺ uptake. SERCA-mediated SR Ca²⁺ uptake was measured in permeabilized control and VF myocytes using SR-entrapped Fluo-5N in the presence of the RyR-antagonist ruthenium red (RR). In these experiments, the SR was first depleted in a Ca²⁺-free solution using caffeine (10 mM) and then SR Ca²⁺ uptake was initiated by addition of 500 nM Ca²⁺ to the bath solution. As shown in Supplementary material online, Figure S3F and G, the rate of SR Ca²⁺ uptake measured as an increase in Fluo-5N fluorescence did not significantly differ between control and VF myocytes. Thus, the intrinsic Ca²⁺ transport activity of SERCA was not altered in VF myocytes.

In large animals, Na⁺/Ca²⁺ exchanger (NCX) contributes significantly to intracellular Ca²⁺ dynamic and hence it could contribute to the generation of Ca²⁺ alternans. NCX activity was assessed by analysing decay kinetics of caffeine-induced Ca²⁺ transients in control and VF myocytes. As shown in Supplementary material online, Figure S4, the rate of decay of caffeine-induced Ca²⁺ transients was not different between control and VF myocytes.

3.6. Role of redox modification of RyR

Redox modification of Ca²⁺ regulatory proteins, including RyRs, is increasingly recognized as a potential factor in the pathophysiology of cardiac diseases, including arrhythmias.^16,24 The levels of ROS in VF and control myocytes were assessed using the ROS-sensitive indicator DCFDA. As shown in Figure 4A and B, ROS production was increased approximately two-fold in VF myocytes compared with controls. Furthermore, measurements with mBB revealed decreased levels of free thiols in RyRs isolated from VF hearts, providing direct evidence of enhanced oxidation of RyRs in VF hearts. RyRs from each group were treated with a specific oxidizer of sulfhydryl groups, 2,2′-dithiodipyridine (DTDP, 200 µM), to assess susceptibility to oxidation or the reducing agent dithiothreitol (DTT, 5 mM) to assess reversibility of oxidation (Figure 4C and D).

Figure 4.

Increased production of ROS and RyR oxidation in VF hearts. (A) Representative images of ROS-sensitive indicator DCFDA loaded into myocytes from normal and VF hearts. (B) Relative normalized DCFDA fluorescence in myocytes from control (n = 25) and VF hearts (n = 31). **P < 0.01 vs. control. Fluorescence values were normalized to the signal measured in the presence of 10 mM of H₂O₂ and presented relative to control (100%). (C) Representative Coomassie Blue-stained gels (upper panels) and corresponding mBB fluorescence intensity (lower panels) of RyR from normal and VF hearts measured under baseline conditions and after 30 min incubation with of 0.2 mM DTDP or 5 mM DTT. (D) Relative free thiol content of RyRs from control vs. VF samples obtained by normalizing mBB fluorescence to RyR amount determined using Coomassie Blue staining of the gels run in parallel. *P < 0.05 baseline VF (n = 7) vs. baseline control (n = 9); ^#P < 0.05 vs. baseline control; ^†P < 0.05 vs. baseline VF.

To determine the functional impact of redox modifications on altered SR Ca²⁺ regulation in VF myocytes, the effects of the reducing agent DTT on SR Ca²⁺ leak were examined in permeabilized VF vs. control myocytes (Figure 5). In these experiments, SR Ca²⁺ leak was estimated as the difference in intra-SR [Ca²⁺] measured with SR-entrapped Fluo-5N in the presence and absence of RR. Since at steady state, [Ca²⁺]_SR is determined by the balance between SR Ca²⁺ leak via RyRs and Ca²⁺ uptake by SERCA, inhibition of RyRs by RR must raise [Ca²⁺]_SR to a degree proportional to the level of Ca²⁺ leak via these channels.²⁵ Consistent with the results of the Ca²⁺ spark measurements, SR Ca²⁺ leak was significantly higher in VF myocytes than in control (Figure 5C). However, treatment of VF cells with the reducing agents DTT or mercaptopropionylglycine (MPG) produced a substantial decrease in leak towards normal levels (Figure 5A–C). These results suggest that RyR-mediated SR Ca²⁺ leak was in part attributable to oxidation of RyRs. Moreover, incubation of intact VF myocytes with the antioxidant MPG led to a marked reduction in the amplitude of Ca²⁺ alternans, restoration of diastolic [Ca²⁺]_SR (Figure 6A–C), and normalization of the steepness of Ca²⁺ release–[Ca²⁺]_SR relationship (Figure 6D and E).

Figure 5.

Reducing agents normalize [Ca²⁺]_SR in permeabilized VF myocytes by inhibiting RR-sensitive SR Ca²⁺ leak. (A) Time-dependent profiles of intra-SR Fluo-5N signals recorded before and after the application of 1 mM DTT and 30 µmol/L RR in control, VF cells, and in VF cells pre-treated with 1 mM N-(2-mercapto-propionyl)glycine (MPG). Representative XY-images of Fluo-5N signal of a control, a VF cell, and a VF cell pre-treated with MPG recorded under specified conditions. (B) Average data of normalized Fluo-5N signal in control (n = 11), VF cells (n = 12), and VF cells pre-treated with MPG (n = 9) recorded in the absence and presence of DTT and RR. *P < 0.05 baseline VF vs. baseline control and VF with MPG. (C) SR Ca²⁺ leak, defined as the difference in Fluo-5N fluorescence recorded with (F_RR) and without (F) RR, under baseline conditions and in the presence of DTT in control, VF, and MPG-treated VF myocytes. *P < 0.05 vs. baseline control; ^#P < 0.05 vs. baseline VF.

Figure 6.

A reducing agent normalizes [Ca²⁺]_SR and lessens the amplitude of Ca²⁺ alternans in patch-clamped VF myocytes. (A) Representative line-scan images and temporal profiles of Rhod-2 and Fluo-5N fluorescence recorded in voltage-clamped VF cells recorded in the absence and presence of MPG, a reducing agent. Cells were stimulated at 1 Hz frequency. Upper traces show voltage protocol used. (B) Average values of end-diastolic [Ca²⁺]_SR were 0.88 ± 0.16, 0.41 ± 0.06, and 0.94 ± 0.22 mM in control (n = 8), in VF myocytes (n = 10), and in VF myocytes treated with 750 µM MPG (n = 9), respectively. *P < 0.05 vs. control. (C) Average amplitude of [Ca²⁺]_c alternans recorded at the indicated frequency of stimulation in voltage-clamped control cells (n = 4–13), VF cells (n = 8–11), and VF cells treated with MPG (n = 6–9). *P < 0.05 vs. 1 Hz control and 1 Hz VF cells treated with MPG. (D) Dependence of the SR Ca²⁺ release gain function ([Ca²⁺]_c amplitude/density of peak I_Ca) on [Ca²⁺]_SR measured for consecutive stimuli in VF cells in the absence and presence of MPG. Cells were paced at 1 Hz. (E) The slope of gain–[Ca²⁺]_SR function, calculated from the data presented (D), was 0.7 ± 0.2 (n = 9) in VF myocytes treated with MPG. *P < 0.05 vs. untreated VF.

4. Discussion

In the present study, the role of, and mechanisms responsible for, abnormal intracellular Ca²⁺ handling in arrhythmogenesis was investigated using a canine model of post-MI SCD with a combination of methodological approaches, including AP mapping in cardiac tissue and monitoring Ca²⁺ levels in the cytosolic and SR compartments of isolated patch-clamped myocytes. The major findings of the present study are as follows: (i) myocytes and tissue obtained from the hearts of animals susceptible to VF exhibited an increased predisposition to arrhythmogenic AP and Ca²⁺ alternans; (ii) these alternans were associated with increased steepness of the Ca²⁺ release–[Ca²⁺]_SR load relationship and hyperactive RyRs; and (iii) the pathological changes in RyRs and in alternans were at least partly due to redox modification(s) of the RyR channel.

4.1. The model

A well-characterized canine model of SCD was used in the present study.¹⁷ This model confers significant advantages over other models, in that it is a large animal model of chronic duration. The model mimics many of the pathological conditions that have been associated with a high risk of SCD including pre-existing ischaemic myocardial injury (i.e. anterior wall MI), acute MI at a site distant from previous ischaemic injury, and altered cardiac autonomic balance (decreased parasympathetic regulation coupled with enhanced sympathetic activation).¹⁷ In addition, it is essential that the lethal arrhythmias must be reliably and reproducibly induced before an accurate assessment of the mechanism responsible for these malignant rhythm disorders can be determined. In this model, VF is reproducibly induced in 95% of animals.¹⁷ Thus, the model used in this study provides the opportunity to investigate the relationship between pathological alterations in Ca²⁺ regulation and the propensity for lethal ventricular arrhythmias following MI in the absence of heart failure.

4.2. Role and mechanisms of Ca²⁺ alternans

Ca²⁺ alternans is increasingly recognized as an important factor in the development of cardiac arrhythmias by generating a substrate for re-entrant excitation.^6,26,27 In the present study, myocytes and tissue obtained from the hearts of animals susceptible to VF exhibited an increased propensity for APD and Ca²⁺ alternans. The underlying factors responsible for Ca²⁺ alternans have not been fully determined, especially in the setting of cardiac disease. Several mechanisms have been suggested, including slowed SERCA-mediated SR Ca²⁺ uptake,^10,11 incomplete recovery of RyR from inactivation,^12,13 and increased steepness of the Ca²⁺ release–SR Ca²⁺ load relationship.^11,14 The relative importance of these mechanisms is currently subject to debate. The effect of slowed Ca²⁺ uptake and prolonged refractoriness would be to decrease Ca²⁺ cycling such that normal Ca²⁺ release from the SR could be obtained only on alternating beats. With a steep Ca²⁺ release–load relationship, which, in general, reflects the stimulatory effects of luminal Ca²⁺ on RyR open probability,²⁸ small differences in [Ca²⁺]_SR would be expected to lead to substantial differences in the amplitude of Ca²⁺ transients, i.e. alternans. In our study, VF-related Ca²⁺ alternans was not attributable to slowed SR Ca²⁺ uptake. SR Ca²⁺ uptake rates were similar in control and VF myocytes (see Supplementary material online, Figure S3F and G). Moreover, Ca²⁺ alternans persisted in VF cells even after exposure to isoproterenol, which induced a significant acceleration of Ca²⁺ transient decay indicative of accelerated SR Ca²⁺ uptake (Figures 2 and 6). Similarly, a slowed recovery of RyR from inactivation did not contribute to alternans. Indeed, the present study demonstrated that both diastolic SR Ca²⁺ release and RyR functional activity were enhanced rather than decreased, consistent with reduced rather than increased inactivation of RyRs. At the same time, supporting a central role for [Ca²⁺]_SR-dependent mechanisms in the genesis of alternans, cytosolic Ca²⁺ transients always alternated in phase with changes in end-diastolic [Ca²⁺]_SR. Moreover, during Ca²⁺ alternans at a given pacing rate, smaller alterations in end-diastolic [Ca²⁺]_SR resulted in larger deviations in cytosolic Ca²⁺ transients (i.e. steeper Ca²⁺ release–[Ca²⁺]_SR relationship) in VF myocytes than in control myocytes. Thus, altered [Ca²⁺]_SR-dependence of release contributed to the increased predisposition to alternans in VF myocytes. Although these studies demonstrate the role of abnormal Ca²⁺ release in the genesis of alternans in our particular arrhythmia model, they do not rule out the possibility that other mechanisms, including defective SR Ca²⁺-reuptake, contribute to alternans in other disease settings. It is also important to note that even though we show significant abnormalities of Ca²⁺ regulation that can create a substrate for arrhythmogenesis, this does not exclude the possibility that repolarization abnormalities (independent of Ca²⁺) also contribute to arrhythmogenic events.^20,29

4.3. Alterations in Ca²⁺ release properties

The present investigation of EC coupling and SR Ca²⁺ release properties provides further insights as to the abnormalities of SR Ca²⁺ signalling in VF myocytes. Our experiments showed that while maximum Ca²⁺ transient amplitude (at 0 mV) was retained, the amplitude of Ca²⁺ transients at low membrane depolarizations was significantly increased, resulting in distinct broadening and flattening of the voltage-dependence of SR Ca²⁺ release in VF myocytes (Figure 3A and B). Furthermore, diastolic [Ca²⁺]_SR was significantly reduced in VF cells resulting in a significant increase in EC gain at a given intra-SR [Ca²⁺] (Figure 3D). Additionally, VF myocytes exhibited a significantly higher RyR-mediated diastolic SR Ca²⁺ leak measured both as Ca²⁺ sparks (see Supplementary material online, Figure S3) and as the ruthenium red-sensitive component of baseline [Ca²⁺]_SR (Figure 5). These results indicate that SR Ca²⁺ release via RyRs was potentiated in VF myocytes during both systole and diastole. RyRs are known to respond positively to elevated luminal Ca²⁺.²⁸ Our finding that the increase in RyR functional activity occurred against the backdrop of reduced [Ca²⁺]_SR is consistent with the hypothesis that RyR responsiveness to luminal Ca²⁺ was increased in VF myocytes. These changes in RyRs could account for, or contribute to, the increased steepness of release–[Ca²⁺]_SR relationship in VF myocytes during Ca²⁺ alternans.

In previous studies, severe and/or large infarctions resulted in diminished rather than enhanced SR Ca²⁺ release. For example, myocytes from the border zone of infarcted hearts and myocytes from hearts with ischaemic cardiomyopathy^30–33 have been reported to have reduced and slowed Ca²⁺ transients, lowered EC coupling gain, and decreased contractility at all membrane potentials in stark contrast to our present findings. These different results suggest that responses to infarction are more heterogeneous and diverse than previously thought and may reflect different stages of cardiac remodelling following infarction and/or qualitatively different types of responses to infarctions of different severities. It is also possible that changes in RyR in the previous studies were masked by other changes such as disruption of the membrane system in models with more severe/larger infarction that induced heart failure. Notably, our model does not exhibit impaired LV systolic function and is a model of arrhythmogenesis without concomitant heart failure.^17,20

4.4. Role of RyR redox modification in altered SR Ca²⁺ release

In the present study, we investigated the hypothesis that abnormal RyR function in VF hearts results from redox modification of the channel protein. This hypothesis was based on the following notions. Sulfhydryl oxidation of reactive cysteine molecules by various oxidizing agents is known to increase RyR open time probability, thus producing ‘leaky’ RyR channels.¹⁶ Previous studies demonstrated increased ROS production and oxidative stress in various ischaemic and non-ischemic cardiac diseases states¹⁵ including models of healed infarction (up to 16 weeks post-MI).³⁴ In the present study, the following findings are consistent with redox-mediated alterations of Ca²⁺ signalling in VF hearts: (i) ROS generation was enhanced in VF myocytes (Figure 4A and B); (ii) the number of free thiols on RyRs decreased in VF hearts compared with control (Figure 4C and D); and (iii) reducing agents normalized Ca²⁺ leak and Ca²⁺ cycling in VF myocytes (Figures 5 and 6).

Even though treatment with antioxidants resulted in significant improvements in intracellular Ca²⁺ handling, the results of the present study do not exclude the possibility that other types of post-translational modification(s), such as phosphorylation by cAMP-dependent protein kinase^35,36 and/or Ca²⁺/calmodulin kinase II,³⁷ also contribute to abnormal Ca²⁺ handling in VF cells. Moreover, these pathways may be linked at the level of CAMKII, which is reportedly activated by increased oxidative stress and contributes to ischaemic myocardial apoptosis³⁸ and arrhythmogenesis.³⁹

4.5. Arrhythmia mechanisms

Previous work illustrated a role for re-entry in the pathogenesis of ventricular tachyarrhythmias in this model.⁴⁰ In addition, Ca²⁺ chelators (BAPTA-AM) and modulators of Ca²⁺ entry (e.g. L-type Ca²⁺ channel blockers) have demonstrated in vivo efficacy in preventing tachyarrhythmias in this model.^17,40,41 Importantly, both have also been shown to inhibit Ca²⁺ alternans.⁵ The results from the present study are consistent with these previous in vivo observations and provide further insights into arrhythmogenic mechanisms in the post-MI heart. Our tissue and myocyte studies demonstrated an increased predisposition to APD and Ca²⁺ alternans in arrhythmic hearts following MI. APD alternans is arrhythmogenic because it can amplify spatial heterogeneities of repolarization, creating a substrate for re-entrant excitation.⁴² Of note, the VF wedge preparations in our study showed increased predisposition to the development of spatially discordant alternans that significantly increased spatial gradients of repolarization and is a known precursor to VF.^6,9

4.6. Limitations

Caution should be used when extrapolating our findings to in vivo settings. Our experiments on isolated cardiac myocytes were performed at the room temperature and ambient O₂ tension (20%) as opposed to physiological temperature (37°C) and O₂ (5%) levels which are known to effect both myocyte Ca²⁺ handling and redox reactions. Although our data suggest that alternans is a potential mechanism of arrhythmogenesis in these VF hearts, our study does not preclude the possibility that other mechanisms such as ectopic activity or conduction block due to preserved structural damage (scar) contribute to arrhythmogenesis.

4.7. Conclusions

Collectively, our data suggest that redox-dependent changes in RyRs contribute to arrhythmogenesis by promoting Ca²⁺ cycling-induced repolarization alternans. Therefore, in the post-infarction heart, normalizing Ca²⁺ release, potentially via targeted antioxidant therapies, may be beneficial in patients at risk for SCD.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.

Funding

This work was supported by the National Institutes of Health Grants [HL074045 and HL063043 to S.G., HL086700 and HL68609 to G.E.B., and HL84142 to K.R.L.].