Cardiac mitochondria and arrhythmias

Abstract

Despite a high prevalence of sudden cardiac death throughout the world, the mechanisms that lead to ventricular arrhythmias are not fully understood. Over the last 20 years, a growing body of evidence indicates that cardiac mitochondria are involved in the genesis of arrhythmia. In this review, we have attempted to describe the role that mitochondria play in altering the heart's electrical function by introducing heterogeneity into the cardiac action potential. Specifically, we have focused on how the energetic status of the mitochondrial network can alter sarcolemmal potassium fluxes through ATP-sensitive potassium channels, creating a ‘metabolic sink’ for depolarizing wave-fronts and introducing conditions that favour catastrophic arrhythmia. Mechanisms by which mitochondria depolarize under conditions of oxidative stress are characterized, and the contributions of several mitochondrial ion channels to mitochondrial depolarization are presented. The inner membrane anion channel in particular opens upstream of other inner membrane channels during metabolic stress, and may be an effective target to prevent the metabolic oscillations that create action potential lability. Finally, we discuss therapeutic strategies that prevent arrhythmias by preserving mitochondrial membrane potential in the face of oxidative stress, supporting the notion that treatments aimed at cardiac mitochondria have significant potential in attenuating electrical dysfunction in the heart.

1. Introduction

Cardiovascular disease is a leading cause of worldwide death in both men and women.¹ Among the manifestations of cardiovascular disease, a significant cause of mortality is sudden cardiac death resulting from malignant ventricular arrhythmia. Despite differences in lifestyle factors across the global population, the frequency of sudden cardiac death is remarkably similar in North America, Europe, and Asia, affecting ∼1 of every 1000 people and accounting for as much as one-third of all cardiac deaths in high-risk populations.^2,3 Novel treatments seeking to decrease the incidence of sudden cardiac death clearly have enormous potential for global health.

Investigations into the electrical function of the heart began over 150 years ago when Kölliker and Müller⁴ demonstrated that the heart produced electricity that was associated with muscle contraction. Building upon Sydney Ringer's initial discoveries of an ionic basis for heart function,^5,6 significant strides have been made in our understanding of the cellular events that can be modulated to influence the heart's rhythm. In the 1960s, compounds such as amiodarone and lidocaine were first used to treat arrhythmia by inhibiting sarcolemmal ion fluxes, and imaging techniques with increasing resolution are constantly improving our insight into tissue-level events that lead to arrhythmia. Despite these technical advances in understanding and diagnosing cardiac rhythm disturbances, the underlying mechanistic bases for cardiac arrhythmias are still being elucidated, reflecting a window for therapeutic potential as these sub-cellular pathways responsible for aberrant conduction are illuminated. In this review, we seek to highlight the role that cardiac mitochondria play in influencing myocyte excitability, emphasizing the potential for emergent therapeutic strategies converging on mitochondria to preserve cardiac electrical function. The majority of our focus herein will concentrate on the etiology of ventricular arrhythmias evoked under conditions of oxidative stress. We will highlight potential preventative approaches taken from the animal literature, with pertinent references from human studies included where appropriate.

2. Action potential heterogeneity and cardiac arrhythmias

As a syncytium, coordinated electrical propagation throughout the heart is obligatory for adequate function. At the cellular level, each individual myocyte must depolarize and repolarize in a specific manner based on anatomical location. Pathological heterogeneity in the cardiac action potential is commonly linked to ventricular arrhythmias, and several sub-cellular factors can contribute to lability in action potential duration. Among the cellular culprits involved in cardiac arrhythmias, ion channels in the sarcolemmal and mitochondrial inner membranes have received considerable attention for their ability to influence action potential duration. Sarcolemmal ion channel mutations leading to prolongation of the action potential (i.e. long QT syndrome), early- or delayed after depolarizations due to activation of calcium channels/exchangers, and altered trans-sarcolemmal ion gradients have all been extensively described in their arrhythmogenic role.⁷ In this article we will discuss the role that cardiac mitochondria play in influencing cardiomyocyte action potential duration, underscoring therapeutic potential for arrhythmia using mitochondria-targeted approaches.

3. Role of sarcolemmal K_ATP channels in arrhythmia

Emerging evidence indicates that the mitochondria induce non-physiological spatiotemporal heterogeneity in the cardiac action potential and predispose the heart to re-entrant arrhythmia. The influence of mitochondrial energetic status on the sarcolemmal action potential is mediated in large part by energy-sensing ATP-sensitive potassium channels (sarcK_ATP) in the sarcolemmal membrane. A significant amount of attention has been devoted to the role that sarcK_ATP channels may play in inducing action potential heterogeneity, leading to cardiac arrhythmias.^8,9 First discovered in the early 1980s,¹⁰ myocardial sarcK_ATP channels are heteromultimers composed of four pore-forming subunits and four accessory subunits, the sulfonylurea receptors, that bind to ATP. Inhibited by intracellular ATP and activated by ADP, Pi, Mg, and/or pH, sarcK_ATP channels open under conditions of oxidative stress to produce an inwardly rectifying background current, typically observed within the first 10 min of ischaemia.¹¹ SarcK_ATP channels are among the most densely populated ion channels in cardiac myocardium,¹² and the opening of even 1% of the total amount of channels in the sarcolemma can significantly shorten the cardiac action potential.¹³

The opening of sarcK_ATP channels may be an endogenous protective mechanism of the myocardial tissue, where channel opening signaled by inadequate ATP supply decreases calcium-mediated cardiac energy demand. As the population of sarcK_ATP opens, the cardiac action potential shortens and reduces the calcium transient. Since calcium overload can lead to necrotic and apoptotic cell death, sarcK_ATP channel opening is believed to be cytoprotective by decreasing the extent contracture by the myofilaments and blunting mitochondrial calcium overload. Several lines of evidence indicate that the expression of functional sarcK_ATP channels is vital to cellular survival in the face of oxidative stress. First, increased sarcK_ATP protein expression correlated with protection against ischaemia/reperfusion injury in female (vs. male) animals^14–17 or following exercise training.^14,18,19 Second, pharmacological block of the sarcK_ATP channel population increased cell death in hearts exposed to ischaemia/reperfusion,^15,18,20 with the block during ischaemia being the critical period leading to increased injury.¹⁵ Third, genetic knockout of the sarcK_ATP channel pore-forming subunit led to animals that were severely intolerant to exercise and displayed enhanced sensitivity to calcium overload.^21–23 Taken together, it appears that there is a physiological role for sarcK_ATP opening in attenuating cell death during ischaemia. Consistent with this notion are observations in humans where diabetic patients taking oral sulfonylureas to control type II diabetes were at a higher disposition for cardiac injury following ischaemia.²⁴

While the opening of sarcK_ATP channels appears to be protective of the viability of ischaemic cardiac myocytes, the consequence of increasing potassium conductance to the whole organ predisposes to electrical dysfunction and in some cases the generation of fatal arrhythmia.^8,25–28 With such a high density of channels in the sarcolemmal membrane, the opening of sarcK_ATP channels can significantly shorten the action potential, and if enough channels open, can render cells inexcitable by holding the membrane potential very close to potassium's Nernst equilibrium potential. This creates a tremendous current sink for the propagating depolarization wave, and arrhythmias may be favoured when there are local regions where the open probability of sarcK_ATP channels is high (i.e. where the energetic status of the cell has been compromised), a phenomenon our group has previously termed ‘metabolic sinks’.^29,30 The presence of metabolic sinks enhances propensity for arrhythmia by influencing the effective refractory period (ERP) of the myocardium, resulting in a shortened excitation wavelength (the product of conduction velocity and refractory period). Pathological heterogeneity in action potential duration increases the ‘dispersion of refractoriness’ within the tissue, and is known to promote re-entry.^31–33 SarcK_ATP opening abbreviates the action potential duration and shortens ERP. SarcK_ATP channel openers^34–37 and blockers³⁸ decrease and increase ERP, respectively, and ERP is also prolonged after knockout of sarcK_ATP pore-forming subunits.³⁹ However, other factors may come into play during ischaemia that alter the relationship between action potential duration and ERP. For example, although ischaemia activates sarcK_ATP and shortens the action potential, a prolonged ERP may occur due to post-repolarization refractoriness,^40,41 presumably due to alterations in Na channel availability.

An arrhythmogenic role for sarcK_ATP has been confirmed in studies using either glibenclamide, which blocks both the mitochondrial and sarcolemmal isoforms of the K_ATP channels, or the sarcolemmal-specific HMR 1833 compounds (or HMR 1098, the sodium salt of HMR 1883). Blocking sarcK_ATP channels with HMR1883 decreased the incidence of ventricular arrhythmia in rat,⁴² rabbit,⁴³ pig,⁴⁴ and dog.²⁶ Importantly, the findings from the animal literature are confirmed in clinical studies where sarcK_ATP channel blockers reduced the incidence of ventricular fibrillation in humans.^45–47

While it seems plausible that the prevention of arrhythmias with sarcK_ATP blockers is due to direct inhibition of sarcKATP currents, sarcK_ATP blockers could theoretically also indirectly prevent arrhythmias. Specifically, by inhibiting sarcK_ATP currents and preventing action potential shortening, the ensuing cellular calcium overload may promote gap junction closure and block re-entrant wave-fronts via cellular uncoupling.⁴⁸ In order to understand the factors driving the opening of sarcK_ATP channels during metabolic stress, an overview of the underlying bioenergetic events leading to sarcK_ATP activation will be presented.

4. Metabolic oscillations

The cardiac mitochondrial network produces over 95% cellular ATP, and accounts for ∼20–30% of myocardial volume in species ranging from mouse to man.⁴⁹ According to the classic chemiosmotic theory as proposed by Mitchell,⁵⁰ mitochondria create a proton motive force by pumping protons out of the mitochondrial matrix, and use this proton electrochemical gradient to liberate the energy needed to phosphorylate ADP to ATP by the F₁F_o-ATPase. The majority of the proton motive force is comprised of the mitochondrial membrane potential (ΔΨ_m), with the magnitude of ΔΨ_m being ∼150 mV in energized mitochondria.⁵¹ Decreases in ΔΨ_m diminish the amount of free energy available to generate ATP, with mitochondria shifting to ATP hydrolysis under pathophysiological conditions when the ΔΨ_m collapses substantially (depicted in Figure 1A).

Figure 1.

Cascade of events where the opening of energy-dissipating anion channels in the mitochondrial inner membrane (IMM) leads to a depolarization of the mitochondrial network, opening of sarcK_ATP channels, and ultimately transition to arrhythmia in the intact organ. (A) Schematic depiction of the IMM under conditions of normoxia associated with sinus rhythm (left) and during metabolic stress (right). Matrix oxidation is characterized by glutathione oxidation and opening of IMAC, which collapses the ΔΨ_m, leading to ROS-induced ROS release in the mitochondrial network. Structures for IMAC and the PTP are speculative and based on previous reports.^77,166 (B) Two-photon images of ΔΨ_m in an intact guinea pig heart under normoxic (left) and oxidative (right) conditions. (C) Recordings of LV pressure (red trace) and ECG in a guinea pig heart subjected to normoxic and oxidative conditions. The time course for (B) and (C) are similar for both images, indicating that collapse of ΔΨ_m was accompanied by transition to fatal ventricular arrhythmia. Panels (B) and (C) are reprinted from Brown et al.⁷⁵ with permission from Elsevier.

The dynamic relationship between K_ATP current and the metabolic status of heart cells was first observed by O'Rourke and colleagues.⁵² Following metabolic stress via substrate deprivation, or in response to increased ADP levels, glibenclamide-sensitive current oscillations were observed in cardiomyocytes. Oscillating sarcK_ATP currents were observed in phase with NADH fluctuations, and were not influenced by changing cytosolic calcium concentrations. Importantly, the vacillating sarcK_ATP currents directly influenced cardiac repolarization and introduced significant lability in the length of the action potential waveform.⁵² Subsequent studies confirmed the initial observation of oscillatory sarcK_ATP currents and action potential duration in cardiac myocytes under conditions of metabolic stress.^53,54

The fluctuations in sarcK_ATP currents, and consequently action potential duration, are intricately linked to the behavior of the mitochondrion. Collapses in ΔΨ_m have been observed in a number of studies where the myocardium is subjected to oxidative stress, with sarcK_ATP current increasing in phase with losses of ΔΨ_m.⁵⁴ Using cationic lipophilic rhodamine fluorescent probes, several studies have noted reversible collapses in ΔΨ_m in isolated cells subjected to oxidative stress via substrate deprivation,⁵⁵ ATP depletion,⁵³ local generation of reactive oxygen species (ROS),⁵⁴ the thiol oxidant diamide,⁵⁶ and respiratory inhibition.⁵³ Recent evidence using two-photon microscopy confirms cellular data as reversible collapses in ΔΨ_m were seen in intact hearts exposed to global ischaemia/reperfusion or diamide.⁵⁷

In addition to nucleotide-dependent activation of sarcK_ATP currents after loss of ΔΨ_m, the collapse of bioenergetics might also activate sarcK_ATP currents through mechanical stretch. In this scenario, the loss of mitochondrial function would quickly preclude development of tension and result in paradoxical segment lengthening of the ischaemic ventricular tissue. Given that both ischaemia and stretch activate sarcK_ATP channels,^58–60 bulging of the myocardium may also contribute to the activation of sarcK_ATP channels. This mechanism of arrhythmogenesis is supported in studies where preventing dyskinesis reduced extracellular potassium accumulation.⁶¹

In order to understand the mechanistic basis for collapses in ΔΨ_m that contribute to arrhythmia, an overview of putative mitochondrial ion channels that may be involved will be discussed.

5. Role of mitochondrial ion channels in cardiac arrhythmias: inner membrane anion channel

Several distinct energy-dissipating ion channels in the inner membrane have been proposed to be involved in the ΔΨ_m collapse, contributing to the generation of arrhythmia. The first of these channels to be discussed is the inner membrane anion channel (IMAC).

Anion flux across the inner mitochondrial membrane was first observed over 40 years ago,^62–64 with early studies primarily interested in the contribution of anion movement on mitochondrial volume regulation. Since the initial observations, the IMAC has been characterized in a number of tissues and is believed to play an important role in anion efflux from energized mitochondria (for review, see^65,66). Although (as with other inner membrane ion channels) the exact structure of the IMAC is not currently known, the sensitivity of the anion channel to regulation by benzodiazepine compounds⁶⁷ suggests that the molecular composition consists of an anion channel subunit that associates with a peripheral benzodiazepine receptor in the outer membrane.

Insights into the factors mediating the collapse in ΔΨ_m have focused on the production of ROS by the mitochondria. ROS-dependent oscillations in ΔΨ_m were first noted by Sollott's group.⁶⁸ In their study, Zorov et al. noted that local generation of ROS produced by laser flash elicited synchronous collapses in ΔΨ_m that were prevented by a ROS scavenger. There is growing evidence that the collapse in ΔΨ_m may be mediated by superoxide anion, leading to cell-wide depolarizations in the myocyardium through a process coined ‘ROS-induced ROS release’.^68,69 According to this theory, ROS produced at the level of a single mitochondrion can stimulate superoxide-mediated depolarization of neighbouring mitochondria. This spatiotemporal behavior among the mitochondrial network led our group to conclude that mitochondria are arranged in a percolation matrix.⁷⁰ According to empirical data (and corroborated by computer simulations), the increase in ROS under conditions of oxidative stress can reach a critical level, after which cell-wide ΔΨ_m oscillations in the mitochondrial network are observed (deemed ‘mitochondrial criticality’).^71,72

The importance of IMAC in influencing the ΔΨ_m was first noted when several distinct ligands to IMAC were found to prevent loss of ΔΨ_m observed in isolated cardiac myocytes. Aon et al.⁵⁴ used a laser flash to induce a local burst of mitochondrial ROS, which causes cell-wide increases in ROS production and oscillations in ΔΨ_m. The reversible collapses in ΔΨ_m (and the cell-wide ROS accumulation) could be prevented with the addition of PK11195, 4-chlorodiazepam, or DIDS, three distinct compounds that have all been previously shown to block the activity of IMAC.^65,73 Importantly, blocking the reversible collapses in ΔΨ_m by targeting the IMAC stopped the oscillations in action potential duration,⁵⁴ providing further cellular evidence that targeting the IMAC may be effective in preventing arrhythmias by stopping ROS-induced ROS release.

A confirmatory role for IMAC involvement in arrhythmia was provided in a series of studies where inhibiting the IMAC prevented arrhythmias in intact mammalian hearts.^29,74,75 Optical mapping of the epicardial surface of guinea pig hearts revealed that blocking IMAC decreased ischaemia-induced action potential shortening and was accompanied by a lack of ventricular tachycardia/fibrillation at the onset of reperfusion.²⁹ Cardioprotection evoked by blocking the IMAC was also observed in isolated rabbit heart and was accompanied by significantly improved left ventricular developed pressure.⁷⁴ Of notable clinical interest, in both studies the reperfusion arrhythmias were prevented when the IMAC was blocked only at the onset of reperfusion (as opposed to pre-treatment).^29,74

6. Mitochondrial permeability transition pore

More attention has been devoted to the activity of the mitochondrial permeability transition pore (PTP) in ischaemia/reperfusion injury than any other mitochondrial inner membrane protein complex. Extensive characterization of the putative composition and importance of the PTP in ischaemia/reperfusion injury has been put forth, and the reader is referred to several excellent reviews in this area.^76–79 It is clear that the opening of the PTP plays a significant role in the generation of necrotic and apoptotic cell death, both of which are involved in the etiology of myocardial infarction.⁸⁰ Administration of cyclosporin-A or sanglifehrin-A, both blockers of the PTP, attenuate several indices of cardiac I/R injury including myocardial infarction,^81–85 left ventricular dysfunction,^86–89 cardiomyocyte death,^90–92 and mitochondrial dysfunction.^93,94 The translation of these studies was recently supported in human data, where administration of cyclosporin-A immediately prior to percutaneous coronary intervention decreased the extent of short-term injury in a small clinical trial.⁹⁵

While the role of PTP opening in tissue death is clear, there is less evidence that the activity of the PTP influences the generation of cardiac arrhythmia, especially those occurring at the onset of reperfusion. In several experiments using isolated cells, collapses in ΔΨ_m observed after substrate deprivation or laser flash were not prevented by the addition of cyclosporin-A.^54,55,68,96 Using two-photon imaging, blocking the PTP was ineffective at preventing the sustained ΔΨ_m collapse in hearts undergoing global ischaemia.⁹⁷ Other investigations confirmed a lack of protection against arrhythmia in rat,⁹⁸ guinea pig,²⁹ and rabbit⁷⁴ hearts. Finally, delivery of a cyclosporin-A bolus prior to stenting did not influence the incidence of ventricular fibrillation in human subjects.⁹⁵

7. MitoK_ATP channels

Evidence for a mitochondrial ATP-sensitive potassium (mitoK_ATP) channel was first observed in rat liver mitohcondria,⁹⁹ and later confirmed in heart.¹⁰⁰ The opening of mitoK_ATP channels may be important in mediating protective interventions given before the onset of ischaemia by partially dissipating the ΔΨ_m, reducing the driving force for calcium into the mitochondria, and improving cellular respiration secondary to mild swelling of the matrix (reviewed in^9,101,102).

Most studies that have examined the cardioprotective effect of mitoK_ATP opening have examined the role of mitoK_ATP in mediating reductions in infarct size elicited by a single preconditioning stimulus.¹⁰² In most (but not all) of these studies, blocking the mitoK_ATP with 5-hydroxydecanoate (5-HD) abolished the reduction in infarct size triggered by the stimulus of interest. While single episodes of preconditioning yield mechanistic insight, it is noteworthy that when repetitive stimuli are administered mitoK_ATP blockade does not reduce the evoked protection, as evidenced by the lack of effect of 5-HD in abolishing the infarct-sparing effects of repetitive ischaemic preconditioning¹⁰³ or chronic exercise.¹⁸

Fewer studies have examined the activity of mitoK_ATP channels in cardiac arrhythmia. As with the infarction literature, a role for mitoK_ATP in protecting against arrhythmia is apparent where mitoK_ATP blockers abolished the anti-arrhythmic phenotype provided by a preconditioning stimulus such as ischaemic preconditioning,^104,105 adenosine,¹⁰⁶ delta opioid agonists,^107,108 estrogen,¹⁰⁹ 3-nitropropionic acid,¹¹⁰ nitroglycerin,¹¹¹ noradrenaline,¹¹² or endothelin receptor agonists.¹¹³ Although mitoK_ATP channels appear to be important in mediating the anti-arrhythmic effects of some preconditioning models, their activity is not attributed to all models of preconditioning. For example, blocking the mitoK_ATP during preconditioning from bradykinin,¹¹⁴ low-flow ischaemia,¹¹⁴ peroxynitrite,¹¹⁵ or estradiol¹¹⁶ did not attenuate the anti-arrhythmic protection.

Protection against arrhythmias via direct activation of mitoK_ATP channels prior to index ischaemia has yielded opposing results, with some investigators showing protection from arrhythmia^106,117 and others showing no beneficial effect.^43,103 One putative explanation for the discordant findings is that the pharmacological agents used to open mitoK_ATP were different among these studies (minoxidil, diazoxide, and/or BMS-191095), and some of these compounds are plagued by non-specificity (addressed below).

While the preconditioning literature provides interesting mechanistic insight regarding anti-arrhythmic strategies administered before index ischaemia, the clinical relevance of these strategies must be questioned. To the clinician, attenuation of arrhythmias must often be attempted after the onset of ischaemia. Targeting mitoK_ATP channels after the onset of metabolic stress seemed promising based on cellular experiments, where administration of mitoK_ATP blockers stopped oscillations in ΔΨ_m evoked by halting respiration,⁵³ and mitoK_ATP opening (with diazoxide) improved cellular survival and mitochondrial integrity during cellular reoxygenation.¹¹⁸ Despite these encouraging cellular data, post-ischaemic administration of mitoK_ATP openers does not decrease arrhythmias,¹¹⁷ and post-conditioning interventions have been shown to be independent of the activity of mitoK_ATP channels.⁹⁸ Indeed, the investigators that observed beneficial effects of diazoxide on isolated cells¹¹⁸ found that the cytoprotective properties of the drug were independent of mitochondrial potassium flux.¹¹⁹ The non-specificity of commonly used mitoK_ATP openers (such as diazoxide) and blockers (such as 5-HD) has received a significant amount of attention in the literature, and several papers have addressed this issue in more detail.^{18,102,120–123}

8. Mitochondrial calcium uniporter

The role that intracellular calcium concentration plays in the generation of arrhythmia has been extensively characterized.^124,125 Early studies going back almost 50 years indicated that decreasing cytosolic calcium fluxes lowered the incidence of arrhythmia,^126,127 paving the way for Class IV anti-arrhythmic agents that decrease arrhythmias by lowering intracellular calcium.

The role of mitochondrial calcium fluxes in the generation of arrhythmia is much less clear. Mitochondrial calcium homeostasis is believed to involve calcium influx into the matrix via the mitochondrial calcium uniporter (MCU), with the major efflux pathway being the mitochondrial sodium–calcium exchanger.¹²⁸ Attempts to decrease arrhythmias by blocking MCU with ruthenium compounds have been somewhat effective but only when given prior to ischaemia. Pre-ischaemic administration of both ruthenium red and Ru360 significantly decreased the incidence of ventricular fibrillation in anesthetized rats,¹²⁹ and both ruthenium red and Ru360 effectively converted ventricular fibrillation to ventricular tachycardia (although neither compound led to the reversion of the ECG to sinus rhythm).¹³⁰

Speculation regarding the mechanism whereby MCU protects against arrhythmia involves keeping matrix calcium concentrations low, ultimately leading to decreased open probability of the PTP.¹²⁹ While this mechanism is likely involved in influencing the tissue survivability, it seems unlikely to play a prominent role in arrhythmogenesis since blockers of the PTP have not been particularly effective in preventing arrhythmia (addressed above). These findings are supported by experiments in myocytes, where the reversible collapse in ΔΨ_m induced during ROS-induced ROS release was not prevented by either ruthenium red⁵⁵ or Ru360.⁶⁸

At present, it is difficult to draw conclusions about the role of the calcium uniporter in arrhythmogenesis due to the confounding effects of the ruthenium compounds on cellular calcium fluxes.¹³¹ Ruthenium red has been shown to block calcium release from the SR^132–134 and L-type calcium channels,¹³⁵ suggesting that the effects of this compound in preventing arrhythmias may be from lowering overall cellular calcium and not by directly acting on the mitochondrion.¹³⁶ Ru360 appears to be more specific for the MCU, but whole-heart experiments are confounded by permeability issues, with some investigators showing the Ru360 effectively enters cardiac cells¹³⁰ and others indicating that it is not permeable.^137,138 Consistent with their ability to reduce cytosolic calcium transients, both ruthenium compounds are potent negative inotropes at concentrations shown to protect against arrhythmias,^139,140 an undesirable side effect when the overall purpose of administering the compound is to improve cardiac function. Future research with novel compounds that lack these pleiotropic/permeability issues will provide better insight into the role of the MCU in reperfusion arrhythmias.

To date, studies examining mitochondrial calcium fluxes have mostly concentrated on the influx of calcium into the matrix via the MCU. One recent study suggested that pressure-puff-induced intracellular Ca²⁺ releases were mediated by the mitochondrial efflux pathway, the mitochondrial sodium–calcium exchanger, which could potentially contribute to cardiac electrical dysfunction.¹⁴¹

9. Contribution of mitochondrial redox status to collapses in Δψ_m

As addressed above, the redox status of heart cells directly influences the cellular excitability. An oxidative shift in the cellular redox potential can promote action potential heterogeneity by modulating several different ion channels. Increased oxidation has been shown to directly activate sarcK_ATP channels,^142,143 alter the inactivation kinetics of L-type calcium channels via increased calcium ‘leak’ from the ryanodine receptor,¹⁴⁴ and influence the state of channels on the mitochondrial inner membrane.

Bursts of ROS are observed within the first few minutes of reperfusion, when the propensity for arrhythmia is extremely high.^145,146 Several experiments have induced ventricular arrhythmias under normoxic conditions with delivery of ROS bursts,^147,148 and attempts to scavenge ROS with superoxide dismutase mimetics¹⁴⁹ or mitochondrial-targeted anti-oxidant peptides¹⁵⁰ were successful in decreasing the incidence of arrhythmia. Future experiments that optimize effective delivery of ROS-scavenging agents to mitochondria clearly have significant potential in abrogating electrical dysfunction.

Among the cellular anti-oxidant defenses, several studies have examined the role of the myocardial glutathione (GSH) pool in arrhythmogenesis. Myocardial GSH is the largest anti-oxidant pool in the heart,¹⁵¹ with the majority of GSH being the reduced (GSH) vs. the oxidized (GSSG) form in healthy tissues. Commonly observed GSH/GSSG ratios in the mammalian heart are ∼200–300:1,^56,75 with a 50–70% decrease typically observed under conditions of oxidative stress.^75,152–154 Administration of either GSH or N-acetylcysteine (NAC), a glutathione precursor, has been shown to significantly reduce reperfusion arrhythmias.^155–157

Increasing evidence supports the notion that myocardial GSH is a key regulator of mitochondrial ROS-induced ROS release. Experiments in isolated cardiac myocytes showed that oscillations in ΔΨ_m could be evoked with the thiol-oxidants diamide⁵⁶ or diethylacetate,⁶⁸ both of which are known to deplete the GSH pool.^75,158,159 Aon et al.⁵⁶ altered the GSH/GSSG ratio in permeabilized myocytes and induced oscillations in ΔΨ_m (beginning at a GSH/GSSG ratio of 150:1), with the absolute concentration of GSSG being of primary importance in inducing ΔΨ_m collapses. Consistent with the notion that IMAC opens ‘upstream’ of the PTP and is a crucial therapeutic target, irreversible collapses in ΔΨ_m indicative of PTP opening were not observed until GSH/GSSG ratios fell below 50:1. In other studies using picochambers to simulate cellular ischaemia/reperfusion in isolated myocytes, ΔΨ_m depolarized during reoxygenation with step-wise increases in the oxygen tension. The depolarizations were mediated by increased ROS, and the addition of exogenous GSH prevented the collapses in ΔΨ_m with increasing oxygen tension.¹⁶⁰

Subsequent experiments confirmed that GSH oxidation evoked collapses in ΔΨ_m in whole hearts,^57,75 which was accompanied by ventricular tachycardia/fibrillation.⁷⁵ Interestingly, the GSH/GSSG ratio in whole-heart homogenates following diamide administration was very similar to ratios in isolated cells that led to mitochondrial criticality.⁵⁶ Finally, blocking the IMAC during diamide administration completely prevented the loss of ΔΨ_m and protected guinea pig hearts from arrhythmias⁷⁵ (see Figure 1 for mechanistic depiction).

10. Implications for GSH depletion and arrhythmias in humans

The findings from animal studies that highlight the beneficial effect of reduced GSH on stabilizing mitochondrial function are corroborated by human data, where low GSH/GSSG ratios were observed in human heart samples from patients in heart failure¹⁶¹ and with type 2 diabetes,¹⁶² two populations that display high risk for cardiac arrhythmias.² Consistent with this notion, administration of the NAC significantly decreased the incidence of cardiac arrhythmia in humans following cardiac surgery.¹⁶³ While promising, NAC itself is confounded by low bioavailability¹⁶⁴ and anaphylactoid-like reactions in some patients,^164,165 necessitating alternative compounds that can replenish cardiac GSH but lack the potentially harmful side effects of high NAC doses.

11. Conclusions

The cardiac mitochondrial network has emerged as a key target for strategies seeking to decrease arrhythmias. As the ‘hubs’ for cellular metabolism, preserving the integrity of the mitochondria in the face of metabolic stress will significantly improve almost all aspects of cellular function. Expanding our understanding of the molecular composition of inner membrane ion channels, as well as development of agents that home to mitochondria to diminish ROS overload have enormous potential as treatments to preserve ΔΨ_m and prevent lethal ventricular arrhythmias.

Conflict of interest: none declared.

Funding

This work was supported by R37HL54598 and East Carolina University.