Reacting to too much excitement: ROS overproduction elicits arrhythmogenic Ca²⁺ waves in the heart

The cardiac cycle is tightly regulated by a number of proteins that function to transfer the electrical stimulus to the contracting chambers of the heart. Movement of calcium ions (Ca2+) within the cell is central to the excitation–contraction coupling, and a key component in the modulation of the strength of each cycle. Increasing demand for greater cardiac output – through changes in myocardial loading or from exogenous stressors (e.g. exercise) – activates the sympathetic nervous system to initiate compensatory mechanisms to augment inotropic function. This occurs through cardiomyocyte β-adrenoreceptor activation. While this response ensures adequate cardiac output, there is evidence that chronic activation of β-adrenoreceptors can initiate maladaptive responses within contractile cells. A common clinical sequela of chronically stimulated cardiomyocytes is the development of cardiac arrhythmias, a process that primarily occurs via SR Ca2+ leak during diastole. However, the underlying mechanisms that serve to uncouple this normally tightly regulated process remain unclear. Initial theories explaining this phenomenon involved post-translational modifications of ryanodine receptors via intracellular kinases as a primary mechanism. It was suggested that phosphorylation of active sites on the receptor increased its affinity for Ca2+, allowing for background release during diastole, thus impairing normal cardiomyocyte relaxation (Neef & Maier, 2007). However, the work of Györke and colleagues alternatively suggested that post-translational modification of ryanodine receptors by reactive oxygen species (ROS) contributes to SR Ca2+ leak in the failing heart (Terentyev et al. 2008), which may serve as a substrate for arrhythmogenic wave development. In a recent issue of The Journal of Physiology, the study by Bovo et al. (2012) provides further evidence for the role of ROS production in the mechanism underlying the chronic changes in ryanodine receptor function and cardiac arrhythmogenesis. While short-term β-adrenergic stimulation (3 min) with isoproterenol (ISO) induced a positive inotropy effect in rabbit ventricular myocytes, it was found that prolonged β-adrenergic stimulation increased mitochondrial metabolic activity. This resulted in increased electron flux and higher intracellular levels of ROS. The authors found that Ca2+ waves occurred at highest frequency after only 6 min of ISO stimulation, corresponding with ROS-induced oxidation of thiol groups on ryanodine receptors and increased SR Ca2+ leak during diastole, mainly in the form of arrhythmogenic Ca2+ waves. However, the authors reported that scavenging ROS during ISO application normalized SR Ca2+ leak while maintaining increased Ca2+ transient amplitude, suggesting that strategies to attenuate ROS production during β-adrenergic stimulation may prevent the occurrence of arrhythmogenic Ca2+ waves while preserving positive inotropy. These results suggest that if levels of ROS begin to exceed the capacity of intracellular anti-oxidant defences, they could potentially lead to irreversible changes in the structure of cytoplasmic proteins, which may ultimately translate into changes in nascent protein function and an increase in the occurrence of arrhythmogenic Ca2+ waves. This study benefits from a well-constructed design that enabled the authors to identify and isolate the specific signalling pathway and its components underlying cardiac arrhythmogenesis following acute, prolonged β-adrenergic stimulation. Examining the contribution of specific molecules in the β-adrenergic pathway provided a mechanistic link between β-adrenergic stimulation and the post-translational modifications of the ryanodine receptors with the finding that mitochondrial ROS are key mediators of the altered Ca2+ cycling. This redox modification would increase diastolic SR Ca2+ leak, which in turn increases energy consumption as more ATP would be required to maintain a homeostatic pump–leak balance. As Bovo et al. (2012) discuss, these conditions would create a ‘vicious cycle’ in which SR Ca2+ leak is not compensated for by increased sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) 2a re-uptake of Ca2+, increasing ROS production and further enhancing SR Ca2+ leak. Ultimately, this would lead to both an impairment of myocardial relaxation and limit contractility through the diminishment of systolic Ca2+ release. As Ca2+ cycling underlies normal cardiac function, it is not surprising that the increased SR Ca2+ leak observed in this study may also account for the cardiac dysfunction observed in other pathologies in which enhanced β-adrenergic stimulation is implicated. The authors identified the effects of acute, prolonged β-adrenergic stimulation on Ca2+ cycling and the switch from positive inotropy to the generation of arrhythmogenic Ca2+ waves. By identifying the role of increased mitochondrial ROS production in this switch, the authors have filled a mechanistic void which may explain the increasing prevalence of arrhythmias in the general population. However, it remains unclear whether the acute effects of β-adrenergic overstimulation are transient in nature or permanent. For instance, chronic activation of this pathway, as observed in individuals with a long-standing history of endurance sport participation or cardiac pathology, may cause progression from paroxysmal to permanent cardiac arrhythmias. Of particular interest, the incidence of atrial fibrillation, the most common sustained arrhythmia, is increased up to 10-fold (O’Keefe et al. 2012) in individuals with chronic, long-standing participation in prolonged exercise, such as marathoners. Furthermore, current evidence suggests that prolonged intense exercise causes transient cardiac dysfunction, with reductions in left systolic and diastolic function (O’Keefe et al. 2012), which would be expected in the current study based on the increased diastolic [Ca2+] and decreased Ca2+ transient during systole. The salient conclusion drawn from this study is that limiting excess ROS production can represent an important therapeutic strategy to prevent arrhythmogenic Ca2+ waves. However, future studies will have to determine whether such strategies can do so without inhibiting other beneficial ROS-mediated physiological signalling cascades. Excessive production of ROS overcomes normal physiological feedback mechanisms, resulting in a feedforward redox signalling cascade that contributes to cellular dysfunction. The elevated rates of oxygen consumption and ROS generation in the heart compared to other organs, specifically under conditions of prolonged β-adrenergic activity, may limit the ability of cardiac muscle to neutralize ROS, offering a promising target for therapeutic intervention. However, ROS, and specifically mitochondrial ROS, have been implicated in various physiological redox signalling cascades involved in the normal regulation of cell and organ function (Feissner et al. 2009). Thus, there appears to be a fine line between the adaptive signalling pathways initiated by low-to-moderate levels of ROS versus the cytochrome c and cell death pathways initiated at higher levels. This may explain the findings that moderate-intensity exercise training reduces the risk for arrhythmias. Bovo et al. (2012) found that the generation of arrhythmogenic Ca2+ waves was both time- and frequency-dependent. This response may account for the ‘risk–benefit paradox’ of vigorous exercise, with prolonged strenuous exercise potentially exceeding the capacity of the anti-oxidant defence system to scavenge excess ROS following β-adrenergic stimulation. However, ROS produced during low- to moderate-intensity exercise or tachycardia are important mediators of the cardiac preconditioning response through increases in ryanodine-mediated Ca2+ release activity and decreases in ryanodine-mediated SR Ca2+ leak (Sanchez et al. 2008). The latter finding has been shown in a recent study in which moderate-intensity endurance exercise training reduced Ca2+ leakage-associated arrhythmogenesis (Bonilla et al. 2012). Thus, future research can focus on how chronic, repetitive bouts of β-adrenergic overstimulation may modulate the acute response observed in this study. The novel finding of mitochondrial ROS-induced oxidation of the ryanodine receptor increasing SR Ca2+ leak adds to the growing list of pathologies in which ROS have been implicated as an aggravating or initiating factor. The long-term consequence of ROS-mediated changes in protein function may also explain why β-adrenergic-induced heart failure represents a significant challenge in the management of symptoms and peri-operative ischaemia/reperfusion-related injuries, in which ROS play a key role in both the pathogenic and preconditioning/cardioprotective mechanisms. Another clinical implication of the increased mitochondrial ROS production on cardiac function involves the initiation of a pro-inflammatory state. Acute β-adrenergic overstimulation and oxidative stress are associated with the release of different mediators of the inflammatory response, namely interleukin (IL)-6 and tumour necrosis factor (TNF)-α. In particular, TNF-α has been shown to increase cytosolic Ca2+ levels via inositol-1,4,5-trisphosphate-mediated (IP3-mediated) release (Hund et al. 2008), which in turn increases ryanodine receptor-mediated Ca2+ release from the SR. This leads to an increase in mitochondrial Ca2+ content which exacerbates ROS production and further increases extracellular Ca2+ influx. Excessive TNF-α expression and cardiomyocyte TNF receptor type-1 stimulation induces hypertrophy, fibrosis, contractile dysfunction and even cell death (Kleinbongard et al. 2011), suggesting that the prolonged β-adrenergic stimulation observed in this study may induce long-term arrhythmogenic effects through activation of pro-inflammatory pathways. Therefore, attenuation of the pro-inflammatory state may offer an alternative therapeutic strategy to prevent the enhanced cytosolic Ca2+ influx and the occurrence of arrhythmias. Thus, Bovo et al. (2012) provide the foundation for future work in which prolonged β-adrenergic stimulation and altered Ca2+ cycling dynamics can be examined in the context of other cardiac pathologies. Bovo et al. (2012) provide substantial evidence for increased mitochondrial ROS production after acute prolonged β-adrenergic stimulation as a key component involved in the switch from positive inotropy to the generation of arrhythmogenic Ca2+ waves. These novel findings are important to our understanding of how arrhythmogenic substrates arise. Future work should substantiate these findings by looking at both the structural and functional cardiovascular consequences of long-term, chronic β-adrenergic stimulation – specifically assessing whether overstimulation increases the prevalence of arrhythmias associated with cardiac pathologies.

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