Alternating membrane potential/calcium interplay underlies repetitive focal activity in a genetic model of calcium-dependent atrial arrhythmias.

Aberrant diastolic calcium (Ca) release due to leaky ryanodine receptors (RyR2s) has been recently associated with atrial fibrillation (AF) and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, it remains unclear how diastolic Ca release contributes to the rising of rapid repetitive focal activity, which is considered as a common AF triggering mechanism. To address this question, we conducted simultaneous voltage/Ca optical mapping in atrial tissue and one-/two-dimensional confocal imaging in atrial tissue and myocytes from wild-type (WT, n = 15) and CPVT mice lacking calsequestrin 2 (Casq2−/−, n = 45), which promotes diastolic Ca release. During β-adrenergic stimulation (100 nm isoproterenol), only Casq2−/− atrial myocytes showed pacing-induced self-sustained repetitive activity (31 ± 21 s vs. none in WT). Importantly, in atrial tissue, this repetitive activity could translate to Ca-dependent focal arrhythmia. Ectopic action potential (AP) firing during repetitive activity occurred only when diastolic Ca release achieved a sufficient level of synchronization. The AP, in turn, synchronized subsequent diastolic Ca release by temporally aligning multiple sources of Ca waves both within individual myocytes and throughout the atrial tissue. This alternating interplay between AP and diastolic Ca release perpetuates the self-sustaining repetitive activity. In fact, pharmacological disruption of synchronized diastolic Ca release (by ryanodine) prevented aberrant APs; and vice versa, the inhibition of AP (by TTX or 0 Na, 0 Ca solution) de-synchronized diastolic Ca release. Taken together, these results suggest that a cyclical interaction between synchronized diastolic Ca release and AP forms a pathological rhythm generator that is involved in Ca-dependent atrial arrhythmias in CPVT. Key points Atrial fibrillation is often initiated and perpetuated by abnormal electrical pulses repetitively originating from regions outside the heart's natural pacemaker. In this study we examined the causal role of abnormal calcium releases from the sarcoplasmic reticulum in producing repetitive electrical discharges in atrial cells and tissues. Calsequestrin2 is a protein that stabilizes the closed state of calcium release channels, i.e. the ryanodine receptors. In the atria from mice predisposed to abnormal calcium releases secondary to the absence of calsequestrin2, we observed abnormal repetitive electrical discharges that may lead to atrial fibrillation. Here, we report a novel pathological rhythm generator. Specifically, abnormal calcium release leads to electrical activation, which in turn results in another abnormal calcium release. This process repeats itself and thus sustains the repetitive electrical discharges. These results suggest that improving the stability of ryanodine receptors might be useful to treat atrial fibrillation. Introduction Atrial fibrillation (AF) is the most common form of cardiac arrhythmia, which afflicts 3 million individuals in the US alone (Go et al. 2001, 2013; Miyasaka et al. 2006). However, despite extensive research, the underlying mechanisms responsible for the induction and perpetuation of AF remain incompletely understood. AF is often initiated and perpetuated by repetitive focal discharges (Wit & Boyden, 2007; Dobrev & Nattel, 2010). Ablation of such sources of focal activity has often proven to be an effective intervention venue for the treatment of AF (Jais et al. 1997; Haissaguerre et al. 1998). Repetitive focal discharges could either directly drive high frequency electrical oscillations that mark AF or result in AF through initiation of re-entry circuits (Wakili et al. 2011). However, the precise mechanisms underlying how regions outside the specialized normal pacemaker sites (e.g. sinoatrial node and latent atrial pacemaker cells) can generate repetitive firing remains elusive. In recent years AF has been linked to abnormal Ca release from the sarcoplasmic reticulum (SR) via cardiac ryanodine receptors (RyR2s). Specifically, genetic mutations that result in ‘leaky’ RyR2s (Capes et al. 2011) and ventricular tachyarrhythmias (i.e. catecholaminergic polymorphic ventricular tachycardia (CPVT)) have also been associated with AF (Bhuiyan et al. 2007; Sumitomo et al. 2007; Pizzale et al. 2008; Kazemian et al. 2011). Moreover, increased susceptibility to AF was found in genetically modified mice harbouring mutations in the RyR2 complex (Chelu et al. 2009; Shan et al. 2012; Glukhov et al. 2013; Faggioni et al. 2014). It has been suggested (Chelu et al. 2009; Shan et al. 2012; Faggioni et al. 2014) that aberrant diastolic SR Ca release (DCR) due to abnormal gating of RyR2 can activate transient inward current and thereby result in an ectopic depolarization (Kass et al. 1978; Berlin et al. 1989; Venetucci et al. 2008), also known as the delayed afterdepolarization (DAD) (January & Fozzard, 1988; Ter Keurs & Boyden, 2007). However, it is not clear whether and how DCR can repetitively generate discharges that result in repetitive DAD-mediated firing (triggered activity) in atrial cells. Moreover, it is uncertain whether such DCR and DADs potentially arising in individual atrial cells could synchronize across atrial myocardium leading to atrial arrhythmia. Indeed, such synchronization of aberrant Ca signalling and electrical activity is thought to be required to overcome the source–sink mismatch, thus resulting in ectopic drives (Houser, 2000; Xie et al. 2010; Myles et al. 2012, 2014). In this study, we used calsequestrin 2 null (Casq2−/−) mice, which are susceptible to aberrant DCR not only in the ventricular (Knollmann et al. 2006) but also in atrial myocytes (Faggioni et al. 2014), potentially resulting in AF in vivo (Faggioni et al. 2014). Here, we demonstrate that aberrant DCR in isolated atrial myocytes can result in self-sustained repetitive activity, which leads to focal arrhythmia in atrial tissue. We applied an integrative imaging approach to reveal specifically how DCR leads to repetitive activity at both cellular and tissue level in Casq2−/− mice. Our results demonstrated a new rhythm generating system that relies on an alternating interplay between membrane potential (Vm) and Ca for generation of tissue-wide focal arrhythmia.