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Cardiovascular Research 2006 70(3):407-409; doi:10.1016/j.cardiores.2006.04.007
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Copyright © 2006, European Society of Cardiology

Unzipping RyR2 in adult cardiomyocytes: Getting closer to mechanisms of inherited ventricular arrhythmias?

Maria Fernandez-Velasco, Ana Maria Gomez and Sylvain Richard*

Physiopathologie Cardiovasculaire, INSERM, U-637, Université Montpellier1, CHU Arnaud de Villeneuve 34295 Montpellier Cedex 5, France

* Corresponding author. Tel.: +33 467 41 52 41; fax: +33 467 41 52 42. Email address: srichard{at}montp.inserm.fr

Received 4 April 2006; accepted 6 April 2006

See article by Yang et al. [19] (pages 475–485) in this issue.

Malignant ventricular arrhythmias (VA) are a major cause of mortality and morbidity worldwide. Causative mechanisms are multiple and complex. VA can be generated by abnormal or instable repolarization of the action potential (AP) in myocytes. Perverted repolarization results mainly from congenital channelopathies, acquired diseases, including hypertrophy and heart failure (HF), or therapeutic intervention. Inherited arrhythmias are due to mutations, mostly in transmembrane ionic channels (Na+, K+, Ca2+) [1,2]. However, because Ca2+ regulates several ionic currents involved in AP repolarization [3], it was not counterintuitive to find that mutations in proteins regulating intracellular Ca2+ homeostasis can also generate VA and sudden cardiac death (SCD). For example, mutations in the cardiac Ca2+ release channel (ryanodine receptor; RyR2) of the sarcoplasmic reticulum (SR) are involved in type 2 arrhythmogenic right ventricular cardiomyopathy and catecholaminergic polymorphic ventricular tachycardia (CPVT) characterized by effort-induced VA in structurally normal hearts [2,4,5]. CPVT are also caused by mutations in calsequestrin, a protein binding Ca2+ and associated to the RyR2 in the lumenal side of the SR [2,4].


   1. Mechanisms involved in RyR2-linked arrhythmias
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2. Effect of DPc10...
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The intrinsic mechanisms by which changes in [Ca2+]i handling promote arrhythmias are not completely elucidated. Obviously, spontaneous SR Ca2+ release can activate a transient inward current (Iti) mediated mainly by the electrogenic Na+/Ca2+ exchanger (NCX) in the forward mode [6]. Iti contributes to delayed after depolarizations (DADs) in diastole, but also possibly to early after depolarizations (EADs), as shown in cardiac hypertrophy and HF [6,7]. Although RyR2 primarily regulate cardiac contraction through the Ca2+-induced Ca2+-release mechanism, they also influence membrane potential. In addition to inducing Iti, RyR2 control the decay kinetics of the L-type Ca2+ current (ICaL) and the amplitude of the inward rectifier K+ current (IK1) [8]. Last but not least, β-adrenergic responsiveness plays an important role in the triggering of arrhythmias both in HF [6] and CPVT [2,4,5].

Numerous mutations in RyR2 have been identified in CPVT [2,4,5]. How these mutations are translated into channel dysfunction, then into DADs and, ultimately, into triggered arrhythmias is still unclear [4]. Mutations may cause conformational changes resulting in increased RyR2 sensitivity to lumenal [9] and cytoplasmic Ca2+ [2,10] and promote SR Ca2+ leak [2,4,5]. Altered binding of accessory protein FKBP12.6 on RyR2 has been reported [11]. FKBP12.6 is known to stabilize the RyR2 in its closed state [12]. Interestingly, FKBP12.6-deficient mice exhibit exercise-induced VA similar to those observed in CPVT [11], implying that mutations lowering the affinity of FKBP12.6 for RyR2 might be arrhythmogenic, although this concept has been challenged [9,13].

Other causes for dysfunction of RyR2 mutants linked to arrhythmia are intrinsic to the channel [10,14]. In particular, the N-terminal domain and the central domain of the RyR2 interact with each other, what has been named "zipping" [15]. Zipping the two domains stabilizes the channel in its closed state [15–17]. A single mutation in one of these domains weakens the molecular interaction and, thereby, causes unzipping and RyR2 hyperactivation or hypersensitization, resulting in continuous SR Ca2+ leak and arrhythmia induction [17,18]. In wild-type RyR2, unzipping can be caused by a synthetic peptide (DPc10), analogous to a sequence in the central domain that normally interacts with the N-terminal domain. DPc10 directly competes for the N-terminal binding site and produces unzipping [15–17].


   2. Effect of DPc10 in permeabilized ventricular myocytes
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 1. Mechanisms involved in...
 2. Effect of DPc10...
3. Perspectives
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In this issue of Cardiovascular Research, Yang et al. [19] have investigated the functional effect of DPc10 on RyR2 in permeabilized rat ventricular myocytes. Although this approach does not allow to show the link with arrhythmia, it is relevant since previous studies of mutant RyR2 were performed in heterologous expression systems lacking a cardiac intracellular environment with all RyR2 accessory proteins and most Ca2+-handling proteins. The overall result shows that DPc10 mimics the functional effects of the R2474S RyR2 mutation and suggests that domain unzipping causes RyR2 dysfunction in ventricular myocytes.

Yang et al. [19] show complex effects of DPc10. First, DPc10 increased the frequency of Ca2+ sparks, but only transiently, while reducing their amplitude. Low concentrations of caffeine, known to increase RyR2 sensibility to Ca2+, had similar transient effects, consistent with autoregulation of diastolic Ca2+ postulating that RyR2 hyperactivity is compensated by a fall in lumenal SR [Ca2+] [20]. The transient effect of increasing or decreasing RyR2 activity has also been demonstrated in intact cardiac myocytes [21]. Second, DPc10, but not caffeine, specifically promoted a sustained increase in [Ca2+]i probably due to SR Ca2+ leak, although not as Ca2+ sparks. This leak was independent of autoregulation. That is, despite the expected depletion of the SR, there was a continuous increase in [Ca2+]i. Third, DPc10 lowered the cytosolic [Ca2+] threshold for occurrence of spontaneous Ca2+ release. This finding indicates that DPc10 increased the sensitivity of RyR2 to lumenal Ca2+.


   3. Perspectives
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 1. Mechanisms involved in...
 2. Effect of DPc10...
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The approach used by Yang et al. [19], providing that DPc10 adequately mimics the mutation, constitutes an interesting step forward to better understand RyR2 dysfunction in CPVT. Electrophysiological investigations of the effect of DPc10 in intact cardiomyocytes will be important to investigate whether and how DPc10 can trigger DADs (eventually EADs) both in normal resting conditions [14] and when the cells are challenged with β-adrenergic stimulation. In particular, the working hypothesis shown in Fig. 1 could be tested. Fig. 1 shows that, in resting conditions, the risk of triggering DADs in the presence of DPc10 (therefore, presumably in CPVT) is latent. This risk is exacerbated following β-adrenergic stimulation, which enhances SR Ca2+ load. Premature SR Ca2+ release is expected to occur via store overload-induced Ca2+ release [9]. Not only activation of INCX, but also possibly Ca2+-dependent decrease in IK1 [8], would provide conditions for DADs. This paradigm is similar to the one established in HF where changes in INCX, IK1, and retained β-adrenergic responsiveness underlie triggered arrhythmias [6]. The use of transgenic mice, like the knock-in mouse model recently developed by S. Priori and collaborators [22], will also probably contribute to provide decisive information linking the clinical phenotype (CPVT), the details of RyR2 dysfunction, and the type and origin of arrhythmias.


Figure 1
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  		Fig. 1 When opening untimely in diastole, Ca2+ released from RyR2 can activate the electrogenic Na+/Ca2+ exchanger (NCX) and, possibly, inhibit IK1. In normal cells (upper panel), no DAD is induced (left panel), even after enhancement of SR Ca2+ load by β-adrenergic receptor stimulation (right panel). In the presence of DPc10 (lower panel), which lowers the threshold for spontaneous Ca2+ release, β-adrenergic receptor stimulation-inducing SR Ca2+ load is expected to increase propensity for DADs.

 


   Acknowledgements
 
We thank the French "Ministère délégué à la Recherche et aux Nouvelles Technologies" (fellowship to MFV). SR and AMG are scientists from the "Centre National de la Recherche Scientifique". Our research is also funded by the European Union (FPG, Life Science Genomics and Biotechnology for Health, contract CT 2005 N°018802, CONTICA).


   References
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 1. Mechanisms involved in...
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