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Cardiovascular Research 2007 76(2):199-201; doi:10.1016/j.cardiores.2007.08.013
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Copyright © 2007, European Society of Cardiology

Inhibition of SR Ca2+ uptake: A novel action of the RyR2–FKBP12.6 antagonist K201

Andrew F. James*

Department of Physiology and Pharmacology, and Bristol Heart Institute Cardiovascular Research Laboratories, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK
Department of Cell Physiology, National Institute for Physiological Sciences, Nishigonaka 38, Myodaiji-cho, Okazaki 444-8585, Japan

*Department of Physiology and Pharmacology, and Bristol Heart Institute Cardiovascular Research Laboratories, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK. Tel.: +44 117 331 2297; fax: +44 117 331 2288. a.james{at}bristol.ac.uk

Received 20 August 2007; revised 28 August 2007; accepted 28 August 2007

See article by Loughrey et al. [13] (pages 236–246) in this issue.

K201, also known as JTV519 or JTV-519, is a 1,4-benzothiazepine derivative that was developed for its protective action against Ca2+-induced myocardial damage [1]. It was subsequently shown to be protective in models of ischaemia-reperfusion injury [2,3] and heart failure (HF) [4], has been in phase II clinical trials as a protective agent against acute myocardial infarction since 2000, and is suggested to reduce reperfusion injury following percutaneous transluminal coronary angioplasty (see http://www.jti.co.jp/JTI_E/IR/FR/0111/0111-Supplementary.pdf). Although like diltiazem, to which it is structurally related, it blocks L-type-Ca2+-current (ICa,L) [5], it is a relatively non-selective blocker of cation-currents including the Na+-current [5], both background and G-protein-gated inward-rectifier K+ currents [5,6], and the rapidly-activating-delayed-rectifier-current (IKr) [6,7]. K201 is also reported to have anti-arrhythmic effects in models of atrial-fibrillation (AF) through prolongation of the effective refractory period (ERP) [6,8]. Other putative-targets for K201 include the {alpha}1-adrenoceptor [1], annexin-V [1], protein-kinase-C{delta} [3], and NO-synthase [9]. More recently, the effects of K201 on the ryanodine-receptor (RyR2), suggested to ameliorate dramatically the progression of HF and to be anti-arrhythmogenic, have attracted considerable attention [4,10–12]. In this issue of Cardiovascular Research, a report by Loughrey et al. provides evidence that K201 also inhibits sarcoplasmic reticulum (SR) Ca2+ uptake [13].

RyR2 is a large trans-membrane protein localized to the SR that plays a vital role in cardiac excitation–contraction (E–C) coupling as a Ca2+-activated–Ca2+-release-channel [14]. The channels are homo-tetramers of RyR2 subunits that exist as a macromolecular complex in the SR membrane in association with a variety of modulatory proteins, including the immunophilin FKBP12.6 [14]. FKBP12.6 is suggested to play an important role in RyR2-channel gating, stabilising a closed state and reducing the sensitivity of RyR2 to cytosolic Ca2+, thereby ensuring efficient E–C coupling. Equally important to efficient E–C coupling is the SR Ca2+-ATPase (SERCA2), which re-sequesters Ca2+ ions to the SR, thereby producing relaxation and replenishing the SR–Ca2+ store in preparation for the next systole. β1-Adrenoceptor-activated pathways involving protein kinase A (PKA)-dependent phosphorylation play a central role in modulating cardiac function to meet demand, as in exercise, for example. It has been suggested that phosphorylation of RyR2 at serine-2809 (S2809) reduces the affinity of RyR2 for FKBP12.6, thereby increasing the Ca2+-sensitivity of RyR2 and increasing SR-Ca2+-release [15]. It is suggested that in HF ‘hyperphosphorylation’ of RyR2 channels, defined as phosphorylation at S2809 of at least three of the four subunits in a channel tetramer, causes marked dissociation of FKBP12.6 from RyR2, resulting in E–C coupling abnormalities [15]. Similarly, RyR2-mutations that underlie inherited susceptibility to the exercise-induced arrhythmia catecholaminergic-polymorphic-ventricular-tachycardia (CPVT) are suggested to result in marked reduction of FKBP12.6 affinity for S2809-phosphorylated RyR2 and, thereby, increased diastolic SR–Ca2+ leak, delayed-afterdepolarisations (DADs), and triggered-activity [16].

K201 has been suggested to reduce diastolic Ca2+ leak in HF and exercise-induced ventricular tachycardia by restoring the association of FKBP12.6 with RyR2 [4,10–12,17,18]. Of particular note, K201 was reported to be ineffective in FKBP12.6–/– mice [10–12]. However, the role of S2809-phosphorylation and FKBP12.6 in the (patho) physiological modulation of RyR2 remains highly contentious [14]. For example, the sustained elevation of Ca2+ release produced by PKA activation has been demonstrated to occur via increased SERCA2 activity and increased fractional SR release [19]. Moreover, recent evidence suggests that S2809-phosphorylation of 3/4 RyR2 subunits in a tetramer represents a normal state of the channel [20].

It is against this background that Loughrey and colleagues have made a thorough investigation of the actions of K201 by fluorimetric recordings from whole-cell voltage-clamped and β-escin-permeabilised cardiomyocytes isolated from normal rabbits [13]. Loughrey et-al demonstrate not only that K201 antagonises diastolic SR–Ca2+ leak via RyR2 in normal myocytes under conditions of SR–Ca2+ overload, as shown very recently by Hunt et al. [21], but, more notably, present entirely novel evidence that the 1,4-benzothiazepine inhibits SR Ca2+ uptake. K201 (1 and 3 µmol/L) produced a significant reduction in the amplitude of the Ca2+-transient in voltage-clamped myocytes, the effect at 1 µmol/L being without ICa,L blockade. Although 1 µmol/L K201 reduced SR Ca2+ release at a given Ca2+ load, unlike the pure RyR2 antagonist, tetracaine, K201 was without effect on SR Ca2+ load, suggesting an additional effect of the 1,4-benzothiazepine. In β-escin-permeabilised cells, the velocity and frequency of Ca2+ waves induced by SR Ca2+ leak were slowed by 1 µmol/L K201 and abolished by 3 µmol/L K201. Most importantly, K201 slowed SR Ca2+ uptake in myocyte aggregates in a concentration-dependent manner; this demonstrates unequivocally that K201 inhibits SERCA2 and provides an explanation as to how K201 was able to reduce the frequency of spontaneous Ca2+ transients without increasing their amplitude or SR Ca2+ load. Finally, the inhibition of SR Ca2+ waves by K201 in permeabilised cells was unaffected by the adenoviral-mediated over-expression of FKBP12.6.

In summary, this important study demonstrates a novel inhibition of SR Ca2+ uptake in addition to providing further evidence that K201 modulates SR–Ca2+ leak independent of FKBP12.6–RyR2 interaction. This action of K201 to reduce SR Ca2+ overload may both protect against myocardial damage and be anti-arrhythmic through reduction of DADs and triggered activity. Moreover, drugs such as K201, with the novel inhibition of SR Ca2+ uptake demonstrated in this study in combination with the previously demonstrated prolongation of ERP [6,8], may be of particular value as anti-arrhythmic agents in AF, which is associated with SR Ca2+ overload and shortened ERP.


   References
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