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

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 α1-adrenoceptor [1], annexin-V [1], protein-kinase-Cδ [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.