Catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin. We recently found that the drug flecainide inhibits RyR2 channels and prevents CPVT in mice and humans. Here we compared the effects of flecainide and tetracaine, a known RyR2 inhibitor ineffective in CPVT myocytes, on arrhythmogenic Ca 2+ waves and elementary sarcoplasmic reticulum (SR) Ca 2+ release events, Ca 2+ sparks. In ventricular myocytes isolated from a CPVT mouse model, flecainide significantly reduced spark amplitude and spark width, resulting in a 40% reduction in spark mass. Surprisingly, flecainide significantly increased spark frequency. As a result, flecainide had no significant effect on spark-mediated SR Ca 2+ leak or SR Ca 2+ content. In contrast, tetracaine decreased spark frequency and spark-mediated SR Ca 2+ leak, resulting in a significantly increased SR Ca 2+ content. Measurements in permeabilized rat ventricular myocytes confirmed the different effects of flecainide and tetracaine on spark frequency and Ca 2+ waves. In lipid bilayers, flecainide inhibited RyR2 channels by open state block, whereas tetracaine primarily prolonged RyR2 closed times. The differential effects of flecainide and tetracaine on sparks and RyR2 gating can explain why flecainide, unlike tetracaine, does not change the balance of SR Ca 2+ fluxes. We suggest that the smaller spark mass contributes to flecainide's antiarrhythmic action by reducing the probability of saltatory wave propagation between adjacent Ca 2+ release units. Our results indicate that inhibition of the RyR2 open state provides a new therapeutic strategy to prevent diastolic Ca 2+ waves resulting in triggered arrhythmias, such as CPVT.

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Key Words: FKBP12.6 Ⅲ FKBP12 Ⅲ Ca sparks Ⅲ binding properties Ⅲ rapamycin Ⅲ RyR2 C ardiac ryanodine receptors (RyR2) are sarcoplasmic reticulum (SR) Ca release channels, crucial in excitation-contraction coupling. 1 Dysfunctional RyR2, exhibiting enhanced Ca leak has been implicated in arrhythmogenesis and heart failure (HF). 1 Homotetrameric RyR2s have a transmembrane channel domain, and regulatory and scaffolding cytoplasmic domain.In heart, FK506 binding protein (FKBP) isoforms FKBP12 and FKBP12.6 are coexpressed and can bind RyR2 at a stoichiometry of 4 FKBP per RyR tetramer. 2,3 FKBP12 binds RyR2 with much lower affinity, but is much higher in concentration in heart than FKBP12.6. 4 Nevertheless, FKBP12 and 12.6 share 85% sequence homology and similar 3D structures. 5 Human and rat FKBP12 differ in only 3 residues, and human and rat FKBP12.6 are identical. This makes study of human FKBP function in rat myocytes reasonable.Effects of FKBPs on RyR2 activity in myocytes are controversial. Some groups reported that dissociation of FKBP12.6 from RyR2 by immunosuppressants (rapamycin or FK506) activated RyR2 channels and induced subconductance states. 6 -8 RyR2 point mutations associated with cardiac sudden death may also exhibit altered FKBP12.6 interaction, 9 and FK506 can alter resting Ca 2ϩ spark frequency (CaSpF) and SR Ca 2ϩ content. 7,8,10 FKBP12.6 overexpression also increased SR load and enhanced contraction. 11 However, others reported that FKBP12.6 removal had no effect on RyR2 activity, 3,12 and failed to observe RyR2 subconductance states in channels from FKBP12.6 knockout mice. 13,14 Some groups suggest that FKBP12 affects RyR2 differently from FKBP12.6. [15][16][17]20 Therefore, FKBP12/12.6 binding and RyR2 effects remain unclear, especially in the cardiomyocyte environment (as studied here).Altered FKBP-RyR2 interaction is a prominent hypothesis explaining increased SR Ca leak in HF via RyR2 hyperphos- Here we characterize in the myocyte environment the interaction between FKBP12/12.6 and RyR2, its functional consequences, and modulation by PKA-dependent phosphorylation. Using fluorescent FKBP12/12.6 (F-FKBP), we simultaneously assessed physical association-dissociation of FKBP12/12.6 with RyR2, and RyR2 activity (via Ca sparks) in permeabilized ventricular myocytes. We found that: (1) both FKBP12.6 and FKBP12 bound to RyR2 (K d Ϸ1 nmol/L and 200 nmol/L, respectively); (2) FKBP12.6 but not FKBP12 inhibited CaSpF; (3) the binding properties of FKBP12.6/12-RyR2 were not changed by PKA-dependent phosphorylation of RyR2; and (4) endogenous FKBP12 was Ϸ1 mol/L cytosol and FKBP12.6 Ϸ100 nmol/L cytosol. MethodsRat and mouse ventricular myocytes were isolated and permeabilized as previously described (see the Online Data Supplement, available at http://circres.ahajournals.org). 23 F-FKBP12.6 and F-FKBP12 were characterized using circular dichroism spectroscopy and ligandbinding studies (Online Figure I). 24 Consistent with previous reports, 25 F-FKBP constructs have the same secondary structure, RyR ...