Rationale: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide’s efficacy in CPVT. Objective: To determine whether RyR2 block independently contributes to flecainide’s efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo. Methods and Results: We synthesized N-methylated flecainide analogues (QX-flecainide and N -methyl flecainide) and showed that N -methylation reduces flecainide’s inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N -methylation did not alter flecainide’s inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2−/−) CPVT mouse model. In membrane-permeabilized Casq2−/− cardiomyocytes—lacking intact sarcolemma and devoid of sodium channel contribution—flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2−/− cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N -methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2−/− mice, whereas N -methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block. Conclusions: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.
Background: Exercise training, and catecholaminergic stimulation, increase the incidence of arrhythmic events in patients affected with arrhythmogenic right ventricular cardiomyopathy correlated with plakophilin-2 (PKP2) mutations. Separate data show that reduced abundance of PKP2 leads to dysregulation of intracellular Ca 2+ (Ca 2+ i ) homeostasis. Here, we study the relation between exercise, catecholaminergic stimulation, Ca 2+ i homeostasis, and arrhythmogenesis in PKP2-deficient murine hearts. Methods: Experiments were performed in myocytes from a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line (PKP2cKO). For training, mice underwent 75 minutes of treadmill running once per day, 5 days each week for 6 weeks. We used multiple approaches including imaging, high-resolution mass spectrometry, electrocardiography, and pharmacological challenges to study the functional properties of cells/hearts in vitro and in vivo. Results: In myocytes from PKP2cKO animals, training increased sarcoplasmic reticulum Ca 2+ load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)-mediated Ca 2+ release events (sparks), and changed the time course of sarcomeric shortening. Phosphoproteomics analysis revealed that training led to hyperphosphorylation of phospholamban in residues 16 and 17, suggesting a catecholaminergic component. Isoproterenol-induced increase in Ca 2+ i transient amplitude showed a differential response to β-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca 2+ i sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels. Conclusions: Exercise disproportionately affects Ca 2+ i homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular β-adrenergic receptors and hyperphosphorylation of phospholamban. These cellular changes create a proarrhythmogenic state that can be mitigated by ryanodine receptor 2 blockade. Our data unveil an arrhythmogenic mechanism for exercise-induced or catecholaminergic life-threatening arrhythmias in the setting of PKP2 deficit. We suggest that membrane-permeable β-blockers are potentially more efficient for patients with arrhythmogenic right ventricular cardiomyopathy, highlight the potential for ryanodine receptor 2 channel blockers as treatment for the control of heart rhythm in the population at risk, and propose that PKP2-dependent and phospholamban-dependent arrhythmogenic right ventricular cardiomyopathy-related arrhythmias have a common mechanism.
Hit-to-lead studies employ a variety of strategies to optimize binding to a target of interest. When a structure for the target is available, hypothesis-driven structure–activity relationships (SAR) are a powerful strategy for refining the pharmacophore to achieve robust binding and selectivity characteristics necessary to identify a lead compound. Recrafting the three-dimensional space occupied by a small molecule, optimization of hydrogen bond contacts, and enhancing local attractive interactions are traditional approaches in medicinal chemistry. Ring size, however, is rarely able to be leveraged as an independent variable because most hits lack the symmetry required for such a study. Our discovery that the cyclic oligomeric depsipeptide ent-verticilide inhibits mammalian cardiac ryanodine receptor calcium release channels with submicromolar potency provided an opportunity to explore ring size as a variable, independent of other structural or functional group changes. We report here that ring size can be a critical independent variable, suggesting that modest conformational changes alone can dramatically affect potency.
A reinvestigation into the macrocyclooligomerization (MCO) of a tetradepsipeptide is reported, uncovering a paradox in which the MCO of depsipeptide monomers can produce “impossible” ring sizes: a 12-atom chain produced the expected 24-membered ring, alongside unexpected 18- and 30-membered cyclic oligomeric depsipeptides (CODs). We report an alternative preparation of authentic 18- and 36-membered macrocycles for this case using a stepwise synthesis that provides definitive analytical characterization for each ring size. Our investigation yields a recharacterization and reassignment of two macrocycles originally reported in this MCO series, along with updated yields and isothermal titration calorimetry data after implementation of new critical protocols for purification and subsequent analysis. Initial studies to probe this mechanistic conundrum are described.
The accessibility of bromonitromethane has declined in recent years, limiting its viability as a reagent for chemical synthesis. The reinvestigation and optimization of a variety of preparations, and the development of safe operating principles, are described. The reproducible protocol described here leverages the effectiveness of hydroxide for nitromethane bromination while respecting its incompatibility with the product it forms. This careful balance was achieved at scales up to 56 g, resulting in a reproducible procedure that provides straightforward, sustainable, and affordable access to this critical reagent.
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