The mechanism of flecainide action in CPVT does not involve a direct effect on RyR2.

RATIONALE: Flecainide, a class 1c antiarrhythmic, has emerged as an effective therapy in preventing arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) refractory to ß-adrenergic receptor blockade. It has been proposed that the clinical efficacy of flecainide in CPVT is because of the combined actions of direct blockade of ryanodine receptors (RyR2) and Na(+) channel inhibition. However, there is presently no direct evidence to support the notion that flecainide blocks RyR2 Ca(2+) flux in the physiologically relevant (luminal-to-cytoplasmic) direction. The mechanism of flecainide action remains controversial. OBJECTIVE: To examine, in detail, the effect of flecainide on the human RyR2 channel and to establish whether the direct blockade of physiologically relevant RyR2 ion flow by the drug contributes to its therapeutic efficacy in the clinical management of CPVT. METHODS AND RESULTS: Using single-channel analysis, we show that, even at supraphysiological concentrations, flecainide did not inhibit the physiologically relevant, luminal-to-cytosolic flux of cations through the channel. Moreover, flecainide did not alter RyR2 channel gating and had negligible effect on the mechanisms responsible for the sarcoplasmic reticulum charge-compensating counter current. Using permeabilized cardiac myocytes to eliminate any contribution of plasmalemmal Na(+) channels to the observed actions of the drug at the cellular level, flecainide did not inhibit RyR2-dependent sarcoplasmic reticulum Ca(2+) release. CONCLUSIONS: The principal action of flecainide in CPVT is not via a direct interaction with RyR2. Our data support a model of flecainide action in which Na(+)-dependent modulation of intracellular Ca(2+) handling attenuates RyR2 dysfunction in CPVT.

Moving in the right direction: elucidating the mechanisms of interaction between flecainide and the cardiac ryanodine receptor.

Flecainide is used to treat catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmia caused by disrupted cellular Ca2+ handling following ß-adrenergic stimulation. The clinical efficacy of flecainide in this context involves complex effects on multiple ion channels that may be influenced by the disease state. A compelling narrative has been constructed around flecainide's nonselective block of sarcoplasmic reticulum (SR) lumen-to-cytoplasm Ca2+ release through intracellular calcium release channels (RyR2). However, ion fluxes across the SR membrane during heart contraction are bidirectional, and here, we review experimental evidence that flecainide's principal action on RyR2 involves the partial block of ion flow in the cytoplasm-to-lumen direction (i.e., flecainide inhibits RyR2-mediated SR 'countercurrent'). Experimental approaches that could advance new knowledge on the mechanism of RyR2 block by flecainide are proposed. Some impediments to progress in this area, that must be overcome to enable the development of superior drugs to treat CPVT, are also considered.

Malignant hyperthermia mutation sites in the Leu2442-Pro2477 (DP4) region of RyR1 (ryanodine receptor 1) are clustered in a structurally and functionally definable area.

To explain the mechanism of pathogenesis of channel disorder in MH (malignant hyperthermia), we have proposed a model in which tight interactions between the N-terminal and central domains of RyR1 (ryanodine receptor 1) stabilize the closed state of the channel, but mutation in these domains weakens the interdomain interaction and destabilizes the channel. DP4 (domain peptide 4), a peptide corresponding to residues Leu2442-Pro2477 of the central domain, also weakens the domain interaction and produces MH-like channel destabilization, whereas an MH mutation (R2458C) in DP4 abolishes these effects. Thus DP4 and its mutants serve as excellent tools for structure-function studies. Other MH mutations have been reported in the literature involving three other amino acid residues in the DP4 region (Arg2452, Ile2453 and Arg2454). In the present paper we investigated the activity of several mutants of DP4 at these three residues. The ability to activate ryanodine binding or to effect Ca2+ release was severely diminished for each of the MH mutants. Other substitutions were less effective. Structural studies, using NMR analysis, revealed that the peptide has two a-helical regions. It is apparent that the MH mutations are clustered at the C-terminal end of the first helix. The data in the present paper indicates that mutation of residues in this region disrupts the interdomain interactions that stabilize the closed state of the channel.

Effects of peptide C corresponding to the Glu724-Pro760 region of the II-III loop of the DHP (dihydropyridine) receptor alpha1 subunit on the domain- switch-mediated activation of RyR1 (ryanodine receptor 1) Ca2+ channels.

The Leu720-Leu764 region of the II-III loop of the dihydropyridine receptor is believed to be important for both orthograde and retrograde communications with the RyR (ryanodine receptor), but its actual role has not yet been resolved. Our recent studies suggest that voltage-dependent activation of the RyR channel is mediated by a pair of interacting N-terminal and central domains, designated as the 'domain switch'. To investigate the effect of peptide C (a peptide corresponding to residues Glu724-Pro760) on domain- switch-mediated activation of the RyR, we measured Ca2+ release induced by DP (domain peptide) 1 or DP4 (which activates the RyR by mediation of the domain switch) and followed the Ca2+ release time course using a luminal Ca2+ probe (chlortetracycline) under Ca2+-clamped conditions. Peptide C produced a significant potentiation of the domain-switch-mediated Ca2+ release, provided that the Ca2+ concentration was sufficiently low (e.g. 0.1 microM) and the Ca2+ channel was only partially activated by the domain peptide. However, at micromolar Ca2+ concentrations, peptide C inhibits activation. Covalent cross-linking of fluorescently labelled peptide C to the RyR and screening of the fluorescently labelled tryptic fragments permitted us to localize the peptide-C-binding site to residues 450-1400, which may represent the primary region involved in physical coupling. Based on the above findings, we propose that the physiological role of residues Glu724-Pro760 is to facilitate depolarization-induced and domain-switch-mediated RyR activation at sub- or near-threshold concentrations of cytoplasmic Ca2+ and to suppress activation upon an increase of cytoplasmic Ca2+.

Pleiotropic mechanisms of action of perhexiline in heart failure.

INTRODUCTION: The re-purposing of the anti-anginal drug perhexiline (PHX) has resulted in symptomatic improvements in heart failure (HF) patients. The inhibition of carnitine palmitoyltransferase-1 (CPT-1) has been proposed as the primary mechanism underlying the therapeutic benefit of PHX. This hypothesis is contentious. AREAS COVERED: We reviewed the primary literature and patent landscape of PHX from its initial development in the 1960s through to its emergence as a drug beneficial for HF. We focused on its physico-chemistry, molecular targets, tissue accumulation and clinical dosing. EXPERT OPINION: Dogma that the beneficial effects of PHX are due primarily to potent myocardial CPT-1 inhibition is not supported by the literature and all available evidence point to it being extremely unlikely that the major effects of PHX occur via this mechanism. In vivo PHX is much more likely to be an inhibitor of surface membrane ion channels and also to have effects on other components of cellular metabolism and reactive oxygen species (ROS) generation across the cardiovascular system. However, the possibility that minor effects of PHX on CPT-1 underpin disproportionately large effects on myocardial function cannot be entirely excluded, especially given the massive accumulation of the drug in heart tissue.

Effect of flecainide derivatives on sarcoplasmic reticulum calcium release suggests a lack of direct action on the cardiac ryanodine receptor.

BACKGROUND AND PURPOSE: Flecainide is a use-dependent blocker of cardiac Na(+) channels. Mechanistic analysis of this block showed that the cationic form of flecainide enters the cytosolic vestibule of the open Na(+) channel. Flecainide is also effective in the treatment of catecholaminergic polymorphic ventricular tachycardia but, in this condition, its mechanism of action is contentious. We investigated how flecainide derivatives influence Ca(2) (+) -release from the sarcoplasmic reticulum through the ryanodine receptor channel (RyR2) and whether this correlates with their effectiveness as blockers of Na(+) and/or RyR2 channels. EXPERIMENTAL APPROACH: We compared the ability of fully charged (QX-FL) and neutral (NU-FL) derivatives of flecainide to block individual recombinant human RyR2 channels incorporated into planar phospholipid bilayers, and their effects on the properties of Ca(2) (+) sparks in intact adult rat cardiac myocytes. KEY RESULTS: Both QX-FL and NU-FL were partial blockers of the non-physiological cytosolic to luminal flux of cations through RyR2 channels but were significantly less effective than flecainide. None of the compounds influenced the physiologically relevant luminal to cytosol cation flux through RyR2 channels. Intracellular flecainide or QX-FL, but not NU-FL, reduced Ca(2) (+) spark frequency. CONCLUSIONS AND IMPLICATIONS: Given its inability to block physiologically relevant cation flux through RyR2 channels, and its lack of efficacy in blocking the cytosolic-to-luminal current, the effect of QX-FL on Ca(2) (+) sparks is likely, by analogy with flecainide, to result from Na(+) channel block. Our data reveal important differences in the interaction of flecainide with sites in the cytosolic vestibules of Na(+) and RyR2 channels.

SERCA2a gene transfer decreases sarcoplasmic reticulum calcium leak and reduces ventricular arrhythmias in a model of chronic heart failure.

BACKGROUND: Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure and is under evaluation in a clinical trial. A critical question is whether SERCA2a gene therapy predisposes to increased sarcoplasmic reticulum calcium (SR Ca(2+)) leak, cellular triggered activity, and ventricular arrhythmias in the failing heart. METHODS AND RESULTS: We studied the influence of SERCA2a gene therapy on ventricular arrhythmogenesis in a rat chronic heart failure model. ECG telemetry studies revealed a significant antiarrhythmic effect of SERCA2a gene therapy with reduction of both spontaneous and catecholamine-induced arrhythmias in vivo. SERCA2a gene therapy also reduced susceptibility to reentry arrhythmias in ex vivo programmed electrical stimulation studies. Subcellular Ca(2+) homeostasis and spontaneous SR Ca(2+) leak characteristics were measured in failing cardiomyocytes transfected in vivo with a novel AAV9.SERCA2a vector. SR Ca(2+) leak was reduced after SERCA2a gene therapy, with reversal of the greater spark mass observed in the failing myocytes, despite normalization of SR Ca(2+) load. SERCA2a reduced ryanodine receptor phosphorylation, thereby resetting SR Ca(2+) leak threshold, leading to reduced triggered activity in vitro. Both indirect effects of reverse remodeling and direct SERCA2a effects appear to underlie the antiarrhythmic action. CONCLUSIONS: SERCA2a gene therapy stabilizes SR Ca(2+) load, reduces ryanodine receptor phosphorylation and decreases SR Ca(2+) leak, and reduces cellular triggered activity in vitro and spontaneous and catecholamine-induced ventricular arrhythmias in vivo in failing hearts. SERCA2a gene therapy did not therefore predispose to arrhythmias and may represent a novel antiarrhythmic strategy in heart failure.

Amyloid-ß protein impairs Ca2+ release and contractility in skeletal muscle.

Inclusion body myositis (IBM), the most common muscle disorder in the elderly, is partly characterized by dysregulation of ß-amyloid precursor protein (ßAPP) expression and abnormal, intracellular accumulation of full-length ßAPP and ß-amyloid epitopes. The present study examined the effects of ß-amyloid accumulation on force generation and Ca(2+) release in skeletal muscle from transgenic mice harboring human ßAPP and assessed the consequence of Aß(1-42) modulation of the ryanodine receptor Ca(2+) release channels (RyRs). ß-Amyloid laden muscle produced less peak force and exhibited Ca(2+) transients with smaller amplitude. To determine whether modification of RyRs by ß-amyloid underlie the effects observed in muscle, in vitro Ca(2+) release assays and RyR reconstituted in planar lipid bilayer experiments were conducted in the presence of Aß(1-42). Application of Aß(1-42) to RyRs in bilayers resulted in an increased channel open probability and changes in gating kinetics, while addition of Aß(1-42) to the rabbit SR vesicles resulted in RyR-mediated Ca(2+) release. These data may relate altered ßAPP metabolism in IBM to reductions in RyR-mediated Ca(2+) release and muscle contractility.

Peptide probe study of the role of interaction between the cytoplasmic and transmembrane domains of the ryanodine receptor in the channel regulation mechanism.

Ryanodine receptor (RyR) mutations linked with some congenital skeletal and cardiac diseases are localized to three easily definable regions: region 1 (N-terminal domain), region 2 (central domain), and a rather broad region 3 containing the channel pore. As shown in our recent studies, the interdomain interaction between regions 1 and 2 plays a critical role in channel regulation and pathogenesis. Here we present evidence that within region 3 there is a similar channel regulation mechanism mediated by an interdomain interaction. DP15, a peptide corresponding to RyR1 residues 4820-4841, produced significant activation of [3H]ryanodine binding above threshold Ca2+ concentrations (>or=0.3 microM), but MH mutations (L4823P or L4837V) made in DP15 almost completely abolished its channel activating function. To identify the DP15 binding site(s) within RyR1, DP15 (labeled with a fluorescent probe Alexa Fluor 680 and a photoaffinity cross-linker APG) was cross-linked to RyR1, and the site of cross-linking was identified by gel analysis of fluorescently labeled proteolytic fragments with the aid of Western blotting with site-specific antibodies. The shortest fluorescently labeled band was a 96 kDa fragment which was stained with an antibody directed to the region of residues 4114-4142 of RyR1, indicating that the interaction between the region of residues 4820-4841 adjacent to the channel pore and the 96 kDa segment containing the region of residues 4114-4142 is involved in the mechanism of Ca2+-dependent channel regulation. In further support of this concept, anti-DP15 antibody and cardiac counterpart of DP15 produced channel activation similar to that of DP15.

Endoplasmic reticulum stress as a novel therapeutic target in heart diseases.

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for the synthesis and folding of proteins as well as calcium storage and signaling. Perturbations of ER function cause ER stress leading to the unfolded protein response (UPR), which includes inhibition of protein synthesis, protein refolding and clearance of misfolded proteins. The UPR aims at restoring cellular homeostasis, however, prolonged ER stress can trigger apoptosis. ER stress-induced apoptosis has been implicated in the pathogenesis of various diseases such as brain ischemia/reperfusion, neurodegeneration, diabetes and, most recently, myocardial infarction and heart failure. Initial events leading to UPR and apoptosis in the heart include protein oxidation and disturbed calcium handling upon ischemia/reperfusion, and forced protein synthesis during cardiac hypertrophy. While XBP-1 and ATF6-mediated induction of ER chaperones seems to protect the heart from ischemia/reperfusion injury, the PERK/ATF4/CHOP branch of the UPR might transmit proapoptotic signals. The precise mechanism of ER stress-induced cardiomyocyte apoptosis remains elusive, however, recent data suggest that the mitochondrial apoptotic machinery is recruited through the upregulation of Puma, a proapoptotic member of the Bcl-2 family. Importantly, suppression of Puma activity prevented both ER stress and ischemia/reperfusion-induced cardiomyocyte loss, highlighting the ER stress pathways as potential therapeutic targets in cardiovascular diseases.

Dantrolene stabilizes domain interactions within the ryanodine receptor.

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.

Activation of the sheep cardiac Ca2+ release channel by simple heteroaromatics.

The broad range of ligands known to modulate ryanodine receptor activity includes a class of heteroaromatic compounds displaying relatively poor efficacy. Greater understanding of the physicochemical properties that predispose these molecules to interaction with the channel should facilitate the rational design of more potent analogues. To this end we are examining the structure-activity relationship for simple heteroaromatic compounds. Efficacy is assessed by the ability to stimulate [3H]ryanodine binding to heavy sarcoplasmic reticulum vesicles. The propensity to activate the channel requires notably little chemical functionality and is associated with the capacity for charge-transfer complex formation in conjunction with steric bulk.

Removal of clustered positive charge from dihydropyridine receptor II-III loop peptide augments activation of ryanodine receptors.

Peptides based on the skeletal muscle DHPR II-III loop have been shown to regulate ryanodine receptor channel activity. The N-terminal region of this cytoplasmic loop is predicted to adopt an alpha-helical conformation. We have selected a peptide sequence of 26 residues (Ala(667)-Asp(692)) as the minimum sequence to emulate the helical propensity of the corresponding protein sequence. The interaction of this control peptide with skeletal and cardiac RyR channels in planar lipid bilayers was then assessed and was found to lack isoform specificity. At low concentrations peptide A(667)-D(692) increased RyR open probability, whilst at higher concentrations open probability was reduced. By replacing a region of clustered positive charge with a neutral sequence with the same predisposition to helicity, the inhibitory effect was ablated and activation was enhanced. This novel finding demonstrates that activation does not derive from the presence of positively charged residues adjacent in the primary structure and, although it may be mediated by the alignment of basic residues down one face of an amphipathic helix, not all of these residues are essential.