Mark L. Bannister

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 Ca2+) 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 Ca2+ homeostasis and spontaneous SR Ca2+ leak characteristics were measured in failing cardiomyocytes transfected in vivo with a novel AAV9.SERCA2a vector. SR Ca2+ leak was reduced after SERCA2a gene therapy, with reversal of the greater spark mass observed in the failing myocytes, despite normalization of SR Ca2+ load. SERCA2a reduced ryanodine receptor phosphorylation, thereby resetting SR Ca2+ 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 Ca2+ load, reduces ryanodine receptor phosphorylation and decreases SR Ca2+ 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.

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 Ca2+ 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 (KQ) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 °C and was unaffected by calmodulin. [3H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca2+ release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 °C, yet required the presence of calmodulin at 22 °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 Ca2+ channel opening rather than the domain switch itself.

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.

SERCA2a Gene Transfer Decreases SR 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 Ca2+) 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 Ca2+ homeostasis and spontaneous SR Ca2+ leak characteristics were measured in failing cardiomyocytes transfected in vivo with a novel AAV9.SERCA2a vector. SR Ca2+ leak was reduced after SERCA2a gene therapy, with reversal of the greater spark mass observed in the failing myocytes, despite normalization of SR Ca2+ load. SERCA2a reduced ryanodine receptor phosphorylation, thereby resetting SR Ca2+ 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 Ca2+ load, reduces ryanodine receptor phosphorylation and decreases SR Ca2+ 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.

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 β-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.

Amyloid-β 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 β-amyloid precursor protein (βAPP) expression and abnormal, intracellular accumulation of full-length βAPP and β-amyloid epitopes. The present study examined the effects of β-amyloid accumulation on force generation and Ca(2+) release in skeletal muscle from transgenic mice harboring human βAPP and assessed the consequence of Aβ(1-42) modulation of the ryanodine receptor Ca(2+) release channels (RyRs). β-Amyloid laden muscle produced less peak force and exhibited Ca(2+) transients with smaller amplitude. To determine whether modification of RyRs by β-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 Aβ(1-42). Application of Aβ(1-42) to RyRs in bilayers resulted in an increased channel open probability and changes in gating kinetics, while addition of Aβ(1-42) to the rabbit SR vesicles resulted in RyR-mediated Ca(2+) release. These data may relate altered βAPP metabolism in IBM to reductions in RyR-mediated Ca(2+) release and muscle contractility.

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.

The contribution of hydrophobic residues in the pore-forming region of the ryanodine receptor channel to block by large tetraalkylammonium cations and Shaker B inactivation peptides

Although no high-resolution structural information is available for the ryanodine receptor (RyR) channel pore-forming region (PFR), molecular modeling has revealed broad structural similarities between this region and the equivalent region of K+ channels. This study predicts that, as is the case in K+ channels, RyR has a cytosolic vestibule lined with predominantly hydrophobic residues of transmembrane helices (TM10). In K+ channels, this vestibule is the binding site for blocking tetraalkylammonium (TAA) cations and Shaker B inactivation peptides (ShBPs), which are stabilized by hydrophobic interactions involving specific residues of the lining helices. We have tested the hypothesis that the cytosolic vestibule of RyR fulfils a similar role and that TAAs and ShBPs are stabilized by hydrophobic interactions with residues of TM10. Both TAAs and ShBPs block RyR from the cytosolic side of the channel. By varying the composition of TAAs and ShBPs, we demonstrate that the affinity of both species is determined by their hydrophobicity, with variations reflecting alterations in the dissociation rate of the bound blockers. We investigated the role of TM10 residues of RyR by monitoring block by TAAs and ShBPs in channels in which the hydrophobicity of individual TM10 residues was lowered by alanine substitution. Although substitutions changed the kinetics of TAA interaction, they produced no significant changes in ShBP kinetics, indicating the absence of specific hydrophobic sites of interactions between RyR and these peptides. Our investigations (a) provide significant new information on both the mechanisms and structural components of the RyR PFR involved in block by TAAs and ShBPs, (b) highlight important differences in the mechanisms and structures determining TAA and ShBP block in RyR and K+ channels, and (c) demonstrate that although the PFRs of these channels contain analogous structural components, significant differences in structure determine the distinct ion-handling properties of the two species of channel.

Pleiotropic mechanisms of action of perhexiline in heart failure.

ABSTRACT 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.

Questioning flecainide's mechanism of action in the treatment of catecholaminergic polymorphic ventricular tachycardia

Flecainide attenuates cardiac Ca2+ cycling abnormalities in malignant catecholaminetriggered arrhythmias but its mechanism of action remains highly contentious. We read with interest the study of Yang et al. (2016) that used in silico predictions in an attempt to determine the relative contribution of block by flecainide of ryanodine receptor 2 (RyR2) and the Na+ channel to the overall therapeutic effect of this drug in catecholaminergic polymorphic ventricular tachycardia (CPVT). Flecainide’s actions were modelled by considering its inhibition of the Na+ current (INa) and RyR2 in combination and individually. The authors conclude that the effects of the drug on Na+ channels are insufficient to explain its efficacy in CPVT, while its block of RyR2 alone was as effective as the combined block of INa and RyR2. Our concerns about the usefulness of this approach are twofold: