Samantha C. Salvage

Flecainide exerts paradoxical effects on sodium currents and atrial arrhythmia in murine RyR2-P2328S hearts

Cardiac ryanodine receptor mutations are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), and some, including RyR2‐P2328S, also predispose to atrial fibrillation. Recent work associates reduced atrial Nav1.5 currents in homozygous RyR2‐P2328S (RyR2S/S) mice with slowed conduction and increased arrhythmogenicity. Yet clinically, and in murine models, the Nav1.5 blocker flecainide reduces ventricular arrhythmogenicity in CPVT. We aimed to determine whether, and how, flecainide influences atrial arrhythmogenicity in RyR2S/S mice and their wild‐type (WT) littermates.

Arrhythmic substrate, slowed propagation and increased dispersion in conduction direction in the right ventricular outflow tract of murine Scn5a+/- hearts.

To test a hypothesis attributing arrhythmia in Brugada Syndrome to right ventricular (RV) outflow tract (RVOT) conduction abnormalities arising from Nav1.5 insufficiency and fibrotic change.

Multiple targets for flecainide action: implications for cardiac arrhythmogenesis

Flecainide suppresses cardiac tachyarrhythmias including paroxysmal atrial fibrillation, supraventricular tachycardia and arrhythmic long QT syndromes (LQTS), as well as the Ca2+‐mediated, catecholaminergic polymorphic ventricular tachycardia (CPVT). However, flecainide can also exert pro‐arrhythmic effects most notably following myocardial infarction and when used to diagnose Brugada syndrome (BrS). These divergent actions result from its physiological and pharmacological actions at multiple, interacting levels of cellular organization. These were studied in murine genetic models with modified Nav channel or intracellular ryanodine receptor (RyR2)‐Ca2+ channel function. Flecainide accesses its transmembrane Nav1.5 channel binding site during activated, open, states producing a use‐dependent antagonism. Closing either activation or inactivation gates traps flecainide within the pore. An early peak INa related to activation of Nav channels followed by rapid de‐activation, drives action potential (AP) upstrokes and their propagation. This is diminished in pro‐arrhythmic conditions reflecting loss of function of Nav1.5 channels, such as BrS, accordingly exacerbated by flecainide challenge. Contrastingly, pro‐arrhythmic effects attributed to prolonged AP recovery by abnormal late INaL following gain‐of‐function modifications of Nav1.5 channels in LQTS3 are reduced by flecainide. Anti‐arrhythmic effects of flecainide that reduce triggering in CPVT models mediated by sarcoplasmic reticular Ca2+ release could arise from its primary actions on Nav channels indirectly decreasing [Ca2+]i through a reduced [Na+]i and/or direct open‐state RyR2‐Ca2+ channel antagonism. The consequent [Ca2+]i alterations could also modify AP propagation velocity and therefore arrhythmic substrate through its actions on Nav1.5 channel function. This is consistent with the paradoxical differences between flecainide actions upon Na+ currents, AP conduction and arrhythmogenesis under circumstances of normal and increased RyR2 function.

Ion channels, long QT syndrome and arrhythmogenesis in ageing.

Ageing is associated with increased prevalences of both atrial and ventricular arrhythmias, reflecting disruption of the normal sequence of ion channel activation and inactivation generating the propagated cardiac action potential. Experimental models with specific ion channel genetic modifications have helped clarify the interacting functional roles of ion channels and how their dysregulation contributes to arrhythmogenic processes at the cellular and systems level. They have also investigated interactions between these ion channel abnormalities and age‐related processes in producing arrhythmic tendency. Previous reviews have explored the relationships between age and loss‐of‐function Nav1.5 mutations in producing arrhythmogenicity. The present review now explores complementary relationships arising from gain‐of‐function Nav1.5 mutations associated with long QT3 (LQTS3). LQTS3 patients show increased risks of life‐threatening ventricular arrhythmias, particularly after 40 years of age, consistent with such interactions between the ion channel abnormailities and ageing. In turn clinical evidence suggests that ageing is accompanied by structural, particularly fibrotic, as well as electrophysiological change. These abnormalities may result from biochemical changes producing low‐grade inflammation resulting from increased production of reactive oxygen species and superoxide. Experimental studies offer further insights into the underlying mechanisms underlying these phenotypes. Thus, studies in genetically modified murine models for LQTS implicated action potential recovery processes in arrhythmogenesis resulting from functional ion channel abnormalities. In addition, ageing wild type (WT) murine models demonstrated both ion channel alterations and fibrotic changes with ageing. Murine models then suggested evidence for interactions between ageing and ion channel mutations and provided insights into potential arrhythmic mechanisms inviting future exploration.

Calcium-dependent Nedd4-2 upregulation mediates degradation of the cardiac sodium channel Nav1.5: implications for heart failure

Reductions in voltage‐gated sodium channel (Nav1.5) function/expression provide a slowed‐conduction substrate for cardiac arrhythmias. Nedd4‐2, which is activated by calcium, post‐translationally modulates Nav1.5. We aim to investigate whether elevated intracellular calcium ([Ca2+]i) reduces Nav1.5 through Nedd4‐2 and its role in heart failure (HF).

Age-dependent electrocardiographic changes in Pgc-1β deficient murine hearts

Increasing evidence implicates chronic energetic dysfunction in human cardiac arrhythmias. Mitochondrial impairment through Pgc‐1β knockout is known to produce a murine arrhythmic phenotype. However, the cumulative effect of this with advancing age and its electrocardiographic basis have not been previously studied. Young (12‐16 weeks) and aged (>52 weeks), wild type (WT) (n = 5 and 8) and Pgc‐1β−/− (n = 9 and 6), mice were anaesthetised and used for electrocardiographic (ECG) recordings. Time intervals separating successive ECG deflections were analysed for differences between groups before and after β1‐adrenergic (intraperitoneal dobutamine 3 mg/kg) challenge. Heart rates before dobutamine challenge were indistinguishable between groups. The Pgc‐1β−/− genotype however displayed compromised nodal function in response to adrenergic challenge. This manifested as an impaired heart rate response suggesting a functional defect at the level of the sino‐atrial node, and a negative dromotropic response suggesting an atrioventricular conduction defect. Incidences of the latter were most pronounced in the aged Pgc‐1β−/− mice. Moreover, Pgc‐1β−/− mice displayed electrocardiographic features consistent with the existence of a pro‐arrhythmic substrate. Firstly, ventricular activation was prolonged in these mice consistent with slowed action potential conduction and is reported here for the first time. Additionally, Pgc‐1β−/− mice had shorter repolarisation intervals. These were likely attributable to altered K+ conductance properties, ultimately resulting in a shortened QTc interval, which is also known to be associated with increased arrhythmic risk. ECG analysis thus yielded electrophysiological findings bearing on potential arrhythmogenicity in intact Pgc‐1β−/− systems in widespread cardiac regions.

The effects of ageing and adrenergic challenge on electrocardiographic phenotypes in a murine model of long QT syndrome type 3

Long QT Syndrome 3 (LQTS3) arises from gain-of-function Nav1.5 mutations, prolonging action potential repolarisation and electrocardiographic (ECG) QT interval, associated with increased age-dependent risk for major arrhythmic events, and paradoxical responses to β-adrenergic agents. We investigated for independent and interacting effects of age and Scn5a+/ΔKPQ genotype in anaesthetised mice modelling LQTS3 on ECG phenotypes before and following β-agonist challenge, and upon fibrotic change. Prolonged ventricular recovery was independently associated with Scn5a+/ΔKPQ and age. Ventricular activation was prolonged in old Scn5a+/ΔKPQ mice (p = 0.03). We associated Scn5a+/ΔKPQ with increased atrial and ventricular fibrosis (both: p < 0.001). Ventricles also showed increased fibrosis with age (p < 0.001). Age and Scn5a+/ΔKPQ interacted in increasing incidences of repolarisation alternans (p = 0.02). Dobutamine increased ventricular rate (p < 0.001) and reduced both atrioventricular conduction (PR segment-p = 0.02; PR interval-p = 0.02) and incidences of repolarisation alternans (p < 0.001) in all mice. However, in Scn5a+/ΔKPQ mice, dobutamine delayed the changes in ventricular repolarisation following corresponding increases in ventricular rate. The present findings implicate interactions between age and Scn5a+/ΔKPQ in prolonging ventricular activation, correlating them with fibrotic change for the first time, adding activation abnormalities to established recovery abnormalities in LQTS3. These findings, together with dynamic electrophysiological responses to β-adrenergic challenge, have therapeutic implications for ageing LQTS patients.

Regulatory actions of 3′,5′‐cyclic adenosine monophosphate on osteoclast function: possible roles of Epac‐mediated signaling

Alterations in cellular levels of the second messenger 3′,5′‐cyclic adenosine monophosphate ([cAMP]i) regulate a wide range of physiologically important cellular signaling processes in numerous cell types. Osteoclasts are terminally differentiated, multinucleated cells specialized for bone resorption. Their systemic regulator, calcitonin, triggers morphometrically and pharmacologically distinct retraction (R) and quiescence (Q) effects on cell‐spread area and protrusion–retraction motility, respectively, paralleling its inhibition of bone resorption. Q effects were reproduced by cholera toxin‐mediated Gs‐protein activation known to increase [cAMP]i, unaccompanied by the [Ca2+]i changes contrastingly associated with R effects. We explore a hypothesis implicating cAMP signaling involving guanine nucleotide‐exchange activation of the small GTPase Ras‐proximate‐1 (Rap1) by exchange proteins directly activated by cAMP (Epac). Rap1 activates integrin clustering, cell adhesion to bone matrix, associated cytoskeletal modifications and signaling processes, and transmembrane transduction functions. Epac activation enhanced, whereas Epac inhibition or shRNA‐mediated knockdown compromised, the appearance of markers for osteoclast differentiation and motility following stimulation by receptor activator of nuclear factor kappa‐Β ligand (RANKL). Deficiencies in talin and Rap1 compromised in vivo bone resorption, producing osteopetrotic phenotypes in genetically modified murine models. Translational implications of an Epac‐Rap1 signaling hypothesis in relationship to N‐bisphosphonate actions on prenylation and membrane localization of small GTPases are discussed.