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.Medical Research Counci

Flecainide Paradoxically Activates Cardiac Ryanodine Receptor Channels under Low Activity Conditions: A Potential Pro-Arrhythmic Action.

Cardiac ryanodine receptor (RyR2) mutations are implicated in the potentially fatal catecholaminergic polymorphic ventricular tachycardia (CPVT) and in atrial fibrillation. CPVT has been successfully treated with flecainide monotherapy, with occasional notable exceptions. Reported actions of flecainide on cardiac sodium currents from mice carrying the pro-arrhythmic homozygotic RyR2-P2328S mutation prompted our explorations of the effects of flecainide on their RyR2 channels. Lipid bilayer electrophysiology techniques demonstrated a novel, paradoxical increase in RyR2 activity. Preceding flecainide exposure, channels were mildly activated by 1 mM luminal Ca2+ and 1 µM cytoplasmic Ca2+, with open probabilities (Po) of 0.03 ± 0.01 (wild type, WT) or 0.096 ± 0.024 (P2328S). Open probability (Po) increased within 0.5 to 3 min of exposure to 0.5 to 5.0 µM cytoplasmic flecainide, then declined with higher concentrations of flecainide. There were no such increases in a subset of high Po channels with Po ≥ 0.08, although Po then declined with ≥5 µM (WT) or ≥50 µM flecainide (P2328S). On average, channels with Po < 0.08 were significantly activated by 0.5 to 10 µM of flecainide (WT) or 0.5 to 50 µM of flecainide (P2328S). These results suggest that flecainide can bind to separate activation and inhibition sites on RyR2, with activation dominating in lower activity channels and inhibition dominating in more active channels

Cell-Adhesion Properties of β-Subunits in the Regulation of Cardiomyocyte Sodium Channels

Voltage-gated sodium (Nav) channels drive the rising phase of the action potential, essential for electrical signalling in nerves and muscles. The Nav channel α-subunit contains the ion-selective pore. In the cardiomyocyte, Nav1.5 is the main Nav channel α-subunit isoform, with a smaller expression of neuronal Nav channels. Four distinct regulatory β-subunits (β1−4) bind to the Nav channel α-subunits. Previous work has emphasised the β-subunits as direct Nav channel gating modulators. However, there is now increasing appreciation of additional roles played by these subunits. In this review, we focus on β-subunits as homophilic and heterophilic cell-adhesion molecules and the implications for cardiomyocyte function. Based on recent cryogenic electron microscopy (cryo-EM) data, we suggest that the β-subunits interact with Nav1.5 in a different way from their binding to other Nav channel isoforms. We believe this feature may facilitate trans-cell-adhesion between β1-associated Nav1.5 subunits on the intercalated disc and promote ephaptic conduction between cardiomyocytes

Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction

Cardiac arrhythmias are associated with raised intracellular [Ca2+] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca2+-dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca2+-dependent phosphatase, calcineurin. Intracellular [Ca2+] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2. Raised [Ca2 +]i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca2+]i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca2+-independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca2+]i. PP2A had no role. Conduction velocity was reduced by raised [Ca2+]i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca2+] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.Centro de Investigaciones Cardiovasculare

Transcriptional profiles of genes related to electrophysiological function in Scn5a+/− murine hearts

The Scn5a gene encodes the major pore-forming Nav1.5 (α) subunit, of the voltage-gated Na+ channel in cardiomyocytes. The key role of Nav1.5 in action potential initiation and propagation in both atria and ventricles predisposes organisms lacking Scn5a or carrying Scn5a mutations to cardiac arrhythmogenesis. Loss-of-function Nav1.5 genetic abnormalities account for many cases of the human arrhythmic disorder Brugada syndrome (BrS) and related conduction disorders. A murine model with a heterozygous Scn5a deletion recapitulates many electrophysiological phenotypes of BrS. This study examines the relationships between its Scn5a+/− genotype, resulting transcriptional changes, and the consequent phenotypic presentations of BrS. Of 62 selected protein-coding genes related to cardiomyocyte electrophysiological or homeostatic function, concentrations of mRNA transcribed from 15 differed significantly from wild type (WT). Despite halving apparent ventricular Scn5a transcription heterozygous deletion did not significantly downregulate its atrial expression, raising possibilities of atria-specific feedback mechanisms. Most of the remaining 14 genes whose expression differed significantly between WT and Scn5a+/− animals involved Ca2+ homeostasis specifically in atrial tissue, with no overlap with any ventricular changes. All statistically significant changes in expression were upregulations in the atria and downregulations in the ventricles. This investigation demonstrates the value of future experiments exploring for and clarifying links between transcriptional control of Scn5a and of genes whose protein products coordinate Ca2+ regulation and examining their possible roles in BrS

Transcriptional profiles of genes related to electrophysiological function in Scn5a+/- murine hearts.

The Scn5a gene encodes the major pore-forming Nav 1.5 (α) subunit, of the voltage-gated Na+ channel in cardiomyocytes. The key role of Nav 1.5 in action potential initiation and propagation in both atria and ventricles predisposes organisms lacking Scn5a or carrying Scn5a mutations to cardiac arrhythmogenesis. Loss-of-function Nav 1.5 genetic abnormalities account for many cases of the human arrhythmic disorder Brugada syndrome (BrS) and related conduction disorders. A murine model with a heterozygous Scn5a deletion recapitulates many electrophysiological phenotypes of BrS. This study examines the relationships between its Scn5a+/- genotype, resulting transcriptional changes, and the consequent phenotypic presentations of BrS. Of 62 selected protein-coding genes related to cardiomyocyte electrophysiological or homeostatic function, concentrations of mRNA transcribed from 15 differed significantly from wild type (WT). Despite halving apparent ventricular Scn5a transcription heterozygous deletion did not significantly downregulate its atrial expression, raising possibilities of atria-specific feedback mechanisms. Most of the remaining 14 genes whose expression differed significantly between WT and Scn5a+/- animals involved Ca2+ homeostasis specifically in atrial tissue, with no overlap with any ventricular changes. All statistically significant changes in expression were upregulations in the atria and downregulations in the ventricles. This investigation demonstrates the value of future experiments exploring for and clarifying links between transcriptional control of Scn5a and of genes whose protein products coordinate Ca2+ regulation and examining their possible roles in BrS

Arrhythmic effects of Epac-mediated ryanodine receptor activation in Langendorff-perfused murine hearts are associated with reduced conduction velocity.

Recent papers have attributed arrhythmic substrate in murine RyR2-P2328S hearts to reduced action potential (AP) conduction velocities (CV), reflecting acute functional inhibition and/or reduced expression of sodium channels. We explored for acute effects of direct exchange protein directly activated by cAMP (Epac)-mediated ryanodine receptor-2 (RyR2) activation on arrhythmic substrate and CV. Monophasic action potential (MAP) recordings demonstrated that initial steady (8 Hz) extrinsic pacing elicited ventricular tachycardia (VT) in 0 of 18 Langendorff-perfused wild-type mouse ventricles before pharmacological intervention. The Epac activator 8-CPT (8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate) (VT in 1 of 7 hearts), and the RyR2 blocker dantrolene, either alone (0 of 11) or with 8-CPT (0 of 9) did not then increase VT incidence (P>.05). Both progressively increased pacing rates and programmed extrasystolic (S2) stimuli similarly produced no VT in untreated hearts (n=20 and n=9 respectively). 8-CPT challenge then increased VT incidences (5 of 7 and 4 of 8 hearts respectively; P.05). 8-CPT but not dantrolene, whether alone or combined with 8-CPT, correspondingly increased AP latencies (1.14±0.04 (n=7), 1.04±0.03 (n=10), 1.09±0.05 (n=8) relative to respective control values). In contrast, AP durations, conditions for 2:1 conduction block and ventricular effective refractory periods remained unchanged throughout. We thus demonstrate for the first time that acute RyR2 activation reversibly induces VT in specific association with reduced CV.We thank the Medical Research Council (UK) (CLH), Wellcome Trust (UK) (CLH), British Heart Foundation (UK) (CLH), McVeigh Benefaction (CLH and AG), and Sudden Arrhythmic Death Syndrome (SADS) UK (CLH and AG) for support

Gating control of the cardiac sodium channel Nav1.5 by its β3-subunit involves distinct roles for a transmembrane glutamic acid and the extracellular domain.

The auxiliary β3-subunit is an important functional regulator of the cardiac sodium channel Nav1.5, and some β3 mutations predispose individuals to cardiac arrhythmias. The β3-subunit uses its transmembrane α-helix and extracellular domain to bind to Nav1.5. Here, we investigated the role of an unusually located and highly conserved glutamic acid (Glu-176) within the β3 transmembrane region and its potential for functionally synergizing with the β3 extracellular domain (ECD). We substituted Glu-176 with lysine (E176K) in the WT β3-subunit and in a β3-subunit lacking the ECD. Patch-clamp experiments indicated that the E176K substitution does not affect the previously observed β3-dependent depolarizing shift of V½ of steady-state inactivation but does attenuate the accelerated recovery from inactivation conferred by the WT β3-subunit. Removal of the β3-ECD abrogated both the depolarizing shift of steady-state inactivation and the accelerated recovery, irrespective of the presence or absence of the Glu-176 residue. We found that steady-state inactivation and recovery from inactivation involve movements of the S4 helices within the DIII and DIV voltage sensors in response to membrane potential changes. Voltage-clamp fluorometry revealed that the E176K substitution alters DIII voltage sensor dynamics without affecting DIV. In contrast, removal of the ECD significantly altered the dynamics of both DIII and DIV. These results imply distinct roles for the β3-Glu-176 residue and the β3-ECD in regulating the conformational changes of the voltage sensors that determine channel inactivation and recovery from inactivation

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

Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction.

Cardiac arrhythmias are associated with raised intracellular [Ca2+] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca2+-dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca2+-dependent phosphatase, calcineurin. Intracellular [Ca2+] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2.Raised [Ca2+]i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca2+]i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca2+-independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca2+]i. PP2A had no role. Conduction velocity was reduced by raised [Ca2+]i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca2+] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1