An epilepsy-associated KV1.2 charge-transfer-center mutation impairs KV1.2 and KV1.4 trafficking.

We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events along the biosynthesis and secretion pathway impair trafficking. These studies suggested that F233S causes a depolarizing shift of ∼48 mV on KV1.2 voltage dependence. Optical tracking of the KV1.2(F233S) voltage-sensing domain (rescued by wild-type KV1.2 or KV1.4) revealed that it operates with modestly perturbed voltage dependence and retains pore coupling, evidenced by off-charge immobilization. The equivalent mutation in the Shaker K+ channel (F290S) was reported to modestly affect trafficking and strongly affect function: an ∼80-mV depolarizing shift, disrupted voltage sensor activation and pore coupling. Our work exposes the multigenic, molecular etiology of a variant associated with epilepsy and reveals that charge-transfer-center disruption has different effects in KV1.2 and Shaker, the archetypes for potassium channel structure and function.

Two epilepsy-associated variants in KCNA2 (KV 1.2) at position H310 oppositely affect channel functional expression.

Two KCNA2 variants (p.H310Y and p.H310R) were discovered in paediatric patients with epilepsy and developmental delay. KCNA2 encodes KV 1.2-channel subunits, which regulate neuronal excitability. Both gain and loss of KV 1.2 function cause epilepsy, precluding the prediction of variant effects; and while H310 is conserved throughout the KV -channel superfamily, it is largely understudied. We investigated both variants in heterologously expressed, human KV 1.2 channels by immunocytochemistry, electrophysiology and voltage-clamp fluorometry. Despite affecting the same channel, at the same position, and being associated with severe neurological disease, the two variants had diametrically opposite effects on KV 1.2 functional expression. The p.H310Y variant produced 'dual gain of function', increasing both cell-surface trafficking and activity, delaying channel closure. We found that the latter is due to the formation of a hydrogen bond that stabilizes the active state of the voltage-sensor domain. Additionally, H310Y abolished 'ball and chain' inactivation of KV 1.2 by KV ß1 subunits, enhancing gain of function. In contrast, p.H310R caused 'dual loss of function', diminishing surface levels by multiple impediments to trafficking and inhibiting voltage-dependent channel opening. We discuss the implications for KV -channel biogenesis and function, an emergent hotspot for disease-associated variants, and mechanisms of epileptogenesis. KEY POINTS: KCNA2 encodes the subunits of KV 1.2 voltage-activated, K+ -selective ion channels, which regulate electrical signalling in neurons. We characterize two KCNA2 variants from patients with developmental delay and epilepsy. Both variants affect position H310, highly conserved in KV channels. The p.H310Y variant caused 'dual gain of function', increasing both KV 1.2-channel activity and the number of KV 1.2 subunits on the cell surface. H310Y abolished 'ball and chain' (N-type) inactivation of KV 1.2 by KV ß1 subunits, enhancing the gain-of-function phenotype. The p.H310R variant caused 'dual loss of function', diminishing the presence of KV 1.2 subunits on the cell surface and inhibiting voltage-dependent channel opening. As H310Y stabilizes the voltage-sensor active conformation and abolishes N-type inactivation, it can serve as an investigative tool for functional and pharmacological studies.

Diagnostic and Prognostic Value of Cardiovascular Magnetic Resonance in Neuromuscular Cardiomyopathies.

Neuromuscular diseases (NMD) encompass a broad spectrum of diseases with variable type of cardiac involvement and there is lack of clinical data on Cardiovascular Magnetic Resonance (CMR) phenotypes or even prognostic value of CMR in NMD. We explored the diagnostic and prognostic value of CMR in NMD-related cardiomyopathies. The study included retrospective analysis of a cohort of 111 patients with various forms of NMD; mitochondrial: n = 14, Friedreich's ataxia (FA): n = 27, myotonic dystrophy: n = 27, Becker/Duchenne's muscular dystrophy (BMD/DMD): n = 15, Duchenne's carriers: n = 6, other: n = 22. Biventricular volumes and function and myocardial late gadolinium enhancement (LGE) pattern and extent were assessed by CMR. Patients were followed-up for the composite clinical endpoint of death, heart failure development or need for permanent pacemaker/intracardiac defibrillator. The major NMD subtypes, i.e. FA, mitochondrial, BMD/DMD, and myotonic dystrophy had significant differences in the incidence of LGE (56%, 21%, 62% & 30% respectively, chi2 = 9.86, p = 0.042) and type of cardiomyopathy phenotype (chi2 = 13.8, p = 0.008), extent/pattern (p = 0.006) and progression rate of LGE (p = 0.006). In survival analysis the composite clinical endpoint differed significantly between NMD subtypes (p = 0.031), while the subgroup with LGE + and LVEF < 50% had the worst prognosis (Log-rank p = 0.0034). We present data from a unique cohort of NMD patients and provide evidence on the incidence, patterns, and the prognostic value of LGE in NMD-related cardiomyopathy. LGE is variably present in NMD subtypes and correlates with LV remodelling, dysfunction, and clinical outcomes in patients with NMD.

Circuit-Specific Early Impairment of Proprioceptive Sensory Neurons in the SOD1G93A Mouse Model for ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons degenerate, resulting in muscle atrophy, paralysis, and fatality. Studies using mouse models of ALS indicate a protracted period of disease development with progressive motor neuron pathology, evident as early as embryonic and postnatal stages. Key missing information includes concomitant alterations in the sensorimotor circuit essential for normal development and function of the neuromuscular system. Leveraging unique brainstem circuitry, we show in vitro evidence for reflex circuit-specific postnatal abnormalities in the jaw proprioceptive sensory neurons in the well-studied SOD1G93A mouse. These include impaired and arrhythmic action potential burst discharge associated with a deficit in Nav1.6 Na+ channels. However, the mechanoreceptive and nociceptive trigeminal ganglion neurons and the visual sensory retinal ganglion neurons were resistant to excitability changes in age-matched SOD1G93A mice. Computational modeling of the observed disruption in sensory patterns predicted asynchronous self-sustained motor neuron discharge suggestive of imminent reflexive defects, such as muscle fasciculations in ALS. These results demonstrate a novel reflex circuit-specific proprioceptive sensory abnormality in ALS.SIGNIFICANCE STATEMENT Neurodegenerative diseases have prolonged periods of disease development and progression. Identifying early markers of vulnerability can therefore help devise better diagnostic and treatment strategies. In this study, we examined postnatal abnormalities in the electrical excitability of muscle spindle afferent proprioceptive neurons in the well-studied SOD1G93A mouse model for neurodegenerative motor neuron disease, amyotrophic lateral sclerosis. Our findings suggest that these proprioceptive sensory neurons are exclusively afflicted early in the disease process relative to sensory neurons of other modalities. Moreover, they presented Nav1.6 Na+ channel deficiency, which contributed to arrhythmic burst discharge. Such sensory arrhythmia could initiate reflexive defects, such as muscle fasciculations in amyotrophic lateral sclerosis, as suggested by our computational model.

Tracking the motion of the KV1.2 voltage sensor reveals the molecular perturbations caused by a de novo mutation in a case of epilepsy.

KEY POINTS: KV1.2 channels, encoded by the KCNA2 gene, regulate neuronal excitability by conducting K+ upon depolarization. A new KCNA2 missense variant was discovered in a patient with epilepsy, causing amino acid substitution F302L at helix S4, in the KV1.2 voltage-sensing domain. Immunocytochemistry and flow cytometry showed that F302L does not impair KCNA2 subunit surface trafficking. Molecular dynamics simulations indicated that F302L alters the exposure of S4 residues to membrane lipids. Voltage clamp fluorometry revealed that the voltage-sensing domain of KV1.2-F302L channels is more sensitive to depolarization. Accordingly, KV1.2-F302L channels opened faster and at more negative potentials; however, they also exhibited enhanced inactivation: that is, F302L causes both gain- and loss-of-function effects. Coexpression of KCNA2-WT and -F302L did not fully rescue these effects. The proband's symptoms are more characteristic of patients with loss of KCNA2 function. Enhanced KV1.2 inactivation could lead to increased synaptic release in excitatory neurons, steering neuronal circuits towards epilepsy. ABSTRACT: An exome-based diagnostic panel in an infant with epilepsy revealed a previously unreported de novo missense variant in KCNA2, which encodes voltage-gated K+ channel KV1.2. This variant causes substitution F302L, in helix S4 of the KV1.2 voltage-sensing domain (VSD). F302L does not affect KCNA2 subunit membrane trafficking. However, it does alter channel functional properties, accelerating channel opening at more hyperpolarized membrane potentials, indicating gain of function. F302L also caused loss of KV1.2 function via accelerated inactivation onset, decelerated recovery and shifted inactivation voltage dependence to more negative potentials. These effects, which are not fully rescued by coexpression of wild-type and mutant KCNA2 subunits, probably result from the enhancement of VSD function, as demonstrated by optically tracking VSD depolarization-evoked conformational rearrangements. In turn, molecular dynamics simulations suggest altered VSD exposure to membrane lipids. Compared to other encephalopathy patients with KCNA2 mutations, the proband exhibits mild neurological impairment, more characteristic of patients with KCNA2 loss of function. Based on this information, we propose a mechanism of epileptogenesis based on enhanced KV1.2 inactivation leading to increased synaptic release preferentially in excitatory neurons, and hence the perturbation of the excitatory/inhibitory balance of neuronal circuits.

Resurgent Na+ Current Offers Noise Modulation in Bursting Neurons.

Neurons utilize bursts of action potentials as an efficient and reliable way to encode information. It is likely that the intrinsic membrane properties of neurons involved in burst generation may also participate in preserving its temporal features. Here we examined the contribution of the persistent and resurgent components of voltage-gated Na+ currents in modulating the burst discharge in sensory neurons. Using mathematical modeling, theory and dynamic-clamp electrophysiology, we show that, distinct from the persistent Na+ component which is important for membrane resonance and burst generation, the resurgent Na+ can help stabilize burst timing features including the duration and intervals. Moreover, such a physiological role for the resurgent Na+ offered noise tolerance and preserved the regularity of burst patterns. Model analysis further predicted a negative feedback loop between the persistent and resurgent gating variables which mediate such gain in burst stability. These results highlight a novel role for the voltage-gated resurgent Na+ component in moderating the entropy of burst-encoded neural information.

Functional heterogeneity of the four voltage sensors of a human L-type calcium channel.

Excitation-evoked Ca(2+) influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca(2+) entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I-IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of CaV1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in CaV1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.

Recurrent missense mutations in TMEM43 (ARVD5) due to founder effects cause arrhythmogenic cardiomyopathies in the UK and Canada.

AIMS: Autosomal dominant arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) (in the group of arrhythmogenic cardiomyopathies) is a common cause of sudden cardiac death in young adults. It is both clinically and genetically heterogeneous, with 12 loci (ARVC/D1-12) and eight genes identified, the majority of which encode structural proteins of cardiac desmosomes. The most recent gene identified, TMEM43, causes disease due to a missense mutation in a non-desmosomal gene (p.S358L) in 15 extended families from Newfoundland, Canada. To determine whether mutations in TMEM43 cause ARVC/D and arrhythmogenic cardiomyopathy in other populations, we fully re-sequenced TMEM43 on 143 ARVC/D probands (families) from the UK and 55 probands (from 55 families) from Newfoundland. METHODS AND RESULTS: Bidirectional sequencing of TMEM43 including intron-exon boundaries revealed 33 variants, the majority located in non-coding regions of TMEM43. For the purpose of validation, families of probands with rare, potentially deleterious coding variants were subjected to clinical and molecular follow-up. Three missense variants of uncertain significance (p.R28W, p.E142K, p.R312W) were located in highly conserved regions of the TMEM43 protein. One variant (p.R312W) also co-segregated with relatives showing clinical signs of disease. Genotyping and expansion of the disease-associated haplotype in subjects with the p.R312W variant from Newfoundland, Canada, and the UK suggest common ancestry. CONCLUSION: Although the p.R312W variant was found in controls (3/378), identification of an ancestral disease p R312W haplotype suggests that the p.R312W variant is a pathogenic founder mutation.

The contribution of RCK domains to human BK channel allosteric activation.

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca(2+)-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca(2+)-sensing apparatus known as the "gating ring." The allosteric interplay between voltage- and Ca(2+)-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca(2+), we optically resolved VSD activation prompted by Ca(2+) binding to the gating ring. The sudden increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca(2+) sensor located in the RCK2 domain abolished the effect of [Ca(2+)](i) increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca(2+) sensing did not prevent the effect of Ca(2+) release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca(2+) association in two distinct sites, voltage sensor activation, and BK channel opening.

Arrhythmogenic right ventricular cardiomyopathy mimics: role of cardiovascular magnetic resonance.

BACKGROUND: Cardiovascular magnetic resonance (CMR) is commonly used in patients with suspected arrhythmogenic right ventricular cardiomyopathy (ARVC) based on ECG, echocardiogram and Holter. However, various diseases may present with clinical characteristics resembling ARVC causing diagnostic dilemmas. The aim of this study was to explore the role of CMR in the differential diagnosis of patients with suspected ARVC. METHODS: 657 CMR referrals suspicious for ARVC in a single tertiary referral centre were analysed. Standardized CMR imaging protocols for ARVC were performed. Potential ARVC mimics were grouped into: 1) displacement of the heart, 2) right ventricular overload, and 3) non ARVC-like cardiac scarring. For each, a judgment of clinical impact was made. RESULTS: Twenty patients (3.0%) fulfilled imaging ARVC criteria. Thirty (4.6%) had a potential ARVC mimic, of which 25 (3.8%) were considered clinically important: cardiac displacement (n=17), RV overload (n=7) and non-ARVC like myocardial scarring (n=4). One patient had two mimics; one patient had dual pathology with important mimic and ARVC. RV overload and scarring conditions were always thought clinically important whilst the importance of cardiac displacement depended on the degree of displacement from severe (partial absence of pericardium) to epiphenomenon (minor kyphoscoliosis). CONCLUSIONS: Some patients referred for CMR with suspected ARVC fulfil ARVC imaging criteria (3%) but more have otherwise unrecognised diseases (4.6%) mimicking potentially ARVC. Clinical assessment should reflect this, emphasising the assessment and/or exclusion of potential mimics in parallel with the detection of ARVC major and minor criteria.

Operation of the voltage sensor of a human voltage- and Ca2+-activated K+ channel.

Voltage sensor domains (VSDs) are structurally and functionally conserved protein modules that consist of four transmembrane segments (S1-S4) and confer voltage sensitivity to many ion channels. Depolarization is sensed by VSD-charged residues residing in the membrane field, inducing VSD activation that facilitates channel gating. S4 is typically thought to be the principal functional component of the VSD because it carries, in most channels, a large portion of the VSD gating charge. The VSDs of large-conductance, voltage- and Ca(2+)-activated K(+) channels are peculiar in that more gating charge is carried by transmembrane segments other than S4. Considering its "decentralized" distribution of voltage-sensing residues, we probed the BK(Ca) VSD for evidence of cooperativity between charge-carrying segments S2 and S4. We achieved this by optically tracking their activation by using voltage clamp fluorometry, in channels with intact voltage sensors and charge-neutralized mutants. The results from these experiments indicate that S2 and S4 possess distinct voltage dependence, but functionally interact, such that the effective valence of one segment is affected by charge neutralization in the other. Statistical-mechanical modeling of the experimental findings using allosteric interactions demonstrates two mechanisms (mechanical coupling and dynamic focusing of the membrane electric field) that are compatible with the observed cross-segment effects of charge neutralization.

Mutations in the Lamin A/C gene mimic arrhythmogenic right ventricular cardiomyopathy.

AIMS: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease predominantly caused by mutations in desmosomal protein genes. Lamin A/C gene (LMNA) mutations are associated with dilated cardiomyopathy, conduction abnormalities and high incidence of sudden cardiac death. In this study, we screened a large cohort of ARVC patients for LMNA mutations. METHODS AND RESULTS: One hundred and eight patients from unrelated families with borderline (n = 27) or definite (n = 81) diagnosis of ARVC were genetically tested for five desmosomal genes and LMNA. Sixty-one (56.5%) were positive for desmosomal gene mutations. Standard polymerase chain reaction (PCR) amplification of the 12 protein-coding LMNA exons was performed and mutational screening performed by direct sequencing. Four patients (4%) without desmosomal gene mutations carried LMNA variants. Three had severe right ventricular involvement, and during follow-up three died (two suddenly and one from congestive heart failure); all three had conduction abnormalities on resting 12-lead electrocardiogram (ECG). Myocardial tissue from two patients showed myocyte loss and fibro-fatty replacement. In one of these, immunohistochemical staining with antibody to plakoglobin showed reduced/absent staining of the intercalated discs in the myocardium. CONCLUSION: Lamin A/C gene mutations can be found in severe forms of ARVC. Lamin A/C gene should be added to desmosomal genes when genetically testing patients with suspected ARVC, particularly when they also have ECG evidence for conduction disease.

Circadian and Seasonal Pattern of Arrhythmic Events in Arrhythmogenic Cardiomyopathy Patients.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disease associated with an increased risk of life-threatening arrhythmias. The aim of the present study was to evaluate the association of ventricular arrhythmias (VA) with circadian and seasonal variation in ARVC. One hundred two ARVC patients with an implantable cardioverter defibrillator (ICD) were enrolled in the study. Arrhythmic events included (a) any initial ventricular tachycardia (VT) or fibrillation (VF) prompting ICD implantation, (b) any VT or non-sustained VT (NSVT) recorded by the ICD, and (c) appropriate ICD shocks/therapy. Differences in the annual incidence of events across seasons (winter, spring, summer, autumn) and period of the day (night, morning, afternoon, evening) were assessed both for all cardiac events and major arrhythmic events. In total, 67 events prior to implantation and 263 ICD events were recorded. These included 135 major (58 ICD therapies, 57 self-terminating VT, 20 sustained VT) and 148 minor (NSVT) events. A significant increase in the frequency of events was observed in the afternoon versus in the nights and mornings (p = 0.016). The lowest number of events was registered in the summer, with a peak in the winter (p < 0.001). Results were also confirmed when excluding NSVT. Arrhythmic events in ARVC follow a seasonal variation and a circadian rhythm. They are more prevalent in the late afternoon, the most active period of the day, and in the winter, supporting the role of physical activity and inflammation as triggers of events.

Metal-driven operation of the human large-conductance voltage- and Ca2+-dependent potassium channel (BK) gating ring apparatus.

Large-conductance voltage- and Ca(2+)-dependent K(+) (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca(2+) homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the "gating ring"), which confers sensitivity to intracellular Ca(2+) and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca(2+)- and Mg(2+)-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba(2+)-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca(2+)-binding site (the "calcium bowl") reduced the Ca(2+) and abolished the Mg(2+) dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca(2+)-binding site and that Mg(2+) can bind to the calcium bowl with less affinity than Ca(2+). Dynamic light scattering analysis revealed a reversible Ca(2+)-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.

Familial evaluation in arrhythmogenic right ventricular cardiomyopathy: impact of genetics and revised task force criteria.

BACKGROUND: With recognition of disease-causing genes in arrhythmogenic right ventricular cardiomyopathy, mutation analysis is being applied. METHODS AND RESULTS: The role of genotyping in familial assessment for arrhythmogenic right ventricular cardiomyopathy was investigated, including the prevalence of mutations in known causal genes, the penetrance and expressivity in genotyped families, and the utility of the 2010 Task Force criteria in clinical diagnosis. Clinical and molecular genetic evaluation was performed in 210 first-degree and 45 second-degree relatives from 100 families. In 51 families, the proband was deceased. The living probands had a high prevalence of ECG abnormalities (89%) and ventricular arrhythmia (78%) and evidence of more severe disease than relatives. Definite or probable causal mutations were found in 58% of families and 73% of living probands, of whom 28% had an additional desmosomal variant (ie, mutation or polymorphism). Ninety-three relatives had a causal mutation; 33% fulfilled the 2010 criteria, whereas only 19% satisfied the 1994 version (P=0.03). An additional desmosomal gene variant was found in 10% and was associated with a 5-fold increased risk of developing penetrant disease (odds ratio, 4.7; 95% confidence interval, 1.1 to 20.4; P=0.04). CONCLUSIONS: Arrhythmogenic right ventricular cardiomyopathy is a genetically complex disease characterized by marked intrafamilial phenotype diversity. Penetrance is definition dependent and is greater with the 2010 criteria compared with the 1994 criteria. Relatives harboring >1 genetic variant had significantly increased risk of developing clinical disease, potentially an important determinant of the phenotypic heterogeneity seen within families with arrhythmogenic right ventricular cardiomyopathy.

Shaping a new Ca²âº conductance to suppress early afterdepolarizations in cardiac myocytes.

Sudden cardiac death (SCD) due to ventricular fibrillation (VF) is a major world-wide health problem. A common trigger of VF involves abnormal repolarization of the cardiac action potential causing early afterdepolarizations (EADs). Here we used a hybrid biological-computational approach to investigate the dependence of EADs on the biophysical properties of the L-type Ca(2+) current (I(Ca,L)) and to explore how modifications of these properties could be designed to suppress EADs. EADs were induced in isolated rabbit ventricular myocytes by exposure to 600 µmol l(-1) H(2)O(2) (oxidative stress) or lowering the external [K(+)] from 5.4 to 2.0-2.7 mmol l(-1) (hypokalaemia). The role of I(Ca,L) in EAD formation was directly assessed using the dynamic clamp technique: the paced myocyte's V(m) was input to a myocyte model with tunable biophysical parameters, which computed a virtual I(Ca,L), which was injected into the myocyte in real time. This virtual current replaced the endogenous I(Ca,L), which was suppressed with nifedipine. Injecting a current with the biophysical properties of the native I(Ca,L) restored EAD occurrence in myocytes challenged by H(2)O(2) or hypokalaemia. A mere 5 mV depolarizing shift in the voltage dependence of activation or a hyperpolarizing shift in the steady-state inactivation curve completely abolished EADs in myocytes while maintaining a normal Ca(i) transient. We propose that modifying the biophysical properties of I(Ca,L) has potential as a powerful therapeutic strategy for suppressing EADs and EAD-mediated arrhythmias.

Suppression of ventricular arrhythmias by targeting late L-type Ca2+ current.

Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of ICa,L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late ICa,L (Ca2+ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak ICa,L, which is essential for proper excitation-contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late ICa,L with minimal effect on peak current. Scaling our investigation from a human CaV1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces ICa,L noninactivating component in a human CaV1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late ICa,L suppresses EADs and EATs (early after Ca2+ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca2+ transient, and (3) late ICa,L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late ICa,L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of CaV1.2 channels rather than blocking their pore, largely preserving contractility.

Exercise-induced ventricular arrhythmias and risk of sudden cardiac death in patients with hypertrophic cardiomyopathy.

BACKGROUND: Non-sustained ventricular tachycardia (NSVT) during ambulatory electrocardiographic monitoring (typically occurring at rest or during sleep) is associated with an increased risk of sudden cardiac death in patients with hypertrophic cardiomyopathy. The prevalence and prognostic significance of ventricular arrhythmias during exercise is unknown. METHODS AND RESULTS: This was a cohort study, with prospective data collection. We studied 1380 patients, referred to a cardiomyopathy clinic in London, UK [mean age 42 years (SD 15); 62% male; mean follow-up 54 (SD 49) months]. Patients underwent two-dimensional and Doppler echocardiography, upright exercise testing, and Holter monitoring. Twenty-seven patients [mean age 40 (SD 14) years (18-64); 22 (81.5%) male] had NSVT (24) or ventricular fibrillation (VF) (3) during exercise. During exercise, 13 (54.2%) had more than one run of NSVT (maximum 5) with a mean heart rate of 221 (SD 48) b.p.m. Patients with exercise NSVT/VF had more severe hypertrophy (22.6 vs. 19.5 mm, P = 0.009) and larger left atria (47.3 vs. 43.7 mm, P = 0.03). Male gender was significantly associated with exercise NSVT/VF [22 (81.5%) vs. 832 (61.5%), P = 0.03]. Eight (29.6%) of the exercise NSVT/VF patients died or had a cardiac event (SD/ICD discharge/transplant) compared with 150 (11.1%) patients without exercise NSVT/VF, P = 0.008. Patients with NSVT/VF had a 3.73-fold increase in risk of SD/ICD discharge (HR 95% CI: 1.61-8.63, P = 0.002). Exercise NSVT alone was associated with a 2.82-fold increased risk (HR 95% CI: 1.02-7.75, P = 0.049). In multivariable analysis with other risk markers, exercise NSVT/VF (but not NSVT alone) was independently associated with an increased risk of SD/ICD [HR 3.14 (95% CI: 1.29-7.61, P = 0.01)]. CONCLUSION: Ventricular arrhythmia during symptom limited exercise is rare in patients with hypertrophic cardiomyopathy, but is associated with an increased risk of sudden cardiac death.

Antithrombotic Treatment in Cardiomyopathies.

Cardiomyopathies are a heterogeneous group of heart muscle diseases and important cause of heart failure with reduced or preserved ejection fraction. Although there is an increasing body of evidence on the incidence, pathophysiology, and natural history of heart failure (HF) in cardiomyopathies, certain aspects of the therapeutic strategies remain unclear. More particularly, there is no consensus if to whether antithrombotic therapy has a favorable risk: benefit ratio in reducing thromboembolic event rate in patients with cardiomyopathies without suffering from primary valvular disease or atrial fibrillation. Although the observational data on increased venous thromboembolic risk are supported by multiple pathophysiological mechanisms, the role of antithrombotic therapy in these patients remains unclear. This review article provides an overview of epidemiologic, pathophysiologic, clinical, and therapeutic data for the prevention of thromboembolism in heart failure due to cardiomyopathies.

Distinct roles for two histamine receptors (hclA and hclB) at the Drosophila photoreceptor synapse.

Histamine (HA) is the photoreceptor neurotransmitter in arthropods, directly gating chloride channels on large monopolar cells (LMCs), postsynaptic to photoreceptors in the lamina. Two histamine-gated channel genes that could contribute to this channel in Drosophila are hclA (also known as ort) and hclB (also known as hisCl1), both encoding novel members of the Cys-loop receptor superfamily. Drosophila S2 cells transfected with these genes expressed both homomeric and heteromeric histamine-gated chloride channels. The electrophysiological properties of these channels were compared with those from isolated Drosophila LMCs. HCLA homomers had nearly identical HA sensitivity to the native receptors (EC(50) = 25 microM). Single-channel analysis revealed further close similarity in terms of single-channel kinetics and subconductance states ( approximately 25, 40, and 60 pS, the latter strongly voltage dependent). In contrast, HCLB homomers and heteromeric receptors were more sensitive to HA (EC(50) = 14 and 1.2 microM, respectively), with much smaller single-channel conductances ( approximately 4 pS). Null mutations of hclA (ort(US6096)) abolished the synaptic transients in the electroretinograms (ERGs). Surprisingly, the ERG "on" transients in hclB mutants transients were approximately twofold enhanced, whereas intracellular recordings from their LMCs revealed altered responses with slower kinetics. However, HCLB expression within the lamina, assessed by both a GFP (green fluorescent protein) reporter gene strategy and mRNA tagging, was exclusively localized to the glia cells, whereas HCLA expression was confirmed in the LMCs. Our results suggest that the native receptor at the LMC synapse is an HCLA homomer, whereas HCLB signaling via the lamina glia plays a previously unrecognized role in shaping the LMC postsynaptic response.