A liposomal platform for the delivery of ion channel proteins for treatment of channelopathies - Application in therapy of cystic fibrosis.

Channelopathies arise from ion channel dysfunction. Successful treatment entails delivery of functional ion channels to replace dysfunctional ones. Glycine receptor (GlyR)-rich cell membrane fragments (CMF) were previously delivered to target cell membranes using fusogenic liposomes. Here, cystic fibrosis transmembrane conductance regulator (CFTR)-bearing CMF were similarly delivered to target cells. We studied the effect of lipid composition on liposomes' ability to incorporate CMF and fuse with target cell membranes to deliver functional CFTR. Four formulations were prepared using thin-film hydration out of different lecithin sources, egg and soy lecithin (EL and SL), in the presence and absence of cholesterol (CHOL): EL + CHOL, EL-CHOL, SL + CHOL, and SL-CHOL. EL liposomes incorporated more CMF than SL liposomes, with CHOL only increasing CMF incorporation in SL liposomes. SL + CHOL fused better with target cell membranes than EL + CHOL. SL + CHOL and EL + CHOL equally delivered CFTR to target cell membranes, owing to the former's superior fusogenic capacity and the latter's superior CMF-incorporation capacity. SL-CHOL and EL-CHOL delivered CFTR to a lesser extent, indicating the importance of CHOL for fusion. Patch-clamp electrophysiology and confocal laser scanning microscopy (CLSM) confirmed CFTR delivery to target cell membranes by SL + CHOL. Therefore, CMF-bearing fusogenic liposomes offer a promising universal platform for the treatment of channelopathies.

Muscle channelopathies.

Muscle channelopathies encompass a wide range of mainly episodic conditions that are characterized by muscle stiffness and weakness. The myotonic conditions, characterized predominantly by stiffness, include myotonia congenita, paramyotonia congenita, and sodium channel myotonia. The periodic paralysis conditions include hypokalemic periodic paralysis, hyperkalemic periodic paralysis, and Andersen-Tawil syndrome. Clinical history is key, and diagnosis is confirmed by next-generation genetic sequencing of a panel of known genes but can also be supplemented by neurophysiology studies and MRI. As genetic testing expands, so have the spectrum of phenotypes seen including pediatric presentations and congenital myopathies. Management of these conditions requires a multidisciplinary approach with extra support needed when patients require anesthetics or when pregnant. Patients with Andersen-Tawil syndrome will also need cardiac input. Diagnosis is important as symptomatic treatment is available for all of these conditions but need to be tailored to the gene and variant of the patient.

Care Recommendations for the Investigation and Management of Children With Skeletal Muscle Channelopathies.

The field of pediatric skeletal muscle channelopathies has seen major new advances in terms of a wider understanding of clinical presentations and new phenotypes. Skeletal muscle channelopathies cause significant disability and even death in some of the newly described phenotypes. Despite this, there are virtually no data on the epidemiology and longitudinal natural history of these conditions or randomized controlled trial evidence of efficacy or tolerability of any treatment in children, and thus best practice care recommendations do not exist. Clinical history, and to a lesser extent examination, is key to eliciting symptoms and signs that indicate a differential diagnosis of muscle channelopathy. Normal routine investigations should not deter one from the diagnosis. Specialist neurophysiologic investigations have an additional role, but their availability should not delay genetic testing. New phenotypes are increasingly likely to be identified by next-generation sequencing panels. Many treatments or interventions for symptomatic patients are available, with anecdotal data to support their benefit, but we lack trial data on efficacy, safety, or superiority. This lack of trial data in turn can lead to hesitancy in prescribing among doctors or in accepting medication by parents. Holistic management addressing work, education, activity, and additional symptoms of pain and fatigue provides significant benefit. Preventable morbidity and sometimes mortality occurs if the diagnosis and therefore treatment is delayed. Advances in genetic sequencing technology and greater access to testing may help to refine recently identified phenotypes, including histology, as more cases are described. Randomized controlled treatment trials are required to inform best practice care recommendations. A holistic approach to management is essential and should not be overlooked. Good quality data on prevalence, health burden, and optimal treatment are urgently needed.

Skeletal muscle channelopathies: a guide to diagnosis and management.

Skeletal muscle channelopathies are a group of rare episodic genetic disorders comprising the periodic paralyses and the non-dystrophic myotonias. They may cause significant morbidity, limit vocational opportunities, be socially embarrassing, and sometimes are associated with sudden cardiac death. The diagnosis is often hampered by symptoms that patients may find difficult to describe, a normal examination in the absence of symptoms, and the need to interpret numerous tests that may be normal or abnormal. However, the symptoms respond very well to holistic management and pharmacological treatment, with great benefit to quality of life. Here, we review when to suspect a muscle channelopathy, how to investigate a possible case and the options for therapy once a diagnosis is made.

Functional characterization of ABCC9 variants identified in sudden unexpected natural death.

BACKGROUND: Genetic variation in ion channel genes ('channelopathies') are often associated with inherited arrhythmias and sudden death. Genetic testing ('molecular autopsies') of channelopathy genes can be used to assist in determining the likely causes of sudden unexpected death. However, different in silico approaches can yield conflicting pathogenicity predictions and assessing their impact on ion channel function can assist in this regard. METHODS AND RESULTS: We performed genetic testing of cases of sudden expected death in the New York City metropolitan area and found four rare or novel variants in ABCC9, which codes for the regulatory SUR2 subunit of KATP channels. All were missense variants, causing amino acid changes in the protein. Three of the variants (A355S, M941V, and K1379Q) were in cases of infants less than six-months old and one (H1305Y) was in an adult. The predicted pathogenicities of the variants were conflicting. We have introduced these variants into a human SUR2A cDNA, which we coexpressed with the Kir6.2 pore-forming subunit in HEK-293 cells and subjected to patch clamp and biochemical assays. Each of the four variants led to gain-of-function phenotypes. The A355S and M941V variants increased in the overall patch current. The sensitivity of the KATP channels to inhibitory 'cytosolic' ATP was repressed for the M941V, H1305Y and K1379Q variants. None of the variants had any effect on the unitary KATP channel current or the surface expression of KATP channels, as determined with biotinylation assays, suggesting that all of the variants led to an enhanced open state. CONCLUSIONS: All four variants caused a gain-of-function phenotype. Given the expression of SUR2-containing KATP channels in the heart and specialized cardiac conduction, vascular smooth muscle and respiratory neurons, it is conceivable that electrical silencing of these cells may contribute to the vulnerability element, which is a component of the triple risk model of sudden explained death in infants. The gain-of-function phenotype of these ABCC9 variants should be considered when assessing their potential pathogenicity.

Autoimmune and inflammatory K+ channelopathies in cardiac arrhythmias: Clinical evidence and molecular mechanisms.

Cardiac K+ channelopathies account for a significant proportion of arrhythmias and sudden cardiac death (SCD) in subjects without structural heart disease. It is well recognized that genetic defects are key factors in many cases, and in practice, the term cardiac channelopathies currently coincides with inherited cardiac channelopathies. However, mounting evidence demonstrate that not only genetic alterations but also autoimmune and inflammatory factors can cause cardiac K+-channel dysfunction and arrhythmias in the setting of a structurally normal heart. In particular, it has been demonstrated that specific autoantibodies as well as inflammatory cytokines can modulate expression and/or function of different K+ channels in the heart, resulting in a disruption of the cardiac action potential and arrhythmias/sudden cardiac death. Awareness about the existence of these newly recognized forms is essential to identify and adequately manage affected patients. In the present review, we focus on autoimmune and inflammatory K+ channelopathies as a novel mechanism for cardiac arrhythmias and analyze the recent advancements in this topic, providing complementary basic, clinical, and population health perspectives.

Identification of small-molecule ion channel modulators in C. elegans channelopathy models.

Ion channels are important therapeutic targets, but the discovery of ion channel drugs remains challenging due to a lack of assays that allow high-throughput screening in the physiological context. Here we report C. elegans phenotype-based methods for screening ion channel drugs. Expression of modified human ether-a-go-go-related gene (hERG) potassium channels in C. elegans results in egg-laying and locomotive defects, which offer indicators for screening small-molecule channel modulators. Screening in worms expressing hERGA561V, which carries a trafficking-defective mutation A561V known to associate with long-QT syndrome, identifies two functional correctors Prostratin and ingenol-3,20-dibenzoate. These compounds activate PKCε signaling and consequently phosphorylate S606 at the pore region of the channel to promote hERGA561V trafficking to the plasma membrane. Importantly, the compounds correct electrophysiological abnormalities in hiPSC-derived cardiomyocytes bearing a heterozygous CRISPR/Cas9-edited hERGA561V. Thus, we have developed an in vivo high-throughput method for screening compounds that have therapeutic potential in treating channelopathies.

Shank3-deficient thalamocortical neurons show HCN channelopathy and alterations in intrinsic electrical properties.

KEY POINTS: Shank3 increases the HCN channel surface expression in heterologous expression systems. Shank3Δ13-16 deficiency causes significant reduction in HCN2 expression and Ih current amplitude in thalamocortical (TC) neurons. Shank3Δ13-16 - but not Shank3Δ4-9 -deficient TC neurons share changes in basic electrical properties which are comparable to those of HCN2-/- TC neurons. HCN channelopathy may critically mediate events downstream from Shank3 deficiency. ABSTRACT: SHANK3 is a scaffolding protein that is highly enriched in excitatory synapses. Mutations in the SHANK3 gene have been linked to neuropsychiatric disorders especially the autism spectrum disorders. SHANK3 deficiency is known to cause impairments in synaptic transmission, but its effects on basic neuronal electrical properties that are more localized to the soma and proximal dendrites remain unclear. Here we confirmed that in heterologous expression systems two different mouse Shank3 isoforms, Shank3A and Shank3C, significantly increase the surface expression of the mouse hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channel. In Shank3Δ13-16 knockout mice, which lack exons 13-16 in the Shank3 gene (both Shank3A and Shank3C are removed) and display a severe behavioural phenotype, the expression of HCN2 is reduced to an undetectable level. The thalamocortical (TC) neurons from the ventrobasal (VB) complex of Shank3Δ13-16 mice demonstrate reduced Ih current amplitude and correspondingly increased input resistance, negatively shifted resting membrane potential, and abnormal spike firing in both tonic and burst modes. Impressively, these changes closely resemble those of HCN2-/- TC neurons but not of the TC neurons from Shank3Δ4-9 mice, which lack exons 4-9 in the Shank3 gene (Shank3C still exists) and demonstrate moderate behavioural phenotypes. Additionally, Shank3 deficiency increases the ratio of excitatory/inhibitory balance in VB neurons but has a limited impact on the electrical properties of connected thalamic reticular (RTN) neurons. These results provide new understanding about the role of HCN channelopathy in mediating detrimental effects downstream from Shank3 deficiency.

Thyrotoxic Channelopathies.

Thyrotoxic periodic paralysis (TPP), a disorder most commonly seen in Asian men, is characterized by abrupt onset of hypokalemia and paralysis. The condition primarily affects the lower extremities and is secondary to thyrotoxicosis. Early recognition of TPP is vital to initiating appropriate treatment and to avoiding the risk of rebound hyperkalemia that may occur if high-dose potassium replacement is given. Here we present a case of 31 year old male with thyrotoxic periodic paralysis with diagnostic and therapeutic approach.

In Silico Evaluation of the Potential Antiarrhythmic Effect of Epigallocatechin-3-Gallate on Cardiac Channelopathies.

Ion channels are transmembrane proteins that allow the passage of ions according to the direction of their electrochemical gradients. Mutations in more than 30 genes encoding ion channels have been associated with an increasingly wide range of inherited cardiac arrhythmias. In this line, ion channels become one of the most important molecular targets for several classes of drugs, including antiarrhythmics. Nevertheless, antiarrhythmic drugs are usually accompanied by some serious side effects. Thus, developing new approaches could offer added values to prevent and treat the episodes of arrhythmia. In this sense, green tea catechins seem to be a promising alternative because of the significant effect of Epigallocatechin-3-Gallate (E3G) on the electrocardiographic wave forms of guinea pig hearts. Thus, the aim of this study was to evaluate the benefits-risks balance of E3G consumption in the setting of ion channel mutations linked with aberrant cardiac excitability phenotypes. Two gain-of-function mutations, Nav1.5-p.R222Q and Nav1.5-p.I141V, which are linked with cardiac hyperexcitability phenotypes were studied. Computer simulations of action potentials (APs) show that 30 µM E3G reduces and suppresses AP abnormalities characteristics of these phenotypes. These results suggest that E3G may have a beneficial effect in the setting of cardiac sodium channelopathies displaying a hyperexcitability phenotype.

Rescuing cardiac automaticity in L-type Cav1.3 channelopathies and beyond.

Pacemaker activity of the sino-atrial node generates the heart rate. Disease of the sinus node and impairment of atrioventricular conduction induce an excessively low ventricular rate (bradycardia), which cannot meet the needs of the organism. Bradycardia accounts for about half of the total workload of clinical cardiologists. The 'sick sinus' syndrome (SSS) is characterized by sinus bradycardia and periods of intermittent atrial fibrillation. Several genetic or acquired risk factors or pathologies can lead to SSS. Implantation of an electronic pacemaker constitutes the only available therapy for SSS. The incidence of SSS is forecast to double over the next 50 years, with ageing of the general population thus urging the development of complementary or alternative therapeutic strategies. In recent years an increasing number of mutations affecting ion channels involved in sino-atrial automaticity have been reported to underlie inheritable SSS. L-type Cav 1.3 channels play a major role in the generation and regulation of sino-atrial pacemaker activity and atrioventricular conduction. Mutation in the CACNA1D gene encoding Cav 1.3 channels induces loss-of-function in channel activity and underlies the sino-atrial node dysfunction and deafness syndrome (SANDD). Mice lacking Cav 1.3 channels (Cav 1.3-/- ) fairly recapitulate SSS and constitute a precious model to test new therapeutic approaches to handle this disease. Work in our laboratory shows that targeting G protein-gated K+ (IKACh ) channels effectively rescues SSS of Cav 1.3-/- mice. This new concept of 'compensatory' ion channel targeting shines new light on the principles underlying the pacemaker mechanism and may open the way to new therapies for SSS.

Profiling neuronal ion channelopathies with non-invasive brain imaging and dynamic causal models: Case studies of single gene mutations.

Clinical assessments of brain function rely upon visual inspection of electroencephalographic waveform abnormalities in tandem with functional magnetic resonance imaging. However, no current technology proffers in vivo assessments of activity at synapses, receptors and ion-channels, the basis of neuronal communication. Using dynamic causal modeling we compared electrophysiological responses from two patients with distinct monogenic ion channelopathies and a large cohort of healthy controls to demonstrate the feasibility of assaying synaptic-level channel communication non-invasively. Synaptic channel abnormality was identified in both patients (100% sensitivity) with assay specificity above 89%, furnishing estimates of neurotransmitter and voltage-gated ion throughput of sodium, calcium, chloride and potassium. This performance indicates a potential novel application as an adjunct for clinical assessments in neurological and psychiatric settings. More broadly, these findings indicate that biophysical models of synaptic channels can be estimated non-invasively, having important implications for advancing human neuroimaging to the level of non-invasive ion channel assays.

A channelopathy mechanism revealed by direct calmodulin activation of TrpV4.

Ca(2+)-calmodulin (CaM) regulates varieties of ion channels, including Transient Receptor Potential vanilloid subtype 4 (TrpV4). It has previously been proposed that internal Ca(2+) increases TrpV4 activity through Ca(2+)-CaM binding to a C-terminal Ca(2+)-CaM binding domain (CBD). We confirmed this model by directly presenting Ca(2+)-CaM protein to membrane patches excised from TrpV4-expressing oocytes. Over 50 TRPV4 mutations are now known to cause heritable skeletal dysplasia (SD) and other diseases in human. We have previously examined 14 SD alleles and found them to all have gain-of-function effects, with the gain of constitutive open probability paralleling disease severity. Among the 14 SD alleles examined, E797K and P799L are located immediate upstream of the CBD. They not only have increase basal activity, but, unlike the wild-type or other SD-mutant channels examined, they were greatly reduced in their response to Ca(2+)-CaM. Deleting a 10-residue upstream peptide (Δ795-804) that covers the two SD mutant sites resulted in strong constitutive activity and the complete lack of Ca(2+)-CaM response. We propose that the region immediately upstream of CBD is an autoinhibitory domain that maintains the closed state through electrostatic interactions, and adjacent detachable Ca(2+)-CaM binding to CBD sterically interferes with this autoinhibition. This work further supports the notion that TrpV4 mutations cause SD by constitutive leakage. However, the closed conformation is likely destabilized by various mutations by different mechanisms, including the permanent removal of an autoinhibition documented here.

HCN channelopathy and cardiac electrophysiologic dysfunction in genetic and acquired rat epilepsy models.

OBJECTIVE: Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post-status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression. METHODS: Electrocardiography studies were recorded in vivo in rats and in vitro in isolated hearts. Cardiac HCN channel messenger RNA (mRNA) and protein expression were measured using quantitative PCR and Western blotting respectively. RESULTS: Cardiac electrophysiology was significantly altered in adult GAERS, with slower heart rate, shorter QRS duration, longer QTc interval, and greater standard deviation of RR intervals compared to control rats. In the post-SE model, we observed similar interictal changes in several of these parameters, and we also observed consistent and striking bradycardia associated with the onset of ictal activity. Molecular analysis demonstrated significant reductions in cardiac HCN2 mRNA and protein expression in both models, providing a molecular correlate of these electrophysiologic abnormalities. SIGNIFICANCE: These results demonstrate that ion channelopathies and cardiac dysfunction can develop as a secondary consequence of chronic epilepsy, which may have relevance for the pathophysiology of cardiac dysfunction in patients with epilepsy.

Congenital short QT syndrome: landmarks of the newest arrhythmogenic cardiac channelopathy.

Congenital or familial short QT syndrome is a genetically heterogeneous cardiac channelopathy without structural heart disease that has a dominant autosomal or sporadic pattern of transmission affecting the electric system of the heart. Patients present clinically with a spectrum of signs and symptoms including irregular palpitations due to episodes of paroxysmal atrialfibrillation, dizziness and fainting (syncope) and/or sudden cardiac death due to polymorphic ventricular tachycardia and ventricular fibrillation. Electrocardiographic (ECG) findings include extremely short QTc intervals (QTc interval ≤330 ms) not significantly modified with heart rate changes and T waves of great voltage witha narrow base. Electrophysiologic studies are characterized by significant shortening of atrial and ventricular refractory periods and arrhythmias induced by programmed stimulation. A few families have been identified with specific genotypes: 3 with mutations in potassium channels called SQT1 (Iks), SQT2 (Ikr) and SQT3 (Ik1). These 3 potassium channel variants are the "genetic mirror image" of long QT syndrome type 2, type 1 and Andersen-Tawil syndrome respectively because they exert opposite gain-of-function effects on the potassium channels in contrast to the loss-of-function of the potassium channels in the long QT syndromes. Three new variants with overlapping phenotypes affecting the slow inward calcium channels havealso been described. Finally, another variant with mixed phenotype affecting the sodium channel was reported. This review focuses the landmarks of this newest arrhythmogenic cardiac channelopathy on the main clinical, genetic, and proposed ECG mechanisms. In addition therapeutic options and the molecular autopsy of this fascinating primary electrical heart disease are discussed.