The impact of BMI on breast cancer - an updated systematic review and meta-analysis.

BACKGROUND: Breast cancer is the most frequent form of cancer in women all over the world. It is the main cause of cancer death and the most often diagnosed cancer in women in 140 of the world's 184 countries. The link between breast cancer risk and body mass index (BMI) has gotten increasing attention in recent years, although the results are still debatable. Therefore, the current systematic review and meta-analysis evaluate the impact of BMI on breast cancer. METHODS: The current study was carried out as a systematic review and meta-analysis, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We systematically searched Cochrane, Google Scholar, PubMed, EMBASE and Scopus databases to identify eligible articles impact of BMI on breast cancer with the appropriate Medical Subject Headings (MeSH). The Newcastle-Ottawa checklist was used for the risk of assessment for the included studies. Meta-analysis was performed using Review Manager 5.3 software. RESULTS: Forty-six studies were included in the current review, which met the selection criteria of the current review. Among included 46 studies in this review, 50% (n = 23) of the studies found the HER2 type of breast cancer followed by triple-negative and HR-positive. The obesity was significantly higher in the case group compared with the control group (P < .001). Heterogeneity between the 14 studies is medium (I2 = 72%). In this review, there was no significant relation between overweight and breast cancer in women (P > .05). Heterogenecity between the 14 studies is medium (I2 = 89%). However, after removing the publication bias a significant relation between overweightness and breast cancer in women (P = .0005) was observed. CONCLUSION: Obese breast cancer patients are a specific type of patient. They are more likely to develop cancer. Their need to surgery and radiation may cause greater difficulties. Obesity and overweight in women greatly increase the risk of breast cancer, according to the findings of the current meta-analysis. To confirm these findings and understand the pathogenic pathways, more research is required.

The impact of body mass index on prostate cancer: An updated systematic review and meta-analysis.

BACKGROUND: Increasing evidence suggested obesity was associated with the risk of prostate cancer. Also, the association between prostate cancer risk and obesity has received much attention in recent years, but the results are still unclear. Therefore, the current systematic review and meta-analysis aimed to evaluate the impact of body mass index (BMI) on prostate cancer. METHODS: We systematically searched PubMed, Google Scholar, Scopus and Cochrane databases with the appropriate key terms to identify the eligible articles related to the impact of BMI on prostate cancer. The Newcastle-Ottawa checklist was used for the quality assessment of studies, and the meta-analysis was carried out using Review Manager 5.3. RESULTS: The present review includes 23 studies that fulfilled the criteria for inclusion. In the meta-analysis, a significant difference was observed between the obese and normal weight (P < .001) and 54% of obese has a risk compared to normal weight. Heterogeneity between the fifteen studies was high (I2 = 100%). Test for overall effect: Z = 8.77 (P < .001) (odds ratio [OR] = 0.32 confidence interval [CI]: 0.25-0.42). However, there was no significant difference observed between the overweight and normal weight (P = .75). Heterogeneity between the fifteen studies is high (I2 = 100%). CONCLUSION: Prostate cancer is a common malignancy that poses a threat to the health of men. Obesity is associated with a higher risk of death from prostate cancer based on the findings of the included studies. Furthermore, wherever possible, the impact of weight change on prostate cancer patient mortality should be investigated.

NPRL2 Inhibition of mTORC1 Controls Sodium Channel Expression and Brain Amino Acid Homeostasis.

Genetic mutations in nitrogen permease regulator-like 2 (NPRL2) are associated with a wide spectrum of familial focal epilepsies, autism, and sudden unexpected death of epileptics (SUDEP), but the mechanisms by which NPRL2 contributes to these effects are not well known. NPRL2 is a requisite subunit of the GAP activity toward Rags 1 (GATOR1) complex, which functions as a negative regulator of mammalian target of rapamycin complex 1 (mTORC1) kinase when intracellular amino acids are low. Here, we show that loss of NPRL2 expression in mouse excitatory glutamatergic neurons causes seizures before death, consistent with SUDEP in humans with epilepsy. Additionally, the absence of NPRL2 expression increases mTORC1-dependent signal transduction and significantly alters amino acid homeostasis in the brain. Loss of NPRL2 reduces dendritic branching and increases the strength of electrically stimulated action potentials (APs) in neurons. The increased AP strength is consistent with elevated expression of epilepsy-linked, voltage-gated sodium channels in the NPRL2-deficient brain. Targeted deletion of NPRL2 in primary neurons increases the expression of sodium channel Scn1A, whereas treatment with the pharmacological mTORC1 inhibitor called rapamycin prevents Scn1A upregulation. These studies demonstrate a novel role of NPRL2 and mTORC1 signaling in the regulation of sodium channels, which can contribute to seizures and early lethality.

iPSC-derived cardiomyocytes from patients with myotonic dystrophy type 1 have abnormal ion channel functions and slower conduction velocities.

Cardiac complications such as electrical abnormalities including conduction delays and arrhythmias are the main cause of death in individuals with Myotonic Dystrophy type 1 (DM1). We developed a disease model using iPSC-derived cardiomyocytes (iPSC-CMs) from a healthy individual and two DM1 patients with different CTG repeats lengths and clinical history (DM1-1300 and DM1-300). We confirmed the presence of toxic RNA foci and mis-spliced MBNL1/2 transcripts in DM1 iPSC-CMs. In DM1-1300, we identified a switch in the cardiac sodium channel SCN5A from the adult to the neonatal isoform. The down-regulation of adult SCN5A isoforms is consistent with a shift in the sodium current activation to depolarized potentials observed in DM1-1300. L-type calcium current density was higher in iPSC-CMs from DM1-1300, which is correlated with the overexpression of the CaV1.2 transcript and proteins. Importantly, INa and ICaL dysfunctions resulted in prolonged action potentials duration, slower velocities, and decreased overshoots. Optical mapping analysis revealed a slower conduction velocity in DM1-1300 iPSC-CM monolayers. In conclusion, our data revealed two distinct ions channels perturbations in DM1 iPSC-CM from the patient with cardiac dysfunction, one affecting Na+ channels and one affecting Ca2+ channels. Both have an impact on cardiac APs and ultimately on heart conduction.

Interleukin-6 inhibition of hERG underlies risk for acquired long QT in cardiac and systemic inflammation.

Increased proinflammatory interleukin-6 (IL-6) levels are associated with acquired long QT-syndrome (LQTS) in patients with systemic inflammation, leading to higher risks for life-threatening polymorphic ventricular tachycardia such as Torsades de Pointes. However, the functional and molecular mechanisms of this association are not known. In most cases of acquired LQTS, the target ion channel is the human ether-á-go-go-related gene (hERG) encoding the rapid component of the delayed rectifier K current, IKr, which plays a critical role in cardiac repolarization. Here, we tested the hypothesis that IL-6 may cause QT prolongation by suppressing IKr. Electrophysiological and biochemical assays were used to assess the impact of IL-6 on the functional expression of IKr in HEK293 cells and adult guinea-pig ventricular myocytes (AGPVM). In HEK293 cells, IL-6 alone or in combination with the soluble IL-6 receptor (IL-6R), produced a significant depression of IKr peak and tail current densities. Block of IL-6R or Janus kinase (JAK) reversed the inhibitory effects of IL-6 on IKr. In AGPVM, IL-6 prolonged action potential duration (APD) which was further prolonged in the presence of IL-6R. Similar to heterologous cells, IL-6 reduced endogenous guinea pig ERG channel mRNA and protein expression. The data are first to demonstrate that IL-6 inhibition of IKr and the resulting prolongation of APD is mediated via IL-6R and JAK pathway activation and forms the basis for the observed clinical QT interval prolongation. These novel findings may guide the development of targeted anti-arrhythmic therapeutic interventions in patients with LQTS and inflammatory disorders.

Regulation of Cardiac Voltage-Gated Sodium Channel by Kinases: Roles of Protein Kinases A and C.

In the heart, voltage-gated sodium (Nav) channel (Nav1.5) is defined by its pore-forming α-subunit and its auxiliary ß-subunits, both of which are important for its critical contribution to the initiation and maintenance of the cardiac action potential (AP) that underlie normal heart rhythm. The physiological relevance of Nav1.5 is further marked by the fact that inherited or congenital mutations in Nav1.5 channel gene SCN5A lead to altered functional expression (including expression, trafficking, and current density), and are generally manifested in the form of distinct cardiac arrhythmic events, epilepsy, neuropathic pain, migraine, and neuromuscular disorders. However, despite significant advances in defining the pathophysiology of Nav1.5, the molecular mechanisms that underlie its regulation and contribution to cardiac disorders are poorly understood. It is rapidly becoming evident that the functional expression (localization, trafficking and gating) of Nav1.5 may be under modulation by post-translational modifications that are associated with phosphorylation. We review here the molecular basis of cardiac Na channel regulation by kinases (PKA and PKC) and the resulting functional consequences. Specifically, we discuss: (1) recent literature on the structural, molecular, and functional properties of cardiac Nav1.5 channels; (2) how these properties may be altered by phosphorylation in disease states underlain by congenital mutations in Nav1.5 channel and/or subunits such as long QT and Brugada syndromes. Our expectation is that understanding the roles of these distinct and complex phosphorylation processes on the functional expression of Nav1.5 is likely to provide crucial mechanistic insights into Na channel associated arrhythmogenic events and will facilitate the development of novel therapeutic strategies.

Induction of autoimmune response to the extracellular loop of the HERG channel pore induces QTc prolongation in guinea-pigs.

KEY POINTS: Channelopathies of autoimmune origin are novel and are associated with corrected QT (QTc) prolongation and complex ventricular arrhythmias. We have recently demonstrated that anti-SSA/Ro antibodies from patients with autoimmune diseases and with QTc prolongation on the ECG target the human ether-à-go-go-related gene (HERG) K+ channel by inhibiting the corresponding current, IKr , at the pore region. Immunization of guinea-pigs with a peptide (E-pore peptide) corresponding to the extracellular loop region connecting the S5 and S6 segments of the HERG channel induces high titres of antibodies that inhibit IKr , lengthen the action potential and cause QTc prolongation on the surface ECG. In addition, anti-SSA/Ro-positive sera from patients with connective tissue diseases showed high reactivity to the E-pore peptide. The translational impact is the development of a peptide-based approach for the diagnosis and treatment of autoimmune-associated long QT syndrome. ABSTRACT: We recently demonstrated that anti-SSA/52 kDa Ro antibodies (Abs) from patients with autoimmune diseases and corrected QT (QTc) prolongation directly target and inhibit the human ether-à-go-go-related gene (HERG) K+ channel at the extracellular pore (E-pore) region, where homology with SSA/52 kDa Ro antigen was demonstrated. We tested the hypothesis that immunization of guinea-pigs with a peptide corresponding to the E-pore region (E-pore peptide) will generate pathogenic inhibitory Abs and cause QTc prolongation. Guinea-pigs were immunized with a 31-amino-acid peptide corresponding to the E-pore region of HERG. On days 10-62 after immunization, ECGs were recorded and blood was sampled for the detection of E-pore peptide Abs. Serum samples from patients with autoimmune diseases were evaluated for reactivity to E-pore peptide by enzyme-linked immunosorbent assay (ELISA), and histology was performed on hearts using Masson's Trichrome. Inhibition of the HERG channel was assessed by electrophysiology and by computational modelling of the human ventricular action potential. The ELISA results revealed the presence of high titres of E-pore peptide Abs and significant QTc prolongation after immunization. High reactivity to E-pore peptide was found using anti-SSA/Ro Ab-positive sera from patients with QTc prolongation. Histological data showed no evidence of fibrosis in immunized hearts. Simulations of simultaneous inhibition of repolarizing currents by anti-SSA/Ro Ab-positive sera showed the predominance of the HERG channel in controlling action potential duration and the QT interval. These results are the first to demonstrate that inhibitory Abs to the HERG E-pore region induce QTc prolongation in immunized guinea-pigs by targeting the HERG channel independently from fibrosis. The reactivity of anti-SSA/Ro Ab-positive sera from patients with connective tissue diseases with the E-pore peptide opens novel pharmacotherapeutic avenues in the diagnosis and management of autoimmune-associated QTc prolongation.

MTSET modification of D4S6 cysteines stabilize the fast inactivated state of Nav1.5 sodium channels.

The transmembrane S6 segments of Na(+) sodium channels form the cytoplasmic entrance of the channel and line the internal aspects of the aqueous pore. This region of the channel has been implicated in Na(+) channel permeation, gating, and pharmacology. In this study we utilized cysteine substitutions and methanethiosulfonate reagent (MTSET) to investigate the role of the S6 segment of homologous domain 4 (D4S6) in the gating of the cardiac (Nav1.5) channel. D4S6 cysteine mutants were heterologously expressed in tsA201 cells and currents recorded using whole-cell patch clamp. Internal MTSET reduced the peak Na(+) currents, induced hyperpolarizing shifts in steady-state inactivation and slowed the recovery of mutant channels with cysteines inserted near the middle (F1760C, V1763C) and C-terminus (Y1767C) of the D4S6. These findings suggested a link between the MTSET inhibition and fast inactivation. This was confirmed by expressing the V1763C and Y1767C mutations in non-inactivating Nav1.5 channels. Removing inactivation abolished the MTSET inhibition of the V1763C and Y1767C mutants. The data indicate that the MTSET-induced reduction in current primarily results from slower recovery from inactivation that produces hyperpolarizing shifts in fast inactivation and decreases the steady-state availability of the channels. This contrasted with a cysteine inserted near the C-terminus of the D4S6 (I1770C) where MTSET increased the persistent Na(+) current at depolarized voltages consistent with impaired fast inactivation. Covalent modification of D4S6 cysteines with MTSET adduct appears to reduce the mobility of the D4S6 segment and stabilize the channels in the fast inactivated state. These findings indicate that residues located near the middle and C-terminus of the D4S6 play an important role in fast inactivation.

Gating pore currents, a new pathological mechanism underlying cardiac arrhythmias associated with dilated cardiomyopathy.

Voltage-gated ion channels (VGIC) are transmembrane proteins responsible for the generation of electrical signals in excitable cells. VGIC were first described in 1952 by Hodgkin and Huxley, (1) and have since been associated with various physiological functions such as propagating nerve impulses, locomotion, and cardiac excitability. VGIC include channels specialized in the selective passage of K(+), Ca(2+) Na(+), or H(+). They are composed of 2 main structures: the pore domain (PD) and the voltage sensor domain (VSD). The PD ensures the physiological flow of ions and is typically composed of 8 transmembrane segments (TM). The VSD detects voltage variations and is composed of 4 TM (S1-S4). Given their crucial physiological role, VGIC dysfunctions are associated with diverse pathologies known as ion channelopathies. These dysfunctions usually affect the membrane expression of ion channels or voltage-dependent conformational changes of the pore. However, an increasing number of ion channelopathies, including periodic paralysis, dilated cardiomyopathy (DCM) associated with cardiac arrhythmias, and peripheral nerve hyperexcitability (PNH), have been linked to the appearance of a new pathological mechanism involving the creation of an alternative permeation pathway through the normally non-conductive VSD of VGIC. This permeation pathway is called the gating pore or omega pore.

Pathogenesis of the Novel Autoimmune-Associated Long-QT Syndrome.

BACKGROUND: Emerging clinical evidence demonstrates high prevalence of QTc prolongation and complex ventricular arrhythmias in patients with anti-Ro antibody (anti-Ro Ab)-positive autoimmune diseases. We tested the hypothesis that anti-Ro Abs target the HERG (human ether-a-go-go-related gene) K(+) channel, which conducts the rapidly activating delayed K(+) current, IKr, thereby causing delayed repolarization seen as QT interval prolongation on the ECG. METHODS AND RESULTS: Anti-Ro Ab-positive sera, purified IgG, and affinity-purified anti-52kDa Ro Abs from patients with autoimmune diseases and QTc prolongation were tested on IKr using HEK293 cells expressing HERG channel and native cardiac myocytes. Electrophysiological and biochemical data demonstrate that anti-Ro Abs inhibit IKr to prolong action potential duration by directly binding to the HERG channel protein. The 52-kDa Ro antigen-immunized guinea pigs showed QTc prolongation on ECG after developing high titers of anti-Ro Abs, which inhibited native IKr and cross-reacted with guinea pig ERG channel. CONCLUSIONS: The data establish that anti-Ro Abs from patients with autoimmune diseases inhibit IKr by cross-reacting with the HERG channel likely at the pore region where homology between anti-52-kDa Ro antigen and HERG channel is present. The animal model of autoimmune-associated QTc prolongation is the first to provide strong evidence for a pathogenic role of anti-Ro Abs in the development of QTc prolongation. It is proposed that adult patients with anti-Ro Abs may benefit from routine ECG screening and that those with QTc prolongation should receive counseling about drugs that may increase the risk for life-threatening arrhythmias.

Regulation/modulation of sensory neuron sodium channels.

The pseudounipolar sensory neurons of the dorsal root ganglia (DRG) give rise to peripheral branches that convert thermal, mechanical, and chemical stimuli into electrical signals that are transmitted via central branches to the spinal cord. These neurons express unique combinations of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na(+) channels that contribute to the resting membrane potential, action potential threshold, and regulate neuronal firing frequency. The small-diameter neurons (<25 µm) isolated from the DRG represent the cell bodies of C-fiber nociceptors that express both TTX-S and TTX-R Na(+) currents. The large-diameter neurons (>35 µm) are typically low-threshold A-fibers that predominately express TTX-S Na(+) currents. Peripheral nerve damage, inflammation, and metabolic diseases alter the expression and function of these Na(+) channels leading to increases in neuronal excitability and pain. The Na(+) channels expressed in these neurons are the target of intracellular signaling cascades that regulate the trafficking, cell surface expression, and gating properties of these channels. Post-translational regulation of Na(+) channels by protein kinases (PKA, PKC, MAPK) alter the expression and function of the channels. Injury-induced changes in these signaling pathways have been linked to sensory neuron hyperexcitability and pain. This review examines the signaling pathways and regulatory mechanisms that modulate the voltage-gated Na(+) channels of sensory neurons.

Modulation of peripheral Na(+) channels and neuronal firing by n-butyl-p-aminobenzoate.

n-butyl-p-aminobenzoate (BAB), a local anesthetic, is administered epidurally in cancer patients to treat pain that is poorly controlled by other drugs that have a number of adverse effects. The purpose of the study was to unravel the mechanisms underlying the apparent selective pain suppressant effect of BAB. We used the whole-cell patch-clamp technique to record Na(+) currents and action potentials (APs) in dissociated, nociceptive dorsal root ganglion (DRG) cells from rats, two types of peripheral sensory neuron Na(+) channels (Nav1.7 and Nav1.8), and the motor neuron-specific Na(+) channel (Nav1.6) expressed in HEK293 cells. BAB (1-100µM) inhibited, in a concentration-dependent manner, the depolarization evoked repetitive firing in DRG cells, the three types of Na(+) current expressed in HEK293 cells, and the TTXr Na(+) current of the DRG neurons. BAB induced a use-dependent block that caused a shift of the inactivation curve in the hyperpolarizing direction. BAB enhanced the onset of slow inactivation of Nav1.7 and Nav1.8 currents but not of Nav1.6 currents. At clinically relevant concentrations (1-100µM), BAB is thus a more potent inhibitor of peripheral TTX-sensitive TTXs, Nav1.7 and TTX-resistant NaV1.8 Na(+) channels than of motor neuron axonal Nav1.6 Na(+) channels. BAB had similar effects on the TTXr Na(+) channels of rat DRG neurons and Nav1.8 channels expressed in HEK293 cells. The observed selectivity of BAB in treating cancer pain may be due to an enhanced and selective responsiveness of Na(+) channels in nociceptive neurons to this local anesthetic.

The variant hERG/R148W associated with LQTS is a mutation that reduces current density on co-expression with the WT.

BACKGROUND: A variant of the ether-à-go-go related channel (hERG), p.Arg148Trp (R148W) was found at heterozygous state in two infants who died from sudden infant death syndrome (SIDS), one with documented prolonged QTc and Torsade de Pointes (TdP), and in an adult woman with QTc >500 ms, atrioventricular block and TdP. This variant was previously reported in cases of severe ventricular arrhythmia but very rarely in control subjects. Its classification as mutation or polymorphism awaited electrophysiological characterization. METHODS: The properties of this N-terminal, proximal domain, hERG variant were explored in Xenopus oocytes injected with the same amount of RNA encoding for either hERG/WT or hERG/R148W or their equimolar mixture. The human ventricular cell (TNNP) model was used to test the effects of changes in hERG current. RESULTS: R148W alone produced a current similar to the WT (369 ± 76 nA (mean ± SEM), n=13 versus 342 ± 55 nA in WT, n=13), while the co-expression of 1/2 WT+1/2 R148W lowered the current by 29% versus WT (243 ± 35 nA, n=13, p<0.05). The voltage dependencies of steady-state activation and inactivation were not changed in the variant alone or in co-expression with the WT. The time constants of fast recovery from inactivation and of fast and slow deactivation analyzed between -120 and +20 mV were not changed. The voltage-dependent distribution of the current amplitudes among fast-, slow- and non-deactivating fractions was unaltered. A 6.6% increase in APD90 from 323.5 ms to 345 ms was observed using the human cardiac ventricular myocyte model. CONCLUSIONS: Such a decrease in hERG current as evidenced here when co-expressing the hERG/R148W variant with the WT may have predisposed to the observed long QT syndrome and associated TdP. Therefore, the heterozygous carriers of hERG/R148W may be at risk of cardiac sudden death.

Coexisting mutations/polymorphisms of the long QT syndrome genes in patients with repaired Tetralogy of Fallot are associated with the risks of life-threatening events.

Coexisting long QT gene mutations/polymorphisms in Tetralogy of Fallot (TOF) patients may aggravate the repolarization abnormality from cardiac repair. We investigated the impact of these genes on the risk of life-threatening events. Genetic variants of the three common long QT genes were identified from patients with repaired TOF. Life-threatening events were defined as sudden cardiac death and hemodynamic unstable ventricular arrhythmia. Biophysical characterization of the alleles of the genetic variants was performed using a whole-cell voltage clamp with expression in Xenopus oocytes. A total of 84 patients (56.0 % male with 1,215 patients-year follow-up) were enrolled. Six rare variants and six non-synonymous single nucleotide polymorphisms (SNPs) were found in 40 (47.6 %) patients. Life-threatening events occurred in five patients; four received implantable cardioverter defibrillator and one died of sudden cardiac death. Life-threatening events occurred more often in those with genetic variants than those without (5/40 vs. 0/44, P = 0.021); particularly, the hERG or SCN5A gene mutations/polymorphisms (2/5 vs. 3/79, P = 0.027 and 5/27 vs. 0/57, P = 0.003, respectively). Among the five patients with life-threatening events, three had compound variants (hERG p.M645R/SCN5A p.R1193Q, hERG p.K897T/SCN5A p.H558R, and KVLQT1 p.G645S/SCN5A p.P1090L), that also increased the risk of events. Their QTc and JTc were all prolonged. Functional study of the novel variant (hERG gene p.M645R) from patients with life-threatening events revealed a dominant negative effect. In conclusion, in repaired TOF patients, coexisting long QT mutations/polymorphisms might have additive effects on the repolarization abnormality from surgery and thereby increase the risks of life-threatening events.

Y1767C, a novel SCN5A mutation, induces a persistent Na+ current and potentiates ranolazine inhibition of Nav1.5 channels.

Long QT syndrome type 3 (LQT3) has been traced to mutations of the cardiac Na(+) channel (Na(v)1.5) that produce persistent Na(+) currents leading to delayed ventricular repolarization and torsades de pointes. We performed mutational analyses of patients suffering from LQTS and characterized the biophysical properties of the mutations that we uncovered. One LQT3 patient carried a mutation in the SCN5A gene in which the cysteine was substituted for a highly conserved tyrosine (Y1767C) located near the cytoplasmic entrance of the Na(v)1.5 channel pore. The wild-type and mutant channels were transiently expressed in tsA201 cells, and Na(+) currents were recorded using the patch-clamp technique. The Y1767C channel produced a persistent Na(+) current, more rapid inactivation, faster recovery from inactivation, and an increased window current. The persistent Na(+) current of the Y1767C channel was blocked by ranolazine but not by many class I antiarrhythmic drugs. The incomplete inactivation, along with the persistent activation of Na(+) channels caused by an overlap of voltage-dependent activation and inactivation, known as window currents, appeared to contribute to the LQTS phenotype in this patient. The blocking effect of ranolazine on the persistent Na(+) current suggested that ranolazine may be an effective therapeutic treatment for patients with this mutation. Our data also revealed the unique role for the Y1767 residue in inactivating and forming the intracellular pore of the Na(v)1.5 channel.

Characterization of novel KCNH2 mutations in type 2 long QT syndrome manifesting as seizures.

BACKGROUND: Long QT syndrome (LQTS) is characterized by corrected QT interval prolongation leading to torsades de pointes and sudden cardiac death. LQTS type 2 (LQTS2) is caused by mutations in the KCNH2 gene, leading to a reduction of the rapidly activating delayed rectifier K+ current and loss of human ether-à-go-go-related gene (hERG) channel function by different mechanisms. Triggers for life-threatening arrhythmias in LQTS2 are often auditory stimuli. OBJECTIVES: To screen KCNH2 for mutations in patients with LQTS2 on an electrocardiogram and auditory-induced syncope interpreted as seizures and sudden cardiac death, and to analyze their impact on the channel function in vitro. METHODS: The KCNH2 gene was screened for mutations in the index patients of three families. The novel mutations were reproduced in vitro using site-directed mutagenesis and characterized using the Xenopus oocyte expression system in voltage clamp mode. RESULTS: Novel KCNH2 mutations (Y493F, A429P and del234-241) were identified in the index patients with mostly typical LQTS2 features on their electrocardiograms. The biochemical data revealed a trafficking defect. The biophysical data revealed a loss of function when mutated hERG channels were coexpressed with the wild type. CONCLUSIONS: In all families, at least one patient carrying the mutation had a history of seizures after auditory stimuli, which is a major trigger for arrhythmic events in LQTS2. Seizures are likely due to cardiac syncope as a consequence of mutation-induced loss of function of the rapidly activating delayed rectifier K+ current.

Phosphorylation of the consensus sites of protein kinase A on alpha1D L-type calcium channel.

The novel alpha(1D) L-type Ca(2+) channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased alpha(1D) Ca(2+) channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the alpha(1) subunit of the alpha(1D) Ca(2+) channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the alpha(1) subunit of alpha(1D) Ca(2+) channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the alpha(1) subunit of alpha(1D) Ca(2+) channel. These novel findings provide new insights into the autonomic regulation of the alpha(1D) Ca(2+) channel in the heart.

A new C-terminal hERG mutation A915fs+47X associated with symptomatic LQT2 and auditory-trigger syncope.

BACKGROUND: A novel mutation of hERG (A915fs+47X) was discovered in a 32-year-old woman with torsades de pointes, long QTc interval (515 ms), and syncope upon auditory trigger. OBJECTIVE: We explored whether the properties of this mutation could explain the pathology. METHODS: Whole-cell A915fs+47X (del) and wild-type (WT) currents were recorded in transiently transfected COS7 cells or Xenopus oocytes. Western blots and sedimentation analysis of del/WT hERG were used to analyze protein expression, assembly, and trafficking. RESULTS: The tail current density at -40 mV after a 2-s depolarization to +40 mV in COS7 cells expressing del was 36% of that for WT. Inactivation was 1.9-fold to 2.8-fold faster in del versus WT between -60 and +60 mV. In the range -60 to -10 mV, we found that a nondeactivating fraction of current was increased in del at the expense of a rapidly deactivating fraction, with a slowly deactivating fraction being unchanged. In Xenopus oocytes, expression of del alone produced 38% of WT currents, whereas coexpression of 1/2 WT + 1/2 del produced 49.8%. Furthermore, the expression of del protein at the cell surface was reduced by about 50%. This suggests that a partial trafficking defect of del contributes to the reduction in del current densities and to the dominant negative effect when coexpressed with WT. In model simulations, the mutation causes a 10% prolongation of action potential duration. CONCLUSION: Decreased current levels caused by a trafficking defect may explain the long QT syndrome observed in our patient.