Birgit Stallmeyer

Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I

Progressive familial heart block type I (PFHBI) is a progressive cardiac bundle branch disease in the His-Purkinje system that exhibits autosomal-dominant inheritance. In 3 branches of a large South African Afrikaner pedigree with an autosomal-dominant form of PFHBI, we identified the mutation c.19G-->A in the transient receptor potential cation channel, subfamily M, member 4 gene (TRPM4) at chromosomal locus 19q13.3. This mutation predicted the amino acid substitution p.E7K in the TRPM4 amino terminus. TRPM4 encodes a Ca2+-activated nonselective cation (CAN) channel that belongs to the transient receptor potential melastatin ion channel family. Quantitative analysis of TRPM4 mRNA content in human cardiac tissue showed the highest expression level in Purkinje fibers. Cellular expression studies showed that the c.19G-->A missense mutation attenuated deSUMOylation of the TRPM4 channel. The resulting constitutive SUMOylation of the mutant TRPM4 channel impaired endocytosis and led to elevated TRPM4 channel density at the cell surface. Our data therefore revealed a gain-of-function mechanism underlying this type of familial heart block.

A Large Candidate Gene Survey Identifies the KCNE1 D85N Polymorphism as a Possible Modulator of Drug-Induced Torsades de Pointes

Background— Drug-induced long-QT syndrome (diLQTS) is an adverse drug effect that has an important impact on drug use, development, and regulation. We tested the hypothesis that common variants in key genes controlling cardiac electric properties modify the risk of diLQTS. Methods and Results— In a case-control setting, we included 176 patients of European descent from North America and Europe with diLQTS, defined as documented torsades de pointes during treatment with a QT-prolonging drug. Control samples were obtained from 207 patients of European ancestry who displayed <50 ms QT lengthening during initiation of therapy with a QT-prolonging drug and 837 control subjects from the population-based KORA study. Subjects were successfully genotyped at 1424 single-nucleotide polymorphisms (SNPs) in 18 candidate genes including 1386 SNPs tagging common haplotype blocks and 38 nonsynonymous ion channel gene SNPs. For validation, we used a set of cases (n=57) and population-based control subjects of European descent. The SNP KCNE1 D85N (rs1805128), known to modulate an important potassium current in the heart, predicted diLQTS with an odds ratio of 9.0 (95% confidence interval, 3.5–22.9). The variant allele was present in 8.6% of cases, 2.9% of drug-exposed control subjects, and 1.8% of population control subjects. In the validation cohort, the variant allele was present in 3.5% of cases and in 1.4% of control subjects. Conclusions— This high-density candidate SNP approach identified a key potassium channel susceptibility allele that may be associated with the rare adverse drug reaction torsades de pointes.

Mutational spectrum in the Ca2+‐activated cation channel gene TRPM4 in patients with cardiac conductance disturbances

Very recently, mutations in the TRPM4 gene have been identified in four pedigrees as the cause of an autosomal dominant form of cardiac conduction disease. To determine the role of TRPM4 gene variations, the relative frequency of TRPM4 mutations and associated phenotypes was assessed in a cohort of 160 unrelated patients with various types of inherited cardiac arrhythmic syndro‐mes. In eight probands with atrioventricular block or right bundle branch block—five familial cases and three spora‐dic cases—a total of six novel and two published TRPM4 mutations were identified. In patients with sinus node dysfunction, Brugada syndrome, or long‐QT syndrome, no mutations were found. The novel mutations include six amino acid substitutions and appeared randomly distributed through predicted TRPM4 protein. In addition, eight polymorphic sites including two in‐frame deletions were found. Mutations separated from polymorphisms by absence in control individuals and familial cosegregation in some families. In summary, TRPM4 gene mutations appear to play a major role in cardiac conduction disease but not for other related syndromes so far. The phenotypes are variable and clearly suggestive of additional factors modulating the disease phenotype in some patients. Hum Mutat 33:109–117, 2012.

Overlapping Cardiac Phenotype Associated with a Familial Mutation in the Voltage Sensor of the KCNQ1 Channel

Background: Cardiac action potential repolarisation is determined by K+ currents including IKs. IKs channels are heteromeric channels composed of KCNQ1 and KCNE E-subunits. Mutations in KCNQ1 are associated with sinus bradycardia, familial atrial fibrillation (fAF) and/or short QT syndrome as a result of gain-of-function, and long QT syndrome (LQTS) due to loss-of-function in the ventricles. Here, we report that the missense mutation R231C located in S4 voltage sensor domain is associated with a combined clinical phenotype of sinus bradycardia, fAF and LQTS. We aim to understand the molecular basis of the complex clinical phenotype. Methods: We expressed and functionally analyzed the respective channels kinetics in Xenopus laevis oocytes. The molecular nature of the residue R231 was studied by homology modeling and molecular dynamics simulation. Results: As a result, the mutation reduced voltage sensitivity of channels, possibly due to neutralization of the positive charge of the arginine side chain substituted by cysteine. Modeling suggested that the charge carrying side chain of R231 is positioned suitably to transfer transmembrane voltages into conformational energy. Further, the mutation altered the functional interactions with KCNE subunits. Conclusion: The mutation acted in a E-subunit dependent manner, suggesting IKs function altered by the presence of different KCNE subunits in sinus node, atria and ventricles as the molecular basis of sinus bradycardia, fAF and LQTS in mutation carriers.

Human iPS cell model of type 3 long QT syndrome recapitulates drug-based phenotype correction

Long QT syndrome is a potentially life-threatening disease characterized by delayed repolarization of cardiomyocytes, QT interval prolongation in the electrocardiogram, and a high risk for sudden cardiac death caused by ventricular arrhythmia. The genetic type 3 of this syndrome (LQT3) is caused by gain-of-function mutations in the SCN5A cardiac sodium channel gene which mediates the fast Nav1.5 current during action potential initiation. Here, we report the analysis of LQT3 human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These were generated from a patient with a heterozygous p.R1644H mutation in SCN5A known to interfere with fast channel inactivation. LQT3 hiPSC-CMs recapitulated pathognomonic electrophysiological features of the disease, such as an accelerated recovery from inactivation of sodium currents as well as action potential prolongation, especially at low stimulation rates. In addition, unlike previously described LQT3 hiPSC models, we observed a high incidence of early after depolarizations (EADs) which is a trigger mechanism for arrhythmia in LQT3. Administration of specific sodium channel inhibitors was found to shorten action and field potential durations specifically in LQT3 hiPSC-CMs and antagonized EADs in a dose-dependent manner. These findings were in full agreement with the pharmacological response profile of the underlying patient and of other patients from the same family. Thus, our data demonstrate the utility of patient-specific LQT3 hiPSCs for assessing pharmacological responses to putative drugs and for improving treatment efficacies.

Gain-of-function mutations in the calcium channel CACNA1C (Cav1.2) cause non-syndromic long-QT but not Timothy syndrome

Gain-of-function mutations in CACNA1C, encoding the L-type Ca(2+) channel Cav1.2, cause Timothy syndrome (TS), a multi-systemic disorder with dysmorphic features, long-QT syndrome (LQTS) and autism spectrum disorders. TS patients have heterozygous mutations (G402S and G406R) located in the alternatively spliced exon 8, causing a gain-of-function by reduced voltage-dependence of inactivation. Screening 540 unrelated patients with non-syndromic forms of LQTS, we identified six functional relevant CACNA1C mutations in different regions of the channel. All these mutations caused a gain-of-function combining different mechanisms, including changes in current amplitude, rate of inactivation and voltage-dependence of activation or inactivation, similar as in TS. Computer simulations support the theory that the novel CACNA1C mutations prolong action potential duration. We conclude that genotype-negative LQTS patients should be investigated for mutations in CACNA1C, as a gain-of-function in Cav1.2 is likely to cause LQTS and only specific and rare mutations, i.e. in exon 8, cause the multi-systemic TS.

Gain-of-function mutation in TASK-4 channels and severe cardiac conduction disorder

Analyzing a patient with progressive and severe cardiac conduction disorder combined with idiopathic ventricular fibrillation (IVF), we identified a splice site mutation in the sodium channel gene SCN5A. Due to the severe phenotype, we performed whole‐exome sequencing (WES) and identified an additional mutation in the KCNK17 gene encoding the K2P potassium channel TASK‐4. The heterozygous change (c.262G>A) resulted in the p.Gly88Arg mutation in the first extracellular pore loop. Mutant TASK‐4 channels generated threefold increased currents, while surface expression was unchanged, indicating enhanced conductivity. When co‐expressed with wild‐type channels, the gain‐of‐function by G88R was conferred in a dominant‐active manner. We demonstrate that KCNK17 is strongly expressed in human Purkinje cells and that overexpression of G88R leads to a hyperpolarization and strong slowing of the upstroke velocity of spontaneously beating HL‐1 cells. Thus, we propose that a gain‐of‐function by TASK‐4 in the conduction system might aggravate slowed conductivity by the loss of sodium channel function. Moreover, WES supports a second hit‐hypothesis in severe arrhythmia cases and identified KCNK17 as a novel arrhythmia gene.

Estradiol regulates human QT-interval: acceleration of cardiac repolarization by enhanced KCNH2 membrane trafficking

BACKGROUND Modulation of cardiac repolarization by sexual hormones is controversial and hormonal effects on ion channels remain largely unknown. In the present translational study, we therefore assessed the relationship between QTc duration and gonadal hormones and studied underlying mechanisms. METHODS AND RESULTS We measured hormone levels and QTc intervals in women during clomiphene stimulation for infertility and women before, during, and after pregnancy. Three heterozygous LQT-2 patients (KCNH2-p.Arg752Pro missense mutation) and two unaffected family members additionally were studied during their menstrual cycles. A comprehensive cellular and molecular analysis was done to identify the mechanisms of hormonal QT-interval regulation. High estradiol levels, but neither progesterone nor estradiol/progesterone ratio, inversely correlated with QTc. Consistent with clinical data, in vitro estradiol stimulation (60 pmol/L, 48 h) enhanced IKCNH2. This increase was mediated by estradiol receptor-α-dependent promotion of KCNH2-channel trafficking to the cell membrane. To study the underlying mechanism, we focused on heat-shock proteins. The heat-shock protein-90 (Hsp90) inhibitor geldanamycin abolished estradiol-induced increase in IKCNH2. Geldanamycin had no effect on KCNH2 transcription or translation; nor did it affect expression of estradiol receptors and chaperones. Estradiol enhanced the physical interaction of KCNH2-channel subunits with heat-shock proteins and augmented ion-channel trafficking to the membrane. CONCLUSION Elevated estradiol levels were associated with shorter QTc intervals in healthy women and female LQT-2 patients. Estradiol acts on KCNH2 channels via enhanced estradiol-receptor-α-mediated Hsp90 interaction, augments membrane trafficking and thereby increases repolarizing current. These results provide mechanistic insights into hormonal control of human ventricular repolarization and open novel therapeutic avenues.

Genes causing inherited forms of cardiomyopathies. A current compendium.

Cardiomyopathies (diseases of the heart muscle) are a relevant but heterogeneous group of cardiac diseases. The underlying structural myocardial alterations, detectable with contemporary cardiac imaging modalities and also on twelve-lead surface ECG, embody both linkage and differentiation of the respective clinical forms. Most of these are defined to the heart, however, cardiac involvement is also known in the presence of specific metabolic or muscular systemic diseases. In many of the known cardiomyopathies, a genetic background has been elucidated thus indicating a potential familial disease. This, in turn, also harbors the risk that more family members may be affected besides the index patient.ZusammenfassungKardiomyopathien („Herzmuskelerkrankungen“) sind eine wichtige und heterogene Gruppe von Herzerkrankungen. Ihnen gemeinsam sind Strukturveränderungen im Myokard, die sich mittels kardialer Bildgebung und im EKG erfassen lassen. Bei familiären Kardiomyopathien findet sich definitionsgemäß eine positive Familienanamnese. Kardiomyopathien kommen meist isoliert vor, können aber auch Ausdruck einer Systembeteiligung im Rahmen einer muskulären, metabolischen oder anderen Erkrankung sein. Viele Kardiomyopathien sind genetisch bedingt und bergen damit das Risiko, dass weitere Familienmitglieder betroffen sein können.

A homozygous SCN5A mutation in a severe, recessive type of cardiac conduction disease

Cardiac sodium channels are key players in the generation and propagation of action potentials in the human heart. Heterozygous mutations in the SCN5A gene have been found to be associated with long QT syndrome, Brugada syndrome, and sinus node dysfunction (SND). Recently, overlapping arrhythmia phenotypes have been reported as well. Here we describe a novel recessive SCN5A mutation in a family originating from the German minority in White Russia. Four affected children with a history of early cardiac arrhythmia encompassing SND, conduction disease, and severe ventricular arrhythmias, are homozygous carriers of a novel SCN5A missense mutation (p.I230T) in the channel protein. Interestingly, the heterozygous mutation carriers had neither significant ECG abnormalities nor a history of cardiac events. Heterologous expression of SCN5A(I230T) channels revealed normal protein transport but altered biophysical sodium channel properties. Voltage range of both activation and inactivation were shifted in a way that resulted in decreased sodium current and loss of channel function. In conclusion, we describe a rare clinical condition with a novel SCN5A mutation causing a new type of complex cardiac arrhythmia. Unlike most previously reported sodium channelopathies, this overlap syndrome displays recessive inheritance characteristics and does not seem to follow simple Mendelian rules.© 2010 Wiley‐Liss, Inc.