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Abstract Several human diseases, including cancer and neurodegeneration, are associated with excessive mitochondrial fragmentation. In this context, mitochondrial division inhibitor (Mdivi-1) has been tested as a therapeutic to block the fission-related protein dynamin-like protein-1 (Drp1). Recent studies suggest that Mdivi-1 interferes with mitochondrial bioenergetics and complex I function. Here we show that the molecular mechanism of Mdivi-1 is based on inhibition of complex I at the IQ site. This leads to the destabilization of complex I, impairs the assembly of N- and Q-respirasomes, and is associated with increased ROS production and reduced efficiency of ATP generation. Second, the calcium homeostasis of cells is impaired, which for example affects the electrical activity of neurons. Given the results presented here, a potential therapeutic application of Mdivi-1 is challenging because of its potential impact on synaptic activity. Similar to the Complex I inhibitor rotenone, Mdivi-1 may lead to neurodegenerative effects in the long term.

Abstract The TWIK-related spinal cord K + channel (TRESK, K2P18.1) is a K2P channel contributing to the maintenance of membrane potentials in various cells. Recently, physiological TRESK function was identified as a key player in T-cell differentiation rendering the channel a new pharmacological target for treatment of autoimmune diseases. The channel activator cloxyquin represents a promising lead compound for the development of a new class of immunomodulators. Identification of cloxyquin binding site and characterization of the molecular activation mechanism can foster the future drug development. Here, we identify the cloxyquin binding site at the M2/M4 interface by mutational scan and analyze the molecular mechanism of action by protein modeling as well as in silico and in vitro electrophysiology using different permeating ion species (K+ / Rb+). In combination with kinetic analyses of channel inactivation, our results suggest that cloxyquin allosterically stabilizes the inner selectivity filter facilitating the conduction process subsequently activating hTRESK.

Influenza A virus replication relies on host cell-derived ubiquitination of the viral polymerase. Here, Günl et al. show that site-specific ubiquitination of PB1-K578 is acquired during infection and facilitates spatiotemporal control of polymerase dimerization and NP binding.

The binding site and mechanism of action for a benzodiazepine-derivative agonist on Kv7.1 channels is determined for drug development to treat long QT syndrome.

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS. Increasing evidence suggests that vulnerable neurons in MS exhibit fatal metabolic exhaustion over time, a phenomenon hypothesized to be caused by chronic hyperexcitability. Axonal Kv7 (outward-rectifying) and oligodendroglial Kir4.1 (inward-rectifying) potassium channels have important roles in regulating neuronal excitability at and around the nodes of Ranvier. Here, we studied the spatial and functional relationship between neuronal Kv7 and oligodendroglial Kir4.1 channels and assessed the transcriptional and functional signatures of cortical and retinal projection neurons under physiological and inflammatory demyelinating conditions. We found that both channels became dysregulated in MS and experimental autoimmune encephalomyelitis (EAE), with Kir4.1 channels being chronically downregulated and Kv7 channel subunits being transiently upregulated during inflammatory demyelination. Further, we observed that pharmacological Kv7 channel opening with retigabine reduced neuronal hyperexcitability in human and EAE neurons, improved clinical EAE signs, and rescued neuronal pathology in oligodendrocyte–Kir4.1–deficient (OL-Kir4.1–deficient) mice. In summary, our findings indicate that neuron-OL compensatory interactions promoted resilience through Kv7 and Kir4.1 channels and identify pharmacological activation of nodal Kv7 channels as a neuroprotective strategy against inflammatory demyelination.

Tauopathies are a major type of proteinopathies underlying neurodegenerative diseases. Mutations in the tau-encoding MAPT-gene lead to hereditary cases of frontotemporal lobar degeneration (FTLD)-tau, which span a wide phenotypic and pathological spectrum. Some of these mutations, such as the N279K mutation, result in a shift of the physiological 3R/4R ratio towards the more aggregation prone 4R isoform. Other mutations such as V337M cause a decrease in the in vitro affinity of tau to microtubules and a reduced ability to promote microtubule assembly. Whether both mutations address similar downstream signalling cascades remains unclear but is important for potential rescue strategies. Here, we developed a novel and optimised forward programming protocol for the rapid and highly efficient production of pure cultures of glutamatergic cortical neurons from hiPSCs. We apply this protocol to delineate mechanisms of neurodegeneration in an FTLD-tau hiPSC-model consisting of MAPTN279K- or MAPTV337M-mutants and wild-type or isogenic controls. The resulting cortical neurons express MAPT-genotype-dependent dominant proteome clusters regulating apoptosis, ROS homeostasis and mitochondrial function. Related pathways are significantly upregulated in MAPTN279K neurons but not in MAPTV337M neurons or controls. Live cell imaging demonstrates that both MAPT mutations affect excitability of membranes as reflected in spontaneous and stimulus evoked calcium signals when compared to controls, albeit more pronounced in MAPTN279K neurons. These spontaneous calcium oscillations in MAPTN279K neurons triggered mitochondrial hyperpolarisation and fission leading to mitochondrial ROS production, but also ROS production through NOX2 acting together to induce cell death. Importantly, we found that these mechanisms are MAPT mutation-specific and were observed in MAPTN279K neurons, but not in MAPTV337M neurons, supporting a pathological role of the 4R tau isoform in redox disbalance and highlighting MAPT-mutation specific clinicopathological-genetic correlations, which may inform rescue strategies in different MAPT mutations.

Summary: Background: De novo missense variants in KCNQ5, encoding the voltage-gated K+ channel KV7.5, have been described to cause developmental and epileptic encephalopathy (DEE) or intellectual disability (ID). We set out to identify disease-related KCNQ5 variants in genetic generalized epilepsy (GGE) and their underlying mechanisms. Methods: 1292 families with GGE were studied by next-generation sequencing. Whole-cell patch-clamp recordings, biotinylation and phospholipid overlay assays were performed in mammalian cells combined with homology modelling. Findings: We identified three deleterious heterozygous missense variants, one truncation and one splice site alteration in five independent families with GGE with predominant absence seizures; two variants were also associated with mild to moderate ID. All missense variants displayed a strongly decreased current density indicating a loss-of-function (LOF). When mutant channels were co-expressed with wild-type (WT) KV7.5 or KV7.5 and KV7.3 channels, three variants also revealed a significant dominant-negative effect on WT channels. Other gating parameters were unchanged. Biotinylation assays indicated a normal surface expression of the variants. The R359C variant altered PI(4,5)P2-interaction. Interpretation: Our study identified deleterious KCNQ5 variants in GGE, partially combined with mild to moderate ID. The disease mechanism is a LOF partially with dominant-negative effects through functional deficits. LOF of KV7.5 channels will reduce the M-current, likely resulting in increased excitability of KV7.5-expressing neurons. Further studies on network level are necessary to understand which circuits are affected and how this induces generalized seizures. Funding: DFG/FNR Research Unit FOR-2715 (Germany/Luxemburg), BMBF rare disease network Treat-ION (Germany), foundation ‘no epilep’ (Germany).

The number of N-Methyl-D-aspartate receptor (NMDAR) linked neurodegenerative diseases such as Alzheimer’s disease and dementia is constantly increasing. This is partly due to demographic change and presents new challenges to societies. To date, there are no effective treatment options. Current medications are nonselective and can lead to unwanted side effects in patients. A promising therapeutic approach is the targeted inhibition of NMDARs in the brain. NMDARs containing different subunits and splice variants display different physiological properties and play a crucial role in learning and memory, as well as in inflammatory or injury processes. They become overactivated during the course of the disease, leading to nerve cell death. Until now, there has been a lack of understanding of the general functions of the receptor and the mechanism of inhibition, which need to be understood in order to develop inhibitors. Ideal compounds should be highly targeted and even splice-variant-selective. However, a potent and splice-variant-selective NMDAR-targeting drug has yet to be developed. Recently developed 3-benzazepines are promising inhibitors for further drug development. The NMDAR splice variants GluN1-1b-4b carry a 21-amino-acid-long, flexible exon 5. Exon 5 lowers the NMDAR’s sensitivity to allosteric modulators by probably acting as an NMDAR modulator itself. The role of exon 5 in NMDAR modulation is still poorly understood. In this review, we summarize the structure and pharmacological relevance of tetrahydro-3-benzazepines.

Viral myocarditis is pathologically associated with RNA viruses such as coxsackievirus B3 (CVB3), or more recently, with SARS-CoV-2, but despite intensive research, clinically proven treatment is limited. Here, by use of a transgenic mouse strain (TG) containing a CVB3ΔVP0 genome we unravel virus-mediated cardiac pathophysiological processes in vivo and in vitro. Cardiac function, pathologic ECG alterations, calcium homeostasis, intracellular organization and gene expression were significantly altered in transgenic mice. A marked alteration of mitochondrial structure and gene expression indicates mitochondrial impairment potentially contributing to cardiac contractile dysfunction. An extended picture on viral myocarditis emerges that may help to develop new treatment strategies and to counter cardiac failure.

In voltage-gated potassium (KV) channels, the voltage-sensing domain (VSD) undergoes activation states to trigger pore opening via electro–mechanical (E–M) coupling. Here authors show that KV7.1 undergoes a two-stage E–M coupling mechanism during voltage-dependent activation.

Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates (‘organoids’) or 3D structures with less physiological relevance (‘spheroids’). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates. The resulting organoids possess a highly homogeneous morphology, size, global gene expression, cellular composition, and structure. They present significant features of the human midbrain and display spontaneous aggregate-wide synchronized neural activity. By automating the entire workflow from generation to analysis, we enhance the intra- and inter-batch reproducibility as demonstrated via RNA sequencing and quantitative whole mount high-content imaging. This allows assessing drug effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow.

Small RNA viruses only have a very limited coding capacity, thus most viral proteins have evolved to fulfill multiple functions. The highly conserved matrix protein 1 (M1) of influenza A viruses is a prime example for such a multifunctional protein, as it acts as a master regulator of virus replication whose different functions have to be tightly regulated. The underlying mechanisms, however, are still incompletely understood. Increasing evidence points towards an involvement of posttranslational modifications in the spatio-temporal regulation of M1 functions. Here, we analyzed the role of M1 tyrosine phosphorylation in genuine infection by using recombinant viruses expressing M1 phosphomutants. Presence of M1 Y132A led to significantly decreased viral replication compared to wildtype and M1 Y10F. Characterization of phosphorylation dynamics by mass spectrometry revealed the presence of Y132 phosphorylation in M1 incorporated into virions that is most likely mediated by membrane-associated Janus kinases late upon infection. Molecular dynamics simulations unraveled a potential phosphorylation-induced exposure of the positively charged linker domain between helices 4 and 5, supposably acting as interaction platform during viral assembly. Consistently, M1 Y132A showed a defect in lipid raft localization due to reduced interaction with viral HA protein resulting in a diminished structural stability of viral progeny and the formation of filamentous particles. Importantly, reduced M1-RNA binding affinity resulted in an inefficient viral genome incorporation and the production of non-infectious virions that interferes with virus pathogenicity in mice. This study advances our understanding of the importance of dynamic phosphorylation as a so far underestimated level of regulation of multifunctional viral proteins and emphasizes the potential feasibility of targeting posttranslational modifications of M1 as a novel antiviral intervention.

Background/Aims: The hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 contributes significantly to the generation of basic cardiac electrical activity in the sinus node and is a mediator of modulation by β–adrenergic stimulation. Heterologous expression of sick sinus syndrome (SSS) and bradycardia associated mutations within the human HCN4 gene results in altered channel function. The main aim was to describe the functional characterization of three (two novel and one known) missense mutations of HCN4 identified in families with SSS. Methods: Here, the two-electrode voltage clamp technique on Xenopus laevis oocytes and confocal imaging on transfected COS7 cells respectively, were used to analyze the functional effects of three HCN4 mutations; R378C, R550H, and E1193Q. Membrane surface expressions of wild type and the mutant channels were assessed by confocal microscopy, chemiluminescence assay, and Western blot in COS7 and HeLa cells. Results: The homomeric mutant channels R550H and E1193Q showed loss of function through increased rates of deactivation and distinctly reduced surface expression in all three homomeric mutant channels. HCN4 channels containing R550H and E1193Q mutant subunits only showed minor effects on the voltage dependence and rates of activation/deactivation. In contrast, homomeric R378C exerted a left-shifted activation curve and slowed activation kinetics. These effects were reduced in heteromeric co-expression of R378C with wild-type (WT) channels. Conclusion: Dysfunction of homomeric/heteromeric mutant HCN4-R378C, R550H, and E1193Q channels in the present study was primarily caused by loss of function due to decreased channel surface expression.

The T-box transcription factor eomesodermin (EOMES) acts both in endoderm specification as well as heart development, suggesting context-specific function. Here, the authors show that dose-controlled EOMES induction is sufficient for cardiogenic programming of human pluripotent stem cells.

Background/Aims: TASK channels belong to the two-pore-domain potassium (K2P) channel family. TASK-1 is discussed to contribute to chronic atrial fibrillation (AFib) and has been together with uncoupling protein 1 found as a marker protein of brown adipose tissue (BAT) fat. In addition, TASK-1 was linked in a genome-wide association study to an increased body mass index. A recent study showed that TASK-1 inhibition is causing obesity in mice by a BAT whitening and that these effects are linked to the mineralocorticoid receptor pathway, albeit the mechanism remained elusive. Therefore, we aimed to probe whether K2P channels are regulated by serum- and glucocorticoid-inducible kinases (SGKs) which are known to modify many cellular functions by modulating ion channels. Methods: To this end we used functional co-expression studies and chemiluminescence-assays in Xenopus oocytes, together with fluorescence imaging and quantitative PCR experiments. Results: SGKs and proteinkinase B (PKB) induced a strong, dose- and time-dependent current reduction of TASK-1 and TASK-3. SGK co-expression reduced the surface expression of TASK-1/3, leading to a predominant localization of the channels into late endosomes. The down regulation of TASK-3 channels was abrogated by the dynamin inhibitor dynasore, confirming a role of SGKs in TASK-1/3 channel endocytosis. Conclusion: Stress-mediated changes in SGK expression pattern or activation is likely to alter TASK-1/3 expression at the surface membrane. The observed TASK-1 regulation might contribute to the pathogenesis of chronic AFib and provide a mechanistic link between increased mineralocorticoid levels and TASK-1 reduction, both linked to BAT whitening.

Coxsackievirus B3 (CVB3) belongs to the enteroviruses, which are a well-known cause of acute and chronic myocarditis, primarily infecting cardiac myocytes. As primary human cardiomyocytes are difficult to obtain, viral myocarditis is quite frequently studied in vitro in different non-cardiac and cardiac-like cell lines. Recently, cardiomyocytes that have been differentiated from human-induced pluripotent stem cells have been described as a new model system to study CVB3 infection. Here, we compared iCell® Cardiomyocytes with other cell lines that are commonly used to study CVB3 infection regarding their susceptibility and patterns of infection and the mode of cell death. iCell® Cardiomyocytes, HeLa cells, HL-1 cells and H9c2 cells were infected with CVB3 (Nancy strain). The viral load, CVB3 RNA genome localization, VP1 expression (including the intracellular localization), cellular morphology and the expression of cell death markers were compared. The various cell lines clearly differed in their permissiveness to CVB3 infection, patterns of infection, viral load, and mode of cell death. When studying the mode of cell death of CVB3-infected iCell® Cardiomyocytes in more detail, especially regarding the necroptosis key players RIPK1 and RIPK3, we found that RIPK1 is cleaved during CVB3 infection. iCell® Cardiomyocytes represent well the natural host of CVB3 in the heart and are thus the most appropriate model system to study molecular mechanisms of CVB3-induced myocarditis in vitro. Doubts are raised about the suitability of commonly used cell lines such as HeLa cells, HL-1 cells and H9c2 cells to evaluate molecular pathways and processes occurring in vivo in enteroviral myocarditis.

Evidence is emerging that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect various organs of the body, including cardiomyocytes and cardiac endothelial cells in the heart. This review focuses on the effects of SARS-CoV-2 in the heart after direct infection that can lead to myocarditis and an outline of potential treatment options. The main points are: (1) Viral entry: SARS-CoV-2 uses specific receptors and proteases for docking and priming in cardiac cells. Thus, different receptors or protease inhibitors might be effective in SARS-CoV-2-infected cardiac cells. (2) Viral replication: SARS-CoV-2 uses RNA-dependent RNA polymerase for replication. Drugs acting against ssRNA(+) viral replication for cardiac cells can be effective. (3) Autophagy and double-membrane vesicles: SARS-CoV-2 manipulates autophagy to inhibit viral clearance and promote SARS-CoV-2 replication by creating double-membrane vesicles as replication sites. (4) Immune response: Host immune response is manipulated to evade host cell attacks against SARS-CoV-2 and increased inflammation by dysregulating immune cells. Efficiency of immunosuppressive therapy must be elucidated. (5) Programmed cell death: SARS-CoV-2 inhibits programmed cell death in early stages and induces apoptosis, necroptosis, and pyroptosis in later stages. (6) Energy metabolism: SARS-CoV-2 infection leads to disturbed energy metabolism that in turn leads to a decrease in ATP production and ROS production. (7) Viroporins: SARS-CoV-2 creates viroporins that lead to an imbalance of ion homeostasis. This causes apoptosis, altered action potential, and arrhythmia.

Background/Aims: Acquired as well as inherited channelopathies are disorders that are caused by altered ion channel function. A family of channels whose malfunction is associated with different channelopathies is the Kv7 K+ channel family; and restoration of normal Kv7 channel function by small molecule modulators is a promising approach for treatment of these often fatal diseases. Methods: Here, we show the modulation of Kv7 channels by the natural compound Rottlerin heterologously expressed in Xenopus laevis oocytes and on iPSC cardiomyocytes overexpressing Kv7.1 channels. Results: We show that currents carried by Kv7.1 (EC50 = 1.48 μM), Kv7.1/KCNE1 (EC50 = 4.9 μM), and Kv7.4 (EC50 = 0.148 μM) are strongly enhanced by the compound, whereas Kv7.2, Kv7.2/Kv7.3, and Kv7.5 are not sensitive to Rottlerin. Studies on Kv7.1/KCNE1 mutants and in silico modelling indicate that Rottlerin binds to the R-L3-activator site. Rottlerin mediated activation of Kv7.1/KCNE1 channels might be a promising approach in long QT syndrome. As a proof of concept, we show that Rottlerin shortens cardiac repolarisation in iPSC-derived cardiomyocytes expressing Kv7.1.Conclusion: Rottlerin or an optimized derivative holds a potential as QT interval correcting drug.

Cardiac induction of human embryonic stem cells (hESCs) is a process bearing increasing medical relevance, yet it is poorly understood from a developmental biology perspective. Anticipated technological progress in deriving stably expandable cardiac precursor cells or in advancing cardiac subtype specification protocols will likely require deeper insights into this fascinating system. Recent improvements in controlling hESC differentiation now enable a near-homogeneous induction of the cardiac lineage. This is based on an optimized initial stimulation of mesoderm-inducing signaling pathways such as Activin and/or FGF, BMP, and WNT, followed by WNT inhibition as a secondary requirement. Here, we describe a comprehensive data set based on varying hESC differentiation conditions in a systematic manner and recording high-resolution differentiation time-courses analyzed by genome-wide expression profiling (GEO accession number GSE67154). As a baseline, hESCs were differentiated into cardiomyocytes under optimal conditions. Moreover, in additional time-series, individual signaling factors were withdrawn from the initial stimulation cocktail to reveal their specific roles via comparison to the standard condition. Hence, this data set presents a rich resource for hypothesis generation in studying human cardiac induction, as we reveal numbers of known as well as uncharacterized genes prominently marking distinct intermediate stages in the process. These data will also be useful for identifying putative cardiac master regulators in the human system as well as for characterizing expandable cardiac stem cells.

Specificity of inhibitors of voltage-gated ion channels is crucial for their use as therapeutics. Here, the authors show that adamantane derivatives interact with a specific binding site on fenestrations that only become available when accessory subunits are bound to the channel.

Background/Aims: Inherited, autosomal dominant spinocerebellar ataxia type 11 (SCA11) is caused by loss of function mutations of TTBK2 (tau tubulin kinase 2). Mutations observed in patients with SCA11 include truncated TTBK2(450). The present study explored the possibility that TTBK2 influences the function of the glutamate receptor GluK2. Methods: GluK2 was expressed in Xenopus oocytes without and with additional expression of wild type TTBK2, the truncated mutant TTBK2(450), or the kinase dead mutants TTBK2(KD) and TTBK2(450/KD). GluK2 current was determined by dual electrode voltage clamp and GluK2 protein abundance in the cell membrane utilizing confocal microscopy. Results: Glutamate exposure of GluK2 expressing oocytes generated a current, which was significantly lower in oocytes expressing GluK2 together with TTBK2 wt or TTBK2(KD) than in oocytes expressing GluK2 alone or together with either TTBK2(450) or TTBK2(450/KD). According to confocal microscopy of EGFP-tagged GluK2, TTBK2 wt decreased the GluK2 protein abundance in the cell membrane. Overexpression of an inactive RAB5(N133I) mutant but not RAB5wt could reverse the TTBK2 effect on GluK2 suggesting that RAB5 function is required for the effect. Conclusions: TTBK2 down-regulates GluK2 activity by decreasing the receptor protein abundance in the cell membrane via RAB5-dependent endocytosis, an effect that may protect against neuroexcitotoxicity.

Loss-of-function mutations in the PITX2 transcription factor gene have been shown to cause familial atrial fibrillation (AF). To potentially model aspects of AF and unravel PITX2-regulated downstream genes for drug target discovery, we here report the generation of integration-free PITX2-deficient hiPS cell lines. We also show that both PITX2 knockout hiPS cells and isogenic wild-type controls can selectively be differentiated into human atrial cardiomyocytes, to potentially uncover differentially expressed gene sets between these groups.

N-Methyl-D-aspartate (NMDA) receptors (NMDARs) are among the most important excitatory neurotransmitter receptors in the human brain. Autoantibodies to the human NMDAR cause the most frequent form of autoimmune encephalitis involving autoantibody-mediated receptor cross-linking and subsequent internalization of the antibody-receptor complex. This has been deemed to represent the predominant antibody effector mechanism depleting the NMDAR from the synaptic and extra-synaptic neuronal cell membrane. To assess in detail the molecular mechanisms of autoantibody-induced NMDAR endocytosis, vesicular trafficking, and exocytosis we transiently co-expressed rat GluN1-1a-EGFP and GluN2B-ECFP alone or together with scaffolding postsynaptic density protein 95 (PSD-95), wild-type (WT), or dominant-negative (DN) mutant Ras-related in brain (RAB) proteins (RAB5WT, RAB5DN, RAB11WT, RAB11DN) in HEK 293T cells. The cells were incubated with a pH-rhodamine-labeled human recombinant monoclonal GluN1 IgG1 autoantibody (GluN1-aAbpH−rhod) genetically engineered from clonally expanded intrathecal plasma cells from a patient with anti-NMDAR encephalitis, and the pH-rhodamine fluorescence was tracked over time. We show that due to the acidic luminal pH, internalization of the NMDAR-autoantibody complex into endosomes and lysosomes increases the pH-rhodamine fluorescence. The increase in fluorescence allows for mechanistic assessment of endocytosis, vesicular trafficking in these vesicular compartments, and exocytosis of the NMDAR-autoantibody complex under steady state conditions. Using this method, we demonstrate a role for PSD-95 in stabilization of NMDARs in the cell membrane in the presence of GluN1-aAbpH−rhod, while RAB proteins did not exert a significant effect on vertical trafficking of the internalized NMDAR autoantibody complex in this heterologous expression system. This novel assay allows to unravel molecular mechanisms of autoantibody-induced receptor internalization and to study novel small-scale specific molecular-based therapies for autoimmune encephalitis syndromes.

Cardiomyocyte-like cells (CMs) derived from human pluripotent stem cells (hPSCs) present a valuable model for human disease modeling, studying early human development and, potentially, developing cell therapeutic approaches. However, the specification of early hPSC-derived CMs into defined cardiac subtypes such as atrial and ventricular cells is not well understood and, thus, poorly controlled. Moreover, the maturation status of hPSC-CMs is not well defined, yet it is known that these cells undergo at least some degree of maturation upon longer term in vitro culture. To gain insight into this process, and to assess their developmental status, we have recently generated a data set of hPSC-CMs monitoring global changes in gene expression upon long term maintenance in vitro, in comparison to human atrial and ventricular heart samples (GEO accession number GEO: GSE64189). These data present a rich resource for evaluating the maturation status of hPSC-CMs, for identifying suitable markers for subtype-specific gene expression, as well as for the generation of functional hypotheses. Here, we provide additional details and quality checks of this data set, and exemplify how it can be used to identify maturation-associated as well as cardiac subtype-specific markers.

The fight-or-flight response (FFR), a physiological acute stress reaction, involves positive chronotropic and inotropic effects on heart muscle cells mediated through β-adrenoceptor activation. Increased systolic calcium is required to enable stronger heart contractions whereas elevated potassium currents are to limit the duration of the action potentials and prevent arrhythmia. The latter effect is accomplished by an increased functional activity of the Kv7.1 channel encoded by KCNQ1. Current knowledge, however, does not sufficiently explain the full extent of rapid Kv7.1 activation and may hence be incomplete. Using inducible genetic KCNQ1 complementation in KCNQ1-deficient human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we here reinvestigate the functional role of Kv7.1 in adapting human CMs to adrenergic stress. Under baseline conditions, Kv7.1 was barely detectable at the plasma membrane of hiPSC-CMs, yet it fully protected these from adrenergic stress-induced beat-to-beat variability of repolarization and torsade des pointes-like arrhythmia. Furthermore, isoprenaline treatment increased field potential durations specifically in KCNQ1-deficient CMs to cause these adverse macroscopic effects. Mechanistically, we find that the protective action by Kv7.1 resides in a rapid translocation of channel proteins from intracellular stores to the plasma membrane, induced by adrenergic signaling. Gene silencing experiments targeting RAB GTPases, mediators of intracellular vesicle trafficking, showed that fast Kv7.1 recycling under acute stress conditions is RAB4A-dependent.Our data reveal a key mechanism underlying the rapid adaptation of human cardiomyocytes to adrenergic stress. These findings moreover aid to the understanding of disease pathology in long QT syndrome and bear important implications for safety pharmacological screening.

The ultrarapid delayed rectifier K+ current (IKur), mediated by Kv1.5 channels, constitutes a key component of the atrial action potential. Functional mutations in the underlying KCNA5 gene have been shown to cause hereditary forms of atrial fibrillation (AF). Here, we combine targeted genetic engineering with cardiac subtype-specific differentiation of human induced pluripotent stem cells (hiPSCs) to explore the role of Kv1.5 in atrial hiPSC-cardiomyocytes. CRISPR/Cas9-mediated mutagenesis of integration-free hiPSCs was employed to generate a functional KCNA5 knockout. This model as well as isogenic wild-type control hiPSCs could selectively be differentiated into ventricular or atrial cardiomyocytes at high efficiency, based on the specific manipulation of retinoic acid signaling. Investigation of electrophysiological properties in Kv1.5-deficient cardiomyocytes compared to isogenic controls revealed a strictly atrial-specific disease phentoype, characterized by cardiac subtype-specific field and action potential prolongation and loss of 4-aminopyridine sensitivity. Atrial Kv1.5-deficient cardiomyocytes did not show signs of arrhythmia under adrenergic stress conditions or upon inhibiting additional types of K+ current. Exposure of bulk cultures to carbachol lowered beating frequencies and promoted chaotic spontaneous beating in a stochastic manner. Low-frequency, electrical stimulation in single cells caused atrial and mutant-specific early afterdepolarizations, linking the loss of KCNA5 function to a putative trigger mechanism in familial AF. These results clarify for the first time the role of Kv1.5 in atrial hiPSC-cardiomyocytes and demonstrate the feasibility of cardiac subtype-specific disease modeling using engineered hiPSCs.

Background/Aims: Potassium channels are tetrameric proteins providing potassium selective passage through lipid embedded proteinaceous pores with highest fidelity. The selectivity results from binding to discrete potassium binding sites and stabilization of a hydrated potassium ion in a central internal cavity. The four potassium binding sites, generated by the conserved TTxGYGD signature sequence are formed by the backbone carbonyls of the amino acids TXGYG. Residues KV1.5-Val481, KV4.3-Leu368 and KV7.1- Ile 313 represent the amino acids in the X position of the respective channels. Methods: Here, we study the impact of these residues on ion selectivity, permeation and inactivation kinetics as well as the modulation by β-subunits using site-specific mutagenesis, electrophysiological analyses and molecular dynamics simulations. Results: We identify this position as key in modulation of slow inactivation by structurally dissimilar β-subunits in different KV channels. Conclusion: We propose a model in which structural changes accompanying activation and β-subunit modulation allosterically constrain the backbone carbonyl oxygen atoms via the side chain of the respective X-residue in the signature sequence to reduce conductance during slow inactivation.

IntroductionCoxsackievirus B3 (CVB3) is known to induce acute and chronic myocarditis. Most infections are clinically unapparent but some patients suffer from ventricular arrhythmias (VA) and sudden cardiac death (SCD). Studies showed that acute CVB3 infection may cause impaired function of cardiac ion channels, creating a proarrhythmic substrate. However, it is unknown whether low level CVB3+ expression in myocytes may cause altered cardiac electrophysiology leading to VA.MethodsCellular electrophysiology was used to analyze cellular action potentials (APs) and occurrence of afterdepolarizations from isolated cardiomyocytes of wildtype (WT) and transgenic CVB3ΔVP0 (CVB3+) mice. Further, we studied surface ECGs, monophasic APs, ventricular effective refractory period (VERP) and inducibility of VAs in Langendorff-perfused whole hearts. All used cardiomyocytes and whole hearts originated from male mice.ResultsCellular action potential duration (APD) in WT and CVB3+ myocytes was unchanged. No difference in mean occurrence or amplitude of afterdepolarizations in WT and CVB3+ myocytes was found. Interestingly, resting membrane potential in CVB3+ myocytes was significantly hyperpolarized (WT: -90.0±2.2 mV, n = 7; CVB3+: -114.1±3.0 mV, n = 14; pConclusionIn CVB3+ mice, prolonged ventricular refractoriness and hyperpolarized resting membrane potentials in presence of unchanged APD were observed, suggesting that low level CVB3 expression does not promote VA by altered cardiac electrophysiology in this type of chronic myocarditis. These findings may suggest that other mechanisms such as chronic myocardial inflammation or fibrosis may account for arrhythmias observed in patients with chronic enteroviral myocarditis.

The scaling relationship between the size of an appendage or organ and that of the body as a whole is tightly regulated during animal development. If a structure grows at a different rate than the rest of the body, this process is termed allometric growth. The zebrafish another longfin (alf) mutant shows allometric growth resulting in proportionally enlarged fins and barbels. We took advantage of this mutant to study the regulation of size in vertebrates. Here, we show that alf mutants carry gain-of-function mutations in kcnk5b, a gene encoding a two-pore domain potassium (K(+)) channel. Electrophysiological analysis in Xenopus oocytes reveals that these mutations cause an increase in K(+) conductance of the channel and lead to hyperpolarization of the cell. Further, somatic transgenesis experiments indicate that kcnk5b acts locally within the mesenchyme of fins and barbels to specify appendage size. Finally, we show that the channel requires the ability to conduct K(+) ions to increase the size of these structures. Our results provide evidence for a role of bioelectric signaling through K(+) channels in the regulation of allometric scaling and coordination of growth in the zebrafish.

In the voltage-sensitive phosphatase Ci-VSP, conformational changes in the transmembrane voltage sensor domain (VSD) are transduced to the intracellular catalytic domain (CD) leading to its dephosphorylation activity against membrane-embedded phosphoinositides. The linker between both domains is proposed to be crucial for the VSD-CD coupling. With a combined approach of electrophysiological measurements on Xenopus oocytes and molecular dynamics simulations of a Ci-VSP model embedded in a lipid bilayer, we analyzed how conformational changes in the linker mediate the interaction between the CD and the activated VSD. In this way, we identified specific residues in the linker that interact with well-defined amino acids in one of the three loops forming the active site of the protein, named TI loop. With our results, we shed light into the early steps of the coupling process between the VSD and the CD, which are based on fine-tuned electrostatic and hydrophobic interactions between the linker, the membrane and the CD.

The multifunctional protein ß-catenin governs as transcription factor the expression of a wide variety of genes relevant for cell proliferation and cell survival. In addition, ß-catenin is localized at the cell membrane and may influence the function of channels. The present study explored the possibility that ß-catenin participates in the regulation of the HERG K(+) channel. To this end, HERG was expressed in Xenopus oocytes with or without ß-catenin and the voltage-gated current determined utilizing the dual electrode voltage clamp. As a result, expression of ß-catenin markedly upregulated HERG channel activity, an effect not sensitive to inhibition of transcription with actinomycin D (10 µM). According to chemiluminescence, ß-catenin may increase HERG channel abundance within the oocyte cell membrane. Following inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) the decay of current was similar in oocytes expressing HERG together with ß-catenin to oocytes expressing HERG alone. The experiments uncover a novel function of APC/ß-catenin, i.e. the regulation of HERG channels.

We previously showed that the serum- and glucocorticoid-inducible kinase 3 (SGK3) increases the AMPA-type glutamate receptor GluA1 protein in the plasma membrane. The activation of AMPA receptors by NMDA-type glutamate receptors eventually leads to postsynaptic neuronal plasticity. Here, we show that SGK3 mRNA is upregulated in the hippocampus of new-born wild type Wistar rats after NMDA receptor activation. We further demonstrate in the Xenopus oocyte expression system that delivery of GluA1 protein to the plasma membrane depends on the small GTPase RAB11. This RAB-dependent GluA1 trafficking requires phosphorylation and activation of phosphoinositol-3-phosphate-5-kinase (PIKfyve) and the generation of PI(3,5)P(2). In line with this mechanism we could show PIKfyve mRNA expression in the hippocampus of wild type C57/BL6 mice and phosphorylation of PIKfyve by SGK3. Incubation of hippocampal slices with the PIKfyve inhibitor YM201636 revealed reduced CA1 basal synaptic activity. Furthermore, treatment of primary hippocampal neurons with YM201636 altered the GluA1 expression pattern towards reduced synaptic expression of GluA1. Our findings demonstrate for the first time an involvement of PIKfyve and PI(3,5)P(2) in NMDA receptor-triggered synaptic GluA1 trafficking. This new regulatory pathway of GluA1 may contribute to synaptic plasticity and memory.