The M2 proteins of bat influenza A viruses reveal atypical features compared to conventional M2 proteins.

The influenza A virus (IAV) M2 protein has proton channel activity, which plays a role in virus uncoating and may help to preserve the metastable conformation of the IAV hemagglutinin (HA). In contrast to the highly conserved M2 proteins of conventional IAV, the primary sequences of bat IAV H17N10 and H18N11 M2 proteins show remarkable divergence, suggesting that these proteins may differ in their biological function. We, therefore, assessed the proton channel activity of bat IAV M2 proteins and investigated its role in virus replication. Here, we show that the M2 proteins of bat IAV did not fully protect acid-sensitive HA of classical IAV from low pH-induced conformational change, indicating low proton channel activity. Interestingly, the N31S substitution not only rendered bat IAV M2 proteins sensitive to inhibition by amantadine but also preserved the metastable conformation of acid-sensitive HA to a greater extent. In contrast, the acid-stable HA of H18N11 did not rely on such support by M2 protein. When mutant M2(N31S) protein was expressed in the context of chimeric H18N11/H5N1(6:2) encoding HA and NA of avian IAV H5N1, amantadine significantly inhibited virus entry, suggesting that ion channel activity supported virus uncoating. Finally, the cytoplasmic domain of the H18N11 M2 protein mediated rapid internalization of the protein from the plasma membrane leading to low-level expression at the cell surface. However, cell surface levels of H18N11 M2 protein were significantly enhanced in cells infected with the chimeric H18N11/H5N1(6:2) virus. The potential role of the N1 sialidase in arresting M2 internalization is discussed. IMPORTANCE Bat IAV M2 proteins not only differ from the homologous proteins of classical IAV by their divergent primary sequence but are also unable to preserve the metastable conformation of acid-sensitive HA, indicating low proton channel activity. This unusual feature may help to avoid M2-mediated cytotoxic effects and inflammation in bats infected with H17N10 or H18N11. Unlike classical M2 proteins, bat IAV M2 proteins with the N31S substitution mediated increased protection of HA from acid-induced conformational change. This remarkable gain of function may help to understand how single point mutations can modulate proton channel activity. In addition, the cytoplasmic domain was found to be responsible for the low cell surface expression level of bat IAV M2 proteins. Given that the M2 cytoplasmic domain of conventional IAV is well known to participate in virus assembly at the plasma membrane, this atypical feature might have consequences for bat IAV budding and egress.

A detailed analysis of single-channel Nav 1.5 recordings does not reveal any cooperative gating.

Cardiac voltage-gated sodium (Na+ ) channels (Nav 1.5) are crucial for myocardial electrical excitation. Recent studies based on single-channel recordings have suggested that Na+ channels interact functionally and exhibit coupled gating. However, the analysis of such recordings frequently relies on manual interventions, which can lead to bias. Here, we developed an automated pipeline to de-trend and idealize single-channel currents, and assessed possible functional interactions in cell-attached patch clamp experiments in HEK293 cells expressing human Nav 1.5 channels as well as in adult mouse and rabbit ventricular cardiomyocytes. Our pipeline involved de-trending individual sweeps by linear optimization using a library of predefined functions, followed by digital filtering and baseline offset. Subsequently, the processed sweeps were idealized based on the idea that the ensemble average of the idealized current identified by thresholds between current levels reconstructs at best the ensemble average current from the de-trended sweeps. This reconstruction was achieved by non-linear optimization. To ascertain functional interactions, we examined the distribution of the numbers of open channels at every time point during the activation protocol and compared it to the distribution expected for independent channels. We also examined whether the channels tended to synchronize their openings and closings. However, we did not uncover any solid evidence of such interactions in our recordings. Rather, our results indicate that wild-type Nav 1.5 channels are independent entities or exhibit only very weak functional interactions that are probably irrelevant under physiological conditions. Nevertheless, our unbiased analysis will be important for further studies examining whether auxiliary proteins potentiate functional Na+ channel interactions. KEY POINTS: Nav 1.5 channels are critical for cardiac excitation. They are part of macromolecular interacting complexes, and it was previously suggested that two neighbouring channels may functionally interact and exhibit coupled gating. Manual interventions when processing single-channel recordings can lead to bias and inaccurate data interpretation. We developed an automated pipeline to de-trend and idealize single-channel currents and assessed possible functional interactions between Nav 1.5 channels in HEK293 cells and cardiomyocytes during activation protocols using the cell-attached patch clamp technique. In recordings consisting of up to 1000 sweeps from the same patch, our analysis did not reveal any evidence of functional interactions or coupled gating between wild-type Nav 1.5 channels. Our unbiased analysis may be useful in further studies examining how Na+ channel interactions are affected by mutations and auxiliary proteins.

Multiomics uncover the proinflammatory role of Trpm4 deletion after myocardial infarction in mice.

Upon myocardial infarction (MI), ischemia-induced cell death triggers an inflammatory response responsible for removing necrotic material and inducing tissue repair. TRPM4 is a Ca2+-activated ion channel permeable to monovalent cations. Although its role in cardiomyocyte-driven hypertrophy and arrhythmia post-MI has been established, no study has yet investigated its role in the inflammatory process orchestrated by endothelial cells, immune cells, and fibroblasts. This study aims to assess the role of TRPM4 in 1) survival and cardiac function, 2) inflammation, and 3) healing post-MI. We performed ligation of the left coronary artery or sham intervention on 154 Trpm4 WT or KO mice under isoflurane anesthesia. Survival and echocardiographic functions were monitored up to 5 wk. We collected serum during the acute post-MI phase to analyze proteomes and performed single-cell RNA sequencing on nonmyocytic cells of hearts after 24 and 72 h. Lastly, we assessed chronic fibrosis and angiogenesis. We observed no significant differences in survival or cardiac function, even though our proteomics data showed significantly decreased tissue injury markers (i.e., creatine kinase M and VE-cadherin) in KO serum after 12 h. On the other hand, inflammation, characterized by serum amyloid P component in the serum, higher number of recruited granulocytes, inflammatory monocytes, and macrophages, as well as expression of proinflammatory genes, was significantly higher in KO. This correlated with increased chronic cardiac fibrosis and angiogenesis. Since inflammation and fibrosis are closely linked to adverse remodeling, future therapeutic attempts at inhibiting TRPM4 will need to assess these parameters carefully before proceeding with translational studies.NEW & NOTEWORTHY Deletion of Trpm4 increases markers of cardiac and systemic inflammation within the first 24 h after MI, while inducing an earlier fibrotic transition at 72 h and more overall chronic fibrosis and angiogenesis at 5 wk. The descriptive, robust, and methodologically broad approach of this study sheds light on an important caveat that will need to be taken into account in all future therapeutic attempts to inhibit TRPM4 post-MI.

Targeting Ion Channel TRPM4.

The transient receptor potential melastatin 4 (TRPM4) ion channel is ubiquitously expressed. Dysregulation and/or functional mutations of TRPM4 lead to several diseases. Within our studies, we screened for TRPM4 inhibitors and identified small molecules that block TRPM4 in the low µM range. Furthermore, we investigated the pathophysiology of TRPM4 in cardiac conditions, immune diseases and cancer using these novel inhibitors, molecular biology techniques and functional assays.

Deletion of Trpm4 Alters the Function of the Nav1.5 Channel in Murine Cardiac Myocytes.

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca2+-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na+ currents in myocytes. Hearts from Trpm4-/- mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na+ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na+ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Nav1.5 function in murine cardiac myocytes.

Functional characterization of a novel SCN5A variant associated with long QT syndrome and sudden cardiac death.

Sudden arrhythmic death syndrome (SADS) in young individuals is a devastating and tragic event often caused by an undiagnosed inherited cardiac disease. Although post-mortem genetic testing represents a promising tool to elucidate potential disease-causing mechanisms in such autopsy-negative death cases, a variant interpretation is still challenging, and functional consequences of identified sequence alterations often remain unclear. Recently, we have identified a novel heterozygous missense variant (N1774H) in the Nav1.5 channel-encoding gene SCN5A in a 19-year-old female SADS victim. The aim of this study was to perform a co-segregation analysis in family members of the index case and to evaluate the functional consequences of this SCN5A variant. Functional characterization of the SCN5A N1774H variant was performed using patch-clamp techniques in TsA-201 cell line transiently expressing either wild-type or variant Nav1.5 channels. Electrophysiological analyses revealed that variant Nav1.5 channels show a loss-of-function in the peak current densities, but an increased late current compared to the wild-type channels, which could lead to both, loss- and gain-of-function respectively. Furthermore, clinical assessment and genetic testing of the relatives of the index case showed that all N1774H mutation carriers have prolonged QT intervals. The identification of several genotype and phenotype positive family members and the functional implication of the SCN5A N1774H variant support the evidence of the in silico predicted pathogenicity of the here reported sequence alteration.

Functional Consequences of the SCN5A-p.Y1977N Mutation within the PY Ubiquitylation Motif: Discrepancy between HEK293 Cells and Transgenic Mice.

Dysfunction of the cardiac sodium channel Nav1.5 (encoded by the SCN5A gene) is associated with arrhythmias and sudden cardiac death. SCN5A mutations associated with long QT syndrome type 3 (LQT3) lead to enhanced late sodium current and consequent action potential (AP) prolongation. Internalization and degradation of Nav1.5 is regulated by ubiquitylation, a post-translational mechanism that involves binding of the ubiquitin ligase Nedd4-2 to a proline-proline-serine-tyrosine sequence of Nav1.5, designated the PY-motif. We investigated the biophysical properties of the LQT3-associated SCN5A-p.Y1977N mutation located in the Nav1.5 PY-motif, both in HEK293 cells as well as in newly generated mice harboring the mouse homolog mutation Scn5a-p.Y1981N. We found that in HEK293 cells, the SCN5A-p.Y1977N mutation abolished the interaction between Nav1.5 and Nedd4-2, suppressed PY-motif-dependent ubiquitylation of Nav1.5, and consequently abrogated Nedd4-2 induced sodium current (INa) decrease. Nevertheless, homozygous mice harboring the Scn5a-p.Y1981N mutation showed no electrophysiological alterations nor changes in AP or (late) INa properties, questioning the in vivo relevance of the PY-motif. Our findings suggest the presence of compensatory mechanisms, with additional, as yet unknown, factors likely required to reduce the "ubiquitylation reserve" of Nav1.5. Future identification of such modulatory factors may identify potential triggers for arrhythmias and sudden cardiac death in the setting of LQT3 mutations.

Generation of an alveolar epithelial type II cell line from induced pluripotent stem cells.

Differentiation of primary alveolar type II epithelial cells (AEC II) to AEC type I in culture is a major barrier in the study of the alveolar epithelium in vitro. The establishment of an AEC II cell line derived from induced pluripotent stem cells (iPSC) represents a novel opportunity to study alveolar epithelial cell biology, for instance, in the context of lung injury, fibrosis, and repair. In the present study, we generated long-lasting AEC II from iPSC (LL-iPSC-AEC II). LL-iPSC-AEC II displayed morphological characteristics of AEC II, including growth in a cobblestone monolayer, the presence of lamellar bodies, and microvilli, as shown by electron microscopy. Also, LL-iPSC-AEC II expressed AEC type II proteins, such as cytokeratin, surfactant protein C, and LysoTracker DND 26 (a marker for lamellar bodies). Furthermore, the LL-iPSC-AEC II exhibited functional properties of AEC II by an increase of transepithelial electrical resistance over time, secretion of inflammatory mediators in biologically relevant quantities (IL-6 and IL-8), and efficient in vitro alveolar epithelial wound repair. Consistent with the AEC II phenotype, the cell line showed the ability to uptake and release surfactant protein B, to secrete phospholipids, and to differentiate into AEC type I. In summary, we established a long-lasting, but finite AEC type II cell line derived from iPSC as a novel cellular model to study alveolar epithelial cell biology in lung health and disease.

Lateral Membrane-Specific MAGUK CASK Down-Regulates NaV1.5 Channel in Cardiac Myocytes.

RATIONALE: Mechanisms underlying membrane protein localization are crucial in the proper function of cardiac myocytes. The main cardiac sodium channel, NaV1.5, carries the sodium current (INa) that provides a rapid depolarizing current during the upstroke of the action potential. Although enriched in the intercalated disc, NaV1.5 is present in different membrane domains in myocytes and interacts with several partners. OBJECTIVE: To test the hypothesis that the MAGUK (membrane-associated guanylate kinase) protein CASK (calcium/calmodulin-dependent serine protein kinase) interacts with and regulates NaV1.5 in cardiac myocytes. METHODS AND RESULTS: Immunostaining experiments showed that CASK localizes at lateral membranes of cardiac myocytes, in association with dystrophin. Whole-cell patch clamp showed that CASK-silencing increases INa in vitro. In vivo CASK knockdown similarly increased INa recorded in freshly isolated myocytes. Pull-down experiments revealed that CASK directly interacts with the C-terminus of NaV1.5. CASK silencing reduces syntrophin expression without affecting NaV1.5 and dystrophin expression levels. Total Internal Reflection Fluorescence microscopy and biotinylation assays showed that CASK silencing increased the surface expression of NaV1.5 without changing mRNA levels. Quantification of NaV1.5 expression at the lateral membrane and intercalated disc revealed that the lateral membrane pool only was increased upon CASK silencing. The protein transport inhibitor brefeldin-A prevented INa increase in CASK-silenced myocytes. During atrial dilation/remodeling, CASK expression was reduced but its localization remained unchanged. CONCLUSION: This study constitutes the first description of an unconventional MAGUK protein, CASK, which directly interacts with NaV1.5 channel and controls its surface expression at the lateral membrane by regulating ion channel trafficking.

Annotation of functional impact of voltage-gated sodium channel mutations.

Voltage-gated sodium channels are pore-forming transmembrane proteins that selectively allow sodium ions to flow across the plasma membrane according to the electro-chemical gradient thus mediating the rising phase of action potentials in excitable cells and playing key roles in physiological processes such as neurotransmission, skeletal muscle contraction, heart rhythm, and pain sensation. Genetic variations in the nine human genes encoding these channels are known to cause a large range of diseases affecting the nervous and cardiac systems. Understanding the molecular effect of genetic variations is critical for elucidating the pathologic mechanisms of known variations and in predicting the effect of newly discovered ones. To this end, we have created a Web-based tool, the Ion Channels Variants Portal, which compiles all variants characterized functionally in the human sodium channel genes. This portal describes 672 variants each associated with at least one molecular or clinical phenotypic impact, for a total of 4,658 observations extracted from 264 different research articles. These data were captured as structured annotations using standardized vocabularies and ontologies, such as the Gene Ontology and the Ion Channel ElectroPhysiology Ontology. All these data are available to the scientific community via neXtProt at https://www.nextprot.org/portals/navmut.

Cardiac voltage-gated calcium channel macromolecular complexes.

Over the past 20 years, a new field of research, called channelopathies, investigating diseases caused by ion channel dysfunction has emerged. Cardiac ion channels play an essential role in the generation of the cardiac action potential. Investigators have largely determined the physiological roles of different cardiac ion channels, but little is known about the molecular determinants of their regulation. The voltage-gated calcium channel Ca(v)1.2 shapes the plateau phase of the cardiac action potential and allows the influx of calcium leading to cardiomyocyte contraction. Studies suggest that the regulation of Ca(v)1.2 channels is not uniform in working cardiomyocytes. The notion of micro-domains containing Ca(v)1.2 channels and different calcium channel interacting proteins, called macro-molecular complex, has been proposed to explain these observations. The objective of this review is to summarize the currently known information on the Ca(v)1.2 macromolecular complexes in the cardiac cell and discuss their implication in cardiac function and disorder. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Ion channel macromolecular complexes in cardiomyocytes: roles in sudden cardiac death.

The movement of ions across specific channels embedded on the membrane of individual cardiomyocytes is crucial for the generation and propagation of the cardiac electric impulse. Emerging evidence over the past 20 years strongly suggests that the normal electric function of the heart is the result of dynamic interactions of membrane ion channels working in an orchestrated fashion as part of complex molecular networks. Such networks work together with exquisite temporal precision to generate each action potential and contraction. Macromolecular complexes play crucial roles in transcription, translation, oligomerization, trafficking, membrane retention, glycosylation, post-translational modification, turnover, function, and degradation of all cardiac ion channels known to date. In addition, the accurate timing of each cardiac beat and contraction demands, a comparable precision on the assembly and organizations of sodium, calcium, and potassium channel complexes within specific subcellular microdomains, where physical proximity allows for prompt and efficient interaction. This review article, part of the Compendium on Sudden Cardiac Death, discusses the major issues related to the role of ion channel macromolecular assemblies in normal cardiac electric function and the mechanisms of arrhythmias leading to sudden cardiac death. It provides an idea of how these issues are being addressed in the laboratory and in the clinic, which important questions remain unanswered, and what future research will be needed to improve knowledge and advance therapy.

PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function.

BACKGROUND: Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. METHODS AND RESULTS: To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (ΔSIV). ΔSIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. CONCLUSIONS: Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α2δ-1-induced downregulation of Cav1.2 channels.

Availability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits ß2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor ß2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

The dispensability of 14-3-3 proteins for the regulation of human cardiac sodium channel Nav1.5.

BACKGROUND: 14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial. AIMS: In this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5. METHODS AND RESULTS: Amongst the seven 14-3-3 isoforms, only 14-3-3η (encoded by YWHAH gene) weakly co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3η overexpression or inhibition with difopein, and 14-3-3η did not affect physical interaction between Nav1.5 α-α subunits. The current-voltage relationship and the amplitude of Nav1.5-mediated sodium peak current density were also not changed. CONCLUSIONS: Our findings illustrate that the direct implication of 14-3-3 proteins in regulating Nav1.5 is not evident in a transformed human kidney cell line tsA201.

Characterization of 2 genetic variants of Na(v) 1.5-arginine 689 found in patients with cardiac arrhythmias.

Hundreds of genetic variants in SCN5A, the gene coding for the pore-forming subunit of the cardiac sodium channel, Na(v) 1.5, have been described in patients with cardiac channelopathies as well as in individuals from control cohorts. The aim of this study was to characterize the biophysical properties of 2 naturally occurring Na(v) 1.5 variants, p.R689H and p.R689C, found in patients with cardiac arrhythmias and in control individuals. In addition, this study was motivated by the finding of the variant p.R689H in a family with sudden cardiac death (SCD) in children. When expressed in HEK293 cells, most of the sodium current (I(Na)) biophysical properties of both variants were indistinguishable from the wild-type (WT) channels. In both cases, however, an ∼2-fold increase of the tetrodotoxin-sensitive late I(Na) was observed. Action potential simulations and reconstruction of pseudo-ECGs demonstrated that such a subtle increase in the late I(Na) may prolong the QT interval in a nonlinear fashion. In conclusion, despite the fact that the causality link between p.R689H and the phenotype of the studied family cannot be demonstrated, this study supports the notion that subtle alterations of Na(v) 1.5 variants may increase the risk for cardiac arrhythmias.

Knockdown of the TRPM4 channel alters cardiac electrophysiology and hemodynamics in a sex- and age-dependent manner in mice.

TRPM4 is a calcium-activated, voltage-modulated, nonselective ion channel widely expressed in various cells and tissues. TRPM4 regulates the influx of sodium ions, thus playing a role in regulating the membrane potential. In the heart, TRPM4 is expressed in both cardiomyocytes and cells of the conductive pathways. Clinical studies have linked TRPM4 mutations to several cardiac disorders. While data from experimental studies have demonstrated TRPM4's functional significance in cardiac physiology, its exact roles in the heart have remained unclear. In this study, we investigated the role of TRPM4 in cardiac physiology in a newly generated Trpm4 knockdown mouse model. Male and female Trpm4 knockdown (Trpm4-/- ) and wild-type mice of different ages (5- to 12- week-old (young) and 24-week-old or more (adult)) were characterized using a multimodal approach, encompassing surface electrocardiograms (ECG), echocardiography recordings, ex vivo ECGs in isolated heart, endocardial mappings, Western blots, and mRNA quantifications. The assessment of cardiac electrophysiology by surface ECGs revealed no significant differences between wild-type and Trpm4-/- young (5- to 12-week-old) mice of either sex. Above 24 weeks of age, adult male Trpm4-/- mice showed reduced heart rate and increased heart rate variability. Echocardiography revealed that only adult male Trpm4-/- mice exhibited slight left ventricular hypertrophic alterations compared to controls, illustrated by alterations of the mitral valve pressure halftime, the mitral valve E/A ratio, the isovolumetric relaxation time, and the mitral valve deceleration. In addition, an assessment of the right ventricular systolic function by scanning the pulmonary valve highlighted an alteration in pulmonary valve peak velocity and pressure in adult male Trpm4-/- mice. Endocardial mapping recordings showed that applying 5 µM of the new TRPM4 inhibitor NBA triggered a third-degree atrioventricular block on 40% of wild-type hearts. These results confirm the key role of TRPM4 in the proper structure and electrical function of the heart. It also reveals differences between male and female animals that have never been reported. In addition, the investigation of the effects of NBA on heart function confirms the role of TRPM4 in atrioventricular conduction.

Neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) controls the sorting of newly synthesized Ca(V)1.2 calcium channels.

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) proteins are ubiquitin ligases, which attach ubiquitin moieties to their target proteins, a post-translational modification that is most commonly associated with protein degradation. Nedd4 ubiquitin ligases have been shown to down-regulate both potassium and sodium channels. In this study, we investigated whether Nedd4 ubiquitin ligases also regulate Ca(v) calcium channels. We expressed three Nedd4 family members, Nedd4-1, Nedd4-2, and WWP2, together with Ca(v)1.2 channels in tsA-201 cells. We found that Nedd4-1 dramatically decreased Ca(v) whole-cell currents, whereas Nedd4-2 and WWP2 failed to regulate the current. Surface biotinylation assays revealed that Nedd4-1 decreased the number of channels inserted at the plasma membrane. Western blots also showed a concomitant decrease in the total expression of the channels. Surprisingly, however, neither the Ca(v) pore-forming α1 subunit nor the associated Ca(v)ß and Ca(v)α(2)δ subunits were ubiquitylated by Nedd4-1. The proteasome inhibitor MG132 prevented the degradation of Ca(v) channels, whereas monodansylcadaverine and chloroquine partially antagonized the Nedd4-1-induced regulation of Ca(v) currents. Remarkably, the effect of Nedd4-1 was fully prevented by brefeldin A. These data suggest that Nedd4-1 promotes the sorting of newly synthesized Ca(v) channels for degradation by both the proteasome and the lysosome. Most importantly, Nedd4-1-induced regulation required the co-expression of Ca(v)ß subunits, known to antagonize the retention of the channels in the endoplasmic reticulum. Altogether, our results suggest that Nedd4-1 interferes with the chaperon role of Ca(v)ß at the endoplasmic reticulum/Golgi level to prevent the delivery of Ca(v) channels at the plasma membrane.

Identification of a novel loss-of-function calcium channel gene mutation in short QT syndrome (SQTS6).

AIMS: Short QT syndrome (SQTS) is a genetically determined ion-channel disorder, which may cause malignant tachyarrhythmias and sudden cardiac death. Thus far, mutations in five different genes encoding potassium and calcium channel subunits have been reported. We present, for the first time, a novel loss-of-function mutation coding for an L-type calcium channel subunit. METHODS AND RESULTS: The electrocardiogram of the affected member of a single family revealed a QT interval of 317 ms (QTc 329 ms) with tall, narrow, and symmetrical T-waves. Invasive electrophysiological testing showed short ventricular refractory periods and increased vulnerability to induce ventricular fibrillation. DNA screening of the patient identified no mutation in previously known SQTS genes; however, a new variant at a heterozygous state was identified in the CACNA2D1 gene (nucleotide c.2264G > C; amino acid p.Ser755Thr), coding for the Ca(v)α(2)δ-1 subunit of the L-type calcium channel. The pathogenic role of the p.Ser755Thr variant of the CACNA2D1 gene was analysed by using co-expression of the two other L-type calcium channel subunits, Ca(v)1.2α1 and Ca(v)ß(2b), in HEK-293 cells. Barium currents (I(Ba)) were recorded in these cells under voltage-clamp conditions using the whole-cell configuration. Co-expression of the p.Ser755Thr Ca(v)α(2)δ-1 subunit strongly reduced the I(Ba) by more than 70% when compared with the co-expression of the wild-type (WT) variant. Protein expression of the three subunits was verified by performing western blots of total lysates and cell membrane fractions of HEK-293 cells. The p.Ser755Thr variant of the Ca(v)α(2)δ-1 subunit was expressed at a similar level compared with the WT subunit in both fractions. Since the mutant Ca(v)α(2)δ-1 subunit did not modify the expression of the pore-forming subunit of the L-type calcium channel, Ca(v)1.2α1, it suggests that single channel biophysical properties of the L-type channel are altered by this variant. CONCLUSION: In the present study, we report the first pathogenic mutation in the CACNA2D1 gene in humans, which causes a new variant of SQTS. It remains to be determined whether mutations in this gene lead to other manifestations of the J-wave syndrome.

Bioorthogonal site-selective conjugation of fluorescent dyes to antibodies: method and potential applications.

Antibodies are immensely useful tools for biochemical research and have found application in numerous protein detection and purification methods. Moreover, monoclonal antibodies are increasingly utilised as therapeutics or, conjugated to active pharmaceutical ingredients, in targeted chemotherapy. Several reagents and protocols are reported to synthesise fluorescent antibodies for protein target detection and immunofluorescence applications. However, most of these protocols lead to non-selective conjugation, over-labelling or in the worst case antigen binding site modification. Here, we have used the antibody disulphide cleavage and re-bridging strategy to introduce bright fluorescent dyes without loss of the antibody function. The resulting fluorescent IgG1 type antibodies were shown to be effective imaging tools in western blot and direct immunofluorescence experiments.