C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation

International audienceVoltage-gated Na(+) (NaV) channels are key regulators of myocardial excitability, and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na(+) current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts

Molecular Basis of Inward Rectification: Polyamine Interaction Sites Located by Combined Channel and Ligand Mutagenesis

Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter

0134 : Using cardiomyocytes differentiated from urine-derived hiPSCs to recapitulate electrophysiological characteristics of LQT2 syndrome

RationaleHuman genetically inherited cardiac diseases have mainly been studied in heterologous systems or animal models, independently of the patients’ genetic background. Because sources for human cardiomyocytes are extremely limited, the use of urine samples to derive cardiomyocytes would be a non-invasive method to identify cardiac dysfunctions that lead to pathologies within the patients’ specific genetic background.ObjectiveCardiomyocytes differentiated from urine-derived pluripotent stem cells (UhiPS-CMs) were obtained from a patient with long QT syndrome and a mutation in hERG KCNH2 gene (p. A561P), and were characterized.Methods and ResultsCells obtained from urine samples from the A561P patient and his asymptomatic mother carrying no hERG mutation were reprogrammed using the episomal-based method. UhiPS cells were then differentiated into cardiomyocytes using a modified matrix sandwich method. UhiPS-CMs showed proper expression of ventricular cytoskeletal proteins and ion channels. They were electrically functional, with nodal-, atrial- and ventricular-like action potentials (APs) recorded using both high-throughput CellOptiq and patch-clamp techniques. Application of ajmaline, 4-aminopyridine, nifedipine, chromanol 293B or E-4031 to the UhiPS-CMs confirmed that INa, Ito, ICa, IKs and IKr currents, respectively, contributed to the APs. Comparing hERG expression from the patient's UhiPS-CMs to the mother's UhiPS-CMs showed that the mutation led to a trafficking defect that resulted in a reduced IKr current. This phenotype led to APs prolongation that sometimes resulted in arrhythmias (early afterdepolarizations).ConclusionUrine-derived pluripotent stem cells from patients carrying ion channels mutations can be used as novel models to differentiate functional cardiomyocytes that recapitulate cardiac arrhythmia phenotypes

A functional network of highly pure enteric neurons in a dish

The enteric nervous system (ENS) is the intrinsic nervous system that innervates the entire digestive tract and regulates major digestive functions. Recent evidence has shown that functions of the ENS critically rely on enteric neuronal connectivity; however, experimental models to decipher the underlying mechanisms are limited. Compared to the central nervous system, for which pure neuronal cultures have been developed for decades and are recognized as a reference in the field of neuroscience, an equivalent model for enteric neurons is lacking. In this study, we developed a novel model of highly pure rat embryonic enteric neurons with dense and functional synaptic networks. The methodology is simple and relatively fast. We characterized enteric neurons using immunohistochemical, morphological, and electrophysiological approaches. In particular, we demonstrated the applicability of this culture model to multi-electrode array technology as a new approach for monitoring enteric neuronal network activity. This in vitro model of highly pure enteric neurons represents a valuable new tool for better understanding the mechanisms involved in the establishment and maintenance of enteric neuron synaptic connectivity and functional networks

Cryptanalysis of Dedicated Cryptographic Hash Functions

In this thesis we study the security of a number of dedicated cryptographic hash functions against cryptanalytic attacks. We begin with an introduction to what cryptographic hash functions are and what they are used for. This is followed by strict definitions of the security properties often required from cryptographic hash functions. FSB hashes are a class of hash functions derived from a coding theory problem. We attack FSB by modeling the compression function of the hash by a matrix in GF(2). We show that collisions and preimages can easily be found in FSB with the proposed security parameters. We describe a meet-in-the-middle attack against the FORK-256 hash function. The attack requires 2^112.8 operations to find a collision, which is a 38000-fold improvement over the expected 2^128 operations. We then present a method for finding slid pairs for the compression function of SHA-1; pairs of inputs and messages that produce closely related outputs in the compression function. We also cryptanalyse two block ciphers based on the compression function of MD5, MDC-MD5 and the Kaliski-Robshaw "Crab" encryption algorithm. VSH is a hash function based on problems in number theory that are believed to be hard. The original proposal only claims collision resistance; we demonstrate that VSH does not meet the other hash function requirements of preimage resistance, one-wayness, and collision resistance of truncated variants. To explore more general cryptanalytic attacks, we discuss the d-Monomial test, a statistical test that has been found to be effective in distinguishing iterated Boolean circuits from real random functions. The test is applied to the SHA and MD5 hash functions. We present a new hash function proposal, LASH, and its initial cryptanalysis.The LASH design is based on a simple underlying primitive, and some of its security can be shown to be related to lattice problems

Multifocal Ectopic Purkinje-Related Premature Contractions: A New SCN5A-Related Cardiac Channelopathy.: MEPPC: a new SCN5A-related cardiac channelopathy

International audienceOBJECTIVES: The aim of this study was to describe a new familial cardiac phenotype and to elucidate the electrophysiological mechanism responsible for the disease. BACKGROUND: Mutations in several genes encoding ion channels, especially SCN5A, have emerged as the basis for a variety of inherited cardiac arrhythmias. METHODS: Three unrelated families comprising 21 individuals affected by multifocal ectopic Purkinje-related premature contractions (MEPPC) characterized by narrow junctional and rare sinus beats competing with numerous premature ventricular contractions with right and/or left bundle branch block patterns were identified. RESULTS: Dilated cardiomyopathy was identified in 6 patients, atrial arrhythmias were detected in 9 patients, and sudden death was reported in 5 individuals. Invasive electrophysiological studies demonstrated that premature ventricular complexes originated from the Purkinje tissue. Hydroquinidine treatment dramatically decreased the number of premature ventricular complexes. It normalized the contractile function in 2 patients. All the affected subjects carried the c.665G>A transition in the SCN5A gene. Patch-clamp studies of resulting p.Arg222Gln (R222Q) Nav1.5 revealed a net gain of function of the sodium channel, leading, in silico, to incomplete repolarization in Purkinje cells responsible for premature ventricular action potentials. In vitro and in silico studies recapitulated the normalization of the ventricular action potentials in the presence of quinidine. CONCLUSIONS: A new SCN5A-related cardiac syndrome, MEPPC, was identified. The SCN5A mutation leads to a gain of function of the sodium channel responsible for hyperexcitability of the fascicular-Purkinje system. The MEPPC syndrome is responsive to hydroquinidine

Création d'un pacemaker biologique cardiaque

Chez des souris en bloc auriculo-ventriculaire (BAVc), nous avons créé un pacemaker biologique cardiaque grâce à un vecteur non-viral. Cinq jours avant l'obtention du BAVc par ablation du faisceau de His, les plasmides codant le canal ionique HCN2 responsable du courant de pacemaker, If, et le récepteur b2-adrénergique, sont mélangés au vecteur tétronic 304 et injectés dans la paroi du ventricule gauche. Le rythme d'échappement ventriculaire des souris HCN2-Adrb2 est significativement plus rapide que celui des souris sham et est stabilisé jusqu à 40 jours. Ce pacemaker biologique est régulé par la stimulation sympathique et permet d augmenter la survie des souris. Le PI(4,5)P2 régule l activité du canal KCNQ1-KCNE1, responsable du courant potassique IKs, en stabilisant l état d ouverture du canal. De très nombreuses mutations du canal KCNQ1 sont responsables du syndrome du QT long à l origine de troubles du rythme cardiaque. Nous avons démontré que l implication de trois formes mutantes de KCNQ1 (R243H, R555C et R539W) dans le syndrome du QT long, s explique par une diminution de l affinité de KCNQ1 pour le PIP2. L osmosensibilité du canal KCNQ1 est une voie physiologique prépondérante de la réponse des cellules cardiaques aux variations osmotiques. La caractérisation des réponses aux variations d osmolarité du canal KCNQ1-KCNE1 et des trois canaux mutants caractérisés par une faible affinité pour le PIP2, suggère que l osmosensibilité de KCNQ1-KCNE1 ne passe par l étirement de la membrane plasmique mais implique une variation du volume cellulaire provoquant une régulation de la concentration de Mg2+, et donc une variation du PIP2 disponible.In a mouse model of complete atrioventricular block (CAVB), we generate a functional ventricular pacemaker by non-viral gene delivery. Five days before catheter-mediated radiofrequency His bundle ablation, plasmids coding cyclic nucleotide-gated HCN2 channel (Hcn2), responsible for the pacemaker current, If, and the ß2-adrenergic receptor (Adbr2), are mixed with the tetronic 304 vector and injected in the wall of the left ventricle. The escape ventricular rhythm in HCN2-Adrb2 mice is significatively faster than in sham mice and is stabilized until 40 days. This biological pacemaker is regulated by sympathetic input, and improves life expectancy in this mouse model of CAVB. PI(4,5)P2 regulates KCNQ1-KCNE1 potassium channel activity, which underlies the potassium current IKs, by stabilizing the open state of the channel. Various mutations of the KCNQ1 channel are responsible for the long QT syndrome, characterized by cardiac arrhythmias. We demonstrated that the involvement of three mutant forms of KCNQ1 (R243H, R555C and R539W) in the long QT syndrome, can be explained by a reduced PIP2 affinity of KCNQ1 channels. The osmosensitivity of KCNQ1 channel is of physiological relevance in cardiac cells in response to osmotic variations. The characterization of the response to osmotic variations of the wild-type and three mutant KCNQ1-KCNE1 channels characterized by a weak PIP2 affinity, suggests that the osmosensibility of KCNQ1-KCNE1 does not involve membrane stretching but a variation of the cellular volume provoking a variation of the Mg2+ concentration, and thus a regulation of available PIP2.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

hERG S4-S5 linker acts as a voltage-dependent ligand that binds to the activation gate and locks it in a closed state

International audienceDelayed-rectifier potassium channels (hERG and KCNQ1) play a major role in cardiac repolarization. These channels are formed by a tetrameric pore (S5-S6) surrounded by four voltage sensor domains (S1-S4). Coupling between voltage sensor domains and the pore activation gate is critical for channel voltage-dependence. However, molecular mechanisms remain elusive. Herein, we demonstrate that covalently binding, through a disulfide bridge, a peptide mimicking the S4-S5 linker (S4-S5L) to the channel S6 C-terminus (S6T) completely inhibits hERG. This shows that channel S4-S5L is sufficient to stabilize the pore activation gate in its closed state. Conversely, covalently binding a peptide mimicking S6T to the channel S4-S5L prevents its inhibiting effect and renders the channel almost completely voltage-independent. This shows that the channel S4-S5L is necessary to stabilize the activation gate in its closed state. Altogether, our results provide chemical evidence that S4-S5L acts as a voltage-controlled ligand that binds S6T to lock the channel in a closed state, elucidating the coupling between voltage sensors and the gate in delayed rectifier potassium channels and potentially other voltage-gated channels