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Voltage-gated sodium (NaV) channels are responsible for the initiation and propagation of action potentials. In the heart, the predominant NaV1.5 α subunit is composed of four homologous repeats (I–IV) and forms a macromolecular complex with multiple accessory proteins, including intracellular fibroblast growth factors (iFGF). In spite of high homology, each of the iFGFs, iFGF11–iFGF14, as well as the individual iFGF splice variants, differentially regulates NaV channel gating, and the mechanisms underlying these differential effects remain elusive. Much of the work exploring iFGF regulation of NaV1.5 has been performed in mouse and rat ventricular myocytes in which iFGF13VY is the predominant iFGF expressed, whereas investigation into NaV1.5 regulation by the human heart-dominant iFGF12B is lacking. In this study, we used a mouse model with cardiac-specific Fgf13 deletion to study the consequences of iFGF13VY and iFGF12B expression. We observed distinct effects on the voltage-dependences of activation and inactivation of the sodium currents (INa), as well as on the kinetics of peak INa decay. Results in native myocytes were recapitulated with human NaV1.5 heterologously expressed in Xenopus oocytes, and additional experiments using voltage-clamp fluorometry (VCF) revealed iFGF-specific effects on the activation of the NaV1.5 voltage sensor domain in repeat IV (VSD-IV). iFGF chimeras further unveiled roles for all three iFGF domains (i.e., the N-terminus, core, and C-terminus) on the regulation of VSD-IV, and a slower time domain of inactivation. We present here a novel mechanism of iFGF regulation that is specific to individual iFGF isoforms and that leads to distinct functional effects on NaV channel/current kinetics.

Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K+) channels regulate daily oscillations in the rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K+conductance(s) driving these daily rhythms in repetitive firing rates, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K+channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in theKcnh8(Kv12.1−/−) orKcnh3(Kv12.2−/−) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1−/−and Kv12.2−/−, than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1−/−, Kv12.2−/−and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1−/−and Kv12.2−/−SCN neurons. Similar results were obtained within vivoshRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1−/−, Kv12.2−/−, Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.

KCNQ2 and KCNQ3 form the M-channels that are important in regulating neuronal excitability. Inherited mutations that alter voltage-dependent gating of M-channels are associated with neonatal epilepsy. In the homolog KCNQ1 channel, two steps of voltage sensor activation lead to two functionally distinct open states, the intermediate-open (IO) and activated-open (AO), which define the gating, physiological, and pharmacological properties of KCNQ1. However, whether the M-channel shares the same mechanism is unclear. Here, we show that KCNQ2 and KCNQ3 feature only a single conductive AO state but with a conserved mechanism for the electro-mechanical (E-M) coupling between voltage sensor activation and pore opening. We identified some epilepsy-linked mutations in KCNQ2 and KCNQ3 that disrupt E-M coupling. The antiepileptic drug retigabine rescued KCNQ3 currents that were abolished by a mutation disrupting E-M coupling, suggesting that modulating the E-M coupling in KCNQ channels presents a potential strategy for antiepileptic therapy.

Polycystin 2 (PC2 or TRPP1, formerly TRPP2) is a calcium-permeant Transient Receptor Potential (TRP) cation channel expressed primarily on the endoplasmic reticulum (ER) membrane and primary cilia of all cell and tissue types. Despite its ubiquitous expression throughout the body, studies of PC2 have focused primarily on its role in the kidney, as mutations in PC2 lead to the development of autosomal dominant polycystic kidney disease (ADPKD), a debilitating condition for which there is no cure. However, the endogenous role that PC2 plays in the regulation of general cellular homeostasis remains unclear. In this study, we measure how PC2 expression changes in different pathological states, determine that its abundance is increased under conditions of cellular stress in multiple tissues including human disease, and conclude that PC2-deficient cells have increased susceptibility to cell death induced by stress. Our results offer new insight into the normal function of PC2 as a ubiquitous stress-sensitive protein whose expression is up-regulated in response to cell stress to protect against pathological cell death in multiple diseases.

The resurgent component of the voltage-gated sodium current (INaR) is a depolarizing conductance, revealed on membrane hyperpolarizations following brief depolarizing voltage steps, which has been shown to contribute to regulating the firing properties of numerous neuronal cell types throughout the central and peripheral nervous systems. Although mediated by the same voltage-gated sodium (Nav) channels that underlie the transient and persistent Nav current components, the gating mechanisms that contribute to the generation of INaR remain unclear. Here, we characterized Nav currents in mouse cerebellar Purkinje neurons, and used tailored voltage-clamp protocols to define how the voltage and the duration of the initial membrane depolarization affect the amplitudes and kinetics of INaR. Using the acquired voltage-clamp data, we developed a novel Markov kinetic state model with parallel (fast and slow) inactivation pathways and, we show that this model reproduces the properties of the resurgent, as well as the transient and persistent, Nav currents recorded in (mouse) cerebellar Purkinje neurons. Based on the acquired experimental data and the simulations, we propose that resurgent Na+ influx occurs as a result of fast inactivating Nav channels transitioning into an open/conducting state on membrane hyperpolarization, and that the decay of INaR reflects the slow accumulation of recovered/opened Nav channels into a second, alternative and more slowly populated, inactivated state. Additional simulations reveal that extrinsic factors that affect the kinetics of fast or slow Nav channel inactivation and/or impact the relative distribution of Nav channels in the fast- and slow-inactivated states, such as the accessory Navβ4 channel subunit, can modulate the amplitude of INaR.

Sudden cardiac death (SCD), the unexpected death due to acquired or genetic cardiovascular disease, follows distinct 24-hour patterns in occurrence. These 24-hour patterns likely reflect daily changes in arrhythmogenic triggers and the myocardial substrate caused by day/night rhythms in behavior, the environment, and endogenous circadian mechanisms. To better address fundamental questions regarding the circadian mechanisms, the National Heart, Lung, and Blood Institute convened a workshop, Understanding Circadian Mechanisms of Sudden Cardiac Death. We present a 2-part report of findings from this workshop. Part 1 summarizes the workshop and serves to identify research gaps and opportunities in the areas of basic and translational research. Among the gaps was the lack of standardization in animal studies for reporting environmental conditions (eg, timing of experiments relative to the light dark cycle or animal housing temperatures) that can impair rigor and reproducibility. Workshop participants also pointed to uncertainty regarding the importance of maintaining normal circadian rhythmic synchrony and the potential pathological impact of desynchrony on SCD risk. One related question raised was whether circadian mechanisms can be targeted to reduce SCD risk. Finally, the experts underscored the need for studies aimed at determining the physiological importance of circadian clocks in the many different cell types important to normal heart function and SCD. Addressing these gaps could lead to new therapeutic approaches/molecular targets that can mitigate the risk of SCD not only at certain times but over the entire 24-hour period.

Sudden cardiac death (SCD) is the sudden, unexpected death due to abrupt loss of heart function secondary to cardiovascular disease. In certain populations living with cardiovascular disease, SCD follows a distinct 24-hour pattern in occurrence, suggesting day/night rhythms in behavior, the environment, and endogenous circadian rhythms result in daily spans of increased vulnerability. The National Heart, Lung, and Blood Institute convened a workshop, Understanding Circadian Mechanisms of Sudden Cardiac Death to identify fundamental questions regarding the role of the circadian rhythms in SCD. Part 2 summarizes research gaps and opportunities in the areas of population and clinical research identified in the workshop. Established research supports a complex interaction between circadian rhythms and physiological responses that increase the risk for SCD. Moreover, these physiological responses themselves are influenced by several biological variables, including the type of cardiovascular disease, sex, age, and genetics, as well as environmental factors. The emergence of new noninvasive biotechnological tools that continuously measure key cardiovascular variables, as well as the identification of biomarkers to assess circadian rhythms, hold promise for generating large-scale human data sets that will delineate which subsets of individuals are most vulnerable to SCD. Additionally, these data will improve our understanding of how people who suffer from circadian disruptions develop cardiovascular diseases that increase the risk for SCD. Emerging strategies to identify new biomarkers that can quantify circadian health (eg, environmental, behavioral, and internal misalignment) may lead to new interventions and therapeutic targets to prevent the progression of cardiovascular diseases that cause SCD.

The 6TM family of K channels comprises the voltage-gated KV subfamilies, the EAG subfamily (which includes hERG channels), the Ca2+-activated Slo subfamily (actually with 7TM, termed BK) and the Ca2+-activated SK subfamily. These channels possess a pore-forming α subunit that comprise tetramers of identical subunits (homomeric) or of different subunits (heteromeric). Heteromeric channels can only be formed within subfamilies (e.g. Kv1.1 with Kv1.2; Kv7.2 with Kv7.3). The pharmacology largely reflects the subunit composition of the functional channel.

[Figure: see text].

The resurgent component of the voltage-gated sodium current (I

Cone Dystrophy with Supernormal Rod Response (CDSRR) is a rare autosomal recessive disorder leading to severe visual impairment in humans, but little is known about its unique pathophysiology. We have previously shown that CDSRR is caused by mutations in the KCNV2 (Potassium Voltage-Gated Channel Modifier Subfamily V Member 2) gene encoding the Kv8.2 subunit, a modulatory subunit of voltage-gated potassium (Kv) channels. In a recent study, we validated a novel mouse model of Kv8.2 deficiency at a late stage of the disease and showed that it replicates the human electroretinogram (ERG) phenotype. In this current study, we focused our investigation on young adult retinas to look for early markers of disease and evaluate their effect on retinal morphology, electrophysiology and immune response in both the Kv8.2 knockout (KO) mouse and in the Kv2.1 KO mouse, the obligate partner of Kv8.2 in functional retinal Kv channels. By evaluating the severity of retinal dystrophy in these KO models, we demonstrated that retinas of Kv KO mice have significantly higher apoptotic cells, a thinner outer nuclear cell layer and increased activated microglia cells in the subretinal space. Our results indicate that in the murine retina, the loss of Kv8.2 subunits contributes to early cellular and physiological changes leading to retinal dysfunction. These results could have potential implications in the early management of CDSRR despite its relatively nonprogressive nature in humans.

The resurgent component of the voltage-gated sodium current (INaR) is a depolarizing conductance, revealed on membrane hyperpolarizations following brief depolarizing voltage steps, which has been shown to contribute to regulating the firing properties of numerous neuronal cell types throughout the central and peripheral nervous systems. Although mediated by the same voltage-gated sodium (Nav) channels that underlie the transient and persistent Nav current components, the gating mechanisms that contribute to the generation of INaR remain unclear. Here, we characterized Nav currents in mouse cerebellar Purkinje neurons, and used tailored voltage-clamp protocols to define how the voltage and the duration of the initial membrane depolarization affect the amplitudes and kinetics of INaR. Using the acquired voltage-clamp data, we developed a novel Markov kinetic state model with parallel (fast and slow) inactivation pathways and, we show that this model reproduces the properties of the resurgent, as well as the transient and persistent, Nav currents recorded in (mouse) cerebellar Purkinje neurons. Based on the acquired experimental data and the simulations, we propose that resurgent Na+ influx occurs as a result of fast inactivating Nav channels transitioning into an open/conducting state on membrane hyperpolarization, and that the decay of INaR reflects the slow accumulation of recovered/opened Nav channels into a second, alternative and more slowly populated, inactivated state. Additional simulations reveal that extrinsic factors that affect the kinetics of fast or slow Nav channel inactivation and/or impact the relative distribution of Nav channels in the fast- and slow-inactivated states, such as the accessory Navβ4 channel subunit, can modulate the amplitude of INaR.SUMMARYThe resurgent component of the voltage-gated sodium current (INaR) is revealed on membrane hyperpolarizations following brief depolarizing voltage steps that activate the rapidly activating and inactivating, transient Nav current (INaT). To probe the mechanisms contributing to the generation and properties of INaR, we combined whole-cell voltage-clamp recordings from mouse cerebellar Purkinje neurons with computational modeling to develop a novel, blocking particle-independent, model for the gating of INaR that involves two parallel inactivation pathways, and we show that this model recapitulates the detailed biophysical properties of INaR measured in mouse cerebellar Purkinje neurons.

Markov models of ion channel dynamics have evolved as experimental advances have improved our understanding of channel function. Past studies have examined limited sets of various topologies for Markov models of channel dynamics. We present a systematic method for identification of all possible Markov model topologies using experimental data for two types of native voltage-gated ion channel currents: mouse atrial sodium currents and human left ventricular fast transient outward potassium currents. Successful models identified with this approach have certain characteristics in common, suggesting that aspects of the model topology are determined by the experimental data. Incorporating these channel models into cell and tissue simulations to assess model performance within protocols that were not used for training provided validation and further narrowing of the number of acceptable models. The success of this approach suggests a channel model creation pipeline may be feasible where the structure of the model is not specified a priori., Author summary Markov models of ion channel dynamics have evolved as experimental advances have improved our understanding of channel function. Past studies have examined limited sets of various structures for Markov models of channel dynamics. Here, we present a computational routine designed to thoroughly search for Markov model topologies for simulating whole-cell currents. We tested this method on two distinct types of voltage-gated cardiac ion channels and found the number of states and connectivity required to recapitulate experimentally observed kinetics. Successful models identified with this approach have certain characteristics in common, suggesting that model structures are determined by the experimental data. Incorporation of these models into higher scale action potential and cable (an approximation of one-dimensional action potential propagation) simulations, identified key channel phenomena that were required for proper function. These methods provide a route to create functional channel models that can be used for action potential simulation without pre-defining their structure ahead of time.

Lorenzini et al. describe the native phosphorylation sites of NaV1.5 channels in mouse left ventricles. By analyzing expression and function of phosphosilent and phosphomimetic mutants, they identify phosphorylation hot spots regulating channel cell surface expression and gating., Phosphorylation of the voltage-gated Na+ (NaV) channel NaV1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed NaV1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on NaV1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic NaV1.5 mutants revealed the roles of three phosphosites in regulating NaV1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface NaV1.5 expression and peak Na+ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular NaV1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of NaV1.5 channels is highly complex, site specific, and dynamic.

Clippinger et al. examine a mutation in troponin T that causes hypertrophic cardiomyopathy and demonstrate that increased molecular mechanics drive the early disease pathogenesis, leading to secondary activation of mechanobiological signaling pathways., Familial hypertrophic cardiomyopathy (HCM), a leading cause of sudden cardiac death, is primarily caused by mutations in sarcomeric proteins. The pathogenesis of HCM is complex, with functional changes that span scales, from molecules to tissues. This makes it challenging to deconvolve the biophysical molecular defect that drives the disease pathogenesis from downstream changes in cellular function. In this study, we examine an HCM mutation in troponin T, R92Q, for which several models explaining its effects in disease have been put forward. We demonstrate that the primary molecular insult driving disease pathogenesis is mutation-induced alterations in tropomyosin positioning, which causes increased molecular and cellular force generation during calcium-based activation. Computational modeling shows that the increased cellular force is consistent with the molecular mechanism. These changes in cellular contractility cause downstream alterations in gene expression, calcium handling, and electrophysiology. Taken together, our results demonstrate that molecularly driven changes in mechanical tension drive the early disease pathogenesis of familial HCM, leading to activation of adaptive mechanobiological signaling pathways.

Familial hypertrophic cardiomyopathy (HCM), a leading cause of sudden cardiac death, is primarily caused by mutations in sarcomeric proteins. The pathogenesis of HCM is complex, with functional changes that span scales from molecules to tissues. This makes it challenging to deconvolve the biophysical molecular defect that drives the disease pathogenesis from downstream changes in cellular function. Here, we examined a HCM mutation in troponin T, R92Q. We demonstrate that the primary molecular insult driving the disease pathogenesis is mutation-induced alterations in tropomyosin positioning, which causes increased molecular and cellular force generation during calcium-based activation. We demonstrate computationally that these increases in force are direct consequences of the initial molecular insult. This altered cellular contractility causes downstream alterations in gene expression, calcium handling, and electrophysiology. Taken together, our results demonstrate that molecularly driven changes in mechanical tension drive the early disease pathogenesis, leading to activation of adaptive mechanobiological signaling pathways.

Phosphorylation of NaV1.5 channels regulates cardiac excitability, yet the phosphorylation sites regulating channel function and the underlying mechanisms remain largely unknown. Using a systematic quantitative phosphoproteomic approach, we analyzed NaV1.5 channel complexes purified from non-failing and failing mouse left ventricles, and we identified 42 phosphorylation sites on NaV1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic NaV1.5 mutants revealed the roles of three phosphosites in regulating NaV1.5 channel expression and gating. The phosphorylated serines-664 and -667 regulate the voltage-dependence of channel activation in a cumulative manner, whereas phosphorylation of the nearby serine-671, which is increased in failing hearts, decreases cell surface NaV1.5 expression and peak Na+current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, the results demonstrate that ventricular NaV1.5 is highly phosphorylated, and that the phosphorylation-dependent regulation of NaV1.5-encoded channels is highly complex, site-specific and dynamic.AbbreviationsA, alanine; E, glutamate; HEK-293, Human Embryonic Kidney 293 cells; INa, peak Na+current; INaL, late Na+current; IP, immunoprecipitation; mαNaVPAN, anti-NaVchannel subunit mouse monoclonal antibody; MS, Mass Spectrometry; MS1, mass spectrum of peptide precursors; MS2 or MS/MS, fragmentation mass spectrum of peptides selected in narrow mass range (2 Da) from MS1 scan; NaV, voltage-gated Na+channel; pS, phosphoserine; pT, phosphothreonine; S, serine; T, threonine; TAC, Transverse Aortic Constriction; TMT, Tandem Mass Tag.

Rationale: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)–enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. Objective: The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. Methods and Results: RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor β1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor β1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor β1–induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor β1–induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6–mediated transcriptional control. Targeted disruption of Txndc5 in mice ( Txndc5 −/− ) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 ( P Txndc5 −/− versus wild-type mice, respectively. Conclusions: The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against β agonist–induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure.

Voltage-gated Na+ (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound β1 and β3 subunits that regulate channel gating, expression, and pharmacology. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the β subunits affect the conformational dynamics of the cardiac NaV channel (NaV1.5) voltage-sensing domains (VSDs). The pore-forming NaV1.5 α subunit contains four domains (DI–DIV), each with a VSD. Our results show that β1 regulates NaV1.5 by modulating the DIV-VSD, whereas β3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the β3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to β3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that β3 binds proximally to the DIII-VSD. Molecular-level differences in β1 and β3 interaction with the α subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability.

Familial hypertrophic cardiomyopathy (HCM), linked to mutations in myosin, myosin-binding proteins and other sarcolemmal proteins, is associated with increased risk of life threatening ventricular arrhythmias, and a number of animal models have been developed to facilitate analysis of disease progression and mechanisms. In the experiments here, we use the αMHC403/+ mouse line in which one αMHC allele harbors a common HCM mutation (in βMHC, Arg403 Gln). Here, we demonstrate marked prolongation of QT intervals in young adult (10–12 week) male αMHC403/+ mice, well in advance of the onset of measurable left ventricular hypertrophy. Electrophysiological recordings from myocytes isolated from the interventricular septum of these animals revealed significantly (P

This is the second of the two White Papers from the fourth UC Davis Cardiovascular Symposium Systems Approach to Understanding Cardiac Excitation-Contraction Coupling and Arrhythmias (3-4 March 2016), a biennial event that brings together leading experts in different fields of cardiovascular research. The theme of the 2016 symposium was 'K+ channels and regulation', and the objectives of the conference were severalfold: (1) to identify current knowledge gaps; (2) to understand what may go wrong in the diseased heart and why; (3) to identify possible novel therapeutic targets; and (4) to further the development of systems biology approaches to decipher the molecular mechanisms and treatment of cardiac arrhythmias. The sessions of the Symposium focusing on the functional roles of the cardiac K+ channel in health and disease, as well as K+ channels as therapeutic targets, were contributed by Ye Chen-Izu, Gideon Koren, James Weiss, David Paterson, David Christini, Dobromir Dobrev, Jordi Heijman, Thomas O'Hara, Crystal Ripplinger, Zhilin Qu, Jamie Vandenberg, Colleen Clancy, Isabelle Deschenes, Leighton Izu, Tamas Banyasz, Andras Varro, Heike Wulff, Eleonora Grandi, Michael Sanguinetti, Donald Bers, Jeanne Nerbonne and Nipavan Chiamvimonvat as speakers and panel discussants. This article summarizes state-of-the-art knowledge and controversies on the functional roles of cardiac K+ channels in normal and diseased heart. We endeavour to integrate current knowledge at multiple scales, from the single cell to the whole organ levels, and from both experimental and computational studies.

Rationale: Ventricular arrhythmias often arise from the Purkinje–myocyte junction and are a leading cause of sudden cardiac death. Notch activation reprograms cardiac myocytes to an induced Purkinje-like state characterized by prolonged action potential duration and expression of Purkinje-enriched genes. Objective: To understand the mechanism by which canonical Notch signaling causes action potential prolongation. Methods and Results: We find that endogenous Purkinje cells have reduced peak K + current, I to , and I K,slow when compared with ventricular myocytes. Consistent with partial reprogramming toward a Purkinje-like phenotype, Notch activation decreases peak outward K + current density, as well as the outward K + current components I to,f and I K , slow . Gene expression studies in Notch-activated ventricles demonstrate upregulation of Purkinje-enriched genes Contactin-2 and Scn5a and downregulation of K + channel subunit genes that contribute to I to,f and I K,slow . In contrast, inactivation of Notch signaling results in increased cell size commensurate with increased K + current amplitudes and mimics physiological hypertrophy. Notch-induced changes in K + current density are regulated at least in part via transcriptional changes. Chromatin immunoprecipitation demonstrates dynamic RBP-J (recombination signal binding protein for immunoglobulin kappa J region) binding and loss of active histone marks on K + channel subunit promoters with Notch activation, and similar transcriptional and epigenetic changes occur in a heart failure model. Interestingly, there is a differential response in Notch target gene expression and cellular electrophysiology in left versus right ventricular cardiac myocytes. Conclusions: In summary, these findings demonstrate a novel mechanism for regulation of voltage-gated potassium currents in the setting of cardiac pathology and may provide a novel target for arrhythmia drug design.

Mutations in theKCNV2gene, which encodes the voltage-gated K+channel protein Kv8.2, cause a distinctive form of cone dystrophy with a supernormal rod response (CDSRR). Kv8.2 channel subunits only form functional channels when combined in a heterotetramer with Kv2.1 subunits encoded by theKCNB1gene. The CDSRR disease phenotype indicates that photoreceptor adaptation is disrupted. The electroretinogram (ERG) response of affected individuals shows depressed rod and cone activity, but what distinguishes this disease is the supernormal rod response to a bright flash of light. Here, we have utilized knock-out mutations of both genes in the mouse to study the pathophysiology of CDSRR. The Kv8.2 knock-out (KO) mice show many similarities to the human disorder, including a depressed a-wave and an elevated b-wave response with bright light stimulation. Optical coherence tomography (OCT) imaging and immunohistochemistry indicate that the changes in six-month-old Kv8.2 KO retinae are largely limited to the outer nuclear layer (ONL), while outer segments appear intact. In addition, there is a significant increase in TUNEL-positive cells throughout the retina. The Kv2.1 KO and double KO mice also show a severely depressed a-wave, but the elevated b-wave response is absent. Interestingly, in all three KO genotypes, the c-wave is totally absent. The differential response shown here of these KO lines, that either possess homomeric channels or lack channels completely, has provided further insights into the role of K+channels in the generation of the a-, b-, and c-wave components of the ERG.

Multiple types of voltage-gated K(+) and non-voltage-gated K(+) currents have been distinguished in mammalian cardiac myocytes based on differences in time-dependent and voltage-dependent properties and pharmacologic sensitivities. Many of the genes encoding voltage-gated K(+) (Kv) and non-voltage-gated K(+) (Kir and K2P) channel pore-forming and accessory subunits are expressed in the heart, and a variety of approaches have been, and continue to be, used to define the molecular determinants of native cardiac K(+) channels and to explore the molecular mechanisms controlling the diversity, regulation, and remodeling of these channels in the normal and diseased myocardium.

Background Cardiovascular (CV) diseases are major causes of mortality in uremic patients. Conventional risk factors fail to identify uremic patients with increased propensity for adverse CV outcomes. We aimed to test the hypothesis that circulating long noncoding RNAs (lncRNAs) could be a prognostic marker to predict adverse CV outcomes in uremic patients. Methods and results Plasma lncRNAs were profiled in patients with end-stage renal disease (ESRD, n = 28) or chronic kidney disease (CKD, n = 8) and in healthy (n = 12) subjects by RNA sequencing. A total of 179 lncRNAs were significantly dysregulated with ESRD; the expression signature of plasma lncRNAs distinguished ESRD from both CKD and control samples. Analysis on a microarray dataset obtained from renal biopsy samples of patients with advanced kidney disease (GSE66494) revealed that a significant proportion of plasma lncRNAs (30.7%) and mRNAs (49.5%) dysregulated with uremia were similarly dysregulated in diseased kidneys, suggesting that plasma RNA profiles mirror the transcriptomal changes in diseased kidney tissues. Further analyses identified eight plasma lncRNAs as potential predictors of adverse CV outcomes in uremic patients. Validation study in an independent cohort of ESRD patients (n = 111) confirmed that elevated plasma lncRNA DKFZP434I0714 is a significant independent predictor of adverse CV outcomes in uremic patients. Additional experiments demonstrated the functional involvement of DKFZP434I0714 in the pathogenesis of endothelial dysfunction. Conclusions In summary, plasma lncRNA expression signature reflects the disease states of uremia. Elevated plasma levels of lncRNA DKFZP434I0714 in uremic patients portend a worse CV outcome and warrant closer monitoring and more aggressive management.

The four chambers of the human heart play distinct roles in the maintenance of normal cardiac function, and are differentially affected by inherited/acquired cardiovascular disease. To probe the molecular determinants of these functional differences, we examined mRNA and lncRNA expression profiles in the left (LA) and right (RA) atria, the left (LV) and right (RV) ventricles, and the interventricular septum (IVS) of non-failing human hearts (N = 8). Analysis of paired atrial and ventricular samples (n = 40) identified 5,747 mRNAs and 2,794 lncRNAs that were differentially (>1.5 fold; FDR 2,300 mRNAs and >1,200 lncRNAs, corresponding to 17–28% of the total transcripts, were differentially expressed. Heterogeneities in mRNA/lncRNA expression profiles in the LA and RA, as well as in the LV, RV and IVS, were also revealed, although the numbers of differentially expressed transcripts were substantially smaller. Gender differences in mRNA and lncRNA expression profiles were also evident in non-failing human atria and ventricles. Gene ontology classification of differentially expressed gene sets revealed chamber-specific enrichment of numerous signaling pathways.

Background Myocardial, transient, outward currents, I to , have been shown to play pivotal roles in action potential (AP) repolarization and remodeling in animal models. The properties and contribution of I to to left ventricular (LV) repolarization in the human heart, however, are poorly defined. Methods and Results Whole-cell, voltage-clamp recordings, acquired at physiological (35°C to 37°C) temperatures, from myocytes isolated from the LV of nonfailing human hearts identified 2 distinct transient currents, I to,fast ( I to,f ) and I to,slow ( I to,s ), with significantly ( P I to,f recovers in ≈10 ms, 100× faster than I to,s , and is selectively blocked by the Kv4 channel toxin, SNX-482. Current-clamp experiments revealed regional differences in AP waveforms, notably a phase 1 notch in LV subepicardial myocytes. Dynamic clamp-mediated addition/removal of modeled human ventricular I to,f , resulted in hyperpolarization or depolarization, respectively, of the notch potential, whereas slowing the rate of I to,f inactivation resulted in AP collapse. AP-clamp experiments demonstrated that changes in notch potentials modified the time course and amplitudes of voltage-gated Ca 2+ currents, I Ca . In failing LV subepicardial myocytes, I to,f was reduced and I to,s was increased, notch and plateau potentials were depolarized ( P P Conclusions I to,f and I to,s are differentially expressed in nonfailing human LV, contributing to regional heterogeneities in AP waveforms. I to,f regulates notch and plateau potentials and modulates the time course and amplitude of I Ca . Slowing I to,f inactivation results in dramatic AP shortening. Remodeling of I to,f in failing human LV subepicardial myocytes attenuates transmural differences in AP waveforms.

Purkinje neurons, the sole output of the cerebellar cortex, deliver GABA-mediated inhibition to the deep cerebellar nuclei. To subserve this critical function, Purkinje neurons fire repetitively, and at high frequencies, features that have been linked to the unique properties of the voltage-gated sodium (Nav) channels expressed. In addition to the rapidly activating and inactivating, or transient, component of the Nav current (INaT) present in many types of central and peripheral neurons, Purkinje neurons, also expresses persistent (INaP) and resurgent (INaR) Nav currents. Considerable progress has been made in detailing the biophysical properties and identifying the molecular determinants of these discrete Nav current components, as well as defining their roles in the regulation of Purkinje neuron excitability. Here, we review this important work and highlight the remaining questions about the molecular mechanisms controlling the expression and the functioning of Nav currents in Purkinje neurons. We also discuss the impact of the dynamic regulation of Nav currents on the functioning of individual Purkinje neurons and cerebellar circuits.

Neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, display daily rhythms in electrical activity with more depolarized resting potentials and higher firing rates during the day than at night. Although these daily variations in the electrical properties of SCN neurons are required for circadian rhythms in physiology and behavior, the mechanisms linking changes in neuronal excitability to the molecular clock are not known. Recently, we reported that mice deficient for either Kcna4 ( Kv1.4-/-) or Kcnd2 ( Kv4.2-/-; but not Kcnd3, Kv4.3-/-), voltage-gated K+ (Kv) channel pore-forming subunits that encode subthreshold, rapidly activating, and inactivating K+ currents (IA), have shortened (0.5 h) circadian periods in SCN firing and in locomotor activity compared with wild-type (WT) mice. In the experiments here, we used a mouse ( Per2Luc) line engineered with a bioluminescent reporter construct, PERIOD2::LUCIFERASE (PER2::LUC), replacing the endogenous Per2 locus, to test the hypothesis that the loss of Kv1.4- or Kv4.2-encoded IA channels also modifies circadian rhythms in the expression of the clock protein PERIOD2 (PER2). We found that SCN explants from Kv1.4-/-Per2Luc and Kv4.2-/- Per2Luc, but not Kv4.3-/-Per2Luc, mice have significantly shorter (by approximately 0.5 h) circadian periods in PER2 rhythms, compared with explants from Per2Luc mice, revealing that the membrane properties of SCN neurons feedback to regulate clock (PER2) expression. The combined loss of both Kv1.4- and Kv4.2-encoded IA channels in Kv1.4-/-/Kv4.2-/-Per2Luc SCN explants did not result in any further alterations in PER2 rhythms. Interestingly, however, mice lacking both Kv1.4 and Kv4.2 show a striking (approximately 1.8 h) advance in their daily activity onset in a light cycle compared with WT mice, suggesting additional roles for Kv1.4- and Kv4.2-encoded IA channels in controlling the light-dependent responses of neurons within and/or outside of the SCN to regulate circadian phase of daily activity.

International audience; Voltage-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.

Summary: The resurgent component of voltage-gated Na+ (Nav) currents, INaR, has been suggested to provide the depolarizing drive for high-frequency firing and to be generated by voltage-dependent Nav channel block (at depolarized potentials) and unblock (at hyperpolarized potentials) by the accessory Navβ4 subunit. To test these hypotheses, we examined the effects of the targeted deletion of Scn4b (Navβ4) on INaR and on repetitive firing in cerebellar Purkinje neurons. We show here that Scn4b−/− animals have deficits in motor coordination and balance and that firing rates in Scn4b−/− Purkinje neurons are markedly attenuated. Acute, in vivo short hairpin RNA (shRNA)-mediated “knockdown” of Navβ4 in adult Purkinje neurons also reduced spontaneous and evoked firing rates. Dynamic clamp-mediated addition of INaR partially rescued firing in Scn4b−/− Purkinje neurons. Voltage-clamp experiments revealed that INaR was reduced (by ∼50%), but not eliminated, in Scn4b−/− Purkinje neurons, revealing that additional mechanisms contribute to generation of INaR. : Loss of Navβ4 attenuates, but does not eliminate, the resurgent sodium current (INaR) in cerebellar Purkinje neurons, revealing that additional mechanism(s) contribute to the generation of INaR. Ransdell et al. also find that INaR magnitude tunes the firing rate of Purkinje neurons and that Navβ4−/− animals display balance and motor deficits. Keywords: cerebellum, resurgent sodium current, Scn4b−/−, Scn4b-targeted shRNA, dynamic clamp

Voltage-gated NaV channels are modulated by two different noncovalent accessory subunits: β1 and β3. Zhu et al. present data showing that β1 and β3 cause distinct effects on channel gating because they interact with NaV channels at different locations. β3 regulates the voltage sensor in domain III, whereas β1 regulates the one in domain IV., Voltage-gated Na+ (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound β1 and β3 subunits that regulate channel gating, expression, and pharmacology. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the β subunits affect the conformational dynamics of the cardiac NaV channel (NaV1.5) voltage-sensing domains (VSDs). The pore-forming NaV1.5 α subunit contains four domains (DI–DIV), each with a VSD. Our results show that β1 regulates NaV1.5 by modulating the DIV-VSD, whereas β3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the β3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to β3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that β3 binds proximally to the DIII-VSD. Molecular-level differences in β1 and β3 interaction with the α subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability.

Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/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.

Genotype-phenotype relationships are determinants of human diseases. Often, we know little about why so many genes are involved in complex common diseases. We hypothesized that this multigene effect arises from the relationship between genes and physiological dynamics. We tested this hypothesis for arrhythmias as physiological dynamics define this disease. We integrated graph theory analysis of genomic and protein-protein interaction networks with dynamical models of ion channel function to identify the physiological dynamics of genome wide variation for five different arrhythmias. Regulatory networks for the cardiac conduction system and arrhythmias were constructed from GWAS and known disease genes. Electrophysiological models of myocyte action potentials were used to conduct extensive parameter variations to identify robust and fragile kinetic parameters that were then, using regulatory networks, associated with genomic determinants. We find that genome-wide determinants of arrhythmias that represent many cellular processes are selectively associated with fragile physiological dynamics of ion channel kinetics. This association predicts disease propensity. Deep RNA sequencing from human left ventricular tissue of arrhythmia and control subjects confirmed the predictive relationship. Taken together these studies show that the varied multigene effects of arrhythmias arises because of associations with fragile kinetic parameters of cardiac electrophysiology.Significance StatementOur understanding of the genetics of common diseases has advanced exponentially over the past decade. We now know that differences and variation in multiple genes contribute to disease susceptibility with significant heterogeneity in the phenotype. However, how genetic variation contributes to disease phenotypes remains unknown. We hypothesized that the relationships between physiological dynamics and genetic architecture is a fundamental determinant of disease susceptibility and genetic heterogeneity. To test our hypothesis, we integrated mathematical models of cardiac electrophysiology with genetic network models of cardiac arrhythmias. We found that disease related genome variants were selectively associated with fragile kinetic parameters that predict disease propensity and identified several novel cellular processes associated with arrhythmogenesis.

SummaryThe promise of using reprogrammed human neurons for disease modeling and regenerative medicine relies on the ability to induce patient-derived neurons with high efficiency and subtype specificity. We have previously shown that ectopic expression of brain-enriched microRNAs (miRNAs), miR-9/9∗ and miR-124 (miR-9/9∗-124), promoted direct conversion of human fibroblasts into neurons. Here we show that coexpression of miR-9/9∗-124 with transcription factors enriched in the developing striatum, BCL11B (also known as CTIP2), DLX1, DLX2, and MYT1L, can guide the conversion of human postnatal and adult fibroblasts into an enriched population of neurons analogous to striatal medium spiny neurons (MSNs). When transplanted in the mouse brain, the reprogrammed human cells persisted in situ for over 6 months, exhibited membrane properties equivalent to native MSNs, and extended projections to the anatomical targets of MSNs. These findings highlight the potential of exploiting the synergism between miR-9/9∗-124 and transcription factors to generate specific neuronal subtypes.

The Kv2.1 delayed rectifier potassium channel exhibits high-level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity-dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1(-/-) ) mice lacking this channel. Kv2.1(-/-) mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1(-/-) mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1(-/-) mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1(-/-) animals. Field recordings from hippocampal slices of Kv2.1(-/-) mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1(-/-) mice, long-term potentiation at the Schaffer collateral - CA1 synapse is decreased. Kv2.1(-/-) mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1(-/-) mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1(-/-) mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function.

Rapidly activating and inactivating A-type K+currents (IA) encoded by Kv4.2 and Kv4.3 pore-forming (α) subunits of the Kv4 subfamily are key regulators of neuronal excitability. Previous studies have suggested a role for Kv4.1 α-subunits in regulating the firing properties of mouse suprachiasmatic nucleus (SCN) neurons. To test this, we utilized an RNA-interference strategy to knockdown Kv4.1, acutely and selectively, in the SCN. Current-clamp recordings revealed that thein vivoknockdown of Kv4.1 significantly (p< 0.0001) increased mean ± SEM repetitive firing rates in SCN neurons during the day (6.4 ± 0.5 Hz) and at night (4.3 ± 0.6 Hz), compared with nontargeted shRNA-expressing SCN neurons (day: 3.1 ± 0.5 Hz; night: 1.6 ± 0.3 Hz). IAwas also significantly (p< 0.05) reduced in Kv4.1-targeted shRNA-expressing SCN neurons (day: 80.3 ± 11.8 pA/pF; night: 55.3 ± 7.7 pA/pF), compared with nontargeted shRNA-expressing (day: 121.7 ± 10.2 pA/pF; night: 120.6 ± 16.5 pA/pF) SCN neurons. The magnitude of the effect of Kv4.1-targeted shRNA expression on firing rates and IAwas larger at night. In addition, Kv4.1-targeted shRNA expression significantly (p< 0.001) increased mean ± SEM nighttime input resistance (Rin; 2256 ± 166 MΩ), compared to nontargeted shRNA-expressing SCN neurons (1143 ± 93 MΩ). Additional experiments revealed that acute knockdown of Kv4.1 significantly (p< 0.01) shortened, by ∼0.5 h, the circadian period of spontaneous electrical activity, clock gene expression and locomotor activity demonstrating a physiological role for Kv4.1-encoded IAchannels in regulating circadian rhythms in neuronal excitability and behavior.

The axon initial segment (AIS) is highly enriched in the structural proteins ankyrin G and βIV-spectrin, the pore-forming (α) subunits of voltage-gated sodium (Nav) channels, and functional Nav channels, and is critical for the initiation of action potentials. We previously reported that FGF14, a member of the intracellular FGF (iFGF) sub-family, is expressed in cerebellar Purkinje neurons and that the targeted inactivation of Fgf14 in mice (Fgf14(-/-)) results in markedly reduced Purkinje neuron excitability. Here, we demonstrate that FGF14 immunoreactivity is high in the AIS of Purkinje neurons and is distributed in a decreasing, proximal to distal, gradient. This pattern is evident early in the postnatal development of Purkinje neurons and is also observed in many other types of central neurons. In (Scn8a(med)) mice, which are deficient in expression of the Nav1.6 α subunit, FGF14 immunoreactivity is markedly increased and expanded in the Purkinje neuron AIS, in parallel with increased expression of the Nav1.1 (Scn1a) α subunit and expanded expression of βIV-spectrin. Although Nav1.1, FGF14, and βIV-spectrin are affected, ankyrin G immunoreactivity at the AIS of Scn8a(med) and wild type (WT) Purkinje neurons was not significantly different. In Fgf14(-/-) Purkinje neurons, βIV-spectrin and ankyrin G immunoreactivity at the AIS were also similar to WT Purkinje neurons, although both the Nav1.1 and Nav1.6 α subunits are modestly, but significantly (p

Translational studies that assess and extend observations made in animal models of human pathology to elucidate relevant and important determinants of human diseases require the availability of viable human tissue samples. However, there are a number of technical and practical obstacles that must be overcome in order to perform cellular and electrophysiological studies of the human heart. In addition, changing paradigms of how diseases are diagnosed, studied and treated require increasingly complex integration of rigorous disease phenotyping, tissue characterization and detailed delineation of a multitude of "_omics". Realizing the need for quality-controlled human cardiovascular tissue acquisition, annotation, biobanking and distribution, we established the Translational Cardiovascular Biobank & Repository at Washington University School of Medicine. Several critical details are essential for the success of cardiovascular biobanking including coordinated, trained and dedicated staff members; adequate, nonrestrictive informed consent protocols; and fully integrated clinical data management applications for annotating, tracking and sharing of tissue and data resources. Labor and capital investments into growing biobanking resources will facilitate collaborative efforts aimed at limiting morbidity and mortality due to heart disease and improving overall cardiovascular health.

International audience; Intracellular Fibroblast Growth Factor 14 (iFGF14) and the other intracellular FGFs (iFGF11-13) regulate the properties and densities of voltage-gated neuronal and cardiac Na(+) (Nav) channels. Recent studies have demonstrated that the iFGFs can also regulate native voltage-gated Ca(2+) (Cav) channels. In the present study, a mass spectrometry (MS)-based proteomic approach was used to identify the components of native cerebellar iFGF14 complexes. Using an anti-iFGF14 antibody, native iFGF14 complexes were immunoprecipitated from wild type adult mouse cerebellum. Parallel control experiments were performed on cerebellar proteins isolated from mice (Fgf14(-/-)) harboring a targeted disruption of the Fgf14 locus. MS analyses of immunoprecipitated proteins demonstrated that the vast majority of proteins identified in native cerebellar iFGF14 complexes are Nav channel pore-forming (α) subunits or proteins previously reported to interact with Nav α subunits. In contrast, no Cav channel α or accessory subunits were revealed in cerebellar iFGF14 immunoprecipitates. Additional experiments were completed using an anti-PanNav antibody to immunoprecipitate Nav channel complexes from wild type and Fgf14(-/-) mouse cerebellum. Western blot and MS analyses revealed that the loss of iFGF14 does not measurably affect the protein composition or the relative abundance of Nav channel interacting proteins in native adult mouse cerebellar Nav channel complexes.