Jeanne M. Nerbonne - US grants

There is substantial evidence to suggest that the membrane properties of individual cortical neurons are functionally as important in determining the overall behavior of cortical circuits as are the attributes of the interconnections between cells. This conclusion seems valid not only in the context of normal cortical functioning but, also, in certain pathological conditions as well. In some epileptic disorders, for example, in which there is abnormal electrical activity in the cortex, epileptic discharges occur not only in single cells, but, also, sychronously in synaptically connected groups of neurons. The initiation and the spread of activity are apparently determined by a combination of the endogenous membrane properties of individual cells and the specific interactions among the various neuronal components of the circuitry. The goal of the research outlined in this proposal is: (1) to isolate, from defined circuits in rat primary visual cortex, neurons with identified connections and known developmental fates; and (2) to characterize the ionic currents (and transmitter related properties) of these cells. The long range goal of the work is to provide an understanding of the mechanisms controlling bursting activity in identified (single) cells and the sychronized firing of population cells (including identified cells) within defined cortical circuits. Using the whole-cell patch-clamp recording method, we shall study the electrophysiological properties of single corpus callosum projecting, geniculate projecting and intrinsic cortical neurons in dissociated cell culture. These cell types will be identified following postnatal, intracerebral injections and retrograde axonal transport of fluorescent markers. In the initial experiments, particular emphasis will be placed on characterizations and comparisons of voltage-activated currents. Later experiments will focus on the postsynaptic effects of the inhibitory neurotransmitter, GABA, on identified cells. GAD immunocytochemistry will be employed to determine the GABAergic nature of identified neurons and to verify the presence of GABAergic terminals on retrogradely labelled cortical cells in culture.

DESCRIPTION (provided by applicant): Multiple types of voltage-gated K+ (Kv) channels with distinct time- and voltage-dependent properties and pharmacological sensitivities have been identified in the mammalian myocardium. This diversity has a physiological significance in that the various Kv channels play distinct roles in controlling action potential waveforms and refractoriness. Although considerable progress has been made in identifying the Kv channel pore-forming (?) subunits that encode diverse cardiac Kv channels, the functional roles of the Kv channel accessory subunits (minK/ MiRPs, Kv?, KChAP, KChIP, DPPX) are rather poorly understood. Studies in heterologous expression systems suggest that Kv accessory subunits can modulate the properties of a variety of Kv ? subunit encoded channels and that each type of Kv channel likely is modulated by multiple accessory subunits. Other recent studies suggest that cardiac Kv (and other) channels function as components of macromolecular protein complexes, comprising pore-forming and accessory subunits, as well as additional regulatory proteins that influence channel properties and mediate interactions with the actin cytoskeleton and the extracellular matrix. To define the physiological roles of the Kv?1, KChlP2 and DPP6 subunits, the studies proposed here will probe directly the functioning of these subunits in the generation of the native Kv channels, lto,f, Ito.s, IK,slow and Iss, in intact cardiac (mouse ventricular) myocytes. The expression levels or the properties of the accessory subunits will be manipulated in vivo and in vitro, and the functional consequences of these manipulations on the properties and cell surface expression of myocardial lto,f, Ito.s, IK,slow and Iss will be determined directly (and simultaneously). The proposed studies will reveal whether individual Kv channel types are regulated/modulated by multiple Kv accessory subunits. In addition, these studies will allow direct testing of the hypothesis that Kv accessory subunits are multifunctional, regulating/modulating the functioning of multiple types of (Kv a subunit encoded) cardiac Kv channels. We anticipate that these studies will provide fundamentally important new insights into the role of Kv channel accessory subunits in the dynamic regulation of cardiac Kv channel macromolecular complexes.

Neurons are the cells of the brain that are the units for information processing. Understanding the properties of their cell membranes, including excitability and neurotransmission between neurons, is crucial to an understanding of brain function. In complex mammalian brains it has been technically difficult to study membrane properties of identifiable cells, particularly in the cerebral cortex. Conversely, the isolation of single cells in a dish ("in vitro") for membrane recordings makes it difficult to identify the type of neurons these cells were in the normal brain. This project will provide a new approach to try to establish reliable methods to culture and identify cell types or classes isolated from the cortex, and characterize the physiological and pharmacological responses of those neurons. Cells that project across the brain hemispheres, called callosal neurons, can be distinctly labelled by their uptake of non-toxic microscopic fluorescent beads. These cells will then be studied in vitro, either as cultured dissociated cells after removal from newborn rats, or in a thin-slice preparation taken from whole brain. Microelectrodes will be used to test the response of these cells to electrical stimuli across their membranes, to examine the nature of the currents of charged ionic particles that drive the excitable properties of the cells. These studies will compare changes in the cell properties with time since isolation or with time since birth, and will look for possible functional differ- ences among cells that appear morphologically similar. This approach to classify cortical cell types on the basis of membrane properties is technically very demanding, but success will be a breakthrough offering a powerful new way for electrophysiologists to study cortical processing.

The long term goal of this research program is to define the roles of various types of ion channels in the functioning of individual cortical neurons and complex cortical circuits. To achieve this goal, it is necessary to characterize the properties of the ion channels expressed in different cortical cell types, to determine the functional roles of these channels in controlling the firing properties of cortical neurons, and to delineate the mechanisms involved in the regulation and modulation of these channels by membrane voltage, neurotransmitters and intracellular second messengers. Our present focus is on intrinsic membrane properties, and the specific hypothesis being tested is that the presence of various types of ion channels underlies the expression of the "regular-spiking", "intrinsically bursting" and "fast-spiking" electrophysiological phenotypes distinguished in in vivo and in vitro recordings from cortical neurons. To test this hypothesis directly, we have developed methods that enable us to identify callosal-projecting, superior colliculus-projecting and GABAergic, inhibitory (rat) visual cortical neurons in vitro and to characterize the intrinsic membrane properties of these cells in detail. Each of these cell populations was selected to correspond to one of the three phenotypes distinguished in in vitro cortical slice recordings. This research proposal is specifically focussed on examining the properties and the functional roles of the depolarization-activated and Ca++-activated K+ channels expressed in these three, distinct cortical cell types. Using the whole- cell variation of the patch clamp recording technique, initial experiments will characterize the time- and voltage-dependent properties and pharmacological sensitivities of the depolarization-activated K+ currents in isolated, identified callosal-projecting, superior colliculus-projecting and GABAergic inhibitory cortical neurons. Subsequent experiments on isolated cells and on identified cells in in vitro cortical slices will be aimed at determining the role of depolarization-activated K+ currents in shaping the waveforms of action potentials and in controlling the overall firing properties of these three cortical cell types. In the second phase of the project, a similar set and sequence of experiments will be completed to characterize the properties and the functional roles of the Ca++-activated K+ channels expressed in callosal-projecting, superior colliculus-projecting and GABAergic, inhibitory cortical neurons. It is unlikely that any direct clinical applications will result from the studies outlined in this proposal. It is expected, however, that the proposed experiments will clarify the types, distributions, and properties of the depolarization-activated and Ca++-activated K+ channels expressed in diverse neocortical cell types and provide insights into the functional roles of these K+ channels in controlling the firing properties of these cells. In addition, it is anticipated that insights gleaned from these studies will facilitate future functional analysis of the cortical circuits in which these different cell types participate.

DESCRIPTION (provided by applicant): It is well documented that membrane excitability and excitation-contraction coupling are altered in the hypertrophied heart, and that ventricular hypertrophy is a risk factor for the development of life-threatening cardiac arrhythmias. Considerable evidence has accumulated to suggest that "electrical remodelling" occurs in the hypertrophied heart and that this reflects, at least in part, changes in the xpression and/or the properties of the voltage-gated K+ (Kv) currents that underlie myocardial action potential repolarization. The mechanisms involved in Kv channel remodeling in the hypertrophied ventricular myocardium, however, have not been delineated. The experiments proposed here will explore directly the molecular mechanisms underlying Kv channel remodeling in a mouse model of pressure overload-induced left ventricular hypertrophy (LVH). Regional differences in the effects of LVH on the functional expression, the properties and/or the distributions of ventricular Kv channels, particularly the transient outward K+ channels, I(to,f) will be determined, and experiments focused on delineating the roles of elevated intracellular Ca2+, Kv channel accessory subunits (KChIP2 and Kv-beta1) and the actin cytoskeleton in regulating functional I(to,f) channel expression will be completed. A sophisticated combination of electrophysiological, biochemical, molecular genetic, immunohistochemical and imaging techniques will be exploited in mice to achieve the stated aims of this proposal. We anticipate that the studies outlined here will provide fundamentally important new insights into the effects of pressure overload-induced left ventricular hypertrophy on repolarizing Kv channels, as well as into the molecular mechanisms underlying "electrical remodelling" in the hypertrophied heart.. In the long term, these insights should translate into more effective treatment strategies to reduce the risk of sudden death and the mortality and morbidity associated with myocardial hypertrophy and failure.

DESCRIPTION (provided by applicant): In the mammalian nervous system, a variety of voltage-gated potassium (K+) channels with distinct time- and voltage-dependent properties and pharmacological sensitivities have been identified. This heterogeneity has a physiological significance in that the various K+ channels function to control resting membrane potentials, action potential waveforms, repetitive firing patterns and the responses to synaptic inputs. The cloning of voltage-gated K+ channel (Ky) pore-forming (cc) and accessory (B, KChAPs, KChIPs) subunits has revealed even greater potential for generating K+ channel diversity than was expected, and the relationships between these subunits and the K+ channels in mammalian neurons is poorly understood. Here, we propose to exploit molecular genetic strategies to identify the molecular correlates of the voltage-gated K+ currents, 'Al' IAs, 'K' and ISS, in sympathetic neurons isolated from the (rat) superior cervical ganglion (SCG), and to define the functional roles of IAF, 'As' 'K' and ISS. Initial experiments will test the hypothesis that there are two molecularly distinct types of IAf channels (encoded by Kv1 and Kv4 a subunits) and determine the functional consequences of removing all IAf channels on the firing properties of SCG cells. Experiments in aims 2 and 3 will test the hypotheses that Ky alpha subunits of the Kv2 and Kv3 subfamilies underlie 'IK and IAs' respectively, in SCG neurons and define the roles 'K and IAs in shaping action potential waveforms and repetitive firing in these cells. The final aim will explore the role(s) of the accessory KChIP proteins in the generation of functional voltage-gated K+ channels in SCG neurons. We anticipate that the studies outlined in this proposal will provide fundamentally important new insights into the molecular basis of functional voltage-gated K+ channel diversity in mammalian sympathetic neurons and into the roles of these K+ channels in the regulation neuronal excitability. Importantly, it seems likely that the multifaceted experimental approach developed here can also be applied to determine the molecular compositions and functional roles of voltage-gated (and other) K+ channels in other cells.

DESCRIPTION (provided by applicant): This proposal requests continued support of a Training Program in Cardiovascular Biology that was established at Washington University in 1977 to support Predoctoral Students conducting Ph.D. thesis research and Postdoctoral Fellows in the early stages of post-graduate training. The goals of this Program are to provide outstanding research opportunities, well-rounded, multidisciplinary training in Modern Cardiovascular Biology, and mentoring to Predoctoral and Postdoctoral Trainees in the laboratories of the participating faculty to prepare these individuals to be productive, independent scientists. The critical components and the clear strengths of this Program are the 20 participating training faculty and the trainees themselves. Another important strength of this Program and, indeed, of the overall environment at Washington University, is the highly collaborative nature of our research efforts. This interactive environment and these collaborations expand the research, training and mentoring opportunities provided to the trainees. The faculty derive from seven departments, and include nationally and internationally recognized leaders in several specific areas of Modern Cardiovascular Biology including molecular biology, physiology, cell biology, biochemistry, pathology, genetics, and human cardiac disease mechanisms. The faculty are well established, well-funded, experienced and highly productive investigators, and all are committed to providing the training, experience, resources, intellectual enthusiasm and mentoring needed to achieve the overall goals of the Training Program and to facilitate the professional development of the individual Program Trainees. The strengths of this Program are manifest in the quantity and quality of our scientific output, in the quality of our present and past trainees, and in the achievements of our past trainees. In addition to the research training provided in individual and collaborating research laboratories, this Training Program provides instruction in "Ethics in Biomedical Research," and provides trainees the opportunity to participate in the "Core Cardiovascular Biology Curriculum," the weekly "Trainees in Cardiovascular Biology Series" and the weekly "Cardiovascular Research Seminar Series," all developed and operated by this Training Grant. This Training Program is also actively involved in monitoring the progress and professional development and in the mentoring of Predoctoral and Postdoctoral Trainees. (End of Abstract)

DESCRIPTION (provided by applicant): Voltage-gated K+ (Kv) channels are key regulators of neuronal excitability, functioning in the control of resting membrane potentials, action potential waveforms, repetitive firing properties, and in modulating the responses to synaptic inputs and synaptic plasticity. Molecular cloning has provided insights into the basis of neuronal Kv channel diversity with the identification of large numbers of Kv channel pore-forming (1) and accessory (2) subunits. Accumulating evidence suggests that neuronal Kv channels function as components of macromolecular protein complexes, comprising (four) Kv 1 and multiple Kv2 subunits and regulatory proteins, although the roles of accessory and regulatory proteins in controlling neuronal Kv channel expression, distribution and functioning are poorly understood. This new R21 proposal will test the novel hypothesis that voltage-gated Na+ (Nav) channel accessory (Nav2) subunits regulate the functional expression of the voltage- gated K+ (Kv) channels underlying the rapidly activating and inactivating, Kv4.2--encoded "A" currents (IA) in cortical pyramidal neurons rather than, or in addition to, regulating voltage-gated Na+ (Nav) channels. This hypothesis reflects recent biochemical findings demonstrating the Nav21 (SCN1b) and Nav22 (SCN2b) subunits co-immunoprecipitate with Kv4.2 from brain in Kv4.2--encoded IA channel macromolecular protein complexes. There are two related aims in this proposal, and these will be pursued simultaneously. Specifically, the studies here will test the hypothesis that Nav21 functions to regulate Kv4.2-encoded IA channels (aim #1), rather than, or in addition to, Nav channels (aim #2) in cortical pyramidal neurons and determine directly the role of Nav21.in regulating the firing properties of these cells (aim #2). To achieve these aims, the expression of endogenous Nav21 will be manipulated in vitro using targeted gene "knockdown" strategies with small interfering RNAs (siRNAs), and the functional consequences of these manipulations on the properties and the cell surface expression of IA (and Nav) channels in (mouse) cortical pyramidal neurons will be determined. Parallel experiments will be complete on cortical pyramidal neurons from mice (SCN1b-/- ) harboring a targeted disruption of the SCN1b (Nav21) locus. It is anticipated that these studies will provide fundamentally important new insights into the mechanisms that regulate the expression and the functioning of macromolecular neuronal Kv channel complexes. In addition, the results of the studies here will guide future investigates in this and other laboratories focused on delineating the molecular, cellular and systemic mechanisms involved in the dynamic regulation of neuronal membrane excitability and on defining the functional consequences of mutations in SCNxb subunits linked to derangements in neuronal excitability. PUBLIC HEALTH RELEVANCE: Voltage-gated potassium (Kv) channels are key determinants of neuronal membrane excitability, functioning in the control of resting membrane potentials, action potential waveforms, repetitive firing properties, the responses to synaptic inputs and synaptic plasticity. Although accumulating evidence suggests that neuronal Kv channels function as components of macromolecular protein complexes, comprising pore- forming (1) subunits and a variety of accessory (2) subunits that affect channel stability, trafficking and/or properties, very little is presently known about the roles of accessory subunits in the physiological regulation of neuronal Kv channels. Exploiting molecular genetic strategies to manipulate channel subunits in vivo and in vitro, this new research program is focused on defining the physiological role(s) of the Nav2 (SCNxb) accessory subunits in regulating the excitability of cortical neurons and on testing the novel hypothesis that the Nav2 accessory subunits actually function to regulate Kv channels rather than, or in addition to, regulating voltage-gated Na+ (Nav) channels. These studies will provide new and fundamentally important insights into the physiological roles of Nav2 subunits in the regulation of neuronal Kv channels and into the molecular mechanisms controlling neuronal functioning and plasticity.

DESCRIPTION (provided by applicant): Recent studies suggest that FGF14, a member of the intracellular fibroblast growth factor (iFGF) subfamily functions as a novel regulator of neuronal excitability. The major phenotype in mice lacking Fgf14 (Fgf14-/-) is ataxia and mutations in FGF14 in humans cause a progressive spinocerebellar ataxia syndrome, SCA27. It has also been demonstrated that FGF14, and other iFGFs interact with the C-terminal domains of voltage-gated Na+ (Nav) channel pore-forming (1) subunits and modulate the properties of heterologously expressed Nav channels. In addition, exploiting a validated (in Fgf14-/-mice) anti-FGF14 specific antibody, we find that FGF14 co- localizes with Nav channel 1 subunits at the ankyrin G-rich axon initial segments (AIS) in cerebellar Purkinje neurons, These observations led us to hypothesize that loss of FGF14 produces a defect in the firing properties of Purkinje neurons, the sole output neurons of the cerebellar cortex. In preliminary studies focused on exploring this hypothesis directly, we found that spontaneous activity and repetitive firing were decreased significantly in Fgf14-/-, compared with wild type, Purkinje neurons. Additional preliminary studies revealed that the expression and AIS localization of the Nav channel 1 subunit, Nav1.6, was reduced markedly in Fgf14-/- Purkinje neurons, whereas Ankyrin G expression at the AIS was not significantly affected. These findings suggest that FGF14- Nav 1 subunit interactions play a critical role in regulating the expression and/or the AIS localization of Nav channels and in controlling the firing (output) properties of cerebellar Purkinje neurons. The experiments outlined in this proposal will test these hypotheses directly and explore the molecular mechanisms involved in mediating the effects of FGF14 on the expression, localization and functioning of Nav channels in cerebellar Purkinje neurons. Additional experiments will be focused on testing directly the hypothesis that the SCA27-linked FGF14 mutant protein, FGF14F145S, functions in vivo as a dominant negative to disrupt the interaction between the wild type FGF14 protein and Nav channel 1 subunits, thereby reducing Nav channel expression/localization and altering the firing properties of cerebellar Purkinje neurons. It is anticipated that these studies will provide new and fundamentally important insights into the functional roles of FGF14 and into the underlying molecular mechanisms involved in FGF14- mediated effects on neuronal excitability. PUBLIC HEALTH RELEVANCE: Health relatedness statement (two or three sentences, describe the relevance of this research to public health) SCA27 is a dominantly inherited spinocerebellar ataxia (SCA) syndrome caused by mutations in the FGF14 gene. SCA27 is characterized by progressive ataxia, cerebellar degeneration and cognitive impairment, and is phenotypically very similar to mice that lack a functional Fgf14 gene. The molecular, cellular and physiological studies proposed will provide new and fundamentally important insights into the functional roles of FGF14 in regulating neuronal excitability and into the underlying molecular mechanisms by which mutations in FGF14 cause disease in humans.

Description (provided by applicant): This renewal application requests support for a Training Program in Cardiovascular Biology, first established at Washington University in 1977, to support Predoctoral Students conducting Ph.D. thesis research and Postdoctoral Fellows in the early stages of post-graduate training. The goals of this Program are to provide outstanding research opportunities, well-rounded, multidisciplinary training in Modern Cardiovascular Biology, and mentoring to Predoctoral and Postdoctoral Trainees in the laboratories of the participating faculty to prepare these individuals to be productive, independent scientists. The critical components and the clear strengths of this Program are the 22 participating training faculty and the trainees themselves. Another important strength of this Program and, indeed, of the overall environment at Washington University, is the highly collaborative nature of our research efforts. This interactive environment and these collaborations expand the research, training and mentoring opportunities provided to the trainees. The faculty derive from multiple (6) Departments (Biochemistry, Biomedical Engineering, Cell Biology and Physiology, Developmental Biology, Medicicne and Radiology) and (4) Divisions (Cardiology, Chemistry, Endocrinology and Nutritional Science) within the Department of Medicine, and include nationally and internationally recognized leaders in several specific areas of Modern Cardiovascular Biology including molecular biology, physiology, cell biology, biochemistry, modeling, imaging, pathology, genetics, and human cardiovascular disease mechanisms. The faculty are well-established, well-funded, experienced and highly productive investigators, and all are committed to providing the training, experience, resources, intellectual enthusiasm and mentoring needed to achieve the overall goals of the Training Program and to facilitate the professional development of the individual Program Trainees. The strengths of this Program are manifest in the quantity and quality of our scientific output, in the quality of our present and past trainees, and in the achievements of our past trainees. In addition to the research training provided in individual and collaborating research laboratories, this Training Program provides instruction in "Ethics in Biomedical Research," and provides trainees the opportunity to participate in the "Core Cardiovascular Biology Curriculum," the weekly "Trainees in Cardiovascular Biology Series" and the weekly "Cardiovascular Research Seminar Series," all developed and operated by this Training Grant. This Training Program is also actively involved in monitoring the progress and professional development and in the mentoring of Predoctoral and Postdoctoral Trainees. RELEVANCE (See instructions): This Program will provide outstanding research opportunities, training and mentoring to Predoctoral and Postdoctoral Trainees in Modern Cardiovascular Biology, including cardiovascular physiology, biochemistry, cell biology, genetics, imaging, modeling, and disease mechanisms. This Program will contribute to the training and professional development of the next generation of outstanding investigators focused on addressing important, unanswered questions in Cardiovascular Biology and Cardiovascular Disease.

DESCRIPTION (provided by applicant): The amplitudes and durations of cardiac action potentials are largely determined by voltage-gated K+ (Kv) channels, and in most cardiac cells, multiple Kv currents with distinct time- and voltage-dependent properties are co-expressed. Important insights into the potential molecular basis of functional myocardial Kv channel diversity were provided with the identification of large numbers of Kv channel pore-forming (1) and accessory (2) subunits. In addition, accumulating evidence suggests that myocardial Kv channels function as components of macromolecular protein complexes, comprising (four) Kv 1 and multiple Kv2 subunits and regulatory proteins, although the roles of accessory and regulatory proteins in controlling the functional cell surface expression and the properties of myocardial Kv channel are poorly understood. This new R21 proposal will test the novel hypothesis that voltage-gated Na+ (Nav) channel accessory (Nav2) subunits regulate the functional expression of Kv4-encoded fast, transient outward Kv (Ito,f) channels in ventricular myocytes rather than, or in addition to, regulating voltage-gated Na+ (Nav) channels. This hypothesis reflects recent biochemical findings demonstrating that the Nav21 (SCN1b) and Nav22 (SCN2b) subunits co- immunoprecipitate with Kv4 1 subunits in native Kv4-encoded Kv channel macromolecular protein complexes. There are two related aims in this proposal, and these will be pursued in parallel. Specifically, the studies here will test the novel hypothesis that Nav21 functions to regulate the cell surface expression and/or the properties of Kv4-encoded myocardial Ito,f channels (aim #1) rather than, or in addition to, regulating Nav channels (aim #2) and determine directly the role of Nav21 in shaping myocardial action potential waveforms.(aim #2). To achieve these aims, the expression of the endogenous Nav21 subunit will be manipulated in (mouse) ventricular myocytes in vitro using targeted gene "knockdown" strategies with small interfering RNAs (siRNAs), and the functional consequences of these manipulations on the properties and the cell surface expression of Ito,f (and Nav) channels will be determined. Parallel experiments will be completed on myocytes isolated from mice (Scn1b-/- ) harboring a targeted disruption of the Scn1b (Nav21) locus. It is anticipated that these studies will provide new and fundamentally important insights into the mechanisms that control the expression and the functioning of macromolecular Kv channel complexes. In addition, the results of the studies here will guide future investigations focused on delineating the molecular, cellular and systemic mechanisms involved in the dynamic regulation of myocardial membrane excitability and in the derangements in cardiac excitability linked to mutations in SCN1b. PUBLIC HEALTH RELEVANCE: Voltage-gated potassium (Kv) channels control the heights and durations of myocardial action potentials and contribute importantly the generation of normal cardiac rhythms. Changes in Kv channel expression and/or properties are observed in a number of inherited and acquired cardiac diseases, and these changes can have profound physiological consequences, including increasing the risk of potentially life-threatening cardiac arrhythmias. Although accumulating evidence suggests that myocardial Kv channels function as components of macromolecular protein complexes, comprising pore-forming (1) subunits and a variety of accessory (2) subunits that affect channel stability, trafficking and/or properties, very little is presently known about the roles of accessory subunits in the physiological regulation of Kv channels in cardiac myocytes. Exploiting molecular genetics strategies to manipulate channel subunits in vivo and in vitro, this new research program is focused on defining the physiological role(s) of the Nav2 (SCNxb) accessory subunits in regulating the excitability of cardiac myocytes and on testing the novel hypothesis that the Nav2 accessory subunits function to regulate Kv channels rather than, or in addition to, regulating voltage- gated Na+ (Nav) channels. These studies will provide new and fundamentally important insights into the physiological roles of Nav2 subunits in the myocardium and into the molecular mechanisms controlling myocardial membrane excitability.

DESCRIPTION (provided by applicant): Voltage-gated Na+ (Nav) channels are responsible for the rapid upstroke of the action potential in cardiac cells and play critical roles in controlling action potential durations and propagation. The primary Nav pore- forming (1) subunit in the myocardium is Nav1.5, encoded by SCN5A, and mutations in SCN5A have been linked to a number of cardiac rhythm disorders, including Long QT3 syndrome, Brugada syndrome, cardiac conduction disease, sick sinus syndrome, and atrial fibrillation. Accumulating evidence suggests that myocardial Nav channels function in multimeric protein complexes, comprising one Nav1 subunit, accessory (2) subunits and a number other accessory/regulatory proteins, although the roles of accessory and regulatory proteins in controlling channel expression, properties and subcellular distributions are not well understood. This R21 proposal will test the hypothesis that intracellular fibroblast growth factors (iFGFs) function as novel regulators of myocardial Nav1.5-encoded channels. This hypothesis is motivated by recent preliminary studies demonstrating that iFGF13 is expressed in adult and neonatal (mouse) ventricles and that iFGF13-targeted RNA interference markedly attenuates Nav current densities in (neonatal mouse ventricular) myocytes. There are two related aims in this proposal, and these will be pursued in parallel. Specifically, the studies outlined here will test the hypothesis that iFGF13 selectively regulates ventricular Nav currents and plays a physiological role in the generation of ventricular action potentials (aim #1). Parallel studies will explore the hypothesis that iFGF13 functions to regulate the stability, the trafficking and/or the subcellular localization of Nav1.5-encoded ventricular Nav channels (aim #2). To achieve these aims, the expression of iFGF13 will be manipulated in (mouse) ventricular myocytes in vitro using targeted gene "knockdown" strategies with small interfering RNAs (siRNAs), and the functional consequences of these manipulations on the properties and the cell surface expression of Nav (and other) channels will be determined. Parallel experiments will be completed on myocytes isolated from mice (Fgf13-/-) harboring a targeted disruption of the Fgf13 locus. It is anticipated that the studies proposed here will provide new and fundamentally important insights into the role(s) of the iFGFs in the dynamic regulation of myocardial Nav channels. In addition, the results of these studies will guide future investigations focused on delineating the molecular, cellular and systemic mechanisms involved in the dynamic regulation of myocardial membrane excitability and in the derangements in cardiac excitability linked to mutations in SCN5A. In the long term, it is anticipated that these studies will provide important new insights into the potential of the iFGFs as therapeutic targets to modulate Nav channel functioning in inherited and acquired cardiac rhythm disorders. PUBLIC HEALTH RELEVANCE: In the heart, voltage-gated sodium Na+ (Nav) channels are responsible for the rapid upstroke of the action potential and play roles in controlling action potential durations and propagation. These channels, therefore, are critical for the generation of normal cardiac rhythms. Changes in Nav channel expression and/or properties are observed in a number of inherited and acquired cardiac diseases, and these changes can have profound physiological consequences, including increasing the risk of potentially life-threatening cardiac arrhythmias. Accumulating evidence suggests that myocardial Nav channels function as components of macromolecular protein complexes, comprising pore-forming (1) subunits and a variety of accessory (2) subunits, although very little is presently known about the roles of these accessory subunits in the regulation of myocardial Nav channel stability, trafficking and/or properties. Combining in vivo and in vitro molecular genetic strategies with electrophysiological and biochemical approaches, this new research program is focused on defining the physiological role(s) of the novel family of Nav channel regulatory proteins, the intracellular fibroblast growth factors (iFGFs), in the regulation of myocardial Nav channel expression and functioning. These studies will provide new and fundamentally important insights into the physiological roles of the iFGFs in the dynamic regulation of myocardial Nav channels and myocardial membrane excitability. In the long term, these studies are also expected to provide important new insights into the potential of the iFGFs as therapeutic targets to modulate Nav channel functioning in inherited and acquired cardiac rhythm disorders.

This renewal application requests support for a Training Program in Cardiovascular Biology, first established at Washington University in 1977, to support Predoctoral Students conducting Ph.D. thesis research and Postdoctoral Fellows in the early stages of post-graduate training. The goals of this Program are to provide outstanding research opportunities, well-rounded, multidisciplinary training in Modern Cardiovascular Biology, and mentoring to Predoctoral and Postdoctoral Trainees in the laboratories of the participating faculty to prepare these individuals to be productive, independent scientists. The critical components and the clear strengths of this Program are the 22 participating training faculty and the trainees themselves. Another important strength of this Program and, indeed, of the overall environment at Washington University, is the highly collaborative nature of our research efforts. This interactive environment and these collaborations expand the research, training and mentoring opportunities provided to the trainees. The faculty derive from multiple (6) Departments (Biochemistry, Biomedical Engineering, Cell Biology and Physiology, Developmental Biology, Medicicne and Radiology) and (4) Divisions (Cardiology, Chemistry, Endocrinology and Nutritional Science) within the Department of Medicine, and include nationally and internationally recognized leaders in several specific areas of Modern Cardiovascular Biology including molecular biology, physiology, cell biology, biochemistry, modeling, imaging, pathology, genetics, and human cardiovascular disease mechanisms. The faculty are well-established, well-funded, experienced and highly productive investigators, and all are committed to providing the training, experience, resources, intellectual enthusiasm and mentoring needed to achieve the overall goals of the Training Program and to facilitate the professional development of the individual Program Trainees. The strengths of this Program are manifest in the quantity and quality of our scientific output, in the quality of our present and past trainees, and in the achievements of our past trainees. In addition to the research training provided in individual and collaborating research laboratories, this Training Program provides instruction in Ethics in Biomedical Research, and provides trainees the opportunity to participate in the Core Cardiovascular Biology Curriculum, the weekly Trainees in Cardiovascular Biology Series and the weekly Cardiovascular Research Seminar Series, all developed and operated by this Training Grant. This Training Program is also actively involved in monitoring the progress and professional development and in the mentoring of Predoctoral and Postdoctoral Trainees. RELEVANCE (See instructions): This Program will provide outstanding research opportunities, training and mentoring to Predoctoral and Postdoctoral Trainees in Modern Cardiovascular Biology, including cardiovascular physiology, biochemistry, cell biology, genetics, imaging, modeling, and disease mechanisms. This Program will contribute to the training and professional development of the next generation of outstanding investigators focused on addressing important, unanswered questions in Cardiovascular Biology and Cardiovascular Disease.

DESCRIPTION (provided by applicant): The suprachiasmatic nucleus (SCN) is the master circadian pacemaker driving daily rhythms in mammalian physiology and behavior. SCN neurons utilize a transcription/translation feedback loop to generate circadian changes in electrical activity. Although we have known that SCN neurons fire during the day and are silent at night since 1982 and considerable evidence implicates subthreshold K+ conductance(s), the critical K+ conductance(s) have not been identified. In recent studies focused on testing the hypothesis that subthreshold, A-type (IA) voltage-gated K+ (Kv) channels are involved, we found that mice lacking Kv4.2 (Kv4.2-/-) or Kv1.4 (Kv1.4-/-) pore-forming (¿) subunits have markedly shorter circadian periods of locomotor (wheel running) activity than wild-type (WT) mice. Using in vitro extracellular microelectrode recordings, we found that the periods of circadian rhythms in firing are similarly shortened in SCN neurons lacking either Kv4.2 or Kv1.4. Initial experiments here (aim 1) will determine if Kv4.2 and Kv1.4 are the only Kv ¿ subunits contributing to the IA channels that modulate SCN excitability and reveal the effects the combined loss Kv4.2 and Kv1.4 on rhythms in SCN firing and locomotor activity. The goal of aim 2 is to determine if the shorter period of circadian firing in SCN neurons lacking Kv4.2 or Kv1.4 reflects the functioning of IA channels in the synchronization (i.e., network properties) or the cell-autonomous regulation of SCN neuron excitability. This aim will, for the first time, establish whether the critical K+ conductance(s) in different SCN cell types are distinct. A long-standing debate in the field is whether daily changes in membrane potential are required for the generation of circadian rhythms in gene expression. Aim 3 will test directly the hypothesis that Kv4.2- and Kv1.4-encoded IA channel mediated changes in excitability also modulate the period and amplitude of circadian changes in gene expression. Finally, the observation that the cyclic changes in SCN neuron firing and locomotor activity persist (albeit with a shorter period) in the absence of Kv1.4 or Kv4.2 indicates that other K+ conductances regulate the daily oscillations in SCN neuron membrane potentials. In aim 4, we will exploit a novel, high-throughput quantitative Taqman-based RT-PCR based method to quantify the expression levels of multiple K+ channel subunits simultaneously, as a function of circadian time, and to identify the subthreshold K+ conductance(s) that mediates the daily depolarizations and hyperpolarizations in the membrane potentials of SCN neurons. These studies will provide fundamentally important new insights into the roles of specific K+ conductances in regulating/modulating daily rhythms in the excitability of SCN neurons. In addition to guiding further investigations into the molecular, cellular and systemic mechanisms linking daily rhythms in neuronal excitability, gene expression and behavior, these insights will translate to advances in understanding the regulation and dysregulation of circadian rhythms and to the development of novel therapeutic strategies to benefit individuals suffering genetic and environmentally-induced disruptions in circadian rhythms.

This proposal seeks support for the Training in Integrative and Systems Biology of Cardiovascular Disease Training Program in the Center for Cardiovascular Research at Washington University. The goals of this Program are to provide predoctoral and postdoctoral trainees with: outstanding research opportunities; fundamental education and basic training in cardiovascular physiology, pathophysiology and disease mechanisms; opportunities to be introduced to, and potentially to participate in, translational cardiovascular research; and, mentoring to prepare them to be productive, independent scientists, educators and mentors. The critical components and the clear strengths of this Program are the twenty-one (21) participating faculty and the trainees themselves. Another clear strength of this Training Program is the highly interactive and collaborative environment in the Center for Cardiovascular Research, indeed at Washington University in general, which translates to expanded research, education, training and mentoring opportunities for all of our trainees. The participating training faculty derive from seven Departments and four Divisions within the Department of Internal Medicine, and includes nationally and internationally recognized leaders in the three research and training areas of emphasis in this Training Program: 1) Cardiac metabolism, remodeling and cardiomyopathies; 2) Ion channels, cardiac excitability and arrhythmias; and, 3) Vascular biology, inflammation and coronary disease. The participating faculty are all well-funded, highly interactive, collaborative and productive investigators who are all also committed to providing the training, experience, resources, intellectual enthusiasm, and mentoring needed to facilitate the scientific and professional development of the Program trainees. Importantly, the more experienced senior faculty participants are also committed to the mentoring of the more junior Training Program faculty preceptors. In addition to the research training provided in individual and collaborating laboratories, trainees in this Program will also participate in a ?Core Curriculum in Integrative and Systems Cardiovascular Biology?, the weekly Trainees in Cardiovascular Biology and ?Cardiovascular Research? seminar series, the biannual ?Cardiovascular Training Program Distinguished Lectureship,? the annual ?Cardiovascular Research Day?, and have the opportunity to participate in translational cardiovascular research. This Training Program also provides trainees with continuing instruction in Ethics and Research Science, and has outlined formal and informal mechanisms for helping trainees develop individualized educational, training and career development plans and for monitoring the progress, professional development and mentoring of each predoctoral and postdoctoral trainee. Formal mechanisms are also in place for evaluating, and evolving as needed, the Training Program itself going forward.

ABSTRACT Heart failure, which afflicts more than five million adults in the United States alone, is associated with markedly increased risk of ventricular arrhythmias and sudden death. The molecular, cellular and systemic mechanisms linking heart failure to increased sudden death risk, however, are poorly understood and, despite considerable attention and effort, risk stratifying patients remains an enormous challenge. Although numerous experimental (animal/cellular) heart failure models have been developed and extensively studied, only limited insights into human arrhythmia mechanisms have been provided. Motivated to change this and advance the field, we have undertaken a comprehensive research effort aimed at defining the mechanisms involved in the physiological regulation of membrane excitability in the human heart and the pathophysiological electrical remodeling associated with human heart failure. Utilizing the infrastructure developed in the Translational Cardiovascular Biobank and Repository at Washington University for the acquisition of non-failing and failing human hearts, we have established robust, reliable methods for the isolation and in vitro maintenance of human ventricular myocytes. Here, we utilize these unique resources to test directly the hypothesis that there are regional differences in the regulation and remodeling of three voltage-dependent conductance pathways critical for the coordinated propagation of activity through the ventricles and the maintenance of normal cardiac rhythms: the Kv4.3-encoded, fast transient, outward K+ current, Ito,f; the recently identified, novel, non-inactivating Kv current component, Iss; and, the Nav1.5-encoded voltage-gated Na+ current, INa. In aim #1, we will define the functional impact of heterogeneous Ito,f remodeling on LV action potential waveforms, and identify the molecular determinants of native Ito,f channels in non-failing human LV and of Ito,f remodeling in failing human LV. In aim #2, we will test the hypothesis that there are also transmural differences in the expression and the remodeling of Iss, and define the functional consequences of cell type-specific differences in Iss expression and remodeling on LV action potential waveforms. Experiments in aim #3 will test the hypothesis that there are transmural differences in the expression, properties and remodeling of Nav1.5-encoded INa channels, particularly the late component of INa, INa,L, in non-failing and failing human LV myocytes and define the functional impact on LV action potential waveforms of heterogeneous INa,L expression and remodeling. These studies will provide new, clinically relevant, insights into the cellular/molecular mechanisms contributing to the physiological regulation and pathophysiological remodeling of native human ventricular Ito,f, Iss and INa channels. These insights will transform the refinement of human cardiac myocyte and whole heart models and translate to novel, mechanism-based strategies to target specific cell types to reduce the risk of life-threatening ventricular arrhythmias in patients suffering human heart failure.

Voltage-gated Na+ (Nav) channels play key roles in action potential generation and in controlling action potential durations and propagation in the mammalian heart, and these channels are critical for the maintenance of normal cardiac rhythms. Changes in Nav channel expression and properties are prevalent in inherited and acquired cardiac diseases, and these changes can have profound pathophysiological consequences, including increasing the risk of potentially life-threatening cardiac arrhythmias. Although it seems generally accepted that native myocardial Nav channels function in macromolecular protein complexes, comprising the pore-forming Nav1.5 subunit and multiple intracellular and transmembrane accessory subunits, the physiological roles of accessory subunits in regulating Nav channel function and how these roles are altered with myocardial disease are poorly understood. This new collaborative research program is focused on defining the post-transcriptional mechanisms involved in the physiological regulation and pathophysiological dysregulation of myocardial Nav1.5-encoded channels by intracellular Nav channel accessory subunits. A multifaceted experimental strategy has been developed to define the molecular and cellular mechanisms underlying the regulatory effects of intracellular Fibroblast Growth Factor 12B, iFGF12B, the main iFGF variant expressed in non-diseased human heart, on the gating of Nav1.5-encoded Nav channels (aim #1), and test the hypothesis that iFGF12A, which is upregulated in failing human heart, has distinct effects on the biophysical and pharmacological properties of cardiac Nav1.5-encoded channels (aim #2). Additional experiments will test the hypothesis that another intracellular accessory subunit, calmodulin, CaM, which binds to the C terminus of Nav1.5 near the iFGF binding site, modulates iFGF12B/iFGF12A- mediated effects on Nav1.5-encoded channel gating (aim #3). We will also create molecularly-detailed Nav channel gating models that include Nav1.5 regulation by iFGF12A, iFGF12B and CaM and will use these models to delineate the impact of iFGF12-mediated regulation of native Nav currents on myocyte electrophysiology. These studies will provide fundamentally important new insights into the molecular and cellular mechanisms underlying iFGF12-mediated regulation of myocardial Nav1.5-encoded channels and into the physiological roles of iFGF12 in the dynamic regulation of cardiac excitability. These insights will inform efforts to explore the potential of iFGFs and of iFGF-Nav1.5 interactions as new therapeutic targets to modulate Nav channel functioning in inherited and acquired cardiac rhythm disorders.