Peter J. Mohler, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Voltage-gated Nav channel Nav1.5 (encoded by SCN5A) initiates rapid depolarization of the cardiac action potential and is essential for normal cardiac conduction. Human SCN5A mutations may lead to cardiac arrhythmia and sudden death. Nav1.5 function is determined by its channel properties as well as its cellular localization. The identity of the cellular pathway(s) required for Nav1.5 localization at excitable membranes in heart is an important and currently unresolved question. Our long-term goals are to elucidate the cellular pathway(s) and molecular determinants underlying cardiac Nav1.5 targeting. Our specific hypothesis is that ankyrin-G (a membrane adaptor protein) is required for Nav1.5 targeting to intercalated disc and T-tubule membrane domains. We base this hypothesis on previous observations that 1) targeted knockout of ankyrin-G in mouse cerebellum blocks targeting of Nav1.6 and 1.2 in neurons, 2) ankyrin-G binds Nav1.2 through a 9 residue motif on Nav1.2 loop 2, 3) this motif is required Nav1.2 targeting in neurons, and 4) Nav1.5 contains a nearly identical sequence in loop 2. Additionally, our preliminary results support the interaction and co-localization of Nav1.5 and ankyrin-G in heart, and suggest that Nav1.5 requires ankyrin-G-binding for targeting and normal physiological function in humans. Based on these observations, the experiments in this proposal will test a role for an ankyrin-G based pathway for Nav1.5 targeting in heart. We predict that these experiments will supply the first evidence for a cellular pathway required for cardiac Nav1.5 targeting, and provide in vivo evidence for a new class of human Na 'channelopathies' due to abnormal Nav1.5 targeting. The specific aims are to: 1) Determine the structural requirements for ankyrin-G/Nav1.5 interactions and test human Nav1.5 (SCN5A) disease mutants for ankyrin-G loss-of-binding. 2) Evaluate the requirement of ankyrin-G for Nav1.5 expression, targeting, and function in heart. 3) Characterize the ankyrin-G pathway for Nav1.5 targeting in cardiomyocytes including identification of cellular intermediates in Nav1.5 targeting pathway and identification/characterization of ankyrin-G-interacting proteins for effects on ankyrin-G/ Nav1.5 localization and expression. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Defects in cardiac excitability are the basis for human arrhythmia and sudden cardiac death, a leading cause of mortality in developed countries. Recent findings demonstrate a new paradigm for human arrhythmia based on gene mutations that affect the expression/subcellular localization of cardiac ion channels and transporters. Human type 4 long QT syndrome (LQT4) results from loss-of-function mutations in the membrane adapter ankyrin-B (AnkB). Subjects with LQT4, and mice with reduced AnkB expression display similar complex cardiac phenotypes including atrial, ventricular, conduction defects, and risk of sudden cardiac death. However, the molecular identities of AnkB polypeptides, scope of AnkB expression in specialized cardiac cells, and cellular role(s) for AnkB polypeptides for cardiac excitability remain critical, yet unanswered questions. Moreover, the mechanisms underlying AnkB regulation in normal heart, and dysfunction in human arrhythmia remain unsolved. The long-term objective of this research proposal is to understand the molecular basis for AnkB function in the heart. We hypothesize that coordinate dysfunction of AnkB polypeptides throughout the heart create the complex phenotype of human type 4 long QT syndrome due to defects in ion channel/transporter trafficking and membrane stability. The specific aims are to: 1) Characterize the expression and subcellular distribution of AnkB isoforms in diverse excitable cell types of heart. 2) Define the cellular role(s) of AnkB for ion channel/transporter trafficking and localization in adult cardiomyocytes using recently developed lentiviral techniques. 3) Characterize the mechanisms underlying AnkB regulation in heart, and determine how human AnkB loss-of-function mutations associated with fatal arrhythmia affect this regulation. The cellular pathways underlying ion channel and transporter targeting, localization, and stability in cardiomyocytes are essentially unknown but present an exciting new target for future cardiac therapies. We propose to use recently developed expression techniques to elucidate the molecular mechanisms underlying AnkB-dependent cellular pathways for ion channel and transporter targeting, localization, and stability in the physiological context of the primary cardiomyocyte. It is anticipated that this information will advance understanding of mechanisms underlying AnkB-based human fatal human arrhythmia as well as acquired cardiac arrhythmias associated with abnormal Ca2+ homeostasis, and begin to define potential future molecular targets for the regulation of cellular excitability. [unreadable] [unreadable] [unreadable]

Project Summary Ion channels and transporters control the movement of charged ions across cell membranes. In the heart, the coordinate activities of these proteins regulate the transmembrane electrochemical gradient to control depolarization/repolarization, and thus cardiac excitability. Normal function of ion channels/transporters requires defined biophysical properties as well as precise expression, organization, and regulation in defined membrane domains. Our recent findings support a new basis for arrhythmia based not on mutations which affect channel biophysics, but instead on mutations to proteins which are required for proper expression and local regulation of ion channels and transporters at excitable membranes. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional signaling molecule with critical roles in cardiac physiology and heart disease. Similar to protein kinase A (PKA) and protein kinase C (PKC), CaMKII regulates the phosphorylation status of a host of target substrates throughout the cell. However, in striking contrast to PKA- and PKC-dependent local signaling, little is known regarding local CaMKII targeting in myocytes. In fact, while a handful of CaMKII-binding partners have been proposed to regulate local signaling, no CaMKII-targeting proteins have been validated in animal models. Our preliminary data support a novel molecular mechanism for CaMKII targeting to the myocyte intercalated disc. bIV spectrin, an actin- and ankyrin-G-associated molecule, associates with CaMKIId both in vitro and in vivo. bIV spectrin, previously only studied in the nervous system, is expressed in heart, associates with ankyrin-G, and targets CaMKII to the myocyte intercalated disc to directly regulate local voltage-gated Na+ channel activity. Notably, we have identified a previously elusive CaMKII phosphorylation site on Nav1.5 (S571) that regulates channel activity. A targeted mouse model deficient in the bIV spectrin CaMKII-binding motif lacks intercalated disc CaMKII expression resulting in reduced Nav1.5 S571 phosphorylation, altered Nav channel activity, action potential abnormalities, and whole animal cardiovascular phenotypes. These preliminary data strongly support bIV spectrin as a previously unrecognized in vivo CaMKII-targeting protein, as well as our central hypothesis that the bIV spectrin-based intercalated disc complex provides critical signaling and structural roles for normal cardiac function. Furthermore, these data strongly suggest that targeted inhibition of the bIV spectrin/CaMKII interaction would serve as a novel, and highly specific mechanism to suppress persistent INa in the heart to treat arrhythmia. While this proposal will primarily focus on the role of bIV spectrin-targeted CaMKII in regulating Nav1.5, we hypothesize that the bIV spectrin complex will play a broader role in local signaling and structural regulation at the myocyte intercalated disc. We predict that these findings will provide the first data for local CaMKII targeting in vivo, provide the first data for the role of CaMKII and Nav1.5 S571 for cardiac INa regulation in health and disease, define the role of ankyrin-G and bIV spectrin in cardiac signaling, membrane structure, and function, and finally define a novel and potentially therapeutic mechanism to suppress persistent INa in heart to treat arrhythmia.

DESCRIPTION (provided by applicant): Defects in cardiac excitability are the basis for human arrhythmia and sudden cardiac death, a leading cause of mortality in developed countries. Ion channels and transporters control the movement of charged ions across cell membranes. In the heart, the coordinate activities of these proteins regulate the transmembrane electrochemical gradient to control depolarization/repolarization, and thus cardiac excitability. Normal function of ion channels and transporters requires defined biophysical properties as well as precise expression, organization, and regulation in defined membrane domains. Findings generated during our first period of funding support a new paradigm for human cardiac disease (arrhythmia) based on dysfunction in proteins that are required for proper expression and local regulation of ion channels and transporters at specific excitable membranes. Specifically, we discovered that ankyrin proteins, previously considered static membrane adapters, play dynamic roles in ion channel, transporter, and signaling protein targeting in ventricular cardiomyocytes. Patients harboring loss-of-function mutations in the ankyrin-B gene (ANK2) display a severe and complex cardiac phenotype. Phenotypes may include sinus node dysfunction, atrial fibrillation (AF), conduction defects, catecholamine-induced polymorphic ventricular arrhythmia, and/or sudden cardiac death. Moreover, we have learned that common ANK2 gene variants in the general population are associated with QTc alterations and ventricular arrhythmia susceptibility, that AnkB levels are strongly altered in large animal models of cardiovascular disease, and that the ANK2 is a candidate gene for AF susceptibility in the general human population. However, despite these translational studies implicating AnkB as a key player in cardiac excitability, the specific molecular roles of AnkB in heart remain surprisingly unknown. In fact, the identities of the in vivo cellular components of the AnkB-targeting pathway (or other cardiac targeting pathways) are still unknown. Finally, lack of an animal model of AnkB deficiency (global AnkB k/o is embryonic lethal) has prevented efforts to define new roles of AnkB in cardiac physiology and disease. For this first competitive renewal, due to important advances during the first funding cycle and the development of a number of innovative new animal models, we are well-positioned to provide the first in vivo information on the fundamental components (both upstream & downstream) of the entire AnkB-targeting pathway at baseline and in disease. We provide exciting new preliminary data that identifies a novel family of membrane trafficking proteins (EHD proteins) that regulate cardiac membrane excitability and associate with AnkB. We further provide new data that AnkB plays a novel role in targeting select Ca2+ channels in sinus node & atria. Finally, our preliminary data in mice demonstrates novel and unexpected roles of AnkB in cardiac membrane biogenesis and maintenance. Together, our published findings and preliminary data support a central hypothesis that the AnkB-based cellular pathway plays dynamic roles in myocyte membrane excitability and cardiac function. The immediate goals of our research program are to understand the specific cellular role(s) of AnkB in the heart (including upstream regulatory pathways [EHD proteins] and novel downstream targets [Cav1.3]) and determine how AnkB dysfunction leads to complex human cardiac disease. For this first competitive renewal, we present a cast of uncharacterized and innovative animal models, novel molecular tools, innovative technologies, and new antibodies to test the specific roles of the AnkB cellular pathway in vivo.

DESCRIPTION (provided by applicant): The objective of this joint Ohio State University (OSU)-Nationwide Children's Hospital (NCH) institutional T32 postdoctoral program is to provide interdisciplinary, translational and state-of-the-art research training in congenital and acquired heart disease. This plan provides a unique opportunity to focus on a continuum of congenital and acquired heart disease from birth to senescence and to compare disease mechanisms and outcomes between these diverse population. The rationale is that advances in heart disease therapy and prevention are most likely to originate from a translational research program that fosters collaboration between physician-scientists and basic scientists. To address the overall theme, we will maintain and expand ongoing efforts to provide an academic environment to cultivate true 'bidirectional' translational research, so that significant clinical problems identified by our clinician scientists can be addressed in the laboratory, and key findings at the bench can be rapidly translated to larger animal studies and eventually back to the bedside. This new application seeks 5 trainees per year for 5 years with a 2-year commitment. Fellows will be selected from MD, DVM or PhD candidates who have strong interests in cardiovascular research and are committed to careers in disease-oriented investigations. The Program Directors have a strong-track record in translational cardiovascular research and will serve as the primary directors at their respective institutions. The training plan design consists of formal academic courses, seminars and journal clubs to provide trainees with an overview of heart disease and an in-depth cardiac research orientation. Trainees will participate in a tailor-made survival skills course comprised of various modules and workshops related to professional development. Each trainee will perform independent research projects in a mentor's laboratory. The research areas encompassed by the 20 extramurally funded basic- and clinical-science mentors and 22 participating faculty are represented by their individual affiliations with The Heart Center at NCH and The Davis Heart and Lung Institute at OSU. A cross-institution, web-based system will be used to perform trainee and mentor evaluations.

DESCRIPTION (provided by applicant): Membrane excitability and excitation-contraction (EC) coupling in the healthy heart rely upon the proper expression, trafficking, and retention of integral membrane proteins (ion channels, transporters, receptors). All play key roles in governing cardiac contraction and short and long term adaptations to physiological and pathophysiological stimuli. The profile of expressed proteins is dynamic, being tightly synchronized to assure the proper responses to stress1. This is highlighted by a decade of research linking dysfunction in membrane protein trafficking with heart disease. Yet, despite its obvious importance, little is known regarding even the identity of the molecular mechanisms underlying the targeting of integral membrane proteins in the context of the heart. The focus of this multiple PI proposal, is to identify new pathways for membrane protein targeting and regulation in heart with the goal of defining novel mechanisms for the regulation of cardiac membrane excitability as well as its dysregulation in disease. While not well studied in any organ system, Eps15 homology domain-containing (EHD) gene products (EHD1-4) are intracellular proteins that appear to be key regulators of membrane protein trafficking. Previously uncharacterized in the heart, our group (Boyden & Mohler) recently provided evidence that this protein family likely plays indispensible roles in protein trafficking in cardia muscle. Notably, we uncovered a vital role for one of these endosomal proteins, EHD3, in the membrane trafficking of the Na/Ca exchanger (NCX) in heart5. Moreover, we showed that EHD proteins are differentially regulated in large animal models of human cardiovascular disease, suggesting that EHD proteins may play a critical role in the remodeling of membrane proteins following myocardial infarction (post MI). Our initial findings predict a role for EHD proteins in membrane protein trafficking in the healthy and diseased heart. Our overall hypothesis is that EHD proteins are indispensable components in the proper trafficking of integral membrane proteins involved in cardiac excitability and EC coupling, and are involved in the remodeling of the heart over a wide variety of cardiac pathologies. The goal of this proposal is to directly test the role of EHD3 and EHD3 in cardiac structural and electrical activity using innovative in vivo models of EHD protein deficiency.

? DESCRIPTION (provided by applicant): The objective of the competitive renewal of this joint Ohio State University (OSU)-Nationwide Children's Hospital (NCH) institutional T32 postdoctoral program is to provide interdisciplinary, translational, and state- of-the-art research training in congenital and acquired heart disease. This plan provides a unique opportunity to focus on a continuum of congenital and acquired heart disease from fetal life to senescence and to compare disease mechanisms and outcomes between these diverse populations. The rationale is that advances in heart disease therapy and prevention are most likely to originate from a translational research program that fosters collaboration between physician-scientists and basic scientists. We will expand ongoing efforts to provide an academic environment to cultivate true `bidirectional' research, so that significant clinical problems identified by our physician scientits can be addressed in the laboratory, and key findings at the bench can be rapidly translated to larger animal studies and back to the bedside. This renewal application seeks 6 trainees per year for 5 years with a 2-3 year commitment. Fellows will be selected from MD, DVM, Pharm.D. or PhD trainees who have strong interests in cardiovascular research and are committed to careers in disease-oriented investigations. The Program Directors have a strong-track record in translational cardiovascular research and mentoring and will serve as the primary directors at their respective institutions. The training plan design consists of formal academic courses, seminars and journal clubs to provide trainees with an overview of heart disease and an in-depth cardiac research orientation. Trainees will participate in a tailor-made survival skills course comprised of various modules and workshops related to professional development. Each trainee will perform independent research projects in a mentor's laboratory. The research areas encompassed by the 34 extramurally-funded basic- and clinical- science mentors and 3 participating faculty are represented by their individual affiliations with The Heart Center at NCH and The Davis Heart and Lung Research Institute at Ohio State. A new clinical investigator pathway is added to the training program to enhance clinical research opportunities for MD fellows. The pathway will be directed by J. Phil Saul, MD, an internationally recognized physician-scientist and Chair of Pediatrics. An aggressive underrepresented minority recruitment and retention plan is described to increase trainee diversity. A cross-institutional system will be used to perform trainee and mentor evaluations. Overall metrics to determine success include programatic review by internal and external advisory committees and the successful execution of each trainee's individual development program. Trainee-specific metrics include completion of required coursework, annual evaluation by the mentoring team, and research productivity as assessed by the number of peer-reviewed manuscripts and abstracts, presentations at national meetings and grant submissions (NIH K, R21 and/or foundation grants).

Abstract Defects in cardiac excitability are the basis for human arrhythmia and sudden cardiac death, a leading cause of mortality in developed countries. Unfortunately, arguably the last major ?game-changing? breakthroughs in electrical cardiomyocyte biology and cardiac signaling for human health were the beta- blocker (discovered in the 1950s) and `ACE' inhibitor (in the 1970s). On the other hand, therapeutic agents to treat disorders of cardiac excitation (arrhythmias) are plagued by limited efficacy and even off-target pro- arrhythmia. Despite a wealth of negative clinical data, excitable cell researchers have largely remained focused on the same paradigm - pharmacological therapies targeting cardiac ion channels. We contend that improved therapies will only arise through a more sophisticated, working understanding of interactions between structural proteins (such as ankyrins), electrical proteins (ion channels, pumps & exchangers) and signaling systems (kinases, phosphatases, oxidases). Our studies discovered that ankyrin and spectrin proteins, previously considered static membrane adapters, play dynamic roles in ion channel, transporter, and signaling protein targeting in ventricular cardiomyocytes. Further, we have learned that these proteins serve as critical central membrane nodes to regulate normal signaling in heart. Finally, and most importantly, we have learned that dysfunction in these pathways results in potentially fatal forms of both congenital and acquired ventricular arrhythmia. Our long-term goal is to discover novel integrated mechanisms for regulating cardiovascular cell excitability and signaling. We have used the informative case of ankyrins and spectrins as a tractable starting point, but propose to rapidly extend these studies to new systems with diverse interacting structure-electrical- signaling systems. Our laboratory has taken an active lead in the identification of new cellular pathways for regulation of cellular excitability based on human clinical, tissue, and genetic data. In addition, we have pushed innovation in the field through the use of physiologically-relevant model systems to study the mechanisms underlying electrical signaling in the complex vertebrate cardiomyocyte. This approach has ultimately culminated in an ability to not only diagnose new forms of potentially fatal arrhythmia, but to design effective patient-selective therapies for these diseases. If successful in obtaining funding from the NHLBI Outstanding Investigator Award, we will continue to pursue scientific studies with the potential to create new, cell-specific insights for improved understanding of cardiac excitability with direct relevance for congenital and acquired human disease.

Project Summary/Abstract Voltage-gated Na+ channels (Nav) are essential for normal atrial excitability and function, as evidenced by the strong link between dysfunction in the primary cardiac Nav alpha subunit (Nav1.5) and atrial arrhythmias. In particular, congenital or acquired defects in Nav that promote inappropriate ?late? Na+ current (INa,L) commonly result in atrial as well as ventricular arrhythmia. While increased late INa has been linked to atrial fibrillation (AF) in patients and in animal disease models, little is known about the underlying pathways for dysregulation. Moreover, the potential of INa,L as a therapeutic target in AF, while viewed with optimism, remains controversial and difficult to assess due to nonspecific nature of pharmacological agents and limitations of available animal models. Nav1.5 is tightly regulated within local signaling domains that control channel post-translational modification. Nav1.5 phosphorylation is an important pathway for modulating channel function and level of INa,L. In ventricle, changes in Nav1.5 phosphorylation have been linked by our group and others to arrhythmogenic INa,L. In contrast, the roles for Nav1.5 phosphorylation in atria are unknown and essentially unstudied. In fact, despite its clear role in atrial function and atrial arrhythmias we know essentially nothing regarding the molecular mechanisms that control atrial INa phosphoregulation. Perhaps more surprising, almost nothing is known about the pathways underlying Nav1.5 dephosphorylation, in either atria or ventricle. We have identified a novel pathway for specific regulation of atrial INa,L by CaMKII with an important role in arrhythmogenesis. Our preliminary data indicate that this pathway includes a previously unappreciated negative regulatory axis for Nav1.5 mediated by protein phosphatase 2A (PP2A). Furthermore, our new data support that PP2A-dependent antagonism of CaMKII phosphorylation occurs within a macromolecular complex organized by ankyrin-G. Finally, we provide initial evidence that this regulatory pathway is altered in animal models and human AF. Our long-term goal is to define the molecular pathway for CaMKII-dependent phosphoregulation of Nav1.5 in atrial myocytes, to understand the role of this pathway in AF, and test whether it may be manipulated for therapeutic benefit. Our central hypothesis is that dysfunction in the PP2A- dependent regulatory axis exacerbates imbalance in Nav1.5 phosphoregulation induced by CaMKII hyperactivity, leading to increased INa,L and ultimately increased AF susceptibility downstream of defects in Ca2+ homeostasis. We will: 1) Define the molecular pathway controlling CaMKII-dependent phosphorylation of atrial Nav1.5; 2) Determine the role of atrial INa phosphoregulation in modulating atrial Ca2+ homeostasis, excitability, and function; and 3) Determine the impact of Nav1.5 phosphoregulation in AF susceptibility and progression. We assert that a fundamental understanding of atrial INa regulation and downstream function is essential for both our understanding of AF mechanisms and the rational design of new therapies.  

? DESCRIPTION (provided by applicant): The objective of the competitive renewal of this joint Ohio State University (OSU)-Nationwide Children's Hospital (NCH) institutional T32 postdoctoral program is to provide interdisciplinary, translational, and state- of-the-art research training in congenital and acquired heart disease. This plan provides a unique opportunity to focus on a continuum of congenital and acquired heart disease from fetal life to senescence and to compare disease mechanisms and outcomes between these diverse populations. The rationale is that advances in heart disease therapy and prevention are most likely to originate from a translational research program that fosters collaboration between physician-scientists and basic scientists. We will expand ongoing efforts to provide an academic environment to cultivate true `bidirectional' research, so that significant clinical problems identified by our physician scientits can be addressed in the laboratory, and key findings at the bench can be rapidly translated to larger animal studies and back to the bedside. This renewal application seeks 6 trainees per year for 5 years with a 2-3 year commitment. Fellows will be selected from MD, DVM, Pharm.D. or PhD trainees who have strong interests in cardiovascular research and are committed to careers in disease-oriented investigations. The Program Directors have a strong-track record in translational cardiovascular research and mentoring and will serve as the primary directors at their respective institutions. The training plan design consists of formal academic courses, seminars and journal clubs to provide trainees with an overview of heart disease and an in-depth cardiac research orientation. Trainees will participate in a tailor-made survival skills course comprised of various modules and workshops related to professional development. Each trainee will perform independent research projects in a mentor's laboratory. The research areas encompassed by the 34 extramurally-funded basic- and clinical- science mentors and 3 participating faculty are represented by their individual affiliations with The Heart Center at NCH and The Davis Heart and Lung Research Institute at Ohio State. A new clinical investigator pathway is added to the training program to enhance clinical research opportunities for MD fellows. The pathway will be directed by J. Phil Saul, MD, an internationally recognized physician-scientist and Chair of Pediatrics. An aggressive underrepresented minority recruitment and retention plan is described to increase trainee diversity. A cross-institutional system will be used to perform trainee and mentor evaluations. Overall metrics to determine success include programatic review by internal and external advisory committees and the successful execution of each trainee's individual development program. Trainee-specific metrics include completion of required coursework, annual evaluation by the mentoring team, and research productivity as assessed by the number of peer-reviewed manuscripts and abstracts, presentations at national meetings and grant submissions (NIH K, R21 and/or foundation grants).