Isabelle Deschenes - US grants

[unreadable] DESCRIPTION (provided by applicant): Potentially lethal arrhythmias in rare inherited syndromes (idiopathic ventricular fibrillation and Long QT syndrome) have been associated with defects in depolarizing sodium currents. Delineation of the molecular basis and mechanism of the cardiac sodium channel are therefore essential for an accurate understanding of cardiac ventricular depolarization. The determination of the crystal structure of a bacterial inwardly rectifying K channel was a major advance that provided a framework for testing hypotheses concerning the structure of related channels. Nevertheless, crystal structures have the limitation that movement, a major feature of both fast and slow inactivation, is imperceptible. Thus, this proposal will underline vital approaches to structure-function analysis of both fast and slow inactivation of the cardiac sodium channel with an emphasis on understanding the role of these gating mechanisms in inherited arrhythmias. The main focus of this proposal is to develop a library of human cardiac sodium channel constructs containing two fluorescent proteins for later analysis with Fluorescence Resonance Energy Transfer (FRET). FRET and biophysical analysis will be combined to characterize the molecular mechanism and movements of the cardiac sodium channel. By combining patch clamping with FRET, we will be able to measure distance between different regions of the sodium channel in its different gating states under normal a pathological conditions. Therefore this proposal will test the hypotheses that: 1. a library of functional Nav1.5 constructs containing both CFP and YFP can be generated to study motion in cardiac sodium channel using FRET. 2. The C- terminal region of the cardiac sodium channel is involved in fast inactivation and moves during this gating mechanism. 3. Motion in the outer pore mouth underlies slow forms of inactivation of the channel. 4. Mutations in the cardiac sodium channel that cause Long QT syndrome affect regions of the channel involved in inactivation. This project will generate dynamic insights on the structure of the channel that no other approaches, including crystallization have been able to engender thus far. This information on the structure of the channel will be of tremendous help to understand how mutations in this channel are responsible for arrhythmias. [unreadable] [unreadable] [unreadable]

Project Summary Cardiac arrhythmias are a leading cause of morbidity and mortality in developed nations, resulting in more than 300,000 deaths per year in the U.S. alone. These arrhythmias are frequently associated with acquired heart diseases, notably cardiac hypertrophy and heart failure (HF), where the dysregulation of a host of ion channels and transporters is observed. One of the most consistent changes frequently associated with compromised repolarization, is selective reduction in the transient outward potassium current Ito. Ito is generated primarily by the voltage-gated potassium (Kv) channel, Kv4, and its interacting auxiliary subunit known as K Channel Interacting Protein 2 (KChIP2). Under hypertrophy and HF there is consistent loss of KChIP2, thought to cause the reduction in Ito. Intriguingly, the loss in KChIP2 expression has been observed to be one of the earliest and most consistent remodeling events in HF development. The commonality and early state of this remodeling begins to suggest KChIP2 loss might not just be one of the casualties during disease progression, but may represent an initiating factor driving pathogenesis. Emerging evidence suggests KChIP2 may not be limited to cell surface regulation of Kv4. Indeed, since its original discovery, there has been an expansion in the roles of KChIP2 in cardiac ion channel function including modulation of Na, L-type Ca, and Kv1.5 channels. In total, there are four KChIP genes (KChIP1-4) with many alternatively spliced isoforms. Interestingly, KChIP3 (found in the brain), calsenilin and DREAM are encoded by a single gene. These names are the result of three independent discoveries due to different roles: modulation of Kv channels, regulation of the protein presenilin, and critically, calcium-sensitive transcriptional repression through binding to DRE (downstream regulatory element) sequences of genes. While KChIP2 is the only isoform found in the heart, given the homology it shares with KChIP3, it led us to hypothesize that KChIP2 could also perform multiple functions. Indeed, during the previous funding period, we identified a significantly expanded importance of KChIP2 in the heart. We demonstrated novel functions for KChIP2 in regulating calcium currents and RyR2. Importantly, we demonstrated a novel role for KChIP2 where it could regulate the genes at the source of INa and Ito by acting, much like KChIP3/DREAM, as a transcriptional repressor targeting a family of microRNAs. In this renewal, we will elucidate in aim 1 the role of KChIP2 as a transcriptional repressor. In aim 2, we will determine the control mechanisms for KChIP2 trafficking between cytoplasm and nucleus. And in aim 3, we will elucidate how chronic stress affects KChIP2 distribution and function in cardiac myocytes. Collectively, the outcomes of these investigations will demonstrate that KChIP2 actions are dramatically more expansive than modulation of Kv4 channels alone, suggesting that these other KChIP2 functions can be potent contributors to adverse remodeling events characterized in the diseased heart.

DESCRIPTION (provided by applicant): It is well-known that monogenic disorders produce alterations of the cardiac action potential that lead to life threatening arrhythmias. Although this work on inheritable channelopathies has greatly contributed to our understanding of arrhythmia mechanisms, monogenic disorders leading to arrhythmias are rare. Interestingly, it is also recognized that even common forms of arrhythmias, such as atrial and ventricular fibrillation can be familial. However, the pattern of inheritance and clinical phenotypes of these patients are complex. In fact, similar to channelopathies such as Long QT (LQT) or Brugada Syndrome (BrS) which are autosomal dominant diseases, these more common arrhythmias also display variable penetrance. There are several prevailing hypotheses that have attempted to explain why a particular gene expression pattern might produce variable phenotypic expression i.e. genotype-phenotype discordance. Importantly, there is emerging evidence indicating that disease modifying genes such as ion channel polymorphisms, can affect the function of ion channels leading to genotype-phenotype discordance. In fact, the PI recently demonstrated that the common sodium channel polymorphism H558R is a disease modifying gene which contributes to the genotype- phenotype discordance seen in multiple families with BrS and LQT3. This polymorphism was able to restore trafficking of mutant BrS channels, and restore the gating kinetics of mutant LQT3 channels resulting in complete normalization of sodium channel function. Thus, this explains the apparent absence of a clinical phenotype in patients carrying the polymorphism along with a disease causing mutation. Therefore, the general hypothesis for this proposal is that sodium channel polymorphisms contribute to the variable penetrance phenomenon. In this proposal, the PI will shed-light on the genotype-phenotype discordance involved in these diseases by primarily using information obtained from large BrS and LQT3 families. These families affected with relatively rare inherited syndromes that relate to sudden cardiac death provide us with a unique opportunity to ascertain the complex phenomenon of variable phenotypic expression of diseases. The specific aims are to: 1. Determine the role of sodium channel polymorphisms in Brugada and Long QT 3 Syndrome. 2. Investigate the mechanisms by which SCN5A polymorphisms can modulate the function of mutated sodium channels. 3. Develop gene therapy approaches, using genetic polymorphisms, to rescue dysfunctional channels. Assessing variability in susceptibility to LQTS and BrS will provide a framework for analysis of other complex gene-environment interactions that may apply to the more common form of sudden cardiac death. Additionally, understanding the mechanisms by which a polymorphism can modulate a mutated channel will also provide fundamental understanding of ion channels assembly and structure. Importantly, gene polymorphisms could become a target for future therapies aimed at rescuing dysfunctional ion channels.

DESCRIPTION (provided by applicant): Sudden cardiac death (SCD) is a catastrophic event that accounts for up to 450,000 deaths each year in the US. Among patients at high risk for SCD are those with inherited cardiac arrhythmias. Long QT syndrome (LQTS) is one example of a group of inherited cardiac arrhythmias that produces defects in cardiac membrane currents. As a direct consequence, LQTS has been associated with prolongation of the QT interval on the ECG, ventricular arrhythmias, and an increased incidence of SCD. In LQTS2 well over two hundred missense mutations have been identified in the KCNH2 gene encoding hERG with the overwhelming majority thought to be characterized by protein processing and trafficking defects leading to a drastic reduction in potassium currents. However, as commonly observed in many autosomal dominant cardiac channelopathies the pattern of inheritance and clinical phenotypes of these patients are complex and often display incomplete penetrance, where disease-causing mutation carriers are asymptomatic. The causes for this variable clinical expressivity are not well understood but in the present research proposal, we will investigate this question by testing the hypothesis that modifier genes contribute to the variable clinical expressivity. Our multidisciplinary group at MetroHealth and Case Western Reserve University has studied clinically as well as in vitro a large 'Cleveland' LQT2 family carrying the hERG mutation R752W. Out of the 101 family members studied, 26 individuals carried the hERG R752W mutation. However, symptomatic LQTS was present in only 5 of the genetically affected family members thus illustrating incomplete penetrance of the disease. We hypothesize that the presence of disease modifying genes can explain the genotype-phenotype discordance observed in this LQT2 family. In this proposal, we will elucidate the mechanisms of incomplete penetrance in this LQT2 family using exome sequencing and cardiomyocytes differentiated from patient derived induced pluripotent stem cells (iPS). We hypothesize that patient-derived iPS differentiated cardiomyocytes (iPS-CM) faithfully recapitulate the arrythmogenic pathology and that heretofore unknown candidate genes revealed by exome sequencing account for variable phenotypic penetrance. The aims of this proposal are: 1. Identify candidate modifier genes responsible for incomplete penetrance in a LQT2 family. 2. Elucidate electrophysiological variability of human cardiomyocytes derived from LQT2 family members. 3. Determine the phenotype of candidate disease modifying gene variants. We will perform these aims by studying closely related LQT2 hERG R752W carrier pairs (i.e. father/son and sib pair) that display discordant clinical phenotype. We believe that the current proposal will offer a fundamental, mechanistic explanation by which genotype-phenotype discordance can arise in a large LQT2 family. This holds potentially significant ramifications for personalized clinical management and will offer novel targets for personalized pharmacologic intervention aimed at the modulation of dysfunctional ion channels in the heart.

? DESCRIPTION (provided by applicant): Cardiac arrhythmias are a leading cause of morbidity and mortality within developed nations. Often, these arrhythmias are associated with acquired heart diseases, notably cardiac heart failure (HF), where the dysregulation of a host of ion transporters and channels is observed. Particularly, a critical imbalance of both depolarizing INa and repolarizing Ito is observed. Our previous work was the first to support the nascent idea that expression of INa and Ito may share common, yet to be identified regulatory mechanisms involving KChIP2, an accessory subunit of Ito. KChIP2 silencing produced simultaneous mRNA degradation and potential translational block of multiple genes at the source of INa and Ito, suggesting a mechanism of microRNA activity, which led to significant loss of Ito and INa. These results suggested KChIP2 may have additional nuclear functions as a transcription factor to regulate other critical cardiac currents. Indeed, a member of the KChIP family found in neuronal tissues, KChIP3, also known as DREAM, is localized to the nucleus where it acts as a Ca2+-regulated transcriptional repressor. Given KChIP2 shares a high degree of sequence homology with DREAM and that it has been found in the nucleus, one can hypothesize that KChIP2 is capable of similar nuclear roles in cardiac settings. Therefore, we hypothesize that KChIP2 controls expression of depolarizing INa and repolarizing Ito through a novel posttranscriptional mechanism involving microRNAs. Indeed, our preliminary data show evidence demonstrating KChIP2 transcriptionally regulates a family of miRNAs known as miR-34s which we demonstrate targets genes involved in generating both INa and Ito. However the significance of this pathway under pathologic conditions is unknown and is the central focus of this proposal. We hypothesize that KChIP2 loss in the diseased heart (hypertrophy and/or HF) is responsible for dysfunction of cardiac excitability at the level of gene expression. Given that KChIP2 is significantly repressed in HF, our specific aims are to: 1. Define the role of KChIP2 miRNA-dependent regulation in cardiac pathology. 2. Evaluate the influence of restored KChIP2 expression on arrhythmia susceptibility and HF progression. 3. Evaluate the influence of miRNA blockade by antagomirs on arrhythmia susceptibility and heart failure progression. To test these aims rat and guinea pig heart failure models produced by transverse aortic constriction (TAC) and the canine pacing-induced HF model will be used. Delivery of either KChIP2 through viral vectors or miR-34 blockade by injection of miRNA antagomirs will be used to assess influence over INa and Ito expression as well as overall arrhythmia susceptibility and HF progression. The delineation of the molecular basis of KChIP2 regulation is essential for an accurate understanding of cardiac depolarization and repolarization and its implications with lethal ventricular arrhythmias.

Project Summary MicroRNAs (miRs) are small non-coding RNA molecules (~22 nucleotides) known to negatively regulate gene expression through RNA silencing and post-transcriptional regulation. Importantly, miRs are increasingly recognized for the maintenance of cardiac function, modulation of cardiac excitability, and the development of disease states through their classical mechanism of finely controlling the expression of target genes. miR1 is the most predominant miR in the heart and plays a critical role for cardiovascular development and cardiac electrophysiology. In this study, we questioned whether this regulation of cardiac function was solely due to the classical mechanism of miRs or if this could also be attributed to a novel role for miR1 mediated through its physical interaction with cardiac proteins. We first performed an in vitro electron mobility shift assay (EMSA) to investigate if miR1 and miR451a, which are both highly expressed in the heart, can bind with membrane proteins extracted from neonatal and adult mouse hearts. Remarkably, we found that miR1, but not miR451a, could specifically bind with membrane proteins and identified that Kir2.1, an inward rectifier potassium channel, was one of the membrane proteins that miR1 binds to. To establish the potential biophysical consequence of this binding, we performed patch clamp recordings of inward rectifier potassium current (IK1) and delivered miR1 acutely to bypass the transcriptome regulation. Importantly, we found that the current density of IK1 was significantly suppressed by acute expression of miR1. To our knowledge, this is the first discovery of the novel and groundbreaking concept that miRs physically interact with ion channels to modulate their biophysical function. Hence the overall hypothesis of this proposal is that miR1 can also regulate cardiac excitability through direct interaction with cardiac ion channels. Therefore, we propose to investigate the mechanism of miR1-ion channel interaction with the following aims: 1. Define the physical interaction between miR1 and Kir2.1. 2. Evaluate the functional implications of the physical interaction between miR1 and Kir2.1. 3. Identify other cardiac ion channels that physically interact with and are modulated by miR1. Several approaches will be used to identify the ion channels that miR1 interacts with. Importantly, we will investigate the physiological role of this physical interaction in expression systems and in cardiomyocytes. We will study the electrophysiology of cardiomyocytes by patch-clamp with acutely-delivered miR1 to investigate how miR1 modulates cardiac electrophysiology without its transcriptome effect. Our study will provide a mechanistic understanding of how miR1 physically binds with ion channels and directly regulates their functions. We will also investigate if this physical interaction between miR1 and Kir2.1 is a general function of miRs by identifying more miR1-bound ion channels. A better understanding of the biophysical interaction between miR1 and ion-channels will help us to comprehensively recognize the biophysical dysregulation of ion channel in cardiac disease.

Project Summary Ion Channelopathies are diseases resulting from malfunctions of ion channels. More than 20 years ago, mutations in genes encoding ion channels were described for the first time in patients with pulmonary, cardiac, neuronal and neuromuscular disorders defining genetic channelopathies. The field has since grown to include more than a hundred different diseases consistent with the distribution of ion channels throughout the human body. Channelopathies have now been linked to a wide variety of diseases, including epilepsy, migraine, several neurological disorders, blindness, deafness, diabetes, hypertension, several different cardiac arrhythmias, asthma, irritable bowel syndrome, skin diseases and cancer just to name a few. Importantly, considering the central involvement of ion channels, these diseases share a great deal in common in terms of disease mechanisms and potential therapeutic development and yet this is the only meeting that exists allowing the entire Channelopathy community to come together. Therefore, Channelopathy 2020 plans to continue a tradition of international scientific meetings dedicated to the topic of channelopathy that began more than 20 years ago with the SkyTop conference in Pennsylvania. This meeting is now being held biannually and hosts approximatively 150 attendees. Channelopathy 2020 will be held from June 25-27 2020 in Québec City, Canada. The meeting will be chaired by Isabelle Deschênes from The Ohio State University and Mohamed Chahine from Laval University. Together with our organizing committee that includes high profile researchers in the field, we have put an exciting program with 2 internationally recognized plenary speakers, 7 different sessions with 25 invited speakers and we will also have 9 short talks where the speakers will be selected from the abstract submitted and will be focused on inviting junior investigators giving them the opportunity to present their work and getting broad exposure in the field. We will also have two poster sessions. Channelopathy 2020 will place emphasis on comprehensive scientific themes shared across channel types and diseases seeking to catalyze on the knowledge from each channelopathy to benefit the broader field. Some of these themes will include basic mechanisms common to the different channelopathies, disease mechanisms at the organ level, novel therapeutics and precision medicine. The area of precision medicine is extremely germane to channelopathies since patients can respond differently to treatment based on the specific mutations and also genotype-phenotype discordance is an important concern for physicians . Knowledge gained from the different channelopathies covered in this meeting can be applied to other channelopathies and this cross-fertilization between the different presentations could lead ultimately to the development of novel therapeutic strategies applicable to the treatment of these different diseases. The unique format of this conference which includes speakers covering all different channelopathies will allow us to build multidisciplinary networks across these different diseases implicating ion channels.