Cellular and Molecular Electrophysiology
Current research focuses on the molecular and cellular mechanisms of cardiovascular physiology including, cardiac muscle function, electrophysiology, vascular tone, and molecular signaling. Novel and state-of-the-art techniques are used including: optical mapping with fluorescent indicators to measure cell physiology at the level of the cell and intact heart, transgenics and mutagenesis to investigate the expression of genes that regulate cardiac electrophysiology and hemodynamics, and animal models of heart failure, cardiac remodeling, and reentrant arrhythmias.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Cell Repolarization, Alternans, and Arrhythmogenesis

David S. Rosenbaum, M.D. (Principal Investigator), Kenneth R. Laurita, Ph.D., Xiaoping Wan, Ph.D., Elizabeth Kaufman, M.D., Otto Costantini, M.D.

Sudden cardiac death remains a major unresolved public health problem. Recently, there has been extraordinary interest in the possible roles of cardiac repolarization in the mechanism of ventricular arrhythmia (VA). In particular, subtle beat-to-beat alternation of ECG T wave shape (i.e. T-wave alternans) – a significant marker of susceptibility to VA in humans – was recently linked to a potential mechanism of arrhythmogenesis where membrane potentials of neighboring myocytes alternate with opposite phase (i.e. discordant alternans) forming the substrate for block and reentry. Alternans may represent a novel electrophysiological mechanism that transforms physiological into pathophysiological heterogeneities of repolarization which form the substrate for VA. We apply techniques of high-resolution optical mapping to experimental models where key pathophysiological components of VA (e.g. structural barriers, electrophysiological heterogeneities between cells, expression of cardiac gap junctions, left ventricular hypertrophy and failure) are carefully controlled. These studies will improve our understanding of the functional organization of electrical activity in the heart and provide important insights into the mechanisms, diagnosis, and possible treatment of life-threatening VA in humans.


Electrical Heterogeneities in the Heart David S. Rosenbaum, M.D. (Principal Investigator), Kenneth R. Laurita, Ph.D., Xiaoping Wan, Ph.D.

Although there is increasing recognition of important heterogeneities in expression and distribution of ion channels in the heart, the topographical distribution of electrophysiologically distinct cell types, and their role in the mechanisms of cardiac arrhythmias are not well understood. High-resolution optical cardiac mapping, calcium imaging, whole cell and patch clamp recordings, confocal microscopy, and immunoblotting techniques are applied to experimental models of normal, hypertrophic, and failing myocardium to investigate novel mechanisms of arrhythmias. A major emphasis is on understanding how the molecular and biophysical properties of different cardiac cell types leads to arrhythmias occuring at the level of the whole heart.


Electrophysiological Phenotyping of Transgenic Animals David S. Rosenbaum, M.D. (Principal Investigator)

Cell to cell communication is vital to the normal operation of the heart. Gap junctions are protein structures which facilitate intercellular communication through the formation of pores which link the cytoplasm of adjacent cells. While it is well known that gap junctions are vastly altered in many different forms of heart disease, the effects of these changes on the formation of life-threatening arrhythmias is poorly understood. Thanks to the advent of genetic engineering, mice have been bred to mimic many different forms of human cardiac disease. The transgenic mice exhibit many similar traits in disease as their human counterparts, including differences in gap junction communication. One important aspect of transgenic technology is the capability to alter the expression of a gene of interest, in this case the gene responsible for the formation of gap junctions. With our experimental apparatus, we can measure the precise effects of alterations in intercellular communication at the genetic level on the overall electrophysiology of the whole heart. We have found that a reduction of gap junction protein expression of 50%, as seen in human heart failure results in significant conduction slowing, which may contribute to the increased chance of arrhythmia often associated with this disease. Furthermore, we have also found that any disruption of cell-to-cell communication will result in some level of conduction slowing, regardless of other effects of disease. With our transgenic approaches we have the capability to identify important targets at the protein level for future antiarrhythmic therapy.

 


Calcium Mediated Arrhythmogenesis
Kenneth R. Laurita, Ph.D. (Principal Investigator)

Sudden death (SCD) resulting from cardiac arrhythmias is the most devastating manifestation of cardiovascular disease, accounting for approximately 500,000 deaths/year in the US alone. Arrhythmias caused by abnormal impulse formation have been reported in patients with heart disease (e.g. congestive heart failure) and in patients with mutations of calcium regulatory proteins. Normally, when a cell depolarizes, contraction is initiated by calcium release from internal stores (sarcoplasmic reticulum) by the ryanodine receptor (RyR) in a process called calcium-induced calcium-release (CICR). Under certain abnormal conditions, calcium can be spontaneously released by RyR channels from the sarcoplasmic reticulum while the heart should be resting and result in fatal arrhythmias. Using novel optical mapping techniques developed in our laboratory, transmembrane and intracellular calcium can be imaged simultaneously from the intact heart. Using this technique we have investigated the mechanisms of spontaneous calcium release and triggered activity under calcium overload conditions and abnormal RyR channels in the whole heart.


Link Between Genetic Sodium Channel Polymorphisms and Penetrance of the Brugada Syndrome
Isabelle Deschenes, Ph.D. (Principal Investigator)

Arrhythmias are a major cause of morbidity and mortality in the US. Genetic background of an individual may play a crucial role in predisposing them to the development of arrhythmias. In fact, mutations in the human sodium and potassium channels are responsible for the rare inherited clinical phenotypes which include the Long QT syndrome and the Brugada syndrome: life threatening arrhythmias leading to sudden death. Regardless of the mutation, Brugada Syndrome is an autosomal dominant disease, where one allele from either parent results in the phenotypic expression of the disease. Interestingly, Brugada Syndrome is characterized by incomplete penetrance, a phenomenon still incompletely understood. Previously, it was not suspected that subtle differences in the ion channel expression and structure could play a role in pre-disposing individuals to arrhythmias. However, we have recently demonstrated that an individual with an autosomal dominant and relatively rare genetic cardiac sodium channel disease (Brugada Syndrome) is asymptomatic. This individual is a clear example of the incomplete penetrance of the disease, because she carries the disease causing mutation but is asymptomatic. We demonstrated that the patient was asymptomatic because her other allele encodes for a functional sodium channel with a common polymorphism that is present in approximately 30% of the population. Through a combination of FRET, patch-clamping and protein chemistry we concluded that the polymorphism was able to rescue the disease causing mutation by increasing mutant protein trafficking to the plasma membrane. This study suggested the interesting possibility that genetic polymorphisms may be a potential target for future therapies aimed at rescuing dysfunctional protein channels. Therefore, in this project we are focusing on understanding how a sodium channel polymorphism can rescue a disease causing mutation and to study if sodium channel polymorphisms can affect the predisposition to lethal arrhythmias and sudden death.


Structure-Function Characterization of the Cardiac Sodium Channel using FRET
Isabelle Deschenes, Ph.D. (Principal Investigator)

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 aim of this project is to develop a library of human cardiac sodium channel (hNav1.5) constructs containing two fluorescent proteins for later analysis with Fluorescence Resonance Energy Transfer (FRET). FRET and biophysical analysis will be used to characterize the molecular mechanism and movements of hNav1.5. A transposition based approach is used to rapidly create a large number of constructs with insertions in different region of the cardiac sodium channel. Further studies will examine the proximity of these regions during gating of the channel. This library will then become a useful tool for characterizing the structure of the cardiac sodium channel.


Novel Molecular Mechanisms of Drug-Induced Long QT Syndrome
Eckhard Ficker, Ph.D. (Principal Investigator)

Drug-induced, acquired long QT syndrome is often caused by direct blockade of the hERG potassium channel by a wide variety of structurally diverse therapeutic compounds. Based on our studies of hERG chaperones we hypothesized that the possibility exists that proteins in the processing pathway provide targets whose inhibition by therapeutic compounds will lead to a defect in hERG processing, a reduction in cell surface expression of hERG/IKr and ultimately a novel form of acquired long QT syndrome.

To identify novel therapeutic compounds that may produce cardiotoxic site effects by compromising hERG biosynthesis and processing we developed a novel chemiluminesence-based high throughput assay. Briefly, this assay monitors changes in the expression of ion channels at the cell surface as a function of chronic drug exposure. We have identified arsenic, which is used clinically for the treatment of acute promyelocytic leukemia and is known for its cardiotoxicity, as the first cardiotoxic compound that does not block hERG directly but reduces its cell surface expression via an acquired trafficking defect. In addition, we have studied the anti-protozoal drug pentamidine, which is used for the treatment of leishmaniasis, trypanosomiasis as well as Pneumocystis carinii pneumonia as another example of a therapeutic compound that reduces the number of functional hERG channels at the cell surface. Our studies should have immediate impact on the clinical use of compounds with novel liabilities as well as the development of novel therapeutic substances.


Role of Molecular Chaperones in hERG Processing
Eckhard Ficker, Ph.D. (Principal Investigator)

Mutations in the cardiac potassium channel gene hERG (KCNH2) cause inherited long QT syndrome (LQTS2) characterized by a prolonged QT interval on the EKG, torsades de pointes arrhythmias and sudden cardiac arrest. Mutations in hERG reduce the repolarizing cardiac potassium current IKr thereby prolonging the cardiac action potential. Loss-of-function may be produced by different mechanisms including kinetic alteration of channel function or insertion of non-functioning channels into the plasma membrane. An ever-expanding group of LQTS2 mutations, however, produces trafficking-deficient channels that are retained in the endoplasmic reticulum (ER) and are thought to express conformational defects recognized by cellular quality control mechanisms. Trafficking deficient LQTS2 mutants often give rise to channels capable of conducting IKr currents if they can escape the endoplasmic reticulum and reach the cell surface. Recently, a variety of strategies has been explored to restore channel trafficking "in vitro" as a first step to implement similar strategies in patients. Given the large number of misfolded LQTS2 mutants the question arises how cells recognize and retain abnormally folded mutant channels and how they move wildtype (WT) channels along productive folding pathways. In mammalian cells, the primary level of quality control for membrane proteins such as hERG is comprised of molecular chaperones that repeatedly interact with incompletely folded proteins to facilitate native conformations. In this project we explore how cytosolic chaperones interact with newly synthesized WT and LQT2 mutant channels and determine how pharmacological chaperones modify this interaction. We propose to identify the molecular components of the multichaperone machinery associated with hERG WT channels during synthesis, assembly and maturation in the ER using mass spectrometric methods. In addition, we will probe the remodeling of the multichaperone machinery associated with misprocessed LQT2 mutations that are retained in the ER and study the relationship of hERG/chaperone complexes with the ubiquitin/proteasome system to determine how triage decisions towards degradation of channel proteins are made.