Ira S. Cohen, Ph.D., M.D. - US grants

The cardiac Purkinje strand is a syncytium. The observed properties of any syncytium depend on both the membrane permeabilities and the interaction of the membrane current flows with the extracellular geometry. The present grant proposal builds on the previous five years of research in which the canine cardiac Purkinje strand was studied by various techniques including morphometric analysis, voltage clamp, extracellular ion selective microelectrodes, and impedance analysis. From these studies we have a characterization of the properties of the cell membrane as distorted by the extracellular space. One major aim of the present grant is to provide similar voltage clamp and impedance analysis of a new preparation, the acutely isolated canine cardiac Purkinje myocyte. It is hoped that a detailed voltage clamp analysis of this building block of the syncytium should provide the necessary missing link to allow us to accurately interpret our data from the intact Purkinje strands. Proposed studies of the dissociated cells include analysis of both plateau and diastolic delayed rectifiers, characterization of the Na/K pump current, and investigation of the effects of various pharmacologic agents on the steady state current-voltage relationship. A second aim of the present grant proposal is to continue our analysis of various membrane currents in the syncytial preparation. These currents include the Na/K pump current initiated by a period of rapid activity, the slowly inactivating TTX-sensitive "window current" and both plateau and diastolic delayed rectifiers. Some of the proposed experiments employ simultaneous measurements of both the membrane currents under voltage clamp and the extracellular [K]. This should provide additional insight into the importance of changes in cleft [K] in the interpretation of time dependent membrane currents. It is hoped that these experiments will provide an increase in our understanding of the control of both the action potential duration, and pacemaker activity in normal and pathologic conditions.

The past decade has seen a veritable explosion in our knowledge of cardiac membrane conductances, largely due to the ability to obtain healthy isolated cardiac myocytes. Most of this new information has been obtained from ventricular and atrial myocytes because of the ease with which they are dissociated. In contrast much less progress has been made on the Purkinje myocyte although examining its properties is central to understanding the origin of many life threatening arrhythmias. Our laboratory has developed a reliable technique for the dissociation of Purkinje myocytes from their collagenous matrix. We study the electrophysiologic properties of the isolated Purkinje myocytes and also those of the Purkinje fibers. In the present application we propose to continue our studies of three Purkinje membrane currents: the inward rectifier current iK1, the pacemaker to a number of important questions. With respect to iK1 these questions include the origin or rectification, the role played by internal K in gating iK1, and the control of the inactivation of this conductance by B stimulation. Our investigations of the pacemaker current will center on our recent finding that acetylcholine can reverse B agonist effects on if without having direct effects of its own. We will also investigate the origin of the pacemaker activation delay. Our studies of the Na/K pump current will attempt to elucidate the reasons for an order of magnitude difference in the ability of dihydroouabain to inhibit the Na/K pump current in ventricular versus Purkinje myocytes. A second set of experiments examines the interaction of the Na/K pump with the Na/Ca exchanger in response to a calcium load, or in response to prolonged blockade of the Na/K pump by cardiac glycosides. These studies should provide us with important insights into the normal electrophysiology and pharmacology of the Purkinje myocyte, and how this normal function can be modified.

The past decade has seen a veritable explosion in our knowledge of cardiac membrane conductances, largely due to the ability to obtain healthy isolated cardiac myocytes. Most of this new information has been obtained from ventricular and atrial myocytes because of the ease with which they are dissociated. In contrast much less progress has been made on the Purkinje myocyte although examining its properties is central to understanding the origin of many life threatening arrhythmias. Our laboratory has developed a reliable technique for the dissociation of Purkinje myocytes from their collagenous matrix. We study the electrophysiologic properties of the isolated Purkinje myocytes and also those of the Purkinje fibers. In the present application we propose to continue our studies of three Purkinje membrane currents: the inward rectifier current iK1, the pacemaker to a number of important questions. With respect to iK1 these questions include the origin or rectification, the role played by internal K in gating iK1, and the control of the inactivation of this conductance by B stimulation. Our investigations of the pacemaker current will center on our recent finding that acetylcholine can reverse B agonist effects on if without having direct effects of its own. We will also investigate the origin of the pacemaker activation delay. Our studies of the Na/K pump current will attempt to elucidate the reasons for an order of magnitude difference in the ability of dihydroouabain to inhibit the Na/K pump current in ventricular versus Purkinje myocytes. A second set of experiments examines the interaction of the Na/K pump with the Na/Ca exchanger in response to a calcium load, or in response to prolonged blockade of the Na/K pump by cardiac glycosides. These studies should provide us with important insights into the normal electrophysiology and pharmacology of the Purkinje myocyte, and how this normal function can be modified.

DESCRIPTION: (Adapted from the Investigator's Abstract) Although acute modulation of cardiac membrane currents has been the subject of intensive investigation since the advent of the patch clamp technique, long-term modulation has received much less attention. This application proposes to investigate changes in cardiac electrophysiology that occur over a period of hours to days. Two major questions will be investigated: 1) the role played by the renin-angiotensin system in setting up the gradient in electrical properties across the ventricular wall which gives rise to the normal polarity of the T-wave, and 2) the changes induced in ion channels and pumps by long term exposure to alpha or beta adrenergic agonists. The applicant will employ a multidisciplinary approach including 1) biophysical (patch clamping), 2) molecular biology (RNase protection assays), and 3) protein chemistry (radioimmunoassays and phosphoantibodies). The results should provide insight into normal cardiac electrophysiology, as well as the changes that occur in response to elevated levels of angiotensin II or catecholamines. As the proposal includes studies of animals maintained on chronic ACE inhibition, AT1 receptor blockade, alpha adrenergic blockade, and beta adrenergic blockade, these studies should also be relevant to the electrical changes induced in cardiac cells by chronic exposure to these commonly used cardiovascular medications.

DESCRIPTION: (Adapted from the Investigator's Abstract) Although acute modulation of cardiac membrane currents has been the subject of intensive investigation since the advent of the patch clamp technique, long-term modulation has received much less attention. This application proposes to investigate changes in cardiac electrophysiology that occur over a period of hours to days. Two major questions will be investigated: 1) the role played by the renin-angiotensin system in setting up the gradient in electrical properties across the ventricular wall which gives rise to the normal polarity of the T-wave, and 2) the changes induced in ion channels and pumps by long term exposure to alpha or beta adrenergic agonists. The applicant will employ a multidisciplinary approach including 1) biophysical (patch clamping), 2) molecular biology (RNase protection assays), and 3) protein chemistry (radioimmunoassays and phosphoantibodies). The results should provide insight into normal cardiac electrophysiology, as well as the changes that occur in response to elevated levels of angiotensin II or catecholamines. As the proposal includes studies of animals maintained on chronic ACE inhibition, AT1 receptor blockade, alpha adrenergic blockade, and beta adrenergic blockade, these studies should also be relevant to the electrical changes induced in cardiac cells by chronic exposure to these commonly used cardiovascular medications.

DESCRIPTION (provided by applicant): The cellular delivery of siRNA via gap junctions represents a unique and potentially clinically important delivery system. Our previous studies have shown that gap junctions composed of connexin43 (Cx43) are permeable to siRNAs and permeating siRNAs can subsequently reduce the mRNA levels of a specific gene. The aim of the studies proposed here is to characterize the transfer and permeation of siRNA from hMSCs and other communication competent cells into a target tissue. Previously we determined that gap junctions composed of Cx43 transfer siRNA, whereas those composed of Cx32 or Cx26 will not. Hence channel permeability for siRNA depends on the connexin. In aim 1 we will determine the permeability of gap junctions made of Cx40, Cx37 and Cx43, to morpholinos and siRNAs. Cx43, Cx40 and Cx37 are chosen because they are ubiquitously expressed in vivo in many organs. In aim 2 we will determine the synthesis and degradation rates of siRNA targeting HCN2 and GFP. We will also investigate the efficacy and time course of functional silencing of the membrane protein, the pacemaker channel HCN2. We will test the hypothesis that cellular delivery of siRNA via gap junction channels can silence HCN2 channel function in target cells by characterizing the functional silencing of HCN2 via gap junction mediated delivery of siRNA from hMSCs or other communication competent cells to a target cell expressing HCN2. We follow HCN2 mRNA concentration using RT-PCR to allow an estimate of the relative content over time in the presence of siRNA. We will also determine the concentrations of tagged morpholinos/siRNAs to establish the effective concentration necessary to silence a gene (HCN2 or GFP) and also provide parameters for our 2D/3D model to determine penetration within a tissue. In aim 3 we will experimentally assess how far siRNA can penetrate multiple cell layers of a syncytium. In aim 4 we will derive a model for transfer of siRNA along a simple linear chain of cells or geometries in 2 or 3 dimensions. It will be used to predict the number and position of siRNA containing cells (hMSCs) required to silence function in a tissue or an organ/tumor. In aim 5 we will assess siRNA effectiveness in silencing GFP in vivo. We use nude mice and inject a bolus of 10 million cells expressing GFP into the dermis or intramuscularly followed at various times with an injection of hMSCs loaded with siRNA targeting GFP. We will track the GFP fluorescence image over time using whole animal imaging. PUBLIC HEALTH RELEVANCE: Small interfering RNA (siRNA) targets a single protein reducing its expression. As such it has great potential as a highly selective drug. However systems for its in vivo delivery are not optimal. The present application investigates the ability of the immuno-privileged adult mesenchymal stem cell (MSC) as well as other cell types to deliver small interfering RNA (siRNA) to a target cell or tissue. The basis of this cell based delivery is the gap junction channel. These channels connect the intracellular compartments of coupled cells and allow transfer of small molecules without entry into the extracellular space. We have already established that cells that make connexins (the building block of gap junctions) can transfer siRNAs. This application asks whether cells can serve as a delivery system for siRNA. By a combination of experiment and mathematical modeling we seek to determine the ability of cellular delivery of siRNA to penetrate tissues in vitro and in vivo.

Project Summary: The long term goal of this application is to create a pure population of cardiac pacemaker cells from an easily accessible autologous cell type, the human hair follicle keratinocyte (HFKT-pacemakers), to characterize their pacemaker mechanism, their ability to integrate into the cardiac syncytium and their potential to function as an in vivo biological pacemaker. If successful in the long term, the health benefit of such an approach will be to substitute for the more than 350,000 electronic pacemakers implanted or reimplanted in patients in the United States each year. The project has four specific aims: (1) to expand the population of induced pluripotent stem cells created from the HFKTs, enhance their differentiation to a cardiac lineage and select for pacemaker myocytes; (2) to characterize the membrane currents in the HFKT- pacemakers as well as their pacemaker function and gene expression profile, and to compare the HFKT- pacemaker to native cardiac primary and secondary pacemakers in vitro; (3) to determine (a) the ability of HFKT pacemakers to couple to adult heart cells from specific locations (atrium or ventricle) in vitro and whether the pacing rate generated is target dependent (b) which connexins HFKT-pacemakers express and the ability of the HFKT-pacemakers to couple to cells expressing fibroblast connexins (a potentially arrhythmogenic situation); (4) to determine in vivo biological pacemaker function generated by placement of HFKT-pacemakers in the canine atrium or ventricle. Our approach will employ 1) novel methods to enhance selection of pacemaker cells, 2) patch clamping to characterize action potential morphology and membrane currents in the isolated pacemaker cells generated, 3) gene chips to determine the pacemaker cells' expression profile, 4) dual whole cell patch clamp, and biochemical and molecular techniques to determine connexin expression and functional cell to cell coupling 5) Injection of the HFKT- pacemakers into the canine atrium or ventricle to determine in vivo pacemaker function. The experiments will be carried out by a team of long term collaborators at Stony Brook University and at the Technion in Israel. The team has extensive expertise in stem cell biology, induced pluripotent stem cells, cardiac pacemaking, patch clamp,, and in vivo studies of biological pacemaker function. Successful execution of the research plan will enhance selection techniques for cardiac cell lineages from IPSCs, characterize the basis of pacemaker activity in the HFKT- pacemakers and determine their effectiveness as an in vivo biological pacemaker. If the HFKT-pacemaker functions well in the canine heart, a future goal would be to advance this novel autologous, cellular approach towards clinical deployment.

DESCRIPTION (provided by applicant): The overall objective of our proposed research is to use our knowledge of the pathophysiology of reentry and of myocardial infarct-associated ventricular tachycardia to hypothesize innovative, mechanism-based approaches to therapy Our general hypothesis is that gene therapy using adult human mesenchymal stem cells (hMSCs) as platforms and/or using viral vectors can deliver overexpressed ion channel gene constructs to prevent/suppress this arrhythmia. Our proposed 5-year plan incorporates: (1) identification and testing the effect of overexpression of specific gene constructs in viral vectors and in hMSC platforms to modify specific ion channel expression in cell lines; (2) using mathematical modeling, cell systems, and animal models that previously have been validated by us and others to test the mechanism of action, efficacy and proarrhythmic potential of each gene and cell therapy approach we design. We specifically hypothesize that gene and cell therapies can be antiarrhythmic by speeding conduction and/or prolonging refractoriness (but not repolarization) and study these possibilities in the canine heart in situ. Our first two Aims (stated as hypotheses) employ novel approaches to speed conduction. 1: A non-cardiac Na channel that shifts inactivation to more depolarized potentials will enhance Na current density in normal myocytes firing at high rates and preserve Na current density in depolarized myocytes. This should increase action potential (AP) upstroke velocity and conduction velocity, such that antegrade activation is normalized to prevent reentrant arrhythmias and/or the head of the activating wave catches the tail to terminate reentrant arrhythmias. 2: Increasing diastolic K conductance should restore depolarized membrane potentials towards normal and enhance excitability for normal myocytes at high stimulation frequencies. The third strategy is to prolong the effective refractory period (ERP) with regard to AP duration (APD). 3: Here, we hypothesize that overexpression of a mutant hERG with slowed deactivation kinetics should improve rate responsiveness and prolong ERP compared to APD. This should speed conduction at high heart rates while blocking propagation of premature depolarizations, reducing the likelihood of reentry. The significance of our proposed research is seen in the identification of novel ion channel constructs, testing them via in silico modeling and then in cell experiments to understand and fine-tune mechanism of action; using innovative means to administer them in cell systems and finally in intact animals to treat a reentrant rhythm - ventricular tachycardia - that is a major cause of morbidity and mortality in the US today. The selectivity and specificity of these approaches far exceed those of drugs and of ablation and open promising new vistas for arrhythmia treatment and prevention.