William Jonathan Lederer - US grants

The proposed work will examine the cellular basis for regulation of contractile force in heart muscle. This study will examine how specific fundamental cellular mechanisms vary during the cardiac cycle and influence force production. Quantitative examination of the following cellular features will be carried out and their influence on the generation of force assessed: (1) The intracellular activity of three important cations - sodium (aiNa), calcium (aiCa) and hydrogen (pHi); (2) The trans-sarcolemmal movement of calcium and sodium (via the calcium current, the sodium current, the Na/Ca exchanger and the Ca-activated non-selective cation current); (3) The internal stores of calcium (the sarcoplasmic reticulum). This work will be carried out in ventricular muscle and in cardiac Purkinje fibers over five years. Single ventricular muscle cells from rat and guinea pig (enzymatically dissociated) will be voltage-clamped using a single-microelectrode method. A fluorescent indicator will be used to measure aiCa (fura-2), aiNa will be measured using an ion-selective microelectrode and pHi will be measured using both techniques (with BCECF as the optical indicator). Video imaging techniques will be used to measure sarcomere length, cell length and the spatial distribution of intracellular calcium. Sheep Purkinje fibers will be examined using a two-microelectrode voltage-clamp method while measuring tension, aiCa (using the calcium-activated photoprotein aequorin), and aiNa and pHi (using ion-selective microelectrodes). This combination of new techniques and established methods will permit us, for the first time, to examine quantitatively the ionic control of tension in myocardium and in Purkinje fibers under normal conditions and when resting aiCa is elevated ("calcium overload"). The planned experiments will address the following questions: (1) What mechanisms control resting aiCa? How important is the mean level of aiNa in controlling aiCa? (2) How is the peak level of aiCa (during the twitch) regulated and how is it modulated by aiNa? (3) How is peak aiCa related to the resting aiCa? How is this relationship altered during "calcium overload"? (4) How do changes of heart rate and of action potential duration modify the answers to these questions? (5) How do therapeutic agents (e.g. cardiotonic steroids, blockers or Na+ and Ca++ channels) modify the above relationships? The proposed research should broaden our understanding of the cellular mechanisms that govern the development of tension in heart. By revealing the physiological control of contraction in heart and its modification by therapeutic agents, this work should lay the foundation for improved treatment of diverse cardiac disease including heart failure.

The proposed work will examine calcium-dependent and triggered arrhythmias in heart muscle. This study will investigate links between the intracellular concentrations of free calcium ions (aiCa), sodium ions (aiNa) and hydrogen ions (pHi) and normal and arrhythmogenic electrical properties of heart. Examination of tissue under voltage clamp will be carried out to determine which ionic currents have been altered during arrhythmogenesis and to what extent each membrane current component is responsible for the generation of the abnormal electrical activity. Experiments will make use of recently developed methods to study membrane potential, membrane current and aiNa, aiCa and pHi. Two experimental preparations will be used. (1). Single ventricular muscle cells from rat and guinea pig (enzymatically dissociated) will be voltage-clamped using a single-microelectrode method. In the single cell experiments the calcium-sensitive fluorescent indicator fura-2 will be used to measue aiCa while the H+ sensitive fluorescent indicator BCECF will be used to measure pHi. Video imaging techniques will be used to measure levels and spatial variations of aiCa and of phi. (2). Sheep Purkinje fibers will be examined using a two-microelectrode voltage-clamp method while measuring tension, aiCa (using the calcium- activated photoprotein aequorin), and aiNa and pHi (using ion- selective microeletrodes). This combination of new techniques and established methods will permit us, for the first time, to examine quantitatively the ionic control of membrane currents in myocardium and in Purkinje fibers under normal and arrhythmogenic conditions. The planned experiments will address the following questions: 1. What is the role of the calcium-activated current ITI in the genesis of cardiac arrhythmias? 2. What cellular processes give rise to ITI? Is this current generated by a calcium-activated channel? 3. How do interactions between the normal calcium current (ICa) and ITI affect arrhythmogenesis in heart? 4. How do Na-Channel blockers and Ca-channel blockers effect ITI? Do the agents act directly on the ITI current pathway? Do they affect ITI by altering intracellular ion activity? Do they only decrease excitability? The proposed research should broaden our understanding of the cellular mechanisms that regulate the normal heartbeat and underlie the development of arrhythmias. Furthermore, by revealing the ionic and cellular basis of certain cardiac rhythm disturbances, this work should lay the foundation for the development of new drugs and improved application of the currently used therapeutic agents and diagnostic techniques.

The Na-Ca exchanger is the dominant sarcolemmal calcium extrusion mechanism in heart muscle. Despite its importance, only recently have quantitative investigations into its function been carried out. In the proposed experiments, biophysical, biochemical and pharmacologic properties of the Na-Ca exchanger will be examined. The goal of this work is to determine how the Na-Ca exchanger works in heart muscle, how it is modulated by important cations (e.g. Ca2+, Na+, H+) and metabolic factors (e.g. ATP) and how it may be altered by drugs and inorganic inhibitors. Additionally, the planned work will examine specific features of the Na-Ca exchanger to determine how it influences contraction in heart. The proposed experiments will make use of two important new approaches for investigations of the Na-Ca exchanger and will use isolated guinea pig and rat heart cells. (1) The first method has been developed in the PI's laboratory. By integrating a flashlamp into the voltage-clamp fluorescence microscopy system, caged calcium (DM nitrophen and Nitr-5) can be used to produce [Ca2+]i jumps to activate the Na-Ca exchanger. Two components of the Na-Ca exchanger current have been identified. One component (never before seen) arises from a molecular conformational change of the transport protein (Iconf) while the other reflects net transport by the exchanger (INa-Ca). Quantitative data generated by this method is quite novel, having been unobtainable until now and represents an important new source of information about the exchanger in functioning cells and will be used as a new tool in the proposed experiments. Preliminary experiments have been carried out using this method to provide estimates of the density of exchangers in the sarcolemma, the turnover rate of the exchanger under various conditions and the charge on the "naked" exchanger protein (i.e. without sodium or calcium bound). The novelty and power of this method is further enhanced because cell length and [Ca2+}i, or [Na+]i or pHi (using fluorescent indicators) can be measured simultaneously. This method will therefore be used to investigate the properties of the Na-Ca exchanger and to examine the effects of exchanger activity on contraction in heart cells. (2) The second approach uses "giant" sarcolemmal membrane patches to measure Na-Ca exchanger current (Hilgemann, 1989, 1990) providing access to both intracellular and extracellular surfaces of the sarcolemmal membrane while measuring INa-Ca. Specific experiments using the above methods are designed to address two broad questions. (1) How is the Na-Ca exchanger controlled within the heart cell? (2) How does the Na-Ca exchanger participate in excitation- contraction coupling? The proposed work should provide important primary information that will broaden our understanding of how the Na-Ca exchanger functions and how it influences [Ca2+]i SR calcium loading and EC coupling in heart muscle. Accordingly, the planned investigation should provide us with new understanding on the normal and pathologic functioning of the heart.

The Na-Ca exchanger is the dominant sarcolemmal calcium extrusion mechanism in heart muscle. Despite its importance, only recently have quantitative investigations into its function been carried out. In the proposed experiments, biophysical, biochemical and pharmacologic properties of the Na-Ca exchanger will be examined. The goal of this work is to determine how the Na-Ca exchanger works in heart muscle, how it is modulated by important cations (e.g. Ca2+, Na+, H+) and metabolic factors (e.g. ATP) and how it may be altered by drugs and inorganic inhibitors. Additionally, the planned work will examine specific features of the Na-Ca exchanger to determine how it influences contraction in heart. The proposed experiments will make use of two important new approaches for investigations of the Na-Ca exchanger and will use isolated guinea pig and rat heart cells. (1) The first method has been developed in the PI's laboratory. By integrating a flashlamp into the voltage-clamp fluorescence microscopy system, caged calcium (DM nitrophen and Nitr-5) can be used to produce [Ca2+]i jumps to activate the Na-Ca exchanger. Two components of the Na-Ca exchanger current have been identified. One component (never before seen) arises from a molecular conformational change of the transport protein (Iconf) while the other reflects net transport by the exchanger (INa-Ca). Quantitative data generated by this method is quite novel, having been unobtainable until now and represents an important new source of information about the exchanger in functioning cells and will be used as a new tool in the proposed experiments. Preliminary experiments have been carried out using this method to provide estimates of the density of exchangers in the sarcolemma, the turnover rate of the exchanger under various conditions and the charge on the "naked" exchanger protein (i.e. without sodium or calcium bound). The novelty and power of this method is further enhanced because cell length and [Ca2+}i, or [Na+]i or pHi (using fluorescent indicators) can be measured simultaneously. This method will therefore be used to investigate the properties of the Na-Ca exchanger and to examine the effects of exchanger activity on contraction in heart cells. (2) The second approach uses "giant" sarcolemmal membrane patches to measure Na-Ca exchanger current (Hilgemann, 1989, 1990) providing access to both intracellular and extracellular surfaces of the sarcolemmal membrane while measuring INa-Ca. Specific experiments using the above methods are designed to address two broad questions. (1) How is the Na-Ca exchanger controlled within the heart cell? (2) How does the Na-Ca exchanger participate in excitation- contraction coupling? The proposed work should provide important primary information that will broaden our understanding of how the Na-Ca exchanger functions and how it influences [Ca2+]i SR calcium loading and EC coupling in heart muscle. Accordingly, the planned investigation should provide us with new understanding on the normal and pathologic functioning of the heart.

DESCRIPTION (provided by applicant): The elementary unit of Ca2+ release in heart muscle is the calcium spark, discovered by the PI in 1993. The molecular mechanisms that underlie activation, modulation and termination of Ca2+ sparks remain elusive or controversial. The proposed work will use new methods developed by the PI to investigate the biophysical properties of Ca2+ sparks that should enable us to broaden our understanding of cardiac Ca2+ signaling and resolve the conflicts. The proposed patch clamp experiments will use cardiac myocytes from rats and rabbits to examine Ca2+ sparks. Simultaneous confocal calcium imaging and flash photolysis of caged Ca2+ will permit a quantitatively investigation of Ca2+ spark behavior. Sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors or RyR2s) will be studied in parallel experiments using a planar lipid bilayer technique with flash photolysis and rapid solution switching methods. Preliminary results suggest that all proposed experiments can be done and should provide important new information. There are four questions that the proposed work seeks to address. 1 How does the triggering of Ca2+ sparks depend on [Ca2+]i and important cellular regulators of EC coupling? 2. How does RyR2 behavior influence the triggering of Ca2+ sparks? 3. How does membrane potential influence Ca2+ spark triggering? 4. What mechanisms underlie Ca2+ spark restitution? The planned work will provide fundamental new information on how Ca2+ sparks work in heart. It will examine Ca2+-induced Ca2+-release (CICR), the triggering by [Ca2+]i of Ca2+ sparks, the regulation of CICR and Ca2+ spark behavior and how restitution of the Ca2+ spark occurs. It will also link Ca2+ spark properties to the behavior of RyR2s. The proposed work should broaden our understanding of cardiac Ca2+ signaling, excitation-contraction (EC) coupling and spontaneous SR Ca2+ release. This investigation is part of the Pl's long-term goal to understand how normal physiological signaling regulates heart muscle and how molecular and cellular alterations of such signaling systems lead to specific pathologies and novel treatments.

Human heart failure is a clinical syndrome of diverse etiology characterized by weakened cardiac contractions. Despite the many causes, preliminary experiments suggest that heart failure (HF) may have a common pathology at the cellular and molecular levels. The PI has identified a reduction in the ability of Ca2+ influx to trigger the [Ca 2+]i transient in ventricular myocytes from the hearts of animals in HF. This defect, called cellular HF , leads to clinical HF when accompanied by betaAR desensitization and/or downregulation. Our observation in pressure overload HF is strongly supported by preliminary findings with gene targeted mouse models of dilated cardiomyopathy HF (MLP knockout mouse) and viral myocardiopathy HF(CVB3 mouse). This raises the possibility that in animal models of HF, there are at least two features that develop over time: cellular HF and betaAR desensitization and/or down-regulation. Our proposed work will examine this hypothesis at the cellular level using state-of-the-art methods including high-speed confocal [Ca 2+]i imaging and whole cell patch-clamp techniques. We will examine excitation-contraction (EC) coupling by measuring membrane currents while recording whole-cell [Ca 2+]i transients, Ca2+ sparks and cellular contractions. Three specific questions will be addressed during the proposed work. (1). What EC coupling defects underlie and/or contribute to cellular HF? (2). How does betaAR signaling affect the EC coupling defects in HF? (3). How do specific pharmacological and molecular therapies alter the EC coupling defects in HF? This project exploits new methods developed by the PI using (Ca 2+]i imaging at high temporal and spatial resolution to investigate HF. The proposed work should increase specific knowledge of the cellular and molecular defects in HF and lay the foundation for novel therapeutic approaches to the treatment of HF.

Cells control many of their functions through localized (micron to sub- micron scale) concentration changes of intracellular messengers. These changes often occur rapidly (millisecond to sub-millisecond scale). For example, contraction of muscle cells and section from neurons are regulated by transient (1-100 ms) increases in intracellular calcium. The introduction of calcium-sensitive fluorescent indicators like fluo-3 discovered in part by J. Kao (Project I) while working in the laboratory of R.Y. Tsien, opened the way for microscopical studies of calcium transients in living cells. Coupled with the improved spatial resolution of confocal microscopy, calcium imaging studies made it possible to observe the release of calcium from single channels or single "release units" in intracellular stores. These 'calcium sparks' were first discovered by Lederer (Project II) in cardiac (Cheng et al., 1993) and smooth muscle (Nelson et al., 1995) cells, and were subsequently discovered in skeletal muscle cells in collaboration with other campus researchers (Klein et al., 1996). These and related studies have contributed to the University of Maryland's stature as a major research center for studies of intracellular calcium signaling and regulation, which is the primary research focus of more than 20 independent campus laboratories. Further advances in this area require improvements in temporal resolution so that the kinetics of spark formation and propagation, and their relationship to the calcium transients they evoke, can be characterized. The equipment requested by this application is intended to create a multi- user core facility that meets this requirement. A high-speed confocal microscope and dedicated voltage clamp apparatus will enable researchers at the University of Maryland and other area institutions to initial and examine calcium sparks with high spatial and temporal resolution. An integrated photolysis laser and temperature-controlled perfusion system will enable them to manipulate the intracellular and extracellular environment of living cells to allow correlation betweens parks and various stimuli. In addition to enabling new approaches to the study of calcium sparks and other dynamic phenomena in living cells, this core facility will have a significant impact on the teaching mission of the University of Maryland.

DESCRIPTION: Rapid progress has been made over the past several years in understanding the molecular mechanisms of signaling by intracellular calcium i diverse cell types. Recent technical and conceptual advances have revealed tha stimulus-evoked Ca2+ signals are often confined to highly localized domains in the cell, with important implications for both the generation and functional consequences of these signals. An international symposium on "Local Calcium Signaling in Cell Physiology" will be held September 9-11, 1998, at the Marine Biological Laboratory in Woods Hole, Massachusetts, to disseminate the latest advances in this field. This meeting, which is the 52nd Annual Symposium of th Society of General Physiologists, will focus on the mechanisms by which localized calcium signals are generated within cells, and on the diverse consequences of these signals for cell function. The meeting will follow a highly successful format used in the past for SGP Symposia. The latest research findings related to the meeting topic will be presented in four forums. 1) Lectures by 19 leaders in the field will cover aspects of local Ca2+ signaling in diverse systems including both excitable cells (neurons, muscle, neuroendocrine cells) as well as non-excitable cells (exocrine cells, lymphocytes, oocytes). The talks will be arranged in five sessions of roughly three hours each and will cover elementary Ca2+ events in skeletal and cardiac muscle, control of ion channels by local Ca2+, exocytosis Ca2+ domains and postsynaptic signaling, and the influence of local Ca2+ on IP receptors and organellar function. 2) Two poster presentations will encourage active participation by all attendees and promote informal discussion of recen results among speakers and attendees. 3) Six short talks will be devoted to late-breaking results and will be given by younger investigators, with topics to be selected from the abstracts submitted for poster sessions as well as fro the scientific community at large. 4) The conference will end with a keynote lecture by Dr. Edwin Neher, a renowned pioneer and leader in the field of loca Ca2+ signaling and its functional roles in cell physiology. The goal of this unique and timely meeting is to bring together a diverse grou of researchers to discuss and compare the latest findings in the rapidly evolving field of local Ca2+ signaling. Attendance at the meeting, which will be widely publicized, is expected to be 150-250 scientists, including graduate students, postdoctoral scientists, and independent investigators interested in a broad range of signaling mechanisms and physiological systems. Because of it interdisciplinary scope, it is anticipated that the conference will not only educate the attendees with respect to the current state of knowledge, but will also play an important role in identifying new directions for future research in this field.

Ca2+ entry though TTX-sensitive Na+ channels was discovered by the PI in heart cells [154] and called "slip-mode conductance". Activated by protein kinase A (PKA), Ca2+ permeability relative to Na+ (P/Ca/P/Na) increased from near zero to approximately 1.0. While slip-mode conductance was confirmed in HEK293 cells expressing cardiac alpha subunits, beta subunits had to be co-expressed [38] and alpha subunits from neither brain nor skeletal muscle could replace cardiac (see Preliminary Results). This proposal will examine slip-mode conductance of the cardiac Na+ channel, quantify its physiological and determine its molecular basis. The planned experiments will test the hypothesis that slip-mode conductance provides significant Ca2+ influx under physiological conditions. Using confocal Ca2+ imaging and patch clamp methods, the PI will test the hypothesis in cardiac myocytes and in HEK293 cells expressing Na+ channels by addressing two experimental questions. (1). What fraction of the [Ca2+]i transient in heart that is due to slip mode conductance? Slip-mode conductance will be examined quantitatively to determine Ca2+ influx and establish how it is affected by physiologic modulators of Ca2+ signaling in heart (e.g. pH, the amount of Ca2+ in the SR protein kinase C). Cardiac myocytes from rat, mouse and human hearts will be compared. Heart cells from transgenic and knockout mice will enable the investigation of A-kinase anchoring proteins (AKAPs) and the beta subunits in slip-mode conductance. (2). Why is the cardiac Na+ channel uniquely abuse to activate slip-mode conductance? Cardiac-skeletal muscle chimeras of the alpha subunit will be used to determine what part(s) of the cardiac alpha subunit is(are) necessary for slip-mode conductance. Mutations that affect channel kinetics (e.g. fast inactivation) will be used to examine the effects of channel gating on slip-mode conductance. The proposed experiments should broaden our understanding of Ca2+ signaling in heart. We should identify the molecular basis of Ca2+ permeation of cardiac Na+ channels and characterize its physiological importance. The planned work thus supports the PI's long-term plan to broaden our understanding of heart function.

Cardiac arrhythmias are a leading cause of death in humans and occur in diverse conditions. The proposed research seeks to identify and characterize fundamental mechanisms that underlie fatal cardiac arrhythmias. Specific cellular and molecular events that trigger arrhythmias will be examined to test the hypothesis that changes in subcellular calcium signaling contribute to arrhythmogenesis. Animal models of altered electrical activity in the heart will be studied at the single cells level using whole patch clamp methods and confocal calcium imaging. Isolated cardiac myocytes from control and transgenic animals and cells expressing specific constructs will be used in the planned work. Preliminary results have demonstrated calcium-dependent links between altered electrical behavior and the expression of specific cellular proteins that are being examined in Project 1 (Russo), Project 2 (Marks) and Project 3 (Kass). The proteins of particular interest include beta1AR, beta2AR, RyR2, FKB12, FKBP12.6, SCN5A and mutations of these proteins. The proposed work examines how expression of the target proteins affects intracellular [Ca2+]i and also Ca2+-dependent membrane currents. This examination will explore the importance of the action potential shape and duration on [Ca2+]i signaling in the proposed experimental models. Additionally the relationship between SR Ca2+ content and Ca2+ release (as measured by Ca2+ sparks and the global (Ca2+) transient) will be examined with these molecular models. The experiments carried out in this project should thus provide fundamental new information on the arrhythmogenic roles played by the betaAR signaling system, sarcolemmal ion channels and the intracellular Ca2+ release channels.

DESCRIPTION (provided by applicant): Ca2+ signaling dysfunction occurs in heart muscle under many conditions and is associated with diverse cardiac pathologies. This project seeks to characterize quantitatively Ca2+ signaling defects that occur in cardiac ventricular myocytes and to investigate the causes of the dysfunction. Confocal Ca2+ imaging of single ventricular myocytes will be carried out to measure Ca2+ sparks and the cellular Ca2+ signal at high temporal and spatial resolution. Intracellular Ca2+ will be rapidly changed by photorelease of caged Ca2+ while membrane potential is controlled with a whole cell patch clamp method. The planned experiments will reveal the links between the Ca2+ current, the Ca2+ sparks and other components of the Ca2+ signal. Preliminary experiments suggest a central hypothesis: a disruption of the machinery of excitation-contraction coupling may underlie the Ca2+ signaling defects that have been observed. Such remodeling may involve reorganization of the cytoskeletal proteins, altered positioning of L-type Ca2+ channel proteins and/or the ryanodine receptor Ca2+ release channels or re-organization of the transverse-tubules. To investigate the hypothesis, transgenic mice, rat models of disease and new cell culture methods will be used to examine the molecular causes of the Ca2+ signaling defects. Specific experiments will be carried out to characterize the role of cytoskeletal elements in the Ca2+ signaling defects. The planned work should therefore broaden our understanding of Ca2+ signaling in heart and clarify how it may become dysfunctional in disease. Additionally, the work should provide insight into the subcellular organization of the heart cell and illuminate the links between transverse tubules, cytoskeletal structures, and voltage- and ligand-gated channels.

[unreadable] DESCRIPTION (provided by applicant): This application requests funds to purchase a high speed imaging system for confocal fluorescence imaging, subcellular photolysis and patch clamp control of single cells. The confocal imaging system will be used in a new shared instrument facility at the University of Maryland Biotechnology Institute (UMBI). Extensive testing of each element of the system by the users has been carried out and planned work using this novel state-of-the-art imaging system is outlined in the proposed projects. The imaging system involves three components. The confocal instrument is the Zeiss LiveS instrument that is capable of acquiring XY images at 1 kHz at 50 lines by 512 pixels or full frame (512 X 512) at a frame rate of 120 Hz (8.3 ms per image). Linescan images (1 X 512) can be obtained at 62.5 kHz (16 microseconds). The photolysis component of the system is centered on a Spectra Physics HIPPO-355 OEM laser running at 100 kHz with an average power of 5 W. The scan head can be placed on an inverted microscope for optimal single cell patch clamp experiments or on an upright microscope of brain slice or special culturing conditions. The triggering and laser launch optics are customized for use with both the Zeiss Live5 instrument and with the patch clamp electronics. It can be synchronized to microsecond precision, which is adequate for all planned work. The patch clamp component of the system is centered on a HEKA EPC10 patch clamp unit and LIH-1600 computer interface running Patchmaster acquisition software. Researchers at the University System of Maryland in Baltimore (UMBI and UMB) have achieved a three-decade reputation for the study of calcium signaling at high temporal and spatial resolution in living cells. They have discovered Ca2+ sparks in cardiac, skeletal and smooth muscle, have investigated function in these tissues in normal and pathological conditions and have been world- wide leaders in this area. They have a similar reputation in neuronal and brain slice imaging. This proposed new instrumentation is not available anywhere in the Baltimore-Washington area and will allow these scientists to carry out funded research and to make significant advances. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] This application seeks partial funding for the Gordon Research Conference (GRC) on "Cardiac Regulatory Mechanisms" to be held at Colby Sawyer College (Colby-Sawyer, NH) on July 20-25, 2008. Young investigators and established leaders will present the latest unpublished findings and the discussion leaders will promote lively discussions and critical questioning. The topics presented will include cardiac signaling, stem cells and regeneration, molecular control of subcellular Ca2+, transcriptional modulation and mitochondrial signaling dynamics. In addition, myofilament behavior and signaling dysfunction will be presented as will influence of gender and aging. Outstanding speakers have been chosen from those who have not presented at this meeting in the last two years. The meeting seeks to foster discussion and interaction among all participants, consistent with GRC tradition and the 25-year history of this particular conference. Participants will be chosen from the talented and productive scientists at all stages in their careers from diverse disciplines, all with a common interest in the topic of regulation of the normal and diseased heart. A key objective of this conference is to foster an inclusive, intense, lively and interactive atmosphere that creates productive discussions and enables exchanges of novel ideas, where everyone is encouraged to participate. The special informal nature of the GRC has promoted discussions that span disciplines and geography and has enabled new personal and scientific connections to develop. The GRC is also a place for young and established investigators to have meaningful interactions. This is an exciting time in cardiovascular science because of the advent of novel technologies that allow us to explore the cellular and molecular bases of normal cardiac function. We are beginning to translate these new findings into a better understanding of disease mechanisms, diagnosis, prevention and treatment. We have had a good mix of fundamental science related to regulation of normal cardiac electrical and mechanical properties, cardiac myocyte cell signaling, and abnormalities in these processes that contribute to cardiac dysfunction. The proposed presentations (see below) are likely to be both exciting and timely. Late breaking science will be generously represented in the oral session with speaker recruited from poster contributions and recent publications. (End of Abstract) [unreadable] [unreadable] [unreadable]

Address; Animals; Arrhythmia; base; Behavior; Calcium; Cardiac; Cardiac Myocytes; cell growth regulation; Cells; Complex; Death, Sudden, Cardiac; Defect; Deletion Mutation; Equilibrium; Event; Functional disorder; Funding; Genetic; Goals; Heart; Homeostasis; improved; Ion Channel; Kinetics; Macromolecular Complexes; Measures; member; Membrane; Molecular; Molecular Target; mouse model; novel; Plant Roots; Play; Process; programs; Progress Reports; Regulation; Research; Research Personnel; research study; Resolution; Role; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; RyR2; Sarcolemma; Signal Transduction; Surface; Therapeutic; tool; Variant; Work

DESCRIPTION (provided by applicant): This research will be done primarily in Brazil at Universidade Federal de Minas Gerais in collaboration with Dr. Silvia Guatimosim, as an extension of Project 3 of NIH Grant number P01 HL67849 (A.R. Marks), 4/1/2006-3/31/2011. Dr. W. Jonathan Lederer is the project leader. The proposed FIRCA project seeks to enable novel real-time imaging and cell biology experiments for an outstanding research group in Brazil headed by Dr. Silvia Guatimosim. The new work will be made possible by using the PI's state-of-the-art facilities at the University of Maryland Center for Biomedical Engineering and Technology to train Dr. Guatimosim and her colleagues in new methods. Broadly the co-investigators will investigate how the cholinergic tone in heart affects Ca2+ signaling in the cardiac myocytes and mitochondrial function. Imaging and biophysical methods will be used along with a novel mouse line (VaChT KDHOM mice) that has reduced expression of the vesicular acetylcholine transporter. This novel mouse line was developed with previous FIRCA funding to Dr. M. Prado and will permit the co-investigators to characterize the consequences of cholinergic hypofunction on heart cell behavior. Provocative preliminary results by Dr. Guatimosim show that the VAChT KDHOM mouse has heart failure including decreased myocardial force, altered ventricular calcium handling and molecular remodeling (Lara et al., Molecular &Cellular Biology, 2010 in press), including altered mitochondrial biology and enzyme levels and increased production of reactive oxygen species (ROS) (see Preliminary Results). Much of this dysfunction was reversed by treatment with a cholinesterase inhibitor (pyridostigmine). Since mitochondria are considered the powerhouse of the cell, the central hypothesis of this FIRCA project is that alterations in mitochondrial dynamics contribute to cardiac malfunction in VAChT mutant mice. To test this hypothesis, we will measure mitochondrial dynamics in patch-clamped ventricular myocytes from VAChT mutants by using a combination of real-time imaging of mitochondrial Ca2+ levels and membrane potential ([Ca2+]mito )The functional data obtained by real-time imaging will provide an integrated understanding of mitochondria's role on heart disease caused by reduced cholinergic tone. This FIRCA proposal will provide the means to build new research capabilities at the Universidade Federal de Minas Gerais site for the simultaneous patch-clamp and real-time imaging of mitochondria, and to incorporate these state-of-the-art techniques into Dr. Guatimosim's research. Dr. Lederer will provide training in Baltimore for Dr. Guatimosim and her co-workers and on-site instruction in Brazil. PUBLIC HEALTH RELEVANCE: Heart dysfunction is one of leading causes of human morbidity and mortality world-wide. The proposed research examines the surprising finding by the investigators that changes in the balance of the nervous system have profound consequences for heart function. This dysfunction occurs in heart failure and appears to target cellular processes that affect both contraction and the heart rhythm;therefore, the investigators seek to understand the molecular mechanisms of this process and thereby enable new therapies for both heart failure and "sudden cardiac death".

DESCRIPTION (provided by applicant): Calcium (Ca2+) dependent arrhythmias have been identified as a significant health problem leading to ventricular tachycardia, fibrillation and death. The exact mechanism by which these arrhythmias arise remains a critically important yet vexing problem. We hypothesize that these Ca2+ dependent arrhythmias occur through a process in which a propagating wave of elevated calcium travels through heart cells and thereby activates and entrains electrical activity. The proposals PIs (Lederer, Jafri, and Winslow) will combine state-of- the art computational modeling with novel laboratory experiments in a multi-scale approach to determine how the calcium signaling defect develops and critically test the hypothesis. This systems biology investigation will examine the molecular physiology of cardiac Ca2+ signaling at high temporal and spatial resolution under normal and pathological conditions. It will utilize the unusually powerful approach of specifically examining the molecular pathophysiology of the Ca2+ dependent arrhythmia using the molecular disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) caused by an extremely well-defined process - point mutations of critical Ca2+ regulatory proteins. We will examine how mutations in the calcium release channel (ryanodine receptor type 2, RyR2) and the Ca2+ binding protein, calsequestrin (CASQ2) contribute to Ca2+ dependent arrhythmogenesis. Mouse models of these two arrhythmias will be used to enable advanced cell biology investigations. The work will be made more general by also including an examination of Ca2+ overload arrhythmias in mouse and guinea pig. The planned investigation will encompass multiple scales: from the molecular defect, to cellular Ca2+ dysfunction to tissue arrhythmia using both mathematical modeling and biological experiments. The project will address the following four specific aims: 1) How do Ca2+sparks trigger and sustain calcium waves? 2) How do Ca2+waves propagate from cell to cell? How do Ca2+waves entrain electrical activity? 3) How do specific mutations in RyR2 and CASQ2 affect Ca2+sparks, Ca2+waves and the propagation of Ca2+waves from cell to cell? 4) How does the 3D organization of the heart affect Ca2+ entrained arrhythmogenesis? This work should provide fundamental new understanding of the heart and the role of Ca2+ in electrical dysfunction and arrhythmia and lay the foundation for new therapeutic approaches. PUBLIC HEALTH RELEVANCE: Calcium dependent cardiac arrhythmias have been identified as a significant health problem and a major cause of cardiac death. The proposed work will provide new understanding of the molecular and cellular causes of these heart rhythm disturbances and enable new treatments.

DESCRIPTION (provided by applicant): Ca2+ sparks in heart have been shown by the PI to occur under physiological conditions during diastole and systole. They not only underlie the normal [Ca2+]i transient but have been found to be critically important in mediating the cellular response to stress and disease, contributing to contractile and arrhythmic dysfunction in conditions ranging from calcium overload to the cardiomyopathy of muscular dystrophy. Recently, work by the PI shows that physiologic stretch, such as that experienced by a myocyte during diastolic filling, dramatically alters Ca2+ spark occurrence transiently in normal cardiac ventricular myocytes. This behavior depends on microtubules affecting the release mechanisms of the sarcoplasmic reticulum (SR). Despite the importance of this new discovery one year ago, we have only now developed the additional tools needed to investigate how dynamic length changes can affect the triggering of Ca2+ sparks under diverse conditions. Using these new tools, we observe (in preliminary investigations) that stretch-dependent changes in Ca2+ sparks are even larger than previously observed and appear to arise from a transient increase in the sensitivity of ryanodine receptors (RyR2s). Additional preliminary work shows that, surprisingly, this transient increase in Ca2+ sparks underlies the activation of arrhythmogenic Ca2+ waves at a very low rate in heart cells from control mice, but at a much higher rate in myocytes from mdx mice, the murine model of Duchenne muscular dystrophy, or from control mice with excessive calcium in the SR. The tools developed by the PI and his colleagues will enable an innovative state-of-the-art investigation into how cardiac Ca2+ signaling is modulated by physiological stretch. The proposed work seeks to investigate stretch-dependent Ca2+ sparks and Ca2+ waves in 1. control ventricular myocytes;2. ventricular myocytes in which RyR2 properties have been altered;3. ventricular myocytes when microtubules are modulated;4. ventricular myocytes from dystrophin null (mdx) mice. The planned research should reveal for the first time the importance of stretch in normal and pathological Ca2+ signaling of cardiac ventricular myocytes. The work will therefore provide not only fundamental new information on normal cellular behavior but also on mechanisms of arrhythmogenesis. Furthermore it will lay the foundation for novel therapies for diverse heart diseases including Duchenne muscular dystrophy. PUBLIC HEALTH RELEVANCE: Contraction and the heart rhythm are regulated by calcium inside of heart cells. This calcium level is now known to be set in part by changes in the cell length (these changes are also called "stretch"), a surprising result that was discovered recently by the scientists working on this proposal. The planned work will examine such stretch-dependent changes in cellular calcium and function and determine how stretch underlies normal and defective heart function.

DESCRIPTION (provided by applicant): Mitochondria are abundant and critically important subcellular organelles whose organization and ultrastructure are complex but unexplained. This multi-PI multiscale modeling proposal seeks to use a combination of mathematical modeling and biological experiments to test the overall hypothesis that the nanoscopic ultrastructure of cardiac mitochondria accounts for the ultimate success of mitochondrial function. Mitochondria in rat cardiac ventricular myocytes will be used in the planned modeling and biological experiments. Our preliminary experiments and published data indicate that the planned work is feasible and that the three PIs can succeed in this ambitious and challenging proposal. Freshly isolated rat cardiac ventricular myocytes and those in short-term (1-3 days) culture will be prepared in the Lederer lab and imaged with electron microscope (EM) tomography by the Mannella team and analyzed quantitatively and interactively by the three PIs. Living cells will be examined by the Lederer team using confocal and super-resolution Stochastic Optical Reconstruction Microscopy (STORM). The quantitative spatial and functional data obtained from EM tomography and live cell imaging will be used to inform and constrain the multi-scale 3D modeling of mitochondria centered in the Jafri lab. The planned iterative approach to biological experiments and mathematical modeling will enable this investigation to define the structural basis for mitochondrial function for the first time. Finally, the proposed investigation seeks to include modeling of mitochondria under control conditions, when mitochondria are stressed by simple interventions and when mitochondria are altered by pressure-overload heart failure. This work therefore will not only provide fundamental new information on how mitochondria function but will also lay the foundation for novel therapies in mitochondrially involved diseases.

Atrial myocyte cell biology will be examined in isolated single cells in vitro and mice in vivo to characterize quantitatively how chemo-mechanical signaling works in health and disease. This signaling pathway is activated by changes in myocyte shape as happens when the atria fill with blood, and myocytes stretch, during diastolic filling. Using extremely high temporal and spatial resolution imaging the PIs will examine how chemo-mechanical signaling contributes to subcellular changes in Ca2+, excitation-contraction coupling to influence both electrical and Ca2+ instability. Preliminary results suggest that newly identified large axial tubules in atrial myocytes (discovered by the PIs) along with Ca2+ release super-hubs play a role in a unique Ca2+ signaling system found in atrial myocytes. Furthermore, the mechano-chemo X-ROS pathway discovered by the PIs in ventricular myocytes is likely to have a special role to play in atrial myocytes. This signaling pathway links the mechanics of cellular stretch, transmitted through microtubules, to the generation of local subcellular reactive oxygen species (ROS) that likely target multiple Ca2+ signaling proteins such as CaMKII and RyR2. Preliminary results suggest this X-ROS signaling is very active in atrial myocytes and may be linked to the novel structures described by the PIs. The proposed work will identify quantitatively the contributions of the special structures, X-ROS signaling and chemo-mechanical signaling to the normal physiology of atrial myocytes and the contributions to the development of atrial fibrillation (AF). Two very different mouse models of AF will be used along with specific transgenic mice to quantitatively characterize Ca2+ signaling and cellular electrophysiology in atrial myocytes and determine how chemo-mechanical signaling contributes to cellular physiology and pathophysiology. This investigation will provide critically important new information on how atrial myocytes work and fail in health and disease. The likely new discoveries produced by the proposed work will broaden our understanding of atrial cell biology and lay the foundation for innovative, effective and novel therapies for atrial dysfunction and AF.