Arnold Schwartz, Ph.D. - US grants

The general long-term goal of this renewal application continues to be the elucidation of cellular and molecular mechanisms of regulation of the cardiovascular system. Our primary emphasis is on proteins and enzymes which govern the delivery, utilization, and exodus of calcium. The goal of most of the projects is the relationship of structure to function. The grant is divided into Missions which have stable long-term objectives and projects which have aims which may be completed or altered within a shorter period of time. Mission I, Pumps and Channels, emphasizes structural stu- dies on the Na+,K+-ATPase, the Mg2+-ATPase of the t-tubules and the calcium channel proteins using techniques of immunology, protein chemistry, and molecular genetics. Mission II, Electrophysiology, examines the effect of calcium channel modulators on vascular smooth muscle and skeletal muscle. Mission III, Contractile Proteins, has six projects which deal with the mechanism whereby smooth and striated muscle are regulated in the short term and/or how this regulation is altered in the long term by "remodeling" processes. Mission IV, Sarcoplasmic Reticulum, focuses on the rapid kinetic evaluation of the Ca2+-ATPase activity in cardiac and skeletal muscle sarcoplasmic reticulum and on the regulation of the Ca2+-ATPase in cardiac sarcoplasmic reticulum by phosphorylation/dephosphorylation of phospholamban. Mission V is composed of two projects which deal with vascular smooth muscle and blood vessel mechanisms through which vasodilator drugs may act. Mission VI has as its goals determination of the primary structure of the lipid transfer protein complex, definition of the structural features which regulate its activity and the identification of factors which regulate its production in liver. Six Cores, Cardiovascular Models, Protein Chemistry, Computer, Administrative, Molecular Biology and Immunology/ Monoclonal Antibodies serve eighteen projects in the six Missions.

DESCRIPTION (provided by applicant): We request consideration for a renewal of an interdisciplinary pre and postdoctoral training program, taking advantage of a long-standing intellectual environment enhanced by the Cardiovascular Research Center (CVRC). New aspects include the addition of state of the art 25 Core Facilities, including an Affymetrix Gene Chip Core combined with a new Molecular Bioinformatics Analysis Core, a new building, The Genomic Research Institute (GR]), linked to a new Department of Genome Science from which gifted Postdoctoral Fellows recruited will be directed to this training program. A faculty of 26, that includes several new members, joins from 10 departments in the College of Medicine, the Vontz Center for Molecular Studies and the Children's Hospital Medical Center. The program continues to emphasize molecular, regulatory mechanisms of cardiac and lung function but a major emphasis is on a wide range of transgenic mouse cardiovascular disease models, including knock-out, knock-in, over expressed and cross-bred. We use in vivo instrumented animals, echo-analysis of conscious mice, ex vivo isolated organs, animals, and single cell electrophysiology. We do not give short shrift to "traditional" physiology, pharmacology, and biochemistry but as applied to small animals. Areas include contractile proteins, membrane transport, ion channels, gene regulation of development, coagulation, homeobox gene targets, calcium homeostasis and dysfunction, embryogenesis using targeted gene engineering, surfactant protein structure-function and signaling pathways. The autonomic nervous system is a major incentive at the receptor level, proximal and distal signaling pathways particularly in disease-induced mouse models with correlations to human polymorphisms. Exposure to contemporary techniques and ideation are emphasized, but without sacrificing the history of creative discoveries that have led to an understanding of the CV system. Predoctoral fellows are selected from a wide pool arising from department programs, Institutes, Centers, a new "Flex" program, a newly funded PSTP and a Short-Term Medical Student programs. Postdoctoral fellows arise from individual faculty, through the new GRI, and from direct WEB contacts, and selected by the Advisory Board and myself. Selection of mentors is based on research interests, irrespective of department affiliation. This Program "links" the trainees from different disciplines to the CV system, through the weekly Fellow Research Club led by the PI, and by a series of myocardial biology lectures by the faculty. Recruitment of minority candidates is by a "hands-on" approach by the PI and Executive Committee. Clinical correlates are stressed. A close preceptor-student relationship fostered by the PI is characteristic. We have been successful with our present six pre and six postdoctoral slots and despite an increasing number of candidates, we choose to remain with this formula.

voltage gated channel; calcium channel; molecular biology; heart contraction; calcium channel blockers; heart pharmacology; phospholamban; calmodulin dependent protein kinase; molecular site; electrophysiology; phosphorylation; protein kinase; drug receptors; affinity chromatography; laboratory rabbit; human tissue;

Excitation-contraction coupling in the heart confers a tight linkage between the L-type calcium channel and the intracellular environment so that Ca2+ ingress upon depolarization triggers contraction. We have been studying the complex architecture, regulation and receptor drug sites on the subunits that comprise the cardiac calcium channel. Considering the importance of this channel in maintaining normal cardiac function, it is not surprising that defects have been found in some types of human heart failure. This continuing project focuses on subunit functional interaction in vivo on the normal and falling heart. The latter is produced in the mouse by a specific increase of L-type calcium channel subunits in a transgenic mouse, which has provided us with a cardiomyopathy very close to the human type. Transgenic "remodeling" of the heart is not only a convenient method of altering subunit stoichiometry in vivo, but provides a way to approach mechanisms of heart failure in a logical manner. The long-term objective is characterization of calcium channel regulation, in terms of the pore, subunit importance, and calcium antagonist receptor functions. The added feature in this renewal application is the transgenic and knockout approach, encompassing coordinated physiological, biochemical and microanatomical methodology. We hope to provide further molecular information on the normal L-type cardiac channel and its possible role in the diseased heart. The specific aims are: 1) to over-express the human cardiac alpha1 subunit specifically and only in the myocytes of transgenic mice. Physiological function, genotype frequency, and expression levels of the transgene, as well as comprehensive cardiac pathological assessment, hopefully will yield important new information. 2) To eliminate the functional effects of the beta-subunit in mouse heart by generating a dominant negative expression system. The high affinity alpha1 interactive domain (AID) will be over- expressed in a cardiac-specific manner, and this will then act as a trap for beta subunits. Characterization is as in Aim 1. We hope to provide new information n the role of the beta-subunit in vivo. 3) To generate conditional knockout mice lacking the alpha2/delta subunit in the heart. Studying these mice, again on the molecular, cellular and whole organ level, should give us valuable information on the role of this subunit in vivo. 4) To continue studies on the pore-lining region of calcium channels. We suggest that these studies will give us specific information concerning some structural features of the pore that are involved in regulation of the channel.

This is an amended application for a renewal consideration of a Program Project Grant, "Molecular Mechanisms of Contraction." The unifying theme of this renewal application for a Program Project is the same is in previous competitive renewal applications viz., regulation of excitation-contraction- coupling (EC-coupling) with the primary focus on calcium-dependent processes. The projects are designed to elucidate fundamental molecular regulatory processes that characterize normal cardiac function, and events that lead to hypertrophy and heart failure in molecularly engineered mice. The latter exhibit intriguing phenotypes that have led to new insights and processes that may be relevant to human disease. An understanding of the biochemical, physiological and pharmacological factors involved in normal cardiac contractile functions and in disease processes, should provide a framework for the understanding of cardiac regulation on a molecular level and development of rational therapeutic measures to prevent or reverse heart failure. Three projects include studies on the human voltage-dependent calcium channel, the beta-adrenergic system and coupling to G proteins, and isoforms of tropomyosin, a regulatory protein of the thin filament of the cardiac sarcomere. The projects interdigitate and focus on fundamental molecular and cellular mechanisms. Techniques on mouse physiology and pharmacology and receptor binding are emphasized. Potential polymorphisms found in the human population and relationships to cardiac muscle dysfunction form an important part of the Program project. The scientific Core, dedicated specifically to techniques of mouse physiology, pharmacology and morphology/pathology, is the essential linch- pin for this Program Project.

Cardiac excitation-contraction is tightly coupled (EC)via the L-voltage-dependent Ca2+ Channel supplying the required Ca2+ ingress with each beat to initiate contraction. Calcium is the cation that serves as the link between depolarization and contraction. We have been studying the complex architecture and regulation that comprises the L-VDCC, the subunits of which include the alphalC, beta and the alpha2/delta, employing transgenic approaches rather than heterologous systems. In this manner, we are able to delineate in vivo function. Transgenic remodeling of the channel subunits in the heart is not only a convenient way to determine and alter subunit stoichoimetry in vivo, but it provides a model for studying cardiac dysfunction related to calcium. The long-term objective is to characterize the in vivo regulation of the L-voltage-dependent Ca2+ Channel, in terms of calcium regulation. In this revised application, we emphasize the in vivo roles of the pore unit (i.e., the alphalC), the beta- and the alpha2/delta1 subunits in regulation of the pore unit in normal and cardiac hypertrophy/failure in the mouse and human heart. A knockout strategy of the alpha2/delta1 should yield significant and detailed information on regulation of the pore unit in normal heart and possibly in heart failure. These transgenic approaches should provide new information regarding how the the L-VDCC functions in vivo , and how each of the associated subunits contributes to the regulation of the pore unit. Hopefully ,the data will be useful in eventually designing rational pharmacotherapeutic and genetic prevention/treatment strategies. The Specific Aims are: Transgenic remodeling of and delineating in vivo roles for subunit composition of mouse myocardium of L-voltage-dependent Ca2+ Channels: 1) Characterization of the alphalC subunit: molecular mechanisms of cardiac hypertrophy/failure. 2) Roles of the beta-subunits;3) Knockout of the alpha2/delta~ subunit.