1985 — 1998 |
Brown, Arthur M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ionic Movements Across Nerve Cell Body Membranes @ Case Western Reserve University |
0.904 |
1996 — 2000 |
Brown, Arthur M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Inward Rectifier Potassium Channels @ Case Western Reserve University
The long-term objectives are to understand the function and structure of inward rectifier K channels (IRKs). IRKs are important in excitable tissues such as nerve and muscle and our particular emphasis is on heart muscle. Here IRKs regulate resting membrane potential and the terminal phase of cardiac repolarization. Consequently their function is of great importance in evaluation and discovery of cardiac antiarrhythmic drugs, especially Class III antiarrhythmics. Three major hypotheses are examined: first whether specific naturally occurring cytoplasmic polyamines (PAs) regulate IRK function in cardiomyocytes; second whether the present topological model of IRKs is correct; and third whether a human cardiac IRK, hIRK is a major component of I-Kr, the rapid part of I-K the delayed rectifier K+ current of cardiomyocytes. The specific aims are to: 1) localize C-terminus binding sites for both Mg2+ and PAs which together confer the unique property of inward rectification; 2) compare the effective valence and time-dependence of PA block with so-called "intrinsic" gating of IRKs; 3) test the relationship between PA levels and IRK activity in cardiomyocytes; 4) est specific topological models of IRKs using glycosylation site insertion mutagenesis; and 5) identify the binding site for the Class Ill antiarrhythmic dofetilide, on hIRK and clone accessory subunit modifiers of this channel. For Aims 1 and 2 and the first part of Aim 5, the research design uses methods of mutagenesis, heterologous expression and electrophysiology in an iterative manner. For Aim 2 chemical interruption of PA metabolism is used to manipulate PA levels of cardiomyocytes while testing IRK function electrophysiologically. For Aim 4 a genetically engineered IRK protein is overexpressed in a baculovirus-Sf9 system, immunopurified and tested for N-glycosylation at sites predicted to be extracellular by present topological models. Functional tests using patch clamp are performed simultaneously. For the second part of Aim 5, a variety of candidate modifier subunits that we have cloned will be tested for their ability to make hIRK more closely mimic I-Kr.
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0.904 |
1998 — 2002 |
Brown, Arthur M |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Targeting Renal Outer Medullary K+ Channel Romk For New Class of Diuretics @ Case Western Reserve University
Mutations in the renal outer medullary K+ channel ROMK produce an antenatal variant of Bartter's syndrome manifested by hypokalemic alkalosis, polyuria and hypotension. We have shown that ROMK mutations reduce K+ flux and propose this as the pathogenesis for the disease. A corollary is that ROMK blockers should act as diuretics and our long-term goal is to develop such blockers. Our specific aims are: 1) characterize the ROMK mutations that produce Bartter's syndrome to identify important functional domains in the protein and to design mutation-specific therapy; 2) use phage display to generate peptide ligands which regulate ROMK channel function and which will serve as ROMK tags; and 3) determine the status of ROMK glycosylation in kidney using either antibodies or high affinity ligands that we will develop. Aim 3 derives from our finding that K+ currents in un-glycosylated ROMK are markedly reduced. The project will not only provide therapy for the ROMK variant of Bartter's but will also provide a new class of loop diuretics. Experiments are designed to test the effects of ROMK mutations on K+ currents and assembly, trafficking, phosphorylation and proteolysis of ROMK channels. We will map the functional changes to a topological model of ROMK that we have developed using glycosylation site insertion mutagenesis. To discover ROMK-specific peptide ligands we will screen phage display libraries by biopanning with cells expressing ROMK1. To provide another rationale for altering ROMK currents we will examine the glycosylation of ROMK in kidney cells using biochemical and immunocytochemical methods. The research methods include: recombinant DNA to engineer Bartter's mutant, expression of recombinant proteins in Spodoptera frugiperda (Sf9) cells; patch-clamp measurements of K+ currents; biochemical methods for analysis of protein; screening phage display libraries by biopanning; sequencing isolated clones; and immunocytochemistry for localization of ROMK protein in kidney and Sf9 cells.
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0.904 |
1998 — 2002 |
Brown, Arthur M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Iron Induced Congestive Heart Failure @ Case Western Reserve University
DESCRIPTION (Adapted from the applicant's abstract) This multidisciplinary research project is designed to characterize the pathophysiology of iron-induced congestive heart failure using a systematic series of studies of the cardiomyopathy of iron overload in a new animal model, the Mongolian gerbil. Iron-induced myocardial disease is the most frequent cause of death in thalassemia major and is a major life-limiting complication of other transfusion-dependent refractory anemias, hereditary hemochromatosis and other forms of iron overload. The investigators hypothesize that (I) the body iron burden is a principal determinant of the magnitude of cardiac iron deposition in patients with thalassemia major, (ii) the nonuniform pattern of iron deposition in the heart results in variability in iron concentrations within cardiac myocytes, and (iii) increased intracellular iron selectively affects specific ion channels in cardiac myocytes, producing abnormalities in sodium and potassium currents, and damages other cellular components, producing cardiomyocyte dysfunction and heart failure. The proposed research has three specific aims: (1) to determine the effects of chronic iron overload on cardiac function during the development and progression of iron-induced heart failure in the gerbil model of iron overload, using miniaturized assessment of cardiac physiology in vivo in the intact animal, physiological studies of the isolated heart, and cellular studies of freshly isolated cardiomyocytes; (2) to determine the effects of iron-chelating therapy and other pharmacological interventions on the progression and regression of iron-induced heart failure in the gerbil model of iron overload, using similar methods; and (3) to determine the molecular mechanisms by which cardiac Na+ currents are decreased and Ca2+-independent transient outward cardiac K+ currents are increased in iron-induced heart failure, using both freshly isolated cardiomyocytes from the gerbil model of iron-induced cardiomyopathy and rat neonatal cardiomyocytes in culture. This research will furnish the first electrophysiological and functional data from a new experimental model of heart failure. The results will provide fundamental information about the molecular basis for the effects of iron on cardiac ion channels and cardiomyocyte function in the heart failure of iron overload. (End of Abstract)
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0.904 |
2012 — 2013 |
Brown, Arthur M Bruening-Wright, Andrew Hix |
R44Activity Code Description: To support in - depth development of R&D ideas whose feasibility has been established in Phase I and which are likely to result in commercial products or services. SBIR Phase II are considered 'Fast-Track' and do not require National Council Review. |
Optimizing the Action Potential of Stem Cell-Derived Human Cardiomyocytes For Car
DESCRIPTION (provided by applicant): Stem Cell-derived Human Cardiomyocytes (SC-hCMs) offer great potential for improving the accuracy of pre- clinical cardiac safety testing. We have characterized a population of SC-hCMs and have demonstrated that these cells show sensitive pharmacology that accurately predicts clinical responses. However, due to low assay throughput and limited resources, only 15 reference compounds were tested. We now propose to increase throughput in preclinical electrophysiology (EP) screens by utilizing a higher-throughput automated EP instrument. We will expand our preclinical in vitro testing to 77 compounds that have been carefully selected based on their known torsadogenic and/or QT prolonging effects. Results from SC-hCM-based assays will be referenced against complete (8 concentration, 8 replicate) concentration-curves of the same compounds generated from high-throughput screens of cell lines expressing each of the major cardiac ion channels. A statistics-based model will be created in collaboration with the U.S. Food and Drug Administration and a leading in silico modeling firm, Leadscope. This model will be based on the unique databases we create which, together the development of software dedicated to mining public and proprietary cardiac databases will dramatically increase productivity of pre-clinical cardiac safet screening. The set of services and products that will result from this project have the potential t save millions of dollars annually by reducing attrition of marketed but cardiotoxic drugs, to improve the safety of drugs in development, and to increase efficiency of drug development by allowing companies to focus on the most promising and safe drug candidates.
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