Role Of The Unfolded Protein Response In Cardiac Protection
Christopher C Glembotski, Professor And Chair
Biologysan Diego State University
Grant 5R01HL075573-06 from National Heart, Lung, And Blood Institute IRG: MIM
Project start date: 2003-12-17
Project end date: 2012-05-31
Sponsored Links Excellgen http://Excellgen.com
Role Of Unfolded Protein Response In Cardiac Protection
Christopher C Glembotski, Professor And Chair
San Diego State University 5250 Campanile Dr San Diego, Ca 92182
Grant 5R01HL075573-04 from National Heart, Lung, And Blood Institute IRG: CVA
Abstract: Our long-term objectives are to understand signaling pathways that mediate cardiac protection during stress. Many protective pathways studied to date foster myocardial cell growth. Although initially adaptive, this growth leads to tissue remodeling and, eventually, to impaired cardiac function. Accordingly, it would be desirable to identify protective pathways that do not induce cell growth; one such pathway may be the unfolded protein response (UPR). The UPR, which has been virtually unstudied in the cardiac context, is activated by stresses that alter the folding of proteins made in the rough ER. Ischemia/reperfusion (I/R), a well known cardiac stress, activates certain aspects of the UPR in the brain. In model systems, such as HeLa and 3T3 cells, one branch of the UPR, mediated by the recently discovered transcription factor, ATF6, induces genes that promote cell survival (i.e. ER stress response [ERSR] genes), but does not activate cell growth. Protective roles for ATF6 have not been studied in any tissue to date. Our hypothesis is that I/R activates the UPR in isolated cardiac myocytes and in the heart, and that subsequent stimulation of the ATF6 branch of the UPR fosters ERSR gene induction and cardioprotection without hypertrophic growth. To address this hypothesis the Specific Aims are to 1) characterize ATF6 activation and ERSR gene induction in cultured cardiac myocytes and in isolated hearts by simulated and global I/R, respectively, 2) use novel ligand-regulated forms of ATF6 (LR-ATF6) to examine the effects of ATF6 activation on ERSR gene induction, hypertrophic growth and survival in cultured cardiac myocytes during simulated I/R, and 3) assess the ability of ATF6 to mediate ERSR gene induction and cardioprotection in vivo, using transgenic mice featuring cardiac-restricted expression of LR-ATF6. These studies will provide a better understanding of the UPR in the heart, which is required to grasp the importance of this pathway and ATF6 in preserving contractile function in the stressed myocardium.
Keywords: cell growth regulation, cytoprotection, gene induction /repression, injury /disease stressor, myocardial ischemia /hypoxia, transcription factor, cardiac myocyte, cardiovascular injury, protein folding, reperfusion, SDS polyacrylamide gel electrophoresis, echocardiography, genetically modified animal, immunofluorescence technique, laboratory mouse, laboratory rat, organ culture, terminal nick end labeling, tissue /cell culture, western blotting
Project start date: 2003-12-17
Project end date: 2008-11-30
5R01HL075573-04 (2007): $294903
5R01HL075573-03 (2006): $368629
5R01HL075573-02 (2005): $339750
Grants awarded to Christopher C Glembotski
MAP KINASES AS MEDIATORS OF THE CARDIAC STRESS RESPONSE
Christopher C Glembotski, Professor And Chair
San Diego State University 5250 Campanile Dr San Diego, Ca 92182
Grant 5R01NS025037-16 from National Institute Of Neurological Disorders And Stroke IRG: CVB
Abstract: When the heart is exposed to potentially harmful stress an important protective response is initiated. In part, this protective response is characterized by sarcomeric stabilization, increased resistance to apoptosis, and the induction of a subset of cardiac genes, some of which contribute to the cardioprotection. The stress response is initially adaptive and can sometimes result in hypertrophic cardiac myocyte growth. Unfortunately, if the stress persists, there is often a cessation of the growth response and, instead, the heart undergoes a remodeling that can be associated with an eventual loss of muscle mass due to increased apoptosis, leading ultimately to heart failure. Our long-term objective is to understand the signal transduction mechanisms responsible for the cardiac myocyte stress response. We recently showed that one of the stress mitogen activated protein kinases, p38 MAPK, plays a central role in this process. This proposal focuses on how p38 contributes to several key features of the cardiac stress response. Our hypothesis is that p38 can induce certain cardiac stress-activated genes through a unique mechanism involving the recently-discovered transcription factor, ATF6. Further, we believe that p38-governed sarcomere stabilization and protection from apoptosis involves the small heat shock proteins (HSPs), alpha B-crystallin (alphaBC) and hsp27, which serve numerous roles, many of which converge on the promotion of cardiac myocyte survival. This hypothesis will be addressed using a cultured cardiac myocyte model system. Our Specific Aims are 1) to examine the mechanism by which p38 confers cardiac gene induction through ATF6, 2) to investigate the signal transduction events through which p38 mediates alphaBC and hsp27 gene induction-, phosphorylation- and translocation to sarcomeres, and 3) to assess the effects of manipulating the levels of alphaBC and hsp27 on selected features of the stress response using a novel combined antisense oligonucleotide/overexpression approach. These studies employ novel combinations of molecular approaches to unravel the roles of p38 MAP kinase in the cardiac myocyte stress response. The results will provide new information required to move the field forward in the search for new therapeutic strategies aimed at managing the cardiac stress response.
Keywords: cardiac myocyte, cytoprotection, gene induction /repression, mitogen activated protein kinase, stress protein, physiologic stressor, protein structure function, regulatory gene, sarcomere, antisens5R01NS025037-19
Project start date: 1986-12-01
Project end date: 2004-02-29
5R01NS025037-16 (2003): $258230
5R01NS025037-15 (2002): $250707
5R01NS025037-14 (2001): $243404
2R01NS025037-13 (2000): $261016
3R01NS025037-13S1 (2000): $50000
LASER SCANNING CONFOCAL MICROSCOPE
Christopher C Glembotski, Professor And Chair
Biologysan Diego State University
5250 Campanile Dr
san Diego, Ca 92182
Grant 1S10RR014634-01 from National Center For Research Resources IRG: ZRG1
Abstract: We are requesting funding for the purchase of a Laser Scanning Confocal Microscope under the Shared Instrumentation Grant Program. The proposed instrument is a BioRad R2000/KR-3 confocal system equipped with a Nikon Eclipse TE300 microscope. Acquisition of this instrument by the sponsoring institution is required to carry out the following studies 1) investigating the role of p38 MAP kinase signaling complexes as mediators of the cardiac stress response; 2) establishing the structural domains of Emt/Itk that are required for T cell activation; 3) regulation of intracellular calcium and the morphology of cardiac myocytes by MAP kinases; 4) contractile protein isoform function; 5) determination of peroxisomal targeting signals; and 6) structure and functional analyses of mitochondria by electron tomography. The confocal microscope will be housed in the Electron Microscope Facility in the Department of Biology at SDSU. The facility presently houses scanning and transmission electron microscopes, a freeze fracture machine, a Nikon epifluorescence microscope and other related instruments. Daily administration, technical supervision, and training for the proposed instrument will be the responsibility of the full-time director of the Facility, Dr. Steven B. Barlow. An advisory committee, composed of the EM Facility Director and two of the participants on this proposal (Glembotski and Tsoukas), will supervise instrument operation and maintenance. This committee will meet regularly to discuss issues pertaining to operation of the proposed instrument. Use of the confocal microscope will be by appointment, scheduled in advance, on a first-come first-served basis. A user fee will be established by the advisory committee in collaboration with the other participants on this proposal and the Chair of Biology. We estimate that this fee will be approximately $20-30 per hour. Since the salary of the facility director is already covered by the department, the user fees collected will be placed in a fund to be used exclusively for instrument maintenance and repair. It is anticipated that user fees will be sufficient to cover most of the instrument-related maintenance needs. As indicated in the attached letter from the Chair of Biology, any additional required funds will be provided by the department
Keywords: biomedical equipment purchase, confocal scanning microscopy
Project start date: 2000-04-01
Project end date: 2001-03-31
1S10RR014634-01 (2000): $297549
Surgery, Physiology And Immunohistopathology
Christopher C Glembotski, Professor And Chair
San Diego State University 5250 Campanile Dr San Diego, Ca 92182
Grant 1P01HL085577-019002 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: The Surgery, Physiology and Immunohistopathology Core (Core C), which will be used by all of the Projects of this PPG, will offer integrated services for assessing heart function and structure in settings that mimic cardiac pathology in mice. Accordingly, Core C will house and maintain the equipment and provide the staff necessary to offer Project Leaders and other PPG personnel the following services 1) Surgery in vivo myocardial infarction and transaortic constriction, 2) Physiology echocardiography, Langendorff and working mouse hearts, 3) Immunohistopathology state-of-the-art confocal fluorescent microscopic imaging of heart and myocyte structure, and cellular locations of signaling molecules in mouse heart sections and cultured cells. The Physiology, Surgery and Immunohistopathology Core, Core C, will be centrally located in the recently completed SDSL) BioScience Center, which houses the SDSU Heart Institute labs and administrative offices. Core C is composed of fully equipped surgery and physiology suites, dedicated mouse holding rooms, a complete tissue histology lab and a state-of-the-art confocal microscopy imaging facility. PPG investigators at all three institutions, as well as the live-cell imaging center in the Cell Biology Core, will be able to connect to the Core C imaging facility via an open microscopy environment (OME). The OME will optimize image data analysis, transfer and storage for all 5 of the PPG projects, as well as facilitating the integration of fixed sample imaging in Core C with live-cell imaging in Core B. An additional advantage of Core C is that it adjoins the SDSU Heart Institute Mouse Genetics Center, which facilitates the generation and use of genetically modified mice by all of the PPG projects. Core C was designed specifically to serve this multiinstitutional PPG; accordingly, it will maximize collaborations between projects, thus enhancing the synergy of this integrated research program. Core C complements and integrates with Core B by providing the ideal venue for examining cellular structure at the microscopic level, and by serving as the location where principles discovered using cells generated in the Cell Biology Core can be examined in a pathophysiological context that mimics the in vivo setting.
Keywords: biomedical facility, cardiac myocyte, cardiovascular surgery, disease /disorder model, echocardiography, laboratory mouse, cell morphology, confocal scanning microscopy, fluorescence microscopy, genetically modified animal, histology, immunocytochemistry, immunopathology, molecular /cellular imaging, tissue /cell preparation
Project start date: 2006-07-01
Project end date: 2011-06-30
BIOCHEMISTRY OF TRIAL NATRIURETIC PEPTIDE
Christopher C Glembotski, Professor And Chair
San Diego State University 5250 Campanile Dr San Diego, Ca 92182
Grant 5R01NS025037-03 from National Institute Of Neurological Disorders And Stroke IRG: PC
Abstract: The specific aims of the proposed research are 1) to thoroughly investigate the protease(s) involved in the formation of AlphaMSH and Beta-endorphin farom ACTH and BetaLPH, respectively, in rat and bovine intermediate pituitaries 2) to characterize the enzyme(s) in the pituitary, and possibly other peptide secreting tissues, that is (are) responsible for the Alpha-amidation of AlphaMSH, and possibly other neuroendocrine peptides 3) to conduct a more in-depth investigation of the intermediate pituitary secretory granule-associated acetyltransferase including the purification, characterization of the molecular nature, tissue localization, and biosynthetic pathway of the enzyme The general approach involves the use of pituitary secretory granule extracts as a source for these enzyme activities, strategically radiolabeled peptides as substrates, and thorough identification of products primarily by reversed-phase high performance liquid chromatography (RP-HPLC). The purification of the acetyltransferase will be performed with a combination of classical techniques, dye-ligand and affinity chromatography, and HPLC. The long-term objectives involve determining how different tissues can regulate the post-translational processing of identical pro-ACTH/endorphin molecules to form tissue-specific collections of peptide hormones with different biological activities. Learning more about the enzymes responsible for the biosynthesis ACTH- and Beta-endorphin-related peptides will help us discover more about how secretagogues, both natural (dopamine and CRF) and synthetic (dexamethasone), can alter biosynthetic and/or secretion rates of these pituitary peptides.
Keywords: ENDOCRINOLOGY, HORMONES METABOLISM, PEPTIDE-POLYPEPTIDE HORMONES METABOLISM, HEART HORMONES, CHEMICAL STRUCTURE--BIOLOGICAL ACTIVITY, ENDOCRINOLOGY, HORMONES BIOSYNTHESIS, PEPTIDE-POLYPEPTIDE HORMONES BIOSYNTHESIS, ENDOCRINOLOGY, HORMONES, STEROID HORMONES, HEART CELLS, HEART FUNCTION, HEART RATE, PHOSPHOLIPIDS, PHOSPHOGLYCERIDES, PHOSPHOINOSITIDES, PROTEASES AND PEPTIDASES, PROTEIN (PEPTIDE) SEQUENCE, protein structure, ANIMALS, CHORDATES, MAMMALS, LAGOMORPHS, ANIMALS, CHORDATES, MAMMALS, RODENTS, MYOMORPHA, MICE (LABORATORY), ANIMALS, CHORDATES, MAMMALS, RODENTS, MYOMORPHA, RATS (LABORATORY), IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, PHYSICAL SEPARATION, CHROMATOGRAPHY, AFFINITY, PHYSICAL SEPARATION, CHROMATOGRAPHY, GEL FILTRATION (PERMEATION), PHYSICAL SEPARATION, CHROMATOGRAPHY, HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, TISSUE (CELL) CULTURE
Project start date: 1986-12-01
Project end date: 1989-11-30
ENDOCRINE REGULATION OF CARDIAC HORMONE EXPRESSION
Christopher C Glembotski, Professor And Chair
San Diego State University 5250 Campanile Dr San Diego, Ca 92182
Grant 1R01HL056861-01A1 from National Heart, Lung, And Blood Institute IRG: END
Abstract: The regulation of blood pressure and volume is critically dependent on a number of hormones including ANF (atrial natriuretic factor) and BNP (brain natriuretic peptide). ANF and BNP are structurally related cardiac-derived peptides that decrease intravascular volume and arterial pressure. Sustained increases in blood pressure often elicit the synthesis and release of the peptides from the heart as part of a compensatory endocrine response. To better understand blood pressure and volume regulation, as well as the underlying causes of hypertension, it is the broad objective of our research program to elucidate the mechanisms governing the production of ANF and BNP. The specific objective of this proposal is to elucidate the post-transcriptional mechanisms responsible for regulating cardiac natriuretic peptide expression, focusing on how pressor hormones can augment the half-life of the BNP transcript. The Specific Aims are to 1) evaluate the differential effects of pressor compounds (natriuretic peptide inducers) on the stabilities of the BNP and ANF mRNAs in primary neonatal rat myocardial cells 2) identify elements in the BNP and ANF transcripts that influence mRNA half-life 3) characterize the factor(s) that bind to these elements and to determine if inducers alter binding in a manner consistent with involvement of these factors in BNP mRNA stabilization 4) study the signaling mechanisms by which pressor compounds stabilize the BNP mRNA The proposed studies will be the first to investigate the hormonal regulation of transcript stability in cardiac myocytes. Accordingly we expect to produce novel results relating not just to natriuretic peptide expression, but to cardiac gene expression in general. Moreover, the planned studies of the signal transduction pathways by which inducers augment BNP mRNA stability will produce new results pertaining to agonist- regulated transcript half-life in cardiac myocytes as well as other cell types.
Keywords: atrial natriuretic peptide, endocrine gland /system, gene expression, hormone regulation /control mechanism, peptide hormone, peptide hormone biosynthesis, DNA binding protein, biological signal transduction, blood pressure, blood volume, cell type, messenger RNA, myocardium, posttranscriptional RNA processing, regulatory gene, gel mobility shift assay, laboratory rat, tissue /cell culture, transfection
Project start date: 1997-04-05
Project end date: 2000-03-31
1R01HL056861-01A1 (1997): $232419
5R01HL056861-03 (1999): $238860
5R01HL056861-02 (1998): $231947
BIOCHEMISTRY OF ATRIAL NATRIURETIC PEPTIDE
Christopher C Glembotski, Professor And Chair
San Diego State University 5250 Campanile Dr San Diego, Ca 92182
Grant 5R01NS025037-08 from National Institute Of Neurological Disorders And Stroke IRG: PC
Abstract: The long-term objective of our research is to understand the mechanism and regulation of atrial natriuretic factor (ANF) biosynthesis, processing, and secretion. The proposal is focused on various treatments regulate ANF release. Our approach will involve the use of several cultured cell mode] systems and combination with biochemical and molecular biological techniques. Perhaps the greatest importance will be the use of our recently described serum-free long-term cultured heart cell system that mimic ANF processing and secretion as they occur in vivo. Specific Aims 1) To determine the cell type responsible for ANF processing, cardiocytes and cardiac mesenchymal cells (e.g. fibroblasts, endothelial cells) will cultured separately and their abilities to process endogenous (cardiocytes) or exogenous pro-ANF will be assessed. 2) The characteristics of the immunoreactive pro-thrombin associated with the heart and cultured cardiocytes will be studied using Western analysis, peptide mapping, radiosequencing, and message characterization. The possible involvement of the atrial thrombin-like material in pro-ANF processing will be assessed. 3) ANF will be expressed in several types of endocrine cells to determine whether the unusual thrombin-like co-/post-secretional cleavage at a single basic amino acid that occurs during pro-ANF maturation is a tissue- or hormone-specific phenomenon. 4) Adrenergic receptor activation, electrical pacing of contraction rate, and changes in cellular tension will be studied as models of acutely regulating ANF secretion, and it will be determined whether these treatments regulate ANF release at the level of the cardiocyte and what intracellular messengers are involved. The proposed studies will contribute new information to our knowledge of the factors that regulate plasma levels of ANF. Such information will help us understand the hemodynamic roles of ANF in both normal and pathological states, and will provide a foundation for the future rational design of ANF-related antihypertensives.
Keywords: atrial natriuretic peptide, hormone regulation /control mechanism, peptide hormone metabolism, alpha adrenergic receptor, cell type, heart cell, peptide hormone biosynthesis, protein sequence, protein structure function, prothrombin, second messenger, secretion, fibroblast, heart contraction, laboratory rabbit, laboratory rat, mesenchyme, tissue /cell culture, transfection, transposon /insertion element
Project start date: 1986-12-01
Project end date: 1995-05-31
5R01NS025037-08 (1994): $217389
5R01NS025037-07 (1993): $207889
5R01NS025037-06 (1992): $200016
The ER Stress Response And ATF6 Activation In The Heart
Christopher C Glembotski, Professor And Chair
San Diego State University 5250 Campanile Dr San Diego, Ca 92182
Grant 5R01NS025037-20 from National Institute Of Neurological Disorders And Stroke IRG: CVB
Abstract: Our long-term objectives are to understand signaling pathways that protect the heart from stress-induced damage. Such pathways often foster hypertrophic myocardial cell growth, which can eventually lead to impaired cardiac function and heart failure. However, the ER stress response (ERSR), which is activated by stresses that alter protein folding in the RER (e.g. hypoxia), fosters cell survival but not cell growth. One pro-survival branch of the ERSR is mediated by the ER membrane protein, ATF6 alpha. Upon ER stress, 90 kDa (p90) ATF6 alpha is cleaved by regulated intramembranous proteolysis (RIP), the p50 cleavage product becomes a transcription factor that induces ER stress response genes (ERSRGs). ERSRGs encode proteins that resolve the ER stress, promote cell survival, but do not induce cell growth. We have shown that in cardiac myocytes, SR/ER Ca ATPase-2 (SERCA2) is induced in an ER stress- and ATF6 alpha-dependent manner. Thus, in addition to protecting the myocardium from stress by inducing numerous pro-survival genes, via ATF6-mediated SERCA2 induction, the ERSR may also foster preservation of myocardial contractility. The recent discovery of a second form of ATF6, ATF6 beta, and our preliminary results that ATF6 beta inhibits ATF6 alpha-mediated ERSRG induction, support our hypothesis that ATF6 alpha and beta possess opposing properties that provide the basis of a novel mechanism for fine-tuning ERSRG induction and optimal cardiac protection. Specific Aims To address this hypothesis the Specific Aims we propose are to 1) characterize the rates of generation and degradation of the p50 forms of ATF6 alpha and beta in cultured cardiac myocytes exposed to ER stress, 2) map the domains of p50 ATF6 alpha and beta that regulate ERSRG induction and ATF6 degradation, and 3) co-express native or mutated p50 ATF6 beta with native p50 ATF6 alpha in various alpha/beta ratios, and assess the effects on SERCA2 induction, Ca transients, cell growth and survival of cultured cardiac myocytes. The ERSR has gone virtually unstudied in the heart; accordingly, the proposed studies are novel and will enhance our understanding of the ERSR and the roles played by ATF6 alpha and beta in the stressed myocardium.
Keywords: cardiac myocyte, cytoprotection, gene induction /repression, protein structure function, stress protein, transcription factor, calcium flux, cell growth regulation, physiologic stressor, protein degradation, protein isoform, laboratory rat, newborn animal, tissue /cell culture, transfection
Project start date: 1986-12-01
Project end date: 2009-02-28
5R01NS025037-20 (2007): $262897
5R01NS025037-19 (2006): $340982
5R01NS025037-18 (2005): $349188
2R01NS025037-17 (2004): $349188
Role Of Unfolded Protein Response In Cardiac Protection
Christopher C Glembotski, Professor And Chair
San Diego State University 5250 Campanile Dr San Diego, Ca 92182
Grant 1R01HL075573-01 from National Heart, Lung, And Blood Institute IRG: CVA
Abstract: Our long-term objectives are to understand signaling pathways that mediate cardiac protection during stress. Many protective pathways studied to date foster myocardial cell growth. Although initially adaptive, this growth leads to tissue remodeling and, eventually, to impaired cardiac function. Accordingly, it would be desirable to identify protective pathways that do not induce cell growth; one such pathway may be the unfolded protein response (UPR). The UPR, which has been virtually unstudied in the cardiac context, is activated by stresses that alter the folding of proteins made in the rough ER. Ischemia/reperfusion (I/R), a well known cardiac stress, activates certain aspects of the UPR in the brain. In model systems, such as HeLa and 3T3 cells, one branch of the UPR, mediated by the recently discovered transcription factor, ATF6, induces genes that promote cell survival (i.e. ER stress response [ERSR] genes), but does not activate cell growth. Protective roles for ATF6 have not been studied in any tissue to date. Our hypothesis is that I/R activates the UPR in isolated cardiac myocytes and in the heart, and that subsequent stimulation of the ATF6 branch of the UPR fosters ERSR gene induction and cardioprotection without hypertrophic growth. To address this hypothesis the Specific Aims are to 1) characterize ATF6 activation and ERSR gene induction in cultured cardiac myocytes and in isolated hearts by simulated and global I/R, respectively, 2) use novel ligand-regulated forms of ATF6 (LR-ATF6) to examine the effects of ATF6 activation on ERSR gene induction, hypertrophic growth and survival in cultured cardiac myocytes during simulated I/R, and 3) assess the ability of ATF6 to mediate ERSR gene induction and cardioprotection in vivo, using transgenic mice featuring cardiac-restricted expression of LR-ATF6. These studies will provide a better understanding of the UPR in the heart, which is required to grasp the importance of this pathway and ATF6 in preserving contractile function in the stressed myocardium.
Keywords: cell growth regulation, cytoprotection, gene induction /repression, injury /disease stressor, myocardial ischemia /hypoxia, transcription factor, cardiac myocyte, cardiovascular injury, protein folding, reperfusion, SDS polyacrylamide gel electrophoresis, echocardiography, genetically modified animal, immunofluorescence technique, laboratory mouse, laboratory rat, organ culture, terminal nick end labeling, tissue /cell culture, western blotting
Project start date: 2003-12-17
Project end date: 2007-11-30
1R01HL075573-01 (2004): $364240
Conditional Association Of Alpha-Cb-Crystallin With Mitochondria
Christopher C Glembotski, Professor And Chair
San Diego State University 5250 Campanile Dr San Diego, Ca 92182
Grant 1P01HL085577-010003 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: Our long-term objective is to understand signaling pathways that protect the heart from ischemia/reperfusion (I/R) injury. Our short-term objective, and the topic of this proposal, is to examine the mechanism of cardioprotection by the cytosolic small heat shock protein (sHSP), alphaB-crystallin (alphaBC). alphaBC (22 kDa) is expressed predominantly in cell types with high oxidative phosphorylation demands, e.g. cardiac myocytes. In cardiac myocytes, alphaBC is phosphorylated upon stimulation of stress MAP kinases (e.g. p38), which, if chronically activated, also drive increased alphaBC expression. Increasing the level of wt alphaBC, or expressing pseudophosphorylated alphaBC, protects against I/R injury. Our preliminary data indicate that mitochondria serve as an alphaBC-binding target during I/R. Accordingly, our hypothesis is that phosphorylated alphaBC protects cardiac myocytes from I/R injury and a portion of this protection is mediated by the conditional association of phospho-alphaBC with mitochondrial outer membrane (mOM) proteins, such as the MPTP. The Specific Aims that address this hypothesis are to 1) examine the kinetics with which I or I/R increase m-alphaBC, and to assess the phosphorylation status of m-alphaBC, using a combination of immunoblotting, confocal and electron microscopy, 2) assess the effects of alphaBC deletion, or overexpression of wild type or mutant forms of alphaBC on MPTP activation, apoptosis, autophagy and myocardial function in response to I/R, and 3) identify mitochondrial alphaBC binding partners and elucidate l/R-dependent changes in the mitochondrial subproteome. Significance and Innovation The proposed studies are the first to examine the interaction of alphaBC with mitochondria in any tissue type, and the first to study how m-alphaBC preserves mitochondrial function during stress. These studies employ a comprehensive series of experiments using state-of-the-art cellular, molecular genetic and proteomics technologies to discover new information required to understand cellular mechanisms that preserve mitochondrial function during I/R stress.
Keywords: biological signal transduction, cardiac myocyte, heat shock protein, mitochondria, myocardial ischemia /hypoxia, binding site, free radical oxygen, gene expression, gene induction /repression, mitochondrial membrane, mitogen activated protein kinase, phosphorylation, confocal scanning microscopy, electron microscopy, mutant, proteomics, western blotting
Project start date: 2006-07-01
Project end date: 2011-06-30
ROLES FOR P38 MAP KINASES IN CARDIAC MYOCYTE GROWTH
Christopher C Glembotski, Professor And Chair
San Diego State University 5250 Campanile Dr San Diego, Ca 92182
Grant 5R01HL063975-04 from National Heart, Lung, And Blood Institute IRG: CVA
Abstract: Early in post-natal development heart muscle cell division is arrested and subsequent growth results from hypertrophic increases in cardiac myocyte size. Although hypertrophic growth ceases in the mature heart, many cardiac-related disorders, such as hypertension, provoke its reinitiation. This renewed growth is initially compensatory but eventually leads to a decompensatory reduction of cardiac muscle mass due to myocyte apoptosis, decreased cardiac function and, ultimately, to heart failure. Our long-term objective is to understand the signal transduction mechanisms that regulate cardiac growth and apoptosis. This proposal addresses the hypothesis that the p38 mitogen activated protein kinases (MAPK) play pivotal roles in determining the balance between cardiac myocyte growth and apoptosis, and that the relative activity states of p38 isoforms, e.g. p38alpha and p38beta, serves as a crucial determinant of myocyte fate. To address the mechanism by which p38 isoforms can be differentially activated in the heart, we propose the following Specific Aims 1) To identify members of proposed p38 signaling complexes in cardiac myocytes. Using Western- and Northern analyses, as well as yeast two-hybrid screening, proteins in the heart that comprise complexes that direct signals toward growth or apoptosis, including hypothetical scaffold proteins, will be identified and, in the case of newly-discovered molecules, their structures and properties determined. 2) To characterize the cellular functions of p38 signaling complex members. Here, we will use a novel antisense oligonucleotide approach and overexpression of key signaling proteins to selectively perturb levels of signaling participants in cultured cardiac myocytes. Effects on the gene expression and morphological characteristics associated with cardiac hypertrophy and apoptosis will be assessed. 3) To elucidate the effects of manipulating the levels of p38 signaling complex members on heart structure and performance in vivo using cardiac-targeted transgenic mouse models. These studies employ novel combinations of powerful molecular approaches to unravel the roles of the recently-discovered p38 MAP kinases in cardiac myocyte growth. We anticipate that the results will provide new information that is required to move the field forward in the search for gene therapy targets aimed at solving problems related to the unusual hypertrohic growth program exhibited by cardiac myocytes.
Keywords: cardiac myocyte, cell growth regulation, enzyme activity, mitogen activated protein kinase, ventricular hypertrophy, antisense nucleic acid, apoptosis, biological signal transduction, complementary DNA, isozyme, nucleic acid sequence, oligonucleotide, laboratory mouse, laboratory rat, northern blotting, southern blotting, tissue /cell culture, transgenic animal, western blotting, yeast two hybrid system
Project start date: 2000-02-07
Project end date: 2005-01-31
5R01HL063975-04 (2003): $352863
5R01HL063975-03 (2002): $342676
5R01HL063975-02 (2001): $332784
1R01HL063975-01 (2000): $348179
BIOCHEMISTRY OF ATRIAL NATRIURETIC PEPTIDE
Christopher C Glembotski, Professor And Chair
San Diego State University 5250 Campanile Dr San Diego, Ca 92182
Grant 5R01NS025037-12 from National Institute Of Neurological Disorders And Stroke IRG: CVB
Abstract: Adapted from the Investigator s ) Atrial natriuritic factor (ANF) and brain natriuritic peptide (BNP) are structurally related cardiac-derived peptides with a vasorelaxant diurtic and natriuretic activities that oppose the action of substances responsible for increasing volume and blood pressure. While the ANF and BNP genes are co-induced by pressor substances, BNP is induced surprisingly quickly as an immediate early or primary response gene (PRG) while ANF is induced slowly as a late or secondary response gene (SRG). The differential induction of BNP and ANF implies that the peptides may serve distinct physiological roles. The proposal focuses on studies of the molecular mechanisms responsible for the complex induction of BNP and ANP by alpha 1 adrenergic agonists. The hypothesis is that while both genes are transcriptionally induced, stabilization of the normally labile BNP mRNA constitutes an important feature of its induction. The specific aims are (1) to study the 5 cis elements that confer alpha 1 adrenergic agonists inducible transcription, (2) to characterize proteins and interact with these cis-elements and to determine how they mediate inducible transcription, (3) to identify elements in ANF and BNP mRNA that influence transcript half-life and (4) to determine how the cis-regulatory elements contribute to ANF and BNP induction in the intact rat myocardium. These studies will lead not only to a better understanding of the hemodynamic role of ANF and BNP but will also provide new information relating to cardiac specific and hormone inducible gene expression.
Keywords: atrial natriuretic peptide, gene induction /repression, genetic transcription, hormone regulation /control mechanism, peptide hormone, alpha adrenergic agent, alpha adrenergic receptor, genetic regulatory element, heart cell, myocardium, peptide hormone metabolism, protein structure /function, regulatory gene, transcription factor, gel mobility shift assay, laboratory rat, northern blotting, protein sequence, tissue /cell culture, transfection
Project start date: 1986-12-01
Project end date: 2000-02-29
5R01NS025037-12 (1999): $268600
5R01NS025037-11 (1998): $258269
5R01NS025037-10 (1997): $248335
2R01NS025037-09A2 (1996): $228421
HYPERTROPHIC STIMULI IN ATRIAL ANF SECRETION, GENE EXPRESSION AND BIOSYNTHESIS
Christopher C Glembotski
Institution:
Grant 5P01HL046345-050003 from National Heart, Lung, And Blood Institute
Keywords: atrial natriuretic peptide, biological signal transduction, gene expression, peptide hormone biosynthesis, secretion, ventricular hypertrophy, calcium flux, endothelin, exocytosis, genetic promoter element, phenylephrine, protein kinase C, fluorescent dye /probe, laboratory mouse, laboratory rat, newborn animal, patch clamp, tissue /cell culture, transgenic animal