| Program : Biomedical Research and Countermeasures | Ground Research | ||||
| Element : Physiology | |||||
Molecular Signaling in Muscle Plasticity |
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| Principal Investigator: | |||||
Henry F. Epstein, M.D. NB302 Baylor College of Medicine One Baylor Plaza Houston, TX 77030 |
Phone: (713) 798-4629 Email: hepstein@bcm.tmc.edu Fax: (713) 798-3771 Congressional District: TX-25 | ||||
| Co-Investigator(s): | |||||
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| Monitoring Center: NSBRI | Solicitation: NSBRI | ||||
| Initial Funding Date: 1997 | Expiration: 2000 | ||||
| Students Funded Under Research: 0 | Post-Doctoral Associates: 0 | ||||
| Task Description: | |||||
| Extended spaceflight under microgravity conditions leads to significant atrophy of weight-bearing muscles. Atrophy and hypertrophy are the extreme outcomes of the high degree of plasticity exhibited by skeletal muscle. Stimuli which control muscle plasticity include neuronal, hormonal, nutritional, and mechanical inputs. The mechanical stimulus for muscle is directly related to the work or exercise performed against a load. Little or no work is performed by weight-bearing muscles under microgravity conditions. A major hypothesis is that focal adhesion kinase (FAK), which is associated with integrin at the adherens junctions and costameres of all skeletal muscles, is an integral part of the major mechanism for molecular signaling upon mechanical stimulation in all muscle fibers. Additionally, we propose that myotonic protein kinase (DMPK) and dystrophin (DYSTR) also participate in distinct mechanically-stimulated molecular signaling pathways that are most critical in type I and type II muscle fibers, respectively. To test these hypotheses, we will use the paradigms of hindlimb unloading and overloading in mice as models for microgravity conditions and a potential exercise countermeasure, respectively, in mice. We expect that FAK loss-of-function will impair hypertrophy and enhance atrophy in all skeletal muscle fibers whereas DYSTR and DMPK loss-of-function will have similar but more selective effects on Type II and Type I fibers, respectively. Gene expression will be monitored by muscle-specific creatine kinase M promoter-reporter construct activity and specific mRNA and protein accumulation in the soleus (type I primarily) and plantaris (type II primarily) muscles. With these paradigms and assays, the following Specific Project Aims will be tested in genetically altered mice: (1) identify the roles of DYSTR and its pathway; (2) evaluate the roles of the DMPK and its pathway; (3) characterize the roles of FAK and its pathway; and (4) genetically analyze the mechanisms and interactions between the FAK, DYSTR, and DMPK-associated pathways in single and specific combinations of mutants. The identification of potential signaling mechanisms may permit future development of pharmacological countermeasures for amelioration and prevention of microgravity-induced atrophy in extended spaceflight, and the analysis of both overloading and unloading paradigms may provide further support for development of exercise-based countermeasures. Understanding the basic mechanisms of molecular signaling in muscle plasticity may aid our understanding and treatment of skeletal muscle atrophy not only in spaceflight, but in similar problems of the aging population, in prolonged bed rest, and in cachexia associated with chronic disease. | |||||
| Protein Machine Directing Myosin Assembly: The UCS (UNC-45, CRO-1, She4p) proteins have been discovered and partially functionally characterized by several distinct approaches in multiple genetically manipulatable experimental organisms. In Saccharomyces cerevisiae, studies of the differential segregation of determinants between mother cells and buds led to the identification of protein She4p (Swi5p-dependent HO expression). This and other SHE proteins are specifically required for the expression of HO endonuclease in the mother but not the bud. Interestingly from our point of view, an unconventional Class V myosin Myo4p was re-identified as She1p by this screen. She4p was also re-identified by an independent genetic screen for defective endocytosis. CRO1 was found to be involved in the transition between cellular and syncytial forms of the filamentous fungus Podospora anserina. Recently, a rng screen for mutations affecting the assembly of function of the contractile ring in cytokinesis of Schizosaccharomyces pombe has identified another member of the UCS family. Temperature-sensitive mutations in this gene, rng-3, when crossed with partial loss-of-function mutations in myosin II lead to synthetic lethality of the doubly mutant progeny. UNC-45 has been proposed to function in the assembly of myosin into thick filaments. Alleles in the unc-45 locus exhibit three phenotypes: temperature- sensitive (ts), constitutive lethal (let) which are also paralyzed, arrested at two-fold embryonic stage (pat), and maternally rescuable lethal (mr). The ts phenotype is reversible during embryonic and larval development, but not after adulthood. This property suggests that the effect is developmental, or more specifically, assembly than a primary instability of thick filaments or myofibrils. Genetic experiments suggest that the ts mutants produce a "poisoned" myosin B isoform whereas the let mutants produce a loss-of-function resembling nulls of the essential myosin A isoform. The structure of C. elegans thick filaments has been characterized through combined biochemical, genetic, and ultrastructural approaches, primarily in our laboratory. The thick filaments are bipolar tubular assemblies. A core substructure is composed of the myosin rod homologue paramyosin and the filagenins, proteins recently discovered in our laboratory. We have presented a new model for the structure of thick filament backbones more generally, based in part on our nematode work. The outermost layer of the tubule contains two myosin isoforms A and B which are homodimers of the myo-3 and unc-54 encoded myosin heavy chains and which assemble into distinct regions. Myosin A is restricted in wild-type to a medial 1.8 (m long zone whereas myosin B is present in the flanking regions. At the junctions of the medial and flanking regions are two small zones of overlap. Four ts alleles have been mapped to conserved residues within the UCS domain of UNC 45 protein. In the CB286 ts mutant in vivo, the accumulation of thick filaments is decreased by 50% in 25 oC versus 15 0C worms. Thick filaments isolated from 25 oC worms show abnormalities of structure. Myosins A and B become scrambled. These filaments are unstable and depolymerize as evidenced by markedly decreased yields of shortened structures. These experiments coupled with the results of the yeast studies suggest strongly that the UCS domain is involved, directly or indirectly, with myosin assembly. The UNC-45 protein of C. elegans also contains three TPR (tetratricopeptide repeat) motifs near the amino terminal. TPR domains are found in several proteins implicated in protein folding and assembly. The TPR domain of UNC-45 most closely resembles the TPR domain of human protein translocase that binds HSP90. HSP90 is a critical molecule in many protein transactions including folding and assembly. Our working hypothesis for the function of the domains of UNC-45 protein is shown below is schematic form. New Structural and Molecular Interactions in the Myosin Filament: The myosin-containing filaments of muscle (also known as thick filament) have been a classic paradigm of protein assembly and organization. Myosin as a protein has served as an established molecular marker of muscle physiology in space-oriented experiments. Recent work in our laboratory has indicated that the classical model of thick filament structure, that of an all-myosin helical filament, is inaccurate. Rather, the thick filament is a tubule formed by cross- linking of myosin-containing subfilaments. The cross-linking is accomplished by additional, non-myosin proteins. The original structural work leading to this new model was performed in the model organism, Caenorhabditis elegans. The backbone of the C. elegans thick filament is composed of paramyosin, a close homologue of myosin heavy chain rods. Seven subfilaments of paramyosin are coupled together by four newly discovered proteins, the filagenins. This coupling gives the thick filament great rigidity and incompressibility which are important in the functioning of an efficient contractile machine. In mammals including humans, the functional analogues of the filagenins are the myosin binding proteins, especially C-protein. Purified myosin alone does not form as rigid filaments as the co-assembly of myosin and C-protein. This work suggests that the myosin binding proteins of mammalian cardiac and skeletal muscle, many of which are organ or fiber-type-specific isoforms, may play critical roles in the transitions caused by changes in load or by microgravity. Therefore, quantitative analysis of the specific isoforms of C- protein should be conducted as well as the myosin analyses. Regulation of DMPK: Like most protein kinases, DMPK is very likely to itself be regulated by multiple signaling molecules through their distinct mechanisms. These mechanisms include binding to a heat shock protein/chaperone, interaction with members of the Ras superfamily of 21 kDa GTPase proteins, and phosphorylation/dephosphorylation catalyzed by other protein kinases or by protein phosphatases, respectively. The molecules that regulate the physiological activity of DMPK in vitro delineate the signaling pathways that functionally integrate DMPK. Recently, a novel member of the small (16-27 kDa) heat shock protein family, MKBP, has been shown to activate the autophosphorylation and transphosphorylation activities about 3-fold in purified component assays. MKBP is highly expressed in skeletal muscle, is upregulated in DM patient muscle, and exists in high molecular weight oligomers with specific locations in human striated muscles. The exact function and mechanism of MKBP is not fully clear, however, a closely related protein, B crystallin is associated with desmin filaments in striated muscles and when mutated, produces skeletal and cardiac myopathy. DMPK is closely related in both its catalytic domain and - helical/coiled coil/leucine zipper motifs to the well-known RhoA kinases that are active in promoting the assembly of the actin cytoskeleton (stress fibers) and the organization of focal adhesion plaques of the plasma membrane, in various cell culture systems in vitro. Indeed, the effects of overexpression of DMPK in lens cells are very similar to those of RhoA transfection and overexpression in parallel experiments. We have also shown that both RhoA and Rac-1 can be coprecipitated with DMPK in lysates of either recombinant bacteria or transfected COS cells. Rac-1 shows a clear 2-3 fold activation of the phosphorylation of histone H1 by DMPK. This activation is GTP specific. The constitutively active GTP-on Rac-1 mutant (G12V) activates DMPK whereas the inactive GDP-on mutant (S17N) does not. RhoA also shows some activation of DMPK in lysates, but we could not detect a significant difference between the active constitutive GTP-on RhoA mutant (A14V) and the inactive constitutive GDP-off on RhoA mutant (T19N). We have also shown interactions of DMPK with a specific protein kinase and two protein phosphatases. Raf kinase, a key component of the Ras-activated MAP signaling pathway, clearly phosphorylates DMPK. This pathway has been implicated in the cellular differentiation of many cell types in response to specific growth factors. We have obtained inconsistent results as to whether the autophosphorylation and/or the transphosphorylation reactions of DMPK are enhanced or unaffected by incubation with Raf kinase. Raf kinase and DMPK show physical interaction by yeast two hybrid complementation analysis. Protein phosphatases 1 and 2A also interact with DMPK. PP2A binds to DMPK by yeast two hybrid complementation analysis and reduces the extent of DMPK autophosphorylation. PP1 also reduces the extent of DMPK autophosphorylation. We plan to utilize this information to study the effects of these phosphatases upon the interactions of Rac-1, RhoA, and Raf kinase with DMPK. Computer-based analysis of the DMPK amino acid sequence suggests that it contains several potential Protein Kinase C and Casein Kinase II phosphorylation sites. The PKC sites are 225 TVR 227 (catalytic region or PK), 467 TLP 469, and 540 SPR 542 (both helix region or H). The CK II sites are 282 TAE (PK), 348 TLSD 387, 419 TPME 422, 443 SPQD 446, 467 TLRE 470, and 548 SHLD 551 (all H). We plan to test the phosphorylation of DMPK by these protein kinases and its effects upon the interactions of Rac-1, RhoA and Raf kinase with DMPK. Working hypothesis for the regulation of DMPK by multiple signaling molecules. DMPK as a Regulator of Muscle Plasticity: We have established that DMPK is very like a molecular integrator of diverse signals: Ras-activated Raf kinase stimulated by protein factor; receptor interactions and the Rac-1 and RhoA GTPase associated with cell adhesion complexes and actin cytoskeleton, integrin, and focal adhesion kinase. Our task is now to test whether the DMPK-associated protein machine is necessary for the muscle plasticity observed with perturbations in load as a model for spaceflight. The Specific Aim is to test the effects of DMPK knockout mutations in inbred strains of mice upon the well- established paradigm of unloading the hindlimb muscle by tail suspension. We have conducted two series of tests and have now optimized the special SDS-PAGE technique required to separate the I, IIA, IIB, and IID isoforms of myosin heavy chains of mice for the analysis of the tested muscles. All of the mice studied are female because of specific problems with male mice in the paradigm. Drs. Frank Booth and Scott Gordon have collaborated with us in setting up the tail suspension model in mice. Dr. Kenneth Baldwin has been invaluable in advising us on the special SDS-PAGE. We study mice at 3 months of age. Our original test series involved 14 mice: 7 homozygous knockouts and 7 control C57 mice. Differences in the switching of myosin isoforms between the knockouts and the controls were observed in preliminary experiments reported last June at the NSBRI retreat. However, Dr. Michael Reid pointed out to us genetic problems in our approach and convinced us of the importance of comparisons of litters derived from mating brother and sister heterozygotes. The progeny by Mendelian segregation will on the average have 1 homozygous wild-type:2 heterozygous wild- type/knockouts:1 homozygous knockout. We have made a concerted effort to breed enough litters so that this rule, the selection of females, and having each genotype split between sham control and tail suspension conditions. To date we have suspended 8 mice and had 4 controls from two litters. We are currently in the midst of another tail suspension experiment. All of these mice will also be analyzed by SDS-PAGE for their pattern of myosin heavy chain isoform switching. This test will have as an endpoint 18 suspended mice and 18 controls. We anticipate that all of our analysis of the already tested mice will be complete by May 1999. Any differences due to the DMPK knockout mutations should be clear. | |||||
| Application to Learning and Memory: The hippocampal formation of the brain cerebral cortex has been shown to function in learning and memory. Patients with DM (myotonic dystrophy) show deficits from "executive functions" such as decision making to significant mental retardation and intellectual impairment. Dr. Paul Schulz and his laboratory in the Department of Neurology at Baylor College of Medicine have identified a new response to tetanic stimulation in the hippocampus called "ITP (intermediate term potentiation)" in contrast to STP (short term potentiation) and LTP (long term potentiation). Various drugs that inhibit STP or LTP do not affect ITP. In collaboration with our lab, homozygous DMPK knockout mice, heterozygous, and wild-type littermates were examined for their responses to tetanic eliminate. The remarkable finding is that there is a 50% decrement of ITP in the homozygous knockouts but not in the heterozygotes or wild-type mice. STP and LTP were normal in all of the mice studied. This is a double result: the first experimental finding of an effect of DMPK in the central nervous system and a linkage of a newly identified process in learning and memory, ITP, to a specific molecule, DMPK. Understanding how skeletal muscle responds to loading and unloading in terms of molecular mechanisms is necessary for development of pharmacological interventions in treating muscle atrophy of prolonged bedrest, cachexia, and aging.
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