Program :    Biomedical Research and Countermeasures	Ground Research
Element :    Physiology
Molecular Mechanisms Regulating Muscle Fiber Composition Under Microgravity
Principal Investigator:
Nadia A. Rosenthal, Ph.D. Cardiovascular Research Center Massachusetts General Hospital CVRC 149 Harvard Medical School 13th Street, 4th Floor Charlestown, MA 02129	Phone: (617) 724-9560 Email: rosentha@helix.mgh.harvard.edu Fax: (617) 724-9561 Congressional District: MA-8
Co-Investigator(s):
Baldwin, K.M. University of California, Irvine
Monitoring Center: NSBRI	Solicitation: NSBRI
Initial Funding Date: 1997	Expiration: 2000
Students Funded Under Research: 0	Post-Doctoral Associates: 0
Task Description:
Drs. Rosenthal (Harvard) and Baldwin (UC, Irvine) specifically proposed the hypothesis that impairment of load bearing affects the distribution of E protein belonging to the basic helix-loop-helix (bHLH) family of transcription factors and that these proteins control muscle fiber type transitions. Molecular mechanisms underlying the dramatic changes in muscle fiber composition produced by weightlessness will be determined (Rosenthal). Several of the E proteins belonging to the bHLH family of transcription factors are differentially distributed among mammalian fiber types. Moreover, mice carrying homozygous null mutations in several of the E proteins exhibit severe postnatal muscle atrophy in each case due to a decrement in a different subset of fiber types. These preliminary data underscore the potential importance of E proteins in regulating fiber-specific patterns of muscle gene expression, and suggest that they may comprise upstream targets for the effects of weightlessness. E protein composition and distribution will be characterized in normal versus unweighted mouse muscles. Fiber specific expression of a potential E protein target, the Skeletal a-actin promoter-driven transgene, will be evaluated during hindlimb unweighting. The skeletal actin promoter-driven b-galactosidase gene in transgenic mice is severely reduced under conditions of weightlessness. The effects of overexpression in vivo of E2-2 and HEB, E proteins in transgenic mice will be used to evaluate their biological role in controlling fast type IIb and IIx fibers respectively, on the fast fiber atrophy observed in unweighted and space- flown mice. The overall goal of this project is to reveal the molecular mechanisms underlying the selective and debilitating atrophy of specific skeletal muscle fiber types that accompanies sustained conditions of microgravity. Since little is currently known about the regulation of fiber-specific gene expression programs in mammalian muscle, elucidation of the basic mechanisms of fiber diversification is a necessary prerequisite to the generation of therapeutic strategies for attenuation of muscle atrophy on earth or in space. Vertebrate skeletal muscle development involves the fusion of undifferentiated mononucleated myoblasts to form multinucleated myofibers, with a concomitant activation of muscle-specific genes encoding proteins that form the force- generating contractile apparatus. The regulatory circuitry controlling skeletal muscle gene expression has been well studied in a number of vertebrate animal systems. The goal of this project has been to achieve a similar level of understanding of the mechanisms underlying the further specification of muscles into different fiber types, and the role played by innervation and physical activity in the maintenance and adaptation of different fiber phenotypes into adulthood. Our recent research on the genetic basis of fiber specificity has focused on the emergence of mature fiber types and have implicated a group of transcriptional regulatory proteins, known as E proteins, in the control of fiber specificity. The restriction of E proteins to selected muscle fiber types is an attractive hypothetical mechanism for the generation of muscle fiber-specific patterns of gene expression. To date our results support a model wherein different E proteins are selectively expressed in muscle cells to determine fiber-restricted gene expression. These studies are a first step to define the molecular mechanisms responsible for the shifts in fiber type under conditions of microgravity, and to determine the potential importance of E proteins, as upstream targets for the effects of weightlessness. n the past years we have determined that the expression of E Proteins is restricted to specific fiber types by post-transcriptional mechanisms. By far, the most prevalent mechanism of cellular control for achieving post- transcriptional regulation of gene expression is selective proteolysis through the ubiquitin -proteasome pathway. Steady-state levels of HEB message are similar in all fast and slow skeletal muscle fiber types, yet the protein is restricted to TypeIIX fibers. HEB appears to be a nodal point for regulating fiber-specific transcription, as expression of the transcription factor is regulated at the post-transcriptional level. We are currently evaluating the biological role of ubiquitination in fiber specific- gene expression by controlling the post-transcriptional expression of E Proteins. The use of metabolic labeling and pharmacological inhibitors of the ubiquitin pathway are being used to identify the mode of regulation of the Type IIX expression pattern. The potential role of specific kinases in effecting the restriction of HEB expression are being examined by using both inhibitors and activators. The results of these studies will provide the necessary information to evaluate the biological role of E proteins in controlling fiber type transitions, and in potentially attenuating the atrophic effects of microgravity conditions. We have also recently shown that ectopic expression of the HEB protein transactivates the Type IIX-specific sk a-actin reporter. The 218 bp skeletal a- actin promoter drives transgene expression solely in mature Type IIX fibers. A mouse also carrying the transgene MLC1/HEB (which ectopically expresses the E Protein HEB in Type IIB fibers forces expression of the skeletal a-actin reporter gene in Type IIB fibers. We have recently begun an additional related study on the roles of specific isoforms of calcineurin, a calcium activated phosphatase implicated in slow fiber specification as well as in hypertrophy. We have recently found a novel calcineurin variant that appears to be specific to slow and chronically stimulated muscle. This preliminary data has led us to evaluate the role of calcineurin isoforms in the response to microgravity, where slow muscle fibers are compromised. The discovery of a new signal transduction pathway for the specification of slow fibers offers an exciting avenue for intervention in slow fiber atrophy
1. RESEARCH PLAN SUMMARY: A. HYPOTHESES, OBJECTIVES AND SPECIFIC AIMS FROM ORIGINAL PROPOSAL Several important concepts are defined by our preliminary studies on muscle fiber specification. First, muscle genes expressed in multiple fiber types, such as skeletal actin, appear to be regulated by modular cis-acting elements that are individually responsible for transcription in single fiber types, but in concert confer a pan-fiber expression pattern. Second, a novel role for the ubiquitously expressed E protein family of transcriptional regulators is suggested by the selective accumulation of HEB in a specific adult fiber type, and by the dramatic effects on fiber type distribution following targeted E protein mutation in knockout mice. Regulation of E protein accumulation in muscle fiber subsets could occur through either the translation only in permissive myofibers or through selective degradation in nonpermissive myofibers. The following research plan was designed to address the following hypotheses arising from these studies. The first Specific Aim tested the hypothesis that manipulations that affect the function of skeletal muscle to simulate various aspects of spaceflight conditions result in a shift in fiber type that can be monitored by transgenic markers, and are accompanied by perturbations in regulators of these markers, namely E proteins. The second Specific Aim tested the hypothesis that the selective atrophy that muscle undergoes in microgravity conditions may be counteracted by overriding the accompanying down-regulation of selected E proteins, using transgenic E protein over-expression, or virally delivered E protein expression cassettes. These basic studies in a mouse model are designed not only to define the molecular basis of fiber atrophy, but to provide potential therapeutic paradigms for interventional strategies in human muscle. Skeletal muscle undergoes dramatic changes in fiber composition in response to weightlessness- In this project we will define the molecular mechanisms responsible for the shifts in fiber type under conditions of microgravity. Involvement of E proteins, ubiquitously expressed bHLH transcription factors, in the establishment of muscle fiber diversity is supported by the observation that in the case of at least one member of this class, accumulation of the protein is restricted to a single fiber type. Targeted disruption of different E protein genes in mice results in the ablation of specific fiber combinations in neonatal muscle beds. Fiber-specific gene expression can be rescued by transfection. of the appropriate E protein in muscle cell cultures derived from E protein knockout animals. These results support a model in which heterodimerization of distinct E proteins with myogenic bHLH factors in different fiber types provides sufficient selectivity to recognize the modular genetic targets responsible for patterns of fiber-restricted gene expression The potential importance of E proteins in regulating fiber-specific changes in muscle gene expression suggest that they may comprise upstream targets for the effects of weightlessness. To test this hypothesis, E protein composition and distribution will be characterized in normal vs. unweighted muscles of genetically engineered mice. Modulation of several fiber-specific transgenes will be characterized in response to hindlimb unweighting, including the skeletal actin promoter-driven LacZ reporter gene, which is severely repressed during hindlimb suspension. Various E proteins will be over-expressed in living muscles, either systemically by a transgene approach, or by adeno-associated viral delivery to specific muscle groups, to evaluate their biological role in controlling fiber type transitions, and in potentially attenuating the atrophic effects of microgravity conditions. SPECIFIC AIMS Skeletal muscle undergoes dramatic changes in fiber composition in response to weightlessness, that compromise its function. In original project we proposed to define the molecular mechanisms responsible for the shifts in fiber type under conditions of microgravity. Specifically we proposed to investigate the hypothesis that E proteins, ubiquitously expressed bHLH transcription factors, play a critical role in the establishment of muscle fiber diversity. This hypothesis is supported by the observation that in the case of at least one member of the E protein class, accumulation of the protein is restricted to a single fiber type. Moreover, targeted disruption of different E protein genes in mice results in the ablation of specific fast fiber combinations in neonatal muscle beds. Fiber- specific gene expression can be rescued by transfection of the appropriate E protein in muscle cell cultures derived from E protein knockout animals. These results support a model in which hetero-dimerization of distinct E proteins with myogenic bHLH factors in different fiber types provides sufficient selectivity to recognize the modular genetic targets responsible for patterns of fiber- restricted gene expression. The potential importance of E proteins in regulating fiber-specific changes in muscle gene expression suggest that they may comprise upstream targets for the effects of weightlessness. To test this possibility, the following Specific Aims are proposed: Specific Aim 1: To define changes in E protein distribution and fiber-specific gene expression in experimentally manipulated mouse muscles. E protein composition and distribution was to be characterized in normal vs. unweighted, hormonally manipulated, exercised or damaged mouse muscles. These experimental protocols were to be performed using mice carrying various fiber-specific transgenes, including the skeletal actin promoter-driven LacZ reporter gene, which is severely repressed during hindlimb suspension. The purpose of this aim was to define appropriate transgenic markers to monitor fiber transitions in subsequent experimental designs, and to characterize the response of potential transcriptional regulators of these fiber-restricted transgenes to manipulations that cause shifts in fiber type. Specific Aim 2. To determine whether altering E protein composition or abundance can cause a shift in muscle fiber type. E protein expression cassettes were to be introduced into mouse muscles, either systemically as muscle-specific transgenes, or locally using a viral delivery system in specific muscle groups. The purpose of this aim was to evaluate the biological role of E proteins in controlling fiber type transitions, and in modulating the specific effects of other, experimental manipulations designed to attenuate muscle atrophy in microgravity conditions. B. MODIFICATIONS REQUIRED AND RATIONALE FOR MODIFICATIONS The reduction in recommended per annum funding for this project ($153,385 reduced to $90,000) necessitated a corresponding reduction and reprioritization of the research objectives. Personnel: Dr. Antonio Musaro, a postdoctoral fellow in the laboratory, is not supported by this grant. He was awarded a Research Development grant from the Muscular Dystrophy association which covered his stipend. His new work on calcineurin has been included in this progress report, due to its relevance to the specific aims of the project. The effort of a technician was reduced from 100% to 50%. Supplies: The supply budget was reduced from $23,400 to $12,041 to reflect the reduction in personnel. Other: The support for mice was reduced from $12,775 to $4088 (200 to 64 mice). The reduction in budget, particularly in the animal support, had a substantial effect on our ability to complete our two Specific Aims as outlined in the proposal. We have therefore concentrated our efforts to complete Specific Aim 1 (to define changes in E protein distribution and fiber-specific gene expression in experimentally manipulated mouse muscles, and have not addressed Specific Aim 2 (to determine whether altering E protein composition or abundance can cause a shift in muscle fiber type). II. SUMMARY OF PROGRESS IN YEAR 3 Specific Aim 1: To define changes in E protein distribution and fiber-specific gene expression in experimentally manipulated mouse muscles. From our progress to date it is clear that regulation of either E Protein synthesis or degradation is required for the restriction of HEB to Type IIX fibers. Documented instances of regulated protein synthesis are less common in eukaryotes, than the alternative, targeted turnover, and sufficiently difficult to study that further characterization beyond synthesis rate measurement using labeled precursors lies outside of this proposal. By far, the most prevalent post-transcriptional mechanism known for controlling concentrations, of cytosolic proteins is through the ubiquitin-proteasome pathway. Proteins are targeted for degradation, most often by phosphorylation or dephosphorylation, and subsequently modified by the attachment of a poly- ubiquitin chain (a 74 AA residue tag). Proteolysis then occurs in the proteasome, a heterogenous structure composed of 12-15 subunits, some of which are responsible for the recognition of specific substrates. A novel nuclear- localized ubliquitin conjugating enzyme, UbcE2A, has been identified as specifically recognizing a 54 AA region of E12 but not other bHLH proteins. Chimeric proteins consisting of b-galactosidase fused with the 54 AAs of E12 were selectively degraded by proteasomes within the nucleus. The protein turnover was blocked by proteasome inhibitors as well as by antisense RNA against the UbcE2A enzyme. Using this system we are determining whether selected proteolysis is the general mechanism by which E Protein expression is restricted in myofibers, and if so whether the 54 AA residues act as a signal for ubiquitination and subsequent degradation of E12 in Type IIB myofibers by the proteasome. Although beta-galactosidase normally is a cytoplasmic enzyme, as a fusion protein consisting of a mutated estrogen hormone binding domain (HBD) at the amino terminus, it enters the nucleus after binding subpharmacological concentration (<1 M) of RU 486, a synthetic steroid. We have generated constructs with the ubiquitously transcribed UbiC promoter directing express of fusion proteins containing full length E Protein cDNAs for E12 and HEB, as well as the segment encoding the 54 AA containing the degradation signal of E12, fused to the carboxyl terminus of the HBD/b-gal chimeric protein. In addition, a control HBD/b-gal expression cassette (without E Portein sequences) has been generated. The HBD/b-gal/E Protein fusion proteins are currently being tested in cell culture, before generating transgenic mice. Application of the proteeasome inhibitor MG132 to the transgenic animals would also be utilized to detect any cytosolic proteasomes involved in degrading E Proteins. Application of RU 486 will cause translocation of the chimeric proteins to the nucleus, exposing them to potential nuclear degradation pathways. Subsequent determination of the discovered mechanism of regulated protein expression. In a related project we have begun to investigate the role of calcium-mediated signal transduction in fiber type specification. These pathways are likely to lie upstream of the ubiquitination-degreation of E proteins, and may ultimately be more accessible to intervention. We have recently begun an additional relate study on the roles of specific isoforms of calcineurin, a calcium activated phosphatase implicated in slow fiber specification. It has been proposed that tonic motor nerve activity, which sustains high intracellular calcium levels in slow twitch muscle fibers may selectively activate the calcineurin pathway. While this model has many attractive feature, it many not encompass the full spectrum of muscle cell function for which calcineurin is responsible in vivo. Our recent studies (Muscaro, et al., 1999) suggest that both myogenic differentiation and hypertropy are also influenced by the calcineurin pathway. The resolution of these disparate functions has important implication for interventional studies. For this project, we have focused on calcineurin activation and downstream molecular events in intact muscles, where other neural and hormonal influences may modulate its action. We have recently found a novel calcineurin variant that appears to be specific to slow and chronically stimulated muscle. In collaboration with Dr. Susan Hamilton's laboratory, we are in the process of evaluating the role of calcineurin isoforms I the response to microgravity, where slow muscle fibers are compromised, by analyzing the hind limb unweighting mouse mode. The activity of calcineurin can be modulated by small molecules, such as cyclosporin and FK506. It is not clear that these drugs interact with all calcuineurin isoforms, however. Reagents specific to individual calcineurins can be developed for mere directed intervention. The discovery of a new signal transduction pathway for the specification of slow fibers offers an exciting avenue for intervention in slow fiber atrophy under microgravity or related conditions on Earth.
The selective loss of muscle fibers is a common theme in muscle aging, bedrest, and virtually every neuromuscular disorder. Elucidation of the molecular mechanisms underlying muscle fiber specificity is essential for the design of therapeutic interventions to maintain muscle integrity under normal gravity conditions, as well as in bedrest or in situations of prolonged activity.

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