Overview
Pharmacological Inhibitors of the Proteosome in Atrophying Muscles (Also Kenneth Baldwin, Ph.D., UC Irvine, Effects of Unloading on Myosin Content and Isoform Specific Regulation in Skeletal Muscle)
Principal Investigator:
Alfred L. Goldberg, Ph.D.
Organization:
Harvard Medical School
Technical Summary
1. Critical Role of Certain Ubiquitination Enzymes
Various prior observations had suggested that the increased protein breakdown responsible for muscle atrophy in many diseases (e.g. cancer, sepsis, diabetes, or hyperthyroidism) is primarily due to an activation of the Ub-proteasome pathway. During the past three years using homogenates of normal and atrophying muscles, we showed that overall rates of Ub-conjugation increase in atrophying muscles, and these hormone and cytokine-dependent responses are due largely to activation of the N-end rule pathway for Ub-conjugation. Specifically we discovered that Ub-conjugation to endogenous proteins and to a model substrate of the N-end rule pathway (lysozyme) were significantly increased. We found that mRNA for the critical ubiquitination enzymes in the N-end rule pathway, E214k and E3α, are increased about two-fold in the atrophying muscles. Thus, the activation of Ub-conjugation and proteolysis by the "N-end" pathway appears to be a very general feature of atrophying muscles, independent of the cause.
In normal muscle extracts, we found that the N-end rule pathway for ubiquitin conjugation also appears to be responsible for the degradation of most soluble proteins. In contrast to muscle, in extracts of HeLa cells, this system is also present but makes only a minor contribution to overall protein ubiquitination. These findings were quite unexpected, because the ubiquitinating enzymes comprising the N-end rule pathway, and in particular E3α, are believed to recognize abnormal proteins with unusual amino-terminal residues. An important role for E214k and E3α in muscle (or in any other cell) was unanticipated since the synthesis of all proteins begins with a methionine in the N-terminus, and over 80 percent have their N-termini acetylated, which prevents recognition by the E3α.
2. Ubiquitination Conjugation After Hind-Limb Suspension
A similar increase in ubiquitin conjugation was found in extracts of muscle following hind-limb suspension (performed by our collaborator, Dr. Ken Baldwin). These findings suggest that the disuse atrophy in astronauts involves a similar activation of the Ub-proteasome pathway, as we have found in many other types of muscle wasting (although these changes upon hind-limb suspension were less pronounced and therefore harder to study routinely than in some other atrophying models).
3. Possible Use of Protease Inhibitors
One other goal of our work during the initial grant period was to evaluate the possible utility of the newly discovered proteasome inhibitors as possible countermeasures to retard the excessive protein breakdown in atrophying muscles. It is noteworthy that these inhibitors, especially the peptide aldehydes, had much larger effects in reducing proteolysis in atrophying muscles than in control muscles. Thus, as suggested previously, the enhanced proteolysis in many catabolic states is due to a proteasome-dependent pathway. Because inhibition of proteasome function in principle could be a useful approach to reduce muscle wasting, we carried out systematic studies to define the effects of peptide aldehydes and the more selective proteasome inhibitor, lactocystin-β-lactone on intracellular proteolysis. These findings provided definitive support for our prior conclusions that the proteasome is the site for degradation of long-lived as well as short-lived proteins. On the other hand, we and a number of other investigators have found that prolonged exposure of most cells to these various inhibitors (i.e. for 24-32 hours) at concentrations that cause a large reduction in protein breakdown causes cell death by apoptosis. So, although partial inhibition of proteasome function is well tolerated, complete inhibition is dangerous, therefore these types of inhibitors are inappropriate for use by astronauts as countermeasures and can only be used against life-threatening illnesses in a hospital setting. In fact, such clinical trials are in progress now in cancer patients. Consequently, future studies will focus on development of inhibitors of ubiquitination enzymes that are specifically important in atrophy (whose inhibition should be non-toxic and affect primarily atrophying muscles).
4. Patterns of Gene Expression in Atrophying Muscles
It seems likely that (1) muscle atrophy involves a suppression of the same program of gene expression that is activated during work-induced hypertrophy or by IGF in normal growth and (2) that there also exist a number of genes induced during muscle atrophy that are of major importance in the loss of muscle protein and contractile function. Our studies thus far have uncovered several genes (e.g. Ub, proteasome subunits, etc.), whose expression increases approximately two- to three-fold in all types of muscle atrophy studied thus far. The increases in these mRNAs are noteworthy because atrophying muscles generally show a decrease in total mRNA and ribosomal RNA. We propose to call such genes, atrophy genes, since these gene products which we call "atrophins" are likely to play a key role in the process of muscle wasting.
Because of the potential value of information on the nature of these atrophy genes, during the past year, under the NSBRI grant we have initiated a gene microarray analysis to obtain a comprehensive picture of the transcriptional changes occurring during muscle atrophy. This approach allows comparison on a single chip of mRNAs from experimental and control tissues from mouse or human cDNA libraries containing 5-10,000 different cDNAs. In order to validate this approach, in our initial experiments we chose to study the pattern of changes in muscle mRNAs induced by fasting, primarily because of the simplicity of this model and the wealth of prior information on this type of muscle wasting, especially the changes in proteolysis and energy metabolism. Our initial observations have proven very informative and promising. We have found over 100 genes whose levels change by about two-fold or more in fasting. They fall into several categories including mRNAs encoding a) multiple subunits of the 20S proteasome and its 19S regulatory complex, which are coordinately up-regulated (as expected). Most interestingly, there are seven genes (ORFs) whose expression increases most markedly (four- to nine-fold), and surprisingly, the functions of all of them are unknown. We are beginning to clone the most highly induced species in order to analyze their expression, to see if they are induced upon unloading, to prepare antibodies against the encoded proteins, and to explore their functions. The protein encoded by this most highly regulated mRNA, which we term atrophin-l, resembles that a subunit of a new type of E3 (a ubiquitination protein ligase) belonging to the F-Box. These exciting observations suggest that this protein is part of a new ubiquitination enzyme involved in the acceleration of protein breakdown during muscle wasting.
Effects of Unloading on Myosin Content and Isoform Specific regulation in Skeletal Muscle (Dr. Kenneth M. Baldwin)
History of the Project
In the initial funding period supported by the NSBRI (October 1997-September 2000) our original project entitled "Effects of Unloading on Myosin Content and Isoform Specific Regulation in Skeletal Muscle" was jointly submitted along with a project proposed by Dr. Alfred Goldberg's group at Harvard Medical School (K.M. Baldwin and A. L. Goldberg, serving as Co-PIs). The central thrust of the proposal was to examine how unloading (and increased loading) states impact the expression of the MHC protein system by examining transcriptional, translational, and degradative (ubiquitin-proteasome) processes. Although the project was funded, the NSBRI appointed Dr. Goldberg as the PI and the proposal was given a new title with a primary focus on degradative processes associated with the ubiquitin-proteasome pathway. Our group focused on a narrower set of objectives: a) regulation of transcription of the slow MHC gene in response to altered loading states; and b) the delineation of myogenic factors in the control of muscle mass and contractile phenotype.
Progress on the Original Proposal
Significant progress was attained on four interrelated projects: 1) in vivo regulation of the beta (slow) MHC gene in soleus muscle of suspended and weight-bearing rats; 2) changes in markers of myogenesis in overloaded rat muscles; 3) mechanisms on up regulation of fast IIb MHC in unloaded muscle; role of the nerve and thyroid hormone; and 4) quantitation of total MHC and MHC isoforms in response to unloading.
In the first project we demonstrated the feasibility of using direct DNA transfection technology for studies on the in vivo regulation of the type I MHC gene promoter in response to weight bearing activity and hindlimb suspension. In that project, we demonstrated that a) normal (optimal) type I MHC transcriptional activity in antigravity muscles requires the presence of an up-stream enhancer sequence (-3500 to -2900) that likely interacts with response elements in the first 400 bp upstream of the transcription start site (TSS); b) unloading-induced down regulation of type I MHC promoter activity is mediated in the proximal 400 bp upstream of the TSS. We have tentatively identified the negative beta e1 response element as a key factor in this process. Additional studies are in progress to more fully characterize the proximal response elements in response to unloading.
The second project was aimed at identifying markers of putative satellite cell proliferation and differentiation processes in muscles that undergo increases in hypertrophy due to increases in chronic loading. These experiments were performed in the context of increased expression of muscle IGF-I at both the mRNA and peptide level. The central findings of this study indicate that myogenic processes are activated in response to increased loading at early time points (e.g. 12 hrs) and that IGF-I is likely modulating this response. Furthermore, the findings indicated that some myogenic cells are likely differentiating early on in the adaptive process, before events leading to satellite cell proliferation have been initiated.
The third project was aimed at understanding how the ~ de novo expression of fast type IIb MHC gene occurs in antigravity muscles, e.g., muscle-types that do not normally express this gene. This work was predicated on the novel observation that hindlimb unloading requires increased levels of thyroid hormone in order to fully express the IIb MHC gene at both the mRNA and protein levels. Our finding suggest that normal innervation is essential for inducing the unique expression of the IIb MHC in a slow muscle in response to the combination of hindlimb suspension and thyroid hormone; and the up regulation of the myogenic factor, MyoD, may be essential to this process. However, in the denervated muscle, there is a discordance between the regulation of the endogenous IIb MHC gene relative to the exogenous IIb promoter-report construct that is not fully understood at the present time.
In the fourth project we developed techniques to quantitate changes in total as well as isoform specific MHC protein and mRNA content in response to unloading in order to show that during unloading, the myofibril system (and particularly the contractile apparatus) undergoes a remodeling in which there are reductions in the slow MHC content at the protein and mRNA levels which accompanies the general degradation process. In addition, there are also maintenance in protein and increase in mRNA content of fast MHCs (IIx-IIb) that occur in spite of the general atrophy process that predominates during unloading. These findings, in conjunction with project III, clearly show that there is MHC isoform-specific gene regulation in response to altered loading states; and these processes are likely mediated by a coordination between transcriptional, translational, and degradation control points.
In summary, we have made significant progress on several fronts in an attempt to address fundamental issues in the biology of muscle plasticity that are relevant to the mission of the NSBRI. However, in view of the fact that future research concerning muscle structure and function funded by the NSBRI needs to be more closely related to seeking countermeasures for reducing muscle atrophy, we have refocused our research to more specifically address the efficacy and mechanisms concerning the role of resistance training in reducing the muscle atrophy that occurs in response to chronic unloading.