Progress 12/01/04 to 11/30/08
Outputs
OUTPUTS: Myofibrillar proteins must be removed from the myofibril before they can be turned over metabolically in functioning muscle cells. It is uncertain how this removal is done without disrupting the contractile function of the myofibril. It has been proposed that the calpains could remove the outer layer of filaments from myofibrils as a first step in myofibrillar protein turnover. Several studies have found that myofilaments can be removed from myofibrils by trituration in the presence of ATP. These easily releasable myofilaments (ERMs) were proposed to be intermediates in myofibrillar protein turnover. It was unclear, however, whether the ERMs were an identifiable entity in muscle, or whether additional trituration would remove more myofilaments until the myofibril was gone, and whether calpains could release ERMs from intact myofibrils. The present study shows that few ERMs could be obtained from the residue after the first removal of ERMs, and yield of ERMs from well washed myofibrils was reduced, probably because some ERMs had been removed during washing. Mild calpain treatment of myofibrils released filaments that had a polypeptide composition and that were ultrastructurally similar to ERMs. The yield of calpain-released ERMs was 2-3-fold greater than the normal yield. Hence, ERMs are an identifiable entity in myofibrils and calpain releases filaments that are similar to ERMs. The role of ERMs in myofibrillar protein turnover is unclear because only filaments on the surface of the myofibril would turnover, and changes in myofibrillar protein isoforms during development could not occur by the ERM mechanism. PARTICIPANTS: Darrel E. Goll, Girija Neti, Valery F. Thompson, and Stefanie M. Novak. Dr. Neti was a visiting professor from Hyderbad, India. TARGET AUDIENCES: This research will be of interest to anyone interested in the rate of skeletal muscle growth and response of muscle to injury. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The results of this study show that ERMs are distinct entities in rat and bovine skeletal muscle. The results also show that calpains release myofilaments that are ultrastructurally the same as ERMs and have the same alpha-actinin to actin ratio as ERMs. Additional studies are needed to show whether or not release of ERMs are indeed the first step in myofibrillar protein turnover. Metabolic turnover of the myofibrillar proteins the ERMs suggests that only the proteins on the surface of the myofibril are turned over and that the proteins in the interior turn over very slowly if at all. Therefore, it seems likely that there is an additional mechanism for protein turnover in skeletal muscle. The nature of this mechanism is unknown and additional research will be needed to discover how this occurs. The results of this study have been published in one paper and a second paper has been submitted for publication.
Publications
- Goll, DE, Neti G, Mares SW, and Thompson VF (2008) Myofibrillar protein turnover: the proteasome and the calpains. J. Anim. Sci, 86, E19-E35.
- Neti, G., Novak, S. M., Thompson, V. F., and Goll, D. E. (2009) Properties of easily releasable myofilaments (ERMS): Are ERMS the first step in myofibrillar protein turnover (submitted)
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Progress 12/01/06 to 11/30/07
Outputs
OUTPUTS: The mechanism by which myofibrillar proteins are turned over metabolically remains unclear. It is clear that the 20 plus proteins that constitute the myofibril in striated muscle turn over with differing half lives, and that the rate of this turnover varies widely in response to physiological demand. Turn over of myofibrillar proteins, however, must occur without disrupting integrity of the myofibril; any disruption of the myofibril will result in loss of contractility/function of that entire myofibril. It was proposed over 30 years ago that one possible mechanism for turnover of myofibrillar proteins would be removal of the outer layer of filaments on the surface of the mypofibrils with subsequent degradation of the released myofilaments. This would result in a progressively smaller myofibril, but no loss of function. It was further proposed at that time that the calpains may be responsible for the release of the outer layer of myofilaments because the calpains degrade many
of the proteins involved in keeping the myofibril intact. Several years after the release of an outer layer of myofilaments was proposed, it was discoved that muscle contained a group of easily releasable myofilaments that could be removed from myofibrils by gentle agitation in the presence of ATP. These easily releasable myofilaments (ERMs)consituted approximately 10-15% of total myofibrillar protein and seemed to be intermediates in myofibrillar protein turnover,as had been proposed earlier. We initated studies to learn whether ERMs could be obtained from skeletal muscle from domestic animals (the earlier studies had been limited to rats), and whether the ERMs were "real" or were simply the result of agitation in the presence of ATP--i.e., could more and more "ERMs" be produced by repeated agitation in the presence of ATP until the entire myofibril was gone or did the yield of ERMs decease and drop to zero after the first set of ERMs was obtained.A procedure has been developed for
obtaining ERMs from bovine skeletal muscle; the amount of ERMs in bovine skeletal muscle is much less than that reported earlier for rat skeletal muscle, with approximately 0.2-0.8% of all myofibrillar proteins being obtained as ERMs. Repeated agitation in the presence of ATP did not release additional ERMs, indicating that the ERMs are a "real" entity in skeletal muscle and not the result of "scraping" additional myofilaments off the surface of the myofibril. The ratio of alpha actinin to actin in the ERMs is less than 10% of the ratio of alpha actinin to actin in myofibrils as would be predicted if the ERMs are myofilaments that have been released from the remainder of the myofibril and have no Z-disk proteins. Very gentle treatment of myofibrils with purified calpain produces a high yield of myofilaments that resemble the ERMs both ultrastructurally and in their alpha actinin to actin ratios. Hence, ERMs are a distinct entity in skeletal muslce from several different species and
their yield can be increased 5-10-fold by gentle calpain treatment.
PARTICIPANTS: Darrel E. Goll Girija Neti Valery F. Thompson Stefanie M. Novak Dr. Neti was a visiting professor from Hyderabad, India
TARGET AUDIENCES: This research will be of interest to anyone interested in rate of skeletal muscle growth and response of muscle to injury.
Impacts
The results of the studies done this past year on this project have shown that the ERMs are distinct entities in the skeletal muscle of at least two species, the rat and the bovine, and that a population of myofilaments that resemble the ERMs can be released from myofibrils by the calpains. These results are consistent with the hypothesis first proposed over 30 years ago that one mechanism for metabolic turnover of myofibrillar proteins is the release of myofilaments from the surface of the myofibril. Although the amount of ERMs that we obtained form bovine skeletal muscle was much less than the 10-15% of total myofibrillar protein reported earlier in studies using rat skeletal muscle, we found that rat muscle contained less ERM protein than what had been reported earlier, with approximately 1-2% of all myofibrillar protein in rat skeletal muscle being released as ERMs. Additional studies determining whether the amount of ERMs increases in muscle undergoing rapid atropy
such as in unweighted hind limb muscles of rats are needed to show whether the ERMs are intermediates in the metabolic turnover of the myofibrillar proteins. Metabolic turn over of myofibrillar proteins only through ERMs would imply that the proteins in the interior of myofibrils would turn over very slowly if at all; this seems physiologically unlikely, and it possible that myofibrillar proteins turn over via more than one mechanism. The nature of this other mechanism, if it exists, it unknown. A few studies done in the 1980s/1990s suggested that indivudal myofibrillar proteins could exchange with their counterparts in the cell cytoplasm, but it is unclear how this exchange would occur. Additional research comparing myofibrillar protein echange and yields of ERMs are needed to learn whether both mechanisms are involved. The results of the studies done this past year have been submitted for publication; one paper in press and one paper in its final stages of preparation.
Publications
- Camou, J.P., Marchello, J.A., Thompson, V.F., Mares, S.W., and Goll, D.E. 2007. Effect of postmortem storage on activity of mu- and m-calpain in five bovine muscles. J. Anim. Sci. 85, 2670-2681.
- Camou, J.P., Mares. S.W., Marchello, J.A., Vazquez, R., Taylor, M., Thompson, V.F., and Goll, D.E. 2007. Isolation and characterization of mu-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps. J. Anim. Sci. 85, 3400-3414.
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Progress 12/01/05 to 12/01/06
Outputs
Inappropriate calpain activity has been implicated in a wide variety of human pathologies from loss of muscle mass in the muscular dystrophies, tissue damage in the ischemic areas surrounding a blood vessel blockage (myocardial infarcts, stroke), multiple sclerosis, proteolytic tissue damage following brain injury, and even Alzheimer's disease. The calpains have generally assumed to be maximally active at pH values slightly above 7, but the effect of temperature on calpain activity has not been studied carefully. To obtain an accurate estimate of calpain activity at different pH values and temperatures, we have measured the proteolytic activity of u-calpain and m-calpain, the two ubiquitous calpains, at 4 different pH values, 7.5, 7.0, 6.5, and 5.8, and at 3 different temperatures, 4 degrees C, 10 degrees C, and 25 degrees C, by using a sensitive BODIPY microtiter plate assay. At 25 degrees C, pH had little effect on activity of u-calpain until it decreased below pH
6.5; at pH 5.8, u-calpain activity was 50% of the activity at pH 6.5, 7.0, 7.5. At 4 degrees and 10 degrees C, pH had a slightly greater effect on u-calpain activity than at 25 degrees C, but the effect was still less than a 35% decrease. Activity of u-calpain at 4 degrees C was 65% of the activity at 25 degrees C, indicating that both pH and temperature have relatively little effect on u-calpain activity.
Impacts
pH has a much greater effect on m-calpain activity; m-calpain was nearly inactive at pH 5.8 (less than 10% of pH 7.5 activity), and m-calpain activity at pH 7.0 was ~ 75% as high as at pH 7.5, and was 30-40% as high at pH 6.5 as it was at pH 7.0. Temperature had relatively little effect on m-calpain activity in the range we studied (~80% as high at 4 degrees C as at 25 degrees C).
Publications
- Saito, M., Li, H., Thompson, V.F., Kunisaki, N., and Goll, D.E. 2006. Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss. Comparative Biochemistry and Physiology, Part B 146, 445-455.
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Progress 12/01/04 to 11/30/05
Outputs
Myofibrillar proteins present a unique system for metabolic turnover of proteins because organization of the myofibrillar lattice must be maintained if the myofibril is to remain functional. The proteosome is involved in turnover of myofibrillar proteins, but intact myofibrils that are 10-100 um in diameter cannot enter the central catalytic cavity of the proteosome. Hence, some intermediate form of myofibrillar proteins exists between the intact myofibril and proteosomal degradation. It has been proposed that 10-15% of the protein in striated muscle myofibrils can be released in the form of easily releasable myofilaments (ERM) and that these ERM are intermediates between the intact myofibril and proteins that can be accessed by the proteosome for degradation. ERM are released by gentle agitation of myofibrils in the presence of ATP and contain the major myofibrillar proteins, actin and myosin, but do not contain a-actinin, desmin, M-protein, or other proteins that
are not associated with the actin and myosin filaments. We have obtained ERM from rat and bovine skeletal muscle; the ERM from either species contain myosin, actin, titin, tropomyosin, and troponin, but do not contain a-actinin or desmin. The yields of ERM were less than reported: 0.18-0.44% of total myofibrillar protein. Incubation with purified calpain increases the yield of ERM by 10%.
Impacts
Anti-ubiquitin antibodies from several sources labeled myosin and actin in both ERM and in intact myofibrils, but did not label any other proteins in either the ERM or the myofibrils. It is not clear that the ERM we obtained are intermediates in myofibrillar protein turnover.
Publications
- No publications reported this period
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