ACTIVITY-DEPENDENT POLYPEPTIDE ELONGATION IN MUSCLE

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The immediate goal of this project is to determine if a decrease in nascent polypeptide elongation rate causes the rapid decrease in protein synthesis observed in non-weightbearing skeletal muscle. A clear identification of the site-of-action of the initial events in muscle atrophy will help to define the intracellular signals that maintain normal gene expression. These signals may influence the adaptation of skeletal muscle to chronic bedrest, paraplegia, limb immobilization, and weightlessness. Rat soleus muscle loses 80% of its myofibril protein if prevented from weightbearing. Although all levels of gene expression are affected, the most rapid changes occur at the level of translation; within five hours of the onset of non-weightbearing myofibril protein synthesis rate has diminished by at least 25% as it evolves toward an eventual 50% decline. Preliminary data indicate that the initial defect may be a decreased rate of nascent polypeptide synthesis that is manifest as in increased polysome size. The working hypothesis of this proposal is that a slowed rate of elongation causes an accumulation of 80S ribosomes on the mRNA. The experiments will also determine if this form of regulation is specific for contractile proteins. Two experiments will test for more 80S ribosomes per mRNA. The first will measure amount of radioactive amino acid incorporated into the nascent polypeptide pool. Corrected for aminoacyl-tRNA specific activity, this value is proportional to the number of nascent polypeptides (and hence the number of 80S ribosomes) associated with the mRNA. The second experiment will directly measure the number of ribosomal subunits associated with both alpha-actin and cytochrome c mRNAs. To test if the regulation is specific for contractile proteins, non-muscle genes (beta- galactosidase and alcohol dehydrogenase) will be constitutively expressed in the atrophying muscle by retroviral-mediated gene transfer. The mRNAs for these genes will not reflect the polysome size shift relative to alpha- actin if regulation is specific for contractile proteins.