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Rates of protein synthesis and degradation were measured in the isolated rat epitrochlearis muscle by radiotracer techniques, by using the specific radioactivity of tRNA-bound amino acid as precursor for protein synthesis. The tissue maintained linear rates of protein synthesis for 3 h of incubation in the presence of amino acids and glucose and in the absence of insulin. Under these conditions, however, the muscles were in negative nitrogen balance, with rates of protein degradation exceeding rates of protein synthesis. Under steady-state conditions of labelling, the specific radioactivities of tRNA-bound leucine, phenylalanine and valine were significantly less than their respective values in the incubation medium, at concentrations in the medium varying from 1 to 10 times those in normal rat serum. Insulin caused a dose- and time-dependent increase in tRNA-based protein synthesis rates, more than doubling rates at 5 and 50 ng of insulin/ml. At the lower, physiological, concentration of insulin, the stimulation of protein synthesis was not observed until the third hour of incubation with the hormone, whereas the rate of protein synthesis at the higher concentration was elevated during the second hour. There were no delays in the stimulation by insulin of glucose conversion into glycogen. The delayed stimulatory effects of insulin on the rate of protein synthesis brought the tissue to a nitrogen balance near zero. The presence of the hormone also prevented the increase in the rate of protein degradation seen in the third hour of incubation in the absence of the hormone. These studies demonstrate the viability of the incubated rat epitrochlearis muscle with respect to protein metabolism and sensitivity to the protein anabolic effects of physiological concentrations of insulin, and indicate that the preparation is a suitable experimental model for the study of the control of protein metabolism in fast-twitch skeletal muscle.

The synthesis rates of different myosin isoenzymes in a single muscle, and of the same isoenzymes in different muscles (soleus, masseter and plantaris), were measured. The rate of total protein synthesis was significantly higher in the soleus [greater than 95% slow myosin (SM)] than in the plantaris [greater than 95% fast myosin (FM)]. Two fast isoenzymes, FM2 and FM3, were synthesized at different rates in the masseter, and SM was synthesized at a faster rate than FM. Intermediate myosin had a synthesis rate similar to that of FM. There was a small but significant difference between the synthesis rates of the SM isoenzymes of the soleus and masseter muscles. FM3 was synthesized faster in the masseter than in the plantaris, whereas FM2 was synthesized faster in the plantaris than in the masseter.

The patterns of myosin isoenzymes in fast- and slow-twitch muscles of the rat hindlimb were studied, by pyrophosphate/polyacrylamide-gel electrophoresis, with hypertrophy (induced by synergist removal) and with spontaneous running exercise of 4 and 11 weeks duration. At 11 weeks, changes with hypertrophy in the slow-twitch soleus, composed of greater than 95% SM2 (slow myosin 2) in normal muscles, were minor, and consisted of an increase in the SM1 and SM1′, and a loss of intermediate myosin (IM), an isoenzyme characteristic of Type IIa fibres [Fitzsimons & Hoh (1983) J. Physiol. (London) 343, 539-550]. The changes were dramatic, however, in the fast-twitch plantaris muscle. There was a 3-fold increase in the proportion of SM. In addition, IM became the predominant isoenzyme in the profile of hypertrophied plantaris by 4 weeks. These increases were balanced by decreases in the proportion of FM2 (fast myosin 2), with FM1 completely absent from the profile at 11 weeks. The changes in the plantaris with exercise were similar in direction but not as extensive as those with hypertrophy, and FM1 remained present at control levels throughout the study. When hypertrophy and exercise were combined, the increase in slow myosin was equal to the sum of the increases with each treatment alone. Changes at 4 weeks were intermediate between those of control and 11-week muscles. Peptide mapping of individual myosin isoenzymes showed that the heavy chains of IM were different from either fast or slow heavy chains. Furthermore, IM was found to be composed of a mixture of fast and slow light chains. These changes suggest that a transformation of myosin from fast to slow isoforms was in progress in the plantaris in response to hypertrophy, via a Type-IIa-myosin (IM) intermediate stage, a phenomenon similar to that occurring in chronically stimulated fast muscles during fast-to-slow transformation [Brown, Salmons & Whalen (1983) J. Biol. Chem. 258, 14686-14692].