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Myogenic determination factors (MDF) have been implicated in the establishment and maintenance of the fast or slow phenotype in skeletal muscle, with MyoD favoring the fast and myogenin favoring the slow phenotype. Accordingly, contractility-induced changes in muscle phenotype should be accompanied by a change in the MyoD/myogenin ratio. Some reports show such changes, but limitations inherent to in vivo studies complicate interpretation of these data. Here we tested whether a relationship can be found between contractility, MDF expression, and the expression of phenotype-specific muscle proteins in a simple in vitro system of cultured primary myotubes. We show that contractions reduce the MyoD/myogenin ratio by specifically repressing MyoD mRNA expression. This is accompanied by a selective repression at a pretranslational level of the expression of fast-type sarcoplasmic reticulum Ca(2+)-ATPase. These in vitro results support a phenotype-determining role of MDFs as a function of contractile activity and show that cultured myotubes can be a useful model for the analysis of the molecular mechanism of such regulation of muscle phenotype.

Thyroid hormone increased the percentage of fibers expressing fast-type sarcoplasmic reticulum Ca(2+)-ATPase in the slow rat soleus muscle from 17% in the hypothyroid to 100% in the hyperthyroid state. This was accompanied by a 12-fold increase in the fast-type Ca(2+)-ATPase protein content of soleus muscle homogenates, suggesting that also the amount of this protein per muscle fiber was increased. In contrast to the fast-type isoform, a decrease in the percentage of fibers expressing slow-type Ca(2+)-ATPase from 100% to 70% was observed in the transition from the hypothyroid to the hyperthyroid state. Slow-type Ca(2+)-ATPase protein levels in muscle homogenates however did not decrease on the same trajectory, but were even elevated in the euthyroid state. In the fast extensor digitorum longus muscle qualitatively similar changes in Ca(2+)-ATPase isoform expression were observed. The results suggest a dual action of thyroid hormone: 1. increasing slow-type Ca(2+)-ATPase expression in individual fibers 2. decreasing the fraction of slow-type Ca(2+)-ATPase expressing fibers.

A spectrophotometric method is described for the determination of sarcoplasmic reticulum (SR) Ca2(+)-ATPase activity (EC 3.1.6.38) in unfractionated muscle homogenates. Conditions were established that give maximal SR Ca2(+)-ATPase activity, while eliminating Ca2(+)-dependent myofibrillar ATPase activity and reducing Ca2(+)-independent or background ATPase activity. High [Ca2+] (20 mM) could be used to selectively inhibit the SR Ca2+ ATPase. Identification of the Ca2(+)-dependent ATPase activity in muscle homogenates as being SR Ca2+ ATPase was based on a comparison of several parameters using homogenate material and purified SR. The following parameters were compared and found to be the same in homogenate and SR: activation and inactivation between 0 and 20 mM Ca2+, temperature dependence, sensitivity toward Triton X-100, and the maximal level of inhibition of ATPase activity achieved by an antibody specific for SR Ca2+ ATPase. The method is illustrated with the analysis of homogenates prepared from freeze-dried muscle fibers and thin sections of muscles typically used in microscope analyses as well as an analysis of freshly prepared homogenates from various types of muscle, which shows a good correlation over a wide range between SR specific Ca2(+)-uptake and -ATPase activities. In addition, a simple, easily constructed cuvette is described which allows the analysis of less than 5 micrograms of tissue (wet weight) in a volume of 25 microliters.

The effects of the thyroid status on the Ca++-transporting capabilities of rat slow skeletal muscle (m.soleus) were studied. The oxalate supported Ca++-uptake activity and Ca++-loading capacity of muscle homogenates from hyperthyroid rats showed an approximate 4.2 and 2.5 fold increase, respectively, as compared to values found in the hypothyroid group. Muscle homogenates of euthyroid rats gave intermediate values. The specific activity of oxalate supported Ca++ uptake, but not the Ca++-loading capacity, of membrane preparations enriched with respect to sarcoplasmic reticulum (SR) increased in proportion to the thyroid status. This was paralleled by a 3.5 fold increase in the amount of active Ca++ pumps in the SR preparations in the transition from hypothyroidism to hyperthyroidism as determined by measurement of Ca++-dependent 32P incorporation. These observations are not explained by differences in degree of purification of the examined SR preparations. Protein profiles of the membrane preparations obtained by gel electrophoresis indicated a thyroid-hormone dependent increase in Ca++-pump content relative to other SR proteins. The results suggest that thyroid hormone stimulates the proliferation of the SR and possibly also increases the Ca++-pump density in the SR membrane.

A fraction containing a protein-glycogen complex was isolated from rat skeletal muscle in order to study the effect of hypothyroidism on phosphorylase activation in this structural and functional unit of the glycogenolytic process. The total activities of phosphorylase and phosphorylase phosphatase in euthyroids and hypothyroids were the same in the fraction containing the protein-glycogen complex (P2 suspension). Hypothyroidism selectively lowered the maximal phosphorylase kinase activity in glycogen particles in the P2 suspension by 40%. Addition of Mg2+ (10 mM), ATP (2 mM), and Ca2+ (5 mM) rapidly stimulated phosphorylase b to a conversion resulting from phosphorylase kinase activation. Hypothyroidism reduced the rate of phosphorylase a formation by 50-70% in the P2 suspension. Glucose 6-phosphate (0.4-1.4 mM) inhibited the rate of phosphorylase a formation and this inhibition was similar for eu- and hypothyroids. There was a shift from 5.2 to 5.8 in the free Ca2+ concentration (pCaF) for half-maximal activation of phosphorylase in the P2 suspension of hypothyroids. A sixfold higher steady-state level of phosphorylase in euthyroids compared to hypothyroids was observed at a pCaF of 5.5. The Ca2+ sensitivity of the phosphorylase kinase, however, was not changed by hypothyroidism. These results provide further insight into the different time course of the phosphorylase activation in skeletal muscle during tetanic stimulation observed in euthyroidism and hypothyroidism (W. J. Leijendekker et al. (1985) Metabolism 34, 437-441).

A method for the determination of lactate is described in which the esterification of lactate with the uv-absorbing compound alpha-p-dibromoacetophenone is followed by separation and quantitation of the ester by reversed-phase HPLC with detection at 254 nm. The reproducibility, detection limit, and precision of the method are comparable to those of conventional methods which use enzymatic cycling for enhanced performance. The applicability of this rapid and simple method is illustrated with the determination of picomole amounts of lactate in resting and stimulated single muscle fibers.