Palmitate oxidation in suspended skeletal muscle fibers from the rat.

Palmitate oxidation in rat skeletal muscle was investigated with a suspension of intact isolated cells. M. flexor digitorum brevis was dissociated by a 6 h collagenase treatment to yield single myofibers of which 76% were viable. The contributions of 14CO2 and 14C-labeled acid-soluble intermediates to total oxidation products from palmitate were evaluated. The myofiber suspension exhibited a higher total oxidation rate than the isolated whole muscle, due to improved transport of palmitate to the sarcolemma. Addition of cytoplasmic cofactors L-carnitine, CoASH and ATP did not increase the palmitate oxidation. 14CO2 amounted to about 37% of oxidation products. With [1(-14)C]- and [16(-14)C]palmitate, the oxidation rates were equal. These findings indicate that the cellular integrity was well preserved. The oxidation rates were sharply decreased in fibers with damaged sarcolemmas, and in intact fibers when rotenon and antimycin A were applied. The damaged fibers restored the production of acid-soluble intermediates in the presence of cofactors. The results indicate that suspended skeletal myofibers are an adequate in vitro system for measurements of metabolic activities in the resting muscle.

Oxidative metabolism of cultured human skeletal muscle cells in comparison with biopsy material.

Human muscle cell cultures were examined for capacities to oxidize several substrates, and for activities of some enzymes related to intermediate metabolism. The results indicate that mitochondrial activities attained appreciable degrees of maturity. The specific activity of creatine kinase increased during myoblast fusion. In contrast, parameters of oxidative metabolism (palmitate and pyruvate oxidation, and cytochrome c oxidase and citrate synthase) did not significantly change throughout myogenesis and thereafter. In differentiated cells (myotubes) the oxidation capacities were pyruvate greater than 2-oxoglutarate greater than malate (+ acetylcarnitine) greater than malate (+ pyruvate), as in muscle biopsies. With regard to protein the cultured human muscle cells showed higher activities than the original biopsies (= 100%) with respect to citrate synthase (179%), but lower values for cytochrome c oxidase (50%) and creatine kinase (7%). Palmitate oxidation capacities were the same in both systems. The presence of antimycin and rotenon inhibited to a comparable extent the palmitate oxidation in cultured muscle and biopsies.

The contribution of myofibers and fibrocytes to palmitate oxidation in skeletal muscle.

Myofibers and fibrocytes were separated from a cell suspension of rat m. flexor digitorum brevis. The procedure was successful only when myofibers were intentionally damaged prior to separation. Density gradient centrifugation yielded a fraction containing 82% of fibrocytes and 20% of myofibers and a second fraction with the complementary 18% and 80% of fibrocytes and myofibers, respectively. Since the damaged myofibers had a negligible palmitate oxidation activity, the relative contribution of fibrocytes and myofibers to palmitate oxidation in the parent cell suspension with intact myofibers could be established, and amounted to 13% and 87%, respectively.

Transfection of human skeletal muscle cells with SV40 large T antigen gene coupled to a metallothionein promoter.

We have undertaken to increase the proliferative capacity of cultured human skeletal myocytes by transfection with a plasmid construct that contains the immortalizing and transforming large T antigen gene of simian virus 40 (SV40) under the control of a zinc-sensitive metallothionein promoter. This construct was chosen to permit rapid growth of transformants in zinc-containing medium, which induces high levels of T antigen expression, and muscle-specific differentiation after withdrawal of exogenous zinc, which reduces levels of T antigen. When grown in 100 microM Zn2+, transformed myocytes expressed the large T antigen, divided rapidly, and acquired an apparently unlimited proliferative capacity. Transfer of these cells to a zinc-poor medium resulted in decreased T antigen immunofluorescence, growth rate, and saturation density as well as a return to a physiological spindle morphology. Despite transformation, these cells expressed differentiation markers characteristic of myoblasts: the B isoform of creatine kinase, and surface antigens 5.1H11, D5, and Thy 1 in the presence or absence of Zn2+. When grown to high density in a serum-poor medium, these cells differentiated further into typical multinucleated myotubes that expressed the M isoform of creatine kinase and increased levels of surface antigen 5.1H11, creatine kinase, and nicotinic acetylcholine receptors, but no detectable Thy 1 antigen. The specific activity of these differentiation markers was higher when the cells were grown in the absence of added zinc. These results indicate that transformation of human skeletal myocytes with a regulatable SV40 large T antigen gene allows an increase of the proliferative capacity of these cells with preservation of their capacity to differentiate in a physiological manner.

Isolated myofibers from rat skeletal muscle in suspension: cellular morphology and calcium homeostasis.

Myofiber suspensions with 80% viability (morphological intactness) were prepared from rat m. flexor digitorum brevis by a 6-hour collagenase treatment. The viable fibers had intact sarcolemmas and generally showed continuous basal laminae. The intracellular compartments exhibited normal muscular morphology. The suspended myofibers showed good tolerance against extracellular calcium (up to 30 mM), but were susceptible to 15 mM EGTA. Calcium ionophore A23187 induced contraction, with loss of viability in both calcium-containing and nominally calcium-free Krebs-Ringer medium. EGTA prevented the ionophore effect, but the muscle cells collapsed as soon as calcium was replenished. A calcium paradox, as observed with perfused heart, could not be induced in isolated skeletal myocytes. This suspended cell system should provide a useful tool for investigating relationships between metabolic parameters and muscle physiology. The extracellular environment can be manipulated easily, and cellular responses of individual skeletal muscle fibers can be measured and visualized.

Skeletal muscle fibers in suspension: a new approach to the study of oxidative and glycolytic metabolism in differentiated muscle.

A preparation of suspended fibers from m. flexor digitorum brevis of the rat was characterized with respect to morphological features, and its relevance for the study of muscular metabolism investigated. The activities of oxidative (palmitate and pyruvate oxidation) and glycolytic (lactate formation) pathways were enhanced in myofiber suspensions when compared to intact whole muscle. The rate of glycolysis was stimulated about two-fold by insulin in both the myofiber suspensions and intact muscle. Parameters of oxidative metabolism responded similarly to metabolic effectors in the myofiber suspensions and in intact muscle. It is concluded that the myofiber suspensions have distinct advantages over intact muscle for biochemical and pharmacological studies.

Postnatal growth and differentiation in three hindlimb muscles of the rat. Characterization with biochemical and enzyme-histochemical methods.

The postnatal development, between 0 and 90 days, of three hindlimb muscles and diaphragm of the rat was investigated with respect to fiber types and diameter (histochemistry) and substrate oxidation rates and enzyme activities (biochemistry). The process of muscle fiber differentiation into mature patterns was evaluated by visual classification into 3 or 4 groups having different staining intensities for 3 enzyme-histochemical reactions, enabling 26 fiber types to be distinguished. These exhibited specific sizes and growth rates that varied among the muscles. One of the hindleg muscles (flexor digitorum brevis) remained much more immature than soleus and extensor digitorum longus. The histochemical and biochemical findings correlated well. The capacity for pyruvate and palmitate oxidation, and the activities of cytochrome c oxidase and citrate synthase, increased markedly between 9 and 37 days in soleus and extensor digitorum longus (except citrate synthase in the latter) but not in flexor digitorum brevis. Creatine kinase activity increased in all hindlimb muscles. Both the capacity and the activity of pyruvate oxidation (determined in homogenates and intact isolated muscles, respectively), were in accordance with the fiber type composition. In contrast to oxidation capacity, the activity of pyruvate oxidation decreased after birth until the mature stage, when a value of 18-42% of that of early postnatal muscles was recorded.

14CO2 production is no adequate measure of [14C]fatty acid oxidation.

Palmitate oxidation was comparatively assayed in various cell-free and cellular systems by 14CO2 production and by the sum of 14CO2 and 14C-labeled acid-soluble products. The 14CO2 production rate was dependent on incubation time and amount of tissue in contrast to the total oxidation rate. The 14CO2 contribution to the oxidation rate of [1-14C]palmitate varied with homogenates from 1% with rat liver to 28% with rat kidney and amounted to only 2-4% with human muscles. With cellular systems the 14CO2 contribution varied between 20% in human fibroblasts and 70% in rat muscles and myocytes. Addition of cofactors increased the oxidation rate, but decreased the 14CO2 contribution. Various conditions appeared also to influence to a different extent the 14CO2 production and the total oxidation rate with rat tissue homogenates and with rat muscle mitochondria. Incorporation of radioactivity from [1-14C]palmitate into protein was not detectable in cell-free systems and only 2-3% of the sum of 14CO2 and 14C-labeled acid-soluble products in cellular systems. Assay of 14CO2 and 14C-labeled acid-soluble products is a much more accurate and sensitive estimation of fatty acid oxidation than assay of only 14CO2.