Electron probe analysis, cryo-ultramicrotomy, and freeze-substitution were used to determine the nature of vacuolation and the subcellular composition in fatigued frog skeletal muscle fibers. The vacuoles caused by fatigue were part of the T-tubule system and contained high concentrations of NaC1. The calcium concentration in the terminal cisternae was higher than previously measured normal resting values. Mitochondrial calcium content was relatively low (mean i SEM, 2 h 2 mmol/kg dry weight). Fiber NaCl was increased. It is concluded that fatigue is not due to the depletion of calcium stores from the terminal cisternae or to uncoupling of mitochondria due to calcium loading but may be caused by multiple mechanisms including failure of the T-tubule action potential.Prolonged tetanic stimulation of vertebrate striated muscles induced a state (hitherto referred to as fatigue) during which the contractile force first declines and subsequently the muscle becomes mechanically refractory to further stimulation. Fatigue is not due to failure of the action potential mechanism (1-3) or to the depletion of high-energy phosphates (4,5), and it is associated with vacuolation detectable by light microscopy (6, 7). The purpose of the present studies was to establish the ultrastructure of these vacuoles and their contents and to determine whether fatigue was due to the depletion of Ca2+ from the terminal cisternae of the sarcoplasmic reticulum or to abnormal accumulation of Ca2+ by the mitochondria, or to both.The preliminary account of some of these findings has been presented (8). METHODSSingle fibers were isolated from the semitendinosus muscle of Rana temporaria or R. pipiens for the physiological experiments, and bundles of 8-12 fibers of R. pipiens were prepared for cryo-ultramicrotomy and electron probe analysis. The muscles were suspended in frog Ringer's solution in a chamber (9) and stretched to 2.8-,sm sarcomere length. Force was recorded with an RCA 5734 transducer. The stimulation consisted of periodic tetanization at 40-50 supramaximal shocks per sec during 0.3 sec of every 1 sec. until the tension was nearly 0.Intracellular pH measurements were made on both control and fatigued fibers (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) struments Co.) and the outputs were recorded differentially. Before and after each experiment, the pH microelectrode was calibrated against the glass micropipette with four different standard pH solutions.Fiber bundles used for electron probe analysis were mounted with one tendon attached to an end of the stainless steel mesh holder used for freezing and the other to the mechanotransducer. When fatigue was established, the tendon attached to the transducer was released and hooked to the other end of the mesh holder; the holder was rapidly transferred to the air gun, frozen within 60-150 sec in Freon 22 supercooled to -164°j 20, and cryosectioned for electron probe analysis (10,11). The details of the method for quantitative electron probe analysis have been published (I2...
SUMMARY1. Fatigue curves were obtained on muscles contracting at length (L) different from the optimal (L0), and performing isometric tetani of fixed duration at regular intervals. Every sixth tetanus occurred at L., the remainder took place at L. The parameters of the fatigue curve were compared with those of controls contracting always at Lo. Fatigability was measured as the coefficient of exponential decrease of tension with time.2. Fatigability varied linearly with length in shortened and lengthened muscles, but the slopes of the relations were significantly different for the two groups.3. The tension values from single experimental muscles fell on two independent curves, one for those obtained at Lo, another for data at L. The same muscle showed, therefore, two different fatigue processes which followed independent temporal evolutions.4. Points 2 and 3 of this Summary imply that fatigue is a process dependent on the actual setting of the myofilaments during contraction and not distributed uniformly within the sarcomere.5. It is proposed that fatigue results from local changes taking place at discrete reactive points in the myofilament. The experimental results are discussed in terms ofthis hypothesis and ofthe sliding model ofcontraction.
1. The development of fatigue in isolated toad sartorius undergoing short, periodic, isometric tetani of controlled duration, was studied. Stimulation was direct and curare was used. 2. In the great majority of cases extratension decreased very regularly towards a steady value, following a simple exponential equation. 3. The final steady tension (expressed as percentage of the maximum) and the constant of exponential decay varied as simple functions of the mean frequency of stimulation and their product was a constant.
In fatigued frog muscle fibers vacuolation has been observed with light microscopy.lJ In the course of investigating the ultrastructure and composition of these vacuoles with electron probe analysis. we were able to determine the calcium content of the in situ terminal cisternae in fatigued muscle.Bundles of 8-12 fibers of frog semitendinosus were stimulated by periodic tetani of 40-50 shockdsec during 0.3 sec every sec until tension declined to baseline. Light microscopy of single fatigued fibers showed vacuoles and an approximately 80% increase in fiber volume. When fatigue was established, the muscles were shot into supercooled Freon 22 and frozen sections of approximately 100 nm thickness were cut at -130°C on a modified LKB cryoultramicrotome without any cryoprotectants or fixation.3 The sections were dried at Torr below -80°C. Quantitative electron probe analysis of the cryo sections was performed as described p r e~i o u s l y .~ Vacuoles, frequently located in longitudinal rows between the mitochondria and occasionally along the Z lines, were observed in electron micrographs of the cryo sections. These vacuoles contained high concentrations of NaC1. Pretreatment with strophanthidin c r ouabain ( IW4 M) did not prevent the appearance, extent, or high NaCl content of the vacuoles; therefore, it is unlikely that vacuolation was due to a ouabain-sensitive Na pump. Freeze-substitution studies of fatigued muscles showed the same distribution of vacuoles as in cryosections and permitted their identification as being membrane bound, continuous with the T-tubules, and involving predominately the longitudinal T-system.The cytoplasmic elemental concentrations measured with large diameter probes (0.5-10 wm) in fatigued muscles were (mmoleslkg dry wt, mean +-SEM) Na 140 * 8, Mg 47 i 3, P 290 t 3, S 224 t 2. CI 133 t 2, K 332 i 3 , Ca 8 i I (FIGURE I). The relatively high fiber Ca content is in agreement with the reported increase in chemically measured Ca in fatigued muscles.6
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