Composition of vacuoles and sarcoplasmic reticulum in fatigued muscle: electron probe analysis.

Abstract

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...

Skeletal Muscle Fatigue: Cellular Mechanisms

“…With continuous stimulation at 1 Hz for 2-4 h, fast-twitch rat muscles increase their Ca 2ϩ content to ϳ2.5 times the endogenous level, whereas slow-twitch muscle shows little increase (183), consistent with the endogenous Ca 2ϩ and maximum SR capacity observed in such muscle types (176,440). Frog muscle fibers stimulated in vitro to fatigue increased SR Ca 2ϩ content by ϳ10% (190). In contrast, fast-and slow-twitch muscle stimulated in situ in the rat with intermittent 100-Hz tetani for 5 min showed an ϳ40% decline in total Ca 2ϩ content (286).…”

“…It has been frequently suggested that Ca 2ϩ uptake by the mitochondria may play a significant role in overall Ca 2ϩ movements in a fiber, particularly those mitochondria situated close to the SR (223,401), with the effect possibly being more important in frog fibers than in mammalian fibers (68,270). However, the mitochondria do not contain high-capacity Ca 2ϩ binding proteins such as calsequestrin, and the absolute amount of Ca 2ϩ contained within the mitochondria in frog fibers at rest, or after stimulation by either a single tetanus or to fatigue, is Ͻ2% of that contained within the SR (190,408). In humans exercising for 1 h, the amount of Ca 2ϩ in the mitochondria only increased by ϳ50% above the level in rested muscle (297).…”

Properties of caffeine- and potassium-contractures in fatigued frog single twitch muscle fibers.

“…These findings are noted to discuss in more detail the cause of muscle fatigue, which is regarded as the inhibition of E-C coupling. SOMLYO et al (1978) and GoNZALEZ-SERRATOS et al (1978) reported that the amount of the stored calcium in sarcoplasmic reticulum (SR) of fatigued muscle fiber is not different from that of normal muscle fiber. In the present experiment, the peak tension of contractures induced by 3 and 5 mM caffeine in the presence of subthreshold concentration of K+ (25 mM) or 0.01 % Triton X-100 in fatigued muscle fiber was not different from that of control caffeine contractures in normal muscle fiber (Figs.…”

Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron-probe study.

“…Unfortunately, it is not known whether or how the cis-trans polarity of vesicles incorporated into phospholipid bilayers reflects the polarity of the in situ TC. We consider it unlikely that K accumulation into TC during tetanus may have been obscured by volume changes due to water movements or lost through freeze-drying for the following reasons: (a) there was no significant swelling or shrinkage of the TC profiles in freeze-substituted material and the TC/cytoplasmic dry mass ratio (as reflected in continuum counts) was unchanged during tetanus; (b) sharp in vivo ionic gradients across cell membranes are preserved during rapid freezing, cryoultramicrotomy and drying (35,96, and our study), even in intracellular vacuoles (32,96) ; (c) the gain in Mg content of the TC during tetanus was readily demonstrable, although it should be negligible compared to K uptake if the respective permeabilities, as measured in isolated preparations, dominated the influx mechanisms; and (d) the gain in K in the TC during K contracture was preserved by the preparative cryotechniques (our study) as were comparable changes in mitochondria and in nuclei in vascular smooth muscle (89). The magnitude and sign of the electrical potential across the SR membrane has considerable bearing on the mechanism of Ca release.…”