Relationship between depolarization‐induced force responses and Ca2+ content in skeletal muscle fibres of rat and toad.

Abstract

1. The relationship between the total Ca2' content of a muscle fibre and the magnitude of the force response to depolarization was examined in mechanically skinned fibres from the iliofibularis muscle of the toad and the extensor digitorum longus muscle of the rat. The response to depolarization in each skinned fibre was assessed either at the endogenous level of Ca2' content or after depleting the fibre of Ca2! to some degree. Ca2W content was determined by a fibre lysing technique. 2. In both muscle types, the total Ca2+ content could be reduced from the endogenous level of -1 3 mmol F' (expressed relative to intact fibre volume) to -0 25 mmol by either depolarization or caffeine application in the presence of Ca2P chelators, showing that the great majority of the Ca2+ was stored in the sarcoplasmic reticulum (SR). Chelation of Ca2+ in the transverse tubular (T-) system, either by exposure of fibres to EGTA before skinning or by permeabilizing the T-system with saponin after skinning, reduced the lower limit of Ca2+ content to 6 0-12 mmol F7', indicating that 10-20% of the total fibre Ca2+ resided in the T-system. 3. In toad fibres, both the peak and the area (i.e. time integral) of the force response to depolarization were reduced by any reduction in SR Ca2+ content, with both decreasing to zero in an approximately linear manner as the SR Ca2+ content was reduced to < 15 % of the endogenous level. In rat fibres, the peak size of the force response was less affected by small decreases in SR content, but both the peak and area of the response decreased to zero with greater depletion. In partially depleted toad fibres, inhibition of SR Ca2P uptake potentiated the force response to depolarization almost 2-fold. 4. The results show that in this skinned fibre preparation: (a) T-system depolarization and caffeine application can each virtually fully deplete the SR of Ca!+ irrespective of any putative inhibitory effect of SR depletion on channel activation; (b) all of the endogenous level of SR Ca2+ must be released in order to produce a maximal response to depolarization; and (c) a substantial part (-40 %) of the Ca2P released by a depolarization is normally taken back into the SR before it can contribute to force production.It is well known that the release of Ca2+ from the sarcoplasmic reticulum (SR) of a vertebrate skeletal muscle fibre is required for the activation of the contractile apparatus (Ashley, Mulligan & Lea, 1991; Melzer, Herrmann-Frank & Liittgau, 1995). Electron probe analysis has shown that there is -I5 mmol (1 fibre water)`o f Ca2P (i.e. 1 -2 mmol F-', when expressed relative to fibre volume) in the SR terminal cisternae in frog fibres at rest and that -40 % of this is present at the end of a 1-2 s tetanus (Somlyo, GonzalezSerratos, Shuman, McClellan & Somlyo, 1981). However, it is not clear whether the Ca2P still present in the terminal cisternae could be released by depolarization or indeed whether some of it had been already released and re-sequestered by that time. Other studies have used Ca...

Skeletal Muscle Fatigue: Cellular Mechanisms

“…In rested muscle, the total Ca 2ϩ content of both fast-and slow-twitch rat fibers is ϳ1.1 mmol/kg wet wt, with most (ϳ1.0 mmol/kg) contained within the SR, equivalent to ϳ11 and 21 mM when expressed relative to the respective SR volume (176,183,343). Total calcium content in human muscle fibers is slightly lower (ϳ0.8 mmol/kg) (338,393).…”

Effect of 23-day muscle disuse on sarcoplasmic reticulum Ca2+ properties and contractility in human type I and type II skeletal muscle fibers

“…The total Ca 2ϩ content within the fiber at the time of lysis ([Ca 2ϩ ]T), expressed in millimoles per liter total fiber volume [in keeping with previous studies (17,36)], could be calculated as the sum of 1) the Ca 2ϩ bound to BAPTA, 2) the Ca 2ϩ bound to all other high affinity binding sites in the fiber (predominantly TnC), and 3) the free Ca 2ϩ in the myoplasm ([Ca 2ϩ ]), as described in detail previously (26,28 2) The effective [BAPTA] within the fiber was taken as being 1.13 times the [BAPTA] of the preequilibration solution, to account for the swelling of the fiber when initially placed in solution, and also the fiber volume to which BAPTA was not accessible (i.e., that occupied by the SR, t-tubular system and mitochondria, etc.) (17).…”

“…As described comprehensively elsewhere (17,26,28,36), the total amount of Ca 2ϩ contained in a fiber can be quantified by preequilibrating the skinned fiber in a solution with a known concentration of the very fast calcium buffer BAPTA and then transferring the fiber to an emulsion of 10% Triton X-100 and paraffin oil (TX-oil) to lyse all membranous compartments and release any Ca 2ϩ from within the fiber. Briefly, the skinned fiber was first placed in the standard weakly Ca 2ϩ -buffered K-HDTA solution for 2 min to wash out all the diffusible Ca 2ϩ -binding proteins present endogenously in the cytoplasm without altering the Ca 2ϩ content of the fiber.…”

Skeletal muscle fatigue – regulation of excitation–contraction coupling to avoid metabolic catastrophe

“…The fact that Mg 2+ reduces the P o of RyR becomes more important when the concentration of ATP decreases, because as shown in Fig. 1 ] on excitation-contraction coupling (Blazev and Lamb, 1999;Lamb and Stephenson, 1991;Laver et al, 1997;Owen et al, 1997). The general consensus of these studies is that [Mg 2+ ] at rest is a strong inhibitor of RyR, which keeps the channel closed and unresponsive to the strong activating effect of ATP and Ca 2+ until an action potential activates the cell.…”