This study investigated the effects of fibre swelling on force production in rat and human skinned muscle fibres, using osmotic compression to reverse the fibre swelling. In mechanically-skinned fibres, the sarcolemma is removed but normal excitation-contraction coupling remains functional. Force responses in mechanically-skinned fibres were examined with and without osmotic compression by polyvinylpyrrolidone 40 kDa (PVP-40) or Dextran 500 kDa (dextran). Fibre diameter increased to 116 ± 2% (mean ± SEM) when rat skinned type II fibres were immersed in the standard intracellular solution, but remained close to the in situ size when 3% (mass/volume) PVP-40 or 4% Dextran were present. Myofibrillar Ca2+ sensitivity, as indicated by pCa50 (- log10[Ca2+] at half-maximal force), was increased in 4% Dextran (0.072 ± 0.007 pCa50 shift), but was not significantly changed in 3% PVP-40. Maximum Ca2+-activated force increased slightly to 103 ± 1% and 104 ± 1% in PVP-40 and Dextran, respectively. Both tetanic and depolarization-induced force responses in rat skinned type II fibres, elicited by electrical stimulation and ion substitution respectively, were increased by ~ 10 to 15% when the fibres were returned to their normal in situ diameter by addition of PVP-40 or Dextran. Interestingly, the potentiation of these force responses in PVP-40 was appreciably greater than could be explained by potentiation of myofibrillar function alone. These results indicate that muscle fibre swelling, as can occur with intense exercise, decreases evoked force responses by reducing both the Ca2+-sensitivity of the contractile apparatus properties and Ca2+ release from the sarcoplasmic reticulum.

[Ca2+] transients inside the sarcoplasmic reticulum (SR) were recorded in frog skeletal muscle twitch fibers under voltage clamp using the low affinity indicator Mag Fluo 4 (loaded in its AM form) with the purpose of studying the effect on Ca2+ release of extrinsic Ca2+ buffers (i.e. BAPTA) added at high concentration to the myoplasm. When the extrinsic Ca2+ buffer is added to the myoplasm, part of the released Ca2+ binds to it, reducing the Ca2+ signal reported by a myoplasmic indicator. This, in turn, hinders the quantification of the amount of Ca2+ released. Monitoring release by measuring [Ca2+] inside the SR avoids this problem. The application of extrinsic buffers at high concentration reduced the resting [Ca2+] in the SR ([Ca2+]SR) continuously from a starting value close to 400 μM reaching the range of 100 μM in about half an hour. The effect of reducing resting [Ca2+]SR on the Ca2+ permeability of the SR activated by voltage clamp depolarization to 0 mV was studied in cells where the myoplasmic [Ca2+] ([Ca2+]myo) transients were simultaneously recorded with Rhod2. The Ca2+ release flux was calculated from [Ca2+]myo and divided by [Ca2+]SR to obtain the permeability. Peak permeability was significantly reduced, from 0.026 ± 0.005 ms-1 at resting [Ca2+]SR = 372 ± 5 μM to 0.021 ± 0.004 ms-1 at resting [Ca2+]SR = 120 ± 16 μM (n = 4, p = 0.03). The time averaged permeability was not significantly changed (0.009 ± 0.003 and 0.010 ± 0.003 ms-1, at the higher and lower [Ca2+]SR respectively). Once the cells were equilibrated with the high buffer intracellular solution, the change in [Ca2+]SR (Δ[Ca2+]SR) in response to voltage clamp depolarization (0 mV, 200 ms) in 20 mM BAPTA was significantly lower (Δ[Ca2+]SR = 30.2 ± 3.5 μM from resting [Ca2+]SR = 88.8 ± 13.6 μM, n = 5) than in 40 mM EGTA (Δ[Ca2+]SR = 72.2 ± 10.4 μM from resting [Ca2+]SR = 98.2 ± 15.6 μM, n = 4) suggesting that a Ca2+ activated component of release was suppressed by BAPTA.

Raising the intracellular [Ca2+] ([Ca2+]i) was previously found to produce uncoupling between the electrical depolarization of the transverse tubules and contraction in skinned muscle fibers. Here we study the effect of elevated [Ca2+]i in voltage clamped cut fibers of frog skeletal muscle to establish how the charge movement, a measure of the activation of the dihydropyridine receptors (DHPR)-voltage sensors, and Ca2+ release, a consequence of the opening of the ryanodine receptor (RyR)-release channels, were affected. [Ca2+]i was raised by various procedures (pharmacological release from the sarcoplasmic reticulum, application of high [Ca2+]i intracellular solution, permeabilization of the plasma membrane by a Ca2+ ionophore) all of which produced impairment of excitation-contraction coupling. The charge movement was reduced from 20.2 ± 1.24 to 9.9 ± 0.94 nC/μF meanwhile the Ca2+ release flux was reduced from 13.5 + 0.7 to 2.2 ± 0.3 μM/ms (n = 33). This suggests that a significant fraction of the DHPRs that remained functional, could not activate RyRs, and were therefore presumably disconnected. These results are broadly consistent with the original reports in skinned fibers. Uncoupling was prevented by the addition to the intracellular solution of the protease inhibitor leupeptin. In approximately 40 % of the uncoupled cells we observed that the [Ca2+]i transient continued to rise after the voltage clamp pulse was turned off. This loss of control by membrane voltage suggests that the uncoupled release channels might have another mechanism of activation, likely by Ca2+.

We investigated the potential role of selected excitation-contraction coupling processes in females with work-related myalgia (WRM) by comparing WRM with healthy controls (CON) using tissue from extensor carpi radialis brevis (ECRB) and trapezius (TRAP) muscles. For the ECRB, age (mean ± SE) was 29.6 ± 3.5 years for CON (n = 9) and 39.2 ± 2.8 years for WRM (n = 13), while for the TRAP, the values were 26.0 ± 2.1 years for CON (n = 7) and 44.6 ± 2.9 years for WRM (n = 11). For the sarcoplasmic reticulum (SR) of the ECRB, WRM displayed concentrations (nmol·(mg protein)(-1)·min(-1)) that were lower (P < 0.05) for Total (202 ± 4.4 vs 178 ± 7.1), Basal (34 ± 1.6 vs 30.1 ± 1.3), and maximal Ca(2+)-ATPase activity (Vmax, 168 ± 4.9 vs 149 ± 6.3), and Ca(2+)-uptake (5.06 ± 0.31 vs 4.13 ± 0.29), but not SERCA1a and SERCA2a isoforms, by comparison with CON. When age was incorporated as a co-variant, Total, Basal, and Ca(2+)-uptake remained different from CON (P < 0.05), but not Vmax (P = 0.13). For TRAP, none of the ATPase properties differed between groups (P > 0.05) either before or following adjustment for age. No differences (P > 0.05) were observed between the groups for Ca(2+)-release in the SR for either TRAP or ECRB. Similarly, no deficiencies, regardless of muscle, were noted for either the Na(+)-K(+)-ATPase content or the α and β subunit isoform distribution in WRM. This preliminary study provides a basis for further research, with expanded numbers, investigating the hypothesis that abnormalities in SR Ca(2+)-regulation are involved in the cellular etiology of WRM.