Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C045

Oral Communications

Mechanisms underlying increases in Ca2+ leakage from sarcoplasmic reticulum with prolonged low-frequency force depression in rat skeletal muscle

D. Watanabe1, M. Wada2

1. Department of Engineering Science, University of Electric-Communications, Tokyo, Japan. 2. Graduate School of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan.

We have shown that in fast skeletal muscles, Ca2+ release of the sarcoplasmic reticulum (SR) was markedly reduced in the early stage of recovery from prolonged low-frequency force depression (PLFFD), which is defined as a grater loss of force at low than at high stimulation frequencies (1). The contents of Ca2+ released from the SR depend largely on the Ca2+ contents contained in the SR lumen (2) that are primarily determined by the balance between uptake and leak of the SR. The purpose of this study was a) to investigate changes in the Ca2+ uptake and leak properties of the SR in the early stage of recovery from PLFFD and b) to identify the mechanism(s) underlying the changes if recognized. All experimental procedures were approved by the Animal Care Committee of Hiroshima University. Under an anesthesia using the mixture of medetomidine, midazolam and butorphanol with appropriate ratio (3), gastrocnemius muscles from Wistar male rats (8-9 weeks old) were electrically stimulated via the sciatic nerve at 70 Hz until the force was reduced to ~50% of the initial force (fatiguing stimulation) and then allowed to rest for 30 min. Afterwards, mechanically skinned fibres and SR microsomes were prepared from the superficial regions of the gastrocnemius muscles and were used for the assay of SR Ca2+ uptake and leak properties. The contralateral muscles were used as controls. The obtained values (± SE) were tested by t-test and two-way ANOVA as required. The major results of this study are shown in table 1 indicating that fatiguing stimulation, leading to PLFFD, brought about significant increases in Ca2+ leak, but no changes in Ca2+ uptake. Ca2+ leak was alleviated to the same extent in control and stimulated fibres by treatment with 20 μM 2,5-ditert-butylhydroquinone (TBQ: SR Ca2+-ATPase inhibitor) suggesting the increased Ca2+ leakage through the ryanodine receptor (RyR). Conversely, in SR microsome, TBQ-induced Ca2+ leak is reduced in stimulated muscle (46 ± 5 for control vs. 26 ± 4 nmol/min/g wet wt for stimulated microsome, n=7 for each group, P < 0.05). With regard to the conflicting results, the dihydropyridine receptor (DHPR) might be involved in stimulation-related changes in the SR Ca2+ leak properties. To examine this point, the Ca2+ leak properties were measured in the solutions replacing all K+ with Na+ where the voltage sensor was inactivated. Under this condition, the relative contents of Ca2+ leaking out of the SR in stimulated fibres approximated to those in control fibres (Fig. 1). These results indicate that in the early stage of recovery from PLFFD, the contents of Ca2+ leaking through the RyR are increased and suggest that increased SR Ca2+ leak, which presumably involves the DHPR, is responsible, at least partly, for decreased SR Ca2+ release that occurs in fatigued skeletal muscles.

Where applicable, experiments conform with Society ethical requirements