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Quantification of endogenous and maximal sarcoplasmic reticulum calcium content in human vastus lateralis muscle

C.R. Lamboley,1 R.M. Murphy,2 M.J. McKenna1 and G.D. Lamb,2 1Institute of Sport, Exercise and Active Living, Victoria University, PO Box 14428, Melbourne, VIC 8001, Australia. and 2Department of Zoology, La Trobe University, Melbourne, VIC 3086, Australia..

The ability of skeletal muscle to produce strong and repeated contractions depends on a coordinated sequence of molecular processes known as excitation-contraction coupling. Briefly, contraction in skeletal muscle is initiated when action potentials propagate into the transverse tubular (T) system, causing rapid depolarization, which in turn triggers Ca2+ release from the sarcoplasmic reticulum (SR). The subsequent force production is closely related to the amount of Ca2+ released from the SR. Since the latter depends strongly on the content of total Ca2+ in the SR ([CaT]SR) it would be important to have a reliable measurement of [CaT]SR in human skeletal muscle fibres under physiological resting conditions.

The present study examined for the first time, in individual fibres from human skeletal muscle biopsies, whether endogenous SR Ca2+ content and maximal SR Ca2+ capacity are different between fast-twitch (FT) and slow-twitch (ST) fibres.

The study was approved by the Human Ethics Committees at Victoria University and La Trobe University. A muscle biopsy was taken from the vastus lateralis muscle from eleven healthy young adults. After injection of a local anaesthetic into the skin and fascia (1% lidocaine (Xylocaine)), a small incision was made and a muscle sample taken (∼150 mg) using a Bergstrom biopsy needle. Individual fibre segments, obtained from the biopsy, were mechanically skinned under paraffin oil so that they still contained their endogenous Ca2+ content. The total amount of endogenous Ca2+ contained in each fibre could be quantified by pre-equilibrating the fibre in a solution with a known concentration of the very fast calcium-buffer BAPTA for 20 s and then transferring the fibre to an emulsion of 10% Triton X-100 and paraffin oil (TX-oil) in order to lyse all membranous compartments and release any Ca2+ from within the fibre (Fryer & Stephenson, 1996). If the preequilibrating [BAPTA] was chosen such that the fibre produced a finite, non-maximal force response upon lysis, then the total amount of Ca2+ present in the fibre can be calculated from the BAPTA concentration in the equilibration solution and the magnitude of the force response. Furthermore, other fibre segments, prior to the TX-oil lysing, were (1) totally depleted of their endogenous SR Ca2+ content by a 1 minute exposure to a solution containing 30 mM caffeine and 0.05 Mg2+ or (2) loaded to their maximal SR Ca2+ capacity by a 4 minute exposure to a solution containing 0.2 μM free Ca2+ (buffered with 1 mM CaEGTA EGTA). Finally, using Western blotting, each muscle fibre was classified as FT or ST according to the myosin heavy chain isoform present.

When fibres with an endogenous Ca2+ content were assayed, the endogenous [CaT]SR obtained was 0.71±0.03 (n=8) and 0.80±0.02 (n=12) mmol.l-1 (expressed relative to intact fibre volume) for ST and FT fibres, respectively (P<0.05). By loading the SR of the fibres maximally, the study also revealed that the maximal SR Ca2+ capacity for ST and FT fibres was 1.35±0.04 (n=13) and 1.70±0.03 (n=17) mmol.l-1, respectively (P<0.01).

In conclusion, the present results show that the SR properties of the FT human fibres were significantly different from those of the ST fibres. The FT fibres had a larger SR capacity and the endogenous Ca2+ content was a relatively lower percentage of the maximum compared with ST fibres (47 and 53%, respectively). Future studies, using the same technique could reveal whether these SR properties alter with the diverse changes that can occur with ageing, inactivity or chronic diseases.

Fryer M.W. & Stephenson D.G. (1996). Total and sarcoplasmic reticulum calcium contents of skinned fibres from rat skeletal muscle. Journal of Physiology, 493, 357-370.