Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB188

Poster Communications

Glycogenolytic derived ATP is essential for skeletal muscle fibre excitability and Na,K-ATPase activity in the transverse tubular system

N. Ørtenblad1,2, J. Nielsen1, R. Jensen1

1. Department of Sport Science and Clinical Biomechanics, University of Southern Denmark, Odense M, Denmark. 2. School of Kinesiology, University of British Columbia, Vancouver, British Columbia, Canada.

  • Fig. 1. Whole EDL muscle fatigue protocol (0.4 s trains of 60 Hz, 0.1 ms, every 5 s for 900 s) in presence of GP inhibitor or Control.

  • Fig. 2. Fractional distribution of the repriming period in fibers with and without depolarization and GP inhibition. The plotted lines indicate the normal distributions. Increased repriming period indicates decreased Na,K -ATPase function.

Despite the clearly documented importance of glycogen in muscle cell function, the precise mechanism whereby muscle glycogen affects basic cellular events, is far from understood (Ørtenblad et al., 2013). This study examined the effect of glycogen phosphorylase (GP) inhibitors DAB and CP-316,819, omitting the use of glycogen derived energy, on muscle force (F) and fatigue development. Further, we examined whether the Na,K-ATPase function in the T-system is dependent on ATP derived via the glycogenolytic-glycolytic pathway, under conditions with normal cytoplasmic ATP concentration. Whole rat EDL muscles from male sprague dawley rats (n = 9, 134 ± 21 g (mean ± SD)) where incubated in a Krebs-Ringer bicarbonate buffer, with 5 mM glucose, for 3 hrs in presence of 100 µM (CP-316,81) or a vehicle (Control). Muscles then underwent a fatiguing stimulation protocol (Fig. 1). In order to test the effect of glycogenolytic derived ATP on t-tubular system Na,K-ATPase function, single fibers from rat fast-twitch muscle were mechanically skinned, sealing off the t-system and thereby permitting action potential triggered twitch and tetanic force (F) responses. The T-system can then be depolarized and/or the GP activity inhibited by DAB (2 mM) or CP-316,819 (10 µM). In whole muscle GP inhibition caused a 38 ± 20% decrease in F in fresh muscle and a pronounced faster fatigue development, with half Fmax obtained after 109 (CP-316,81) and 201 (control) stimulations (Fig. 1). In the mechanically skinned fibres, tetanic F decreased by 28 ± 3% when depolarized to -67 mV. In the fully polarized fibres, inhibition of GP, decreased tetanic F by 33 ± 3% (CP, P<0.001) and 12 ± 4% (DAB, P=0.004), which was further pronounced in the depolarized fibre (CP 86 ± 3% and DAB 61 ± 10 %, P<0.0001). Similar results were obtained with twitch responses. To evaluate the Na,K-ATPase function one can quantify the membrane ability to respond to two closely spaced AP (elicited with 1-20 ms space). If second pulse generate an AP, it will produce a larger twitch response and this interpulse space is dependent on the membrane repriming time, which in turn is dependent on the Na,K-ATPase function (Dutka and Lamb, 2007). In a fully polarized fibre, the repriming time was 4 ± 0.2 ms (20 fibres), which was increased to 6 ± 0.5 ms (19 fibres, P<0.001) in the depolarized fibre, indicative of a longer time for the Na,K-ATPase to repolarize the fibre (Fig. 2). There was no effect of GP inhibition on RP in the fully polarized fibre. However, when the T-system was depolarized and GP inhibited, the RP increased to 8 ± 1.2 ms (P<0.001) (13 fibres). These findings demonstrate that glycogenolytic derived ATP is vital for normal muscle function and fatigue resistance. This may be fully or partly explained by the observation that glycogenolytic resynthesis of ATP is critical to t-system Na,K-ATPase function, and maintain muscle excitability, even in the presence of high global muscle ATP.

Where applicable, experiments conform with Society ethical requirements