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Effect of doublet stimulation on tetanic Ca2+ responses measured in isolated fast interosseous fibres of the mouse

A.J. Bakker,1 T.R. Cully2 and B.S. Launikonis,2 1School of Anatomy, Physiology & Human Biology, University of Western Australia, Perth, WA 6009, Australia and 2School of Biomedical Sciences, University of Queensland, St Lucia, QLD 4072, Australia.

At the start of tetanic contractions, mammalian skeletal muscle fibres are often excited by high frequency motorneuron double discharges, of 200 Hz or more (Desmedt & Godauxa, 1977). Recently, Cheng et al., 2013 reported that the presence of an initial 200 Hz doublet action potential can result in a 100% increase in peak tetanic Ca2+ compared to control Ca2+ transients elicited at a stimulation rate of 70Hz alone, there was also a faster rise in force (Cheng et al., 2013). However, Cheng et al. tracked Ca2+ release with only ms temporal resolution, leaving the evolution of the Ca2+ transient that underlies the rapidly rising force response unknown. In this study, we imaged Ca2+ release with μs resolution using a Zeiss 5 Live during doublet action potential stimulation in conjunction with the fast, low affinity Ca2+ indicator Mag-Fluo-4.

Mice were killed by cervical dislocation, and the interosseous muscles removed and placed in a collagenase II digestion solution (mg/ml) for 30 min. The muscles were then gently triturated to produce single fibres. Fibres were loaded with Mag-Fluo-4 (5μM for 15 min), and placed in a solution bath containing a HEPES based Kreb’s Ringer, and the myosin inhibitor BTS (100 μM) to prevent fibre movement. Fast fibres were selected based on the shape of elicited tetanic Ca2+ transients (Calderon, Bolanos & Caputo, 2011). In control measurements, fibres were activated by 10 action potentials at 120 Hz using platinum electrodes. Stimulation during the doublet measurements was similar with the exception that the first 2 action potentials were at 200 Hz. Ca2+ fluorescence was captured at ∼10 kHz using a Zeiss 5 Live confocal microscope in linescan mode. Changes in the progression of the Ca2+ response was quantitated by estimating the lowest Ca2+ fluorescence found after each Ca2+ spike, and normalising the value to the fluorescence nadir after the 6th response.

Figure 1

A. Effects of control and doublet stimulation on Mag-fluo-4 fluorescence (AU: arbitrary units). B. Changes in the minimum fluorescence value between Ca2+ transient spikes before (time 0) and after activation under control conditions or with an initial 120 Hz doublet (*P<0.05).

The presence of an initial 200 Hz doublet action potential did not significantly alter the amplitudes of the Ca2+ spikes during the transient. However, after doublet stimulation, the minimum fluorescence value between Ca2+ transient spikes rose to a stable, maximal level after the first response that was approximately 1.8 times greater than the basal Ca2+ fluorescence after the first response under control conditions (control: 61.06 ± 2.82% of 6th response; doublet: 111.27 ± 13.19% of 6th response) (Figure). Furthermore, in controls, the minimum fluorescence value between Ca2+ transient spikes did not reach the maximum normalised value until 16 - 25 ms after initial activation, compared to only 5 ms in fibres exposed to doublet stimulation.

These results indicate that doublet stimulation rapidly increases the minimum fluorescence value between Ca2+ transient spikes in fast twitch muscle fibres. Doublet activation may lead to more rapid saturation of cytosolic Ca2+ binding sites and therefore, faster initiation of cross-bridge cycling in fast skeletal muscle fibres.

Desmedt JE, Godaux E. (1977) Ballistic contractions in man: characteristic recruitment pattern of single motor units of the tibialis anterior muscle. Journal of Physiology 264: 673-93.

Cheng AJ, Place N, Bruton JD, Holmberg HC, Westerblad H. (2013) Doublet discharge stimulation increases sarcoplasmic reticulum Ca2+ release and improves performance during fatiguing contractions in mouse muscle fibres. Journal of Physiology 591: 3739-48.

Calderon JC, Bolanos P, Caputo C. (2011) Kinetic changes in tetanic Ca2+ transients in enzymatically dissociated muscle fibres under repetitive stimulation. Journal of Physiology 589: 5269-83.