Programme Contents

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

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 6^th response.

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 6^th response; doublet: 111.27 ± 13.19% of 6^th 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.