Kinetic changes in tetanic Ca²⁺ transients in enzymatically dissociated muscle fibres under repetitive stimulation

Abstract

Non-technical summary The transient and progressive decrease in skeletal muscle performance during contraction is known as fatigue. One of the phenomena associated with fatigue is an alteration in the excitation-contraction coupling mechanism. Using isolated muscle fibres loaded with the fast Ca 2+ dye Magfluo-4, we have found that after a protocol of repetitive stimulation there are alterations in the ability of the fibres to release and reuptake Ca 2+ , which are more evident and rapidly established in fast fibres compared to slow ones. All alterations were reversed after several minutes of rest and were not related to a phenomenon of inactivation of Ca 2+ release. These data increase our knowledge of the events present during muscle fatigue and will help understand the mechanisms responsible for them.Abstract We used enzymatically dissociated flexor digitorum brevis (FDB) and soleus fibres loaded with the fast Ca 2+ dye Magfluo-4 AM, and adhered to Laminin, to test whether repetitive stimulation induces progressive changes in the kinetics of Ca 2+ release and reuptake in a fibre-type-dependent fashion. We applied a protocol of tetani of 350 ms, 100 Hz, every 4 s to reach a mean amplitude reduction of 25% of the first peak. Morphology type I (MT-I) and morphology type II (MT-II) fibres underwent a total of 96 and 52.8 tetani (P < 0.01 between groups), respectively. The MT-II fibres (n = 18) showed significant reductions of the amplitude (19%), an increase in rise time (8.5%) and a further reduction of the amplitude/rise time ratio (25.5%) of the first peak of the tetanic transient after 40 tetani, while MT-I fibres (n = 5) did not show any of these changes. However, both fibre types showed significant reductions in the maximum rate of rise of the first peak after 40 tetani. Two subpopulations among the MT-II fibres could be distinguished according to Ca 2+ reuptake changes. Fast-fatigable MT-II fibres (fMT-II) showed an increase of 32.2% in the half-width value of the first peak, while for fatigue-resistant MT-II fibres (rMT-II), the increase amounted to 6.9%, both after 40 tetani. Significant and non-significant increases of 36.4% and 11.9% in the first time constant of decay (t 1 ) values were seen after 40 tetani in fMT-II and rMT-II fibres, respectively. MT-I fibres did not show kinetic changes in any of the Ca 2+ reuptake variables. All changes were reversed after an average recovery of 7.5 and 15.4 min for MT-I and MT-II fibres, respectively. Further experiments ruled out the possibility that the differences in the kinetic changes of the first peak of the Ca 2+ transients between fibres MT-I and MT-II could be related to the inactivation of Ca 2+ release mechanism. In conclusion, we established a model of enzymatically dissociated fibres, loaded with Magfluo-4 and adhered to Laminin, to study muscle fatigue and demonstrated fibre-type-dependent, fatigue-induced kinetic changes in both Ca 2+ release and reuptake.

Establishment of a human skeletal muscle-derived cell line: biochemical, cellular and electrophysiological characterization

“…Skeletal muscle is a highly differentiated tissue made up of myofibers, a syncytium of cells filled with myofibrils and containing sarcomeres that generate the force necessary for muscle contraction. During the past few years a number of techniques have been developed to isolated single muscle fibers from small rodents allowing detailed investigations of the functional properties of the excitation contraction (EC) coupling mechanism at the ultrastructural, biochemical and cellular levels in normal and pathological conditions [1][2][3][4]. Because of their high degree of differentiation and specialization, it is difficult to maintain differentiated muscle fibers in culture for more than a few days [5] and it is nearly impossible to obtain mature fibers starting from precursor satellite cells.…”

Tetanic Ca2+ transient differences between slow- and fast-twitch mouse skeletal muscle fibres: a comprehensive experimental approach

“…Ru360 activity was demonstrated, by using confocal microscopy, since it partially inhibited the fatigue-induced mitochondrial Ca 2? uptakenot shown- (Bruton et al 2003;Calderón et al 2011).…”

“…S1 (Calderón et al 2009(Calderón et al , 2010(Calderón et al , 2011. Briefly, fibres types I and IIA share the tetanic Ca 2?…”

“…Tetanic stimuli were of 350 ms, at 100 Hz, unless other conditions specified. Tetani of these characteristics have been amply used before (Westerblad and Allen 1994;Carroll et al 1997;Calderón et al 2010Calderón et al , 2011. The amplifier output was fed into a DigiData 1200 interface (Axon Instruments, Foster City, CA, USA).…”

“…dyes as Mag-Fura-2 and Mag-Fluo-4 can faithfully track the kinetics of Ca 2? signaling, slower dyes as Fluo-3, Rhod-2 or Indo-1, cannot (Carroll et al 1997;Hollingworth et al 2009;Calderón et al 2010Calderón et al , 2011Calderón et al , 2014. Then it is likely that fast dyes can reveal fast and subtle information on the effect of different mechanisms that remove Ca 2?…”