Studying t‐tubular functionality in mechanically skinned fibres to understand the mechanism of muscle fatigue

Many studies on muscle fatigue involve measurements from the interstitial space (e.g. ion concentrations) and surface membrane (e.g. resting and action potential), and of intracellular Ca2+ and force. Ca2+ and force measurements provide well-defined representations of the capacity of the sarcoplasmic reticulum to release Ca2+ and of the sarcomere to generate force, respectively. Measurements from the interstitial space and surface membrane, on the other hand, do not provide such a representation of the causes of less Ca2+ release during fatigue because the latter depends on t-tubular events, i.e. t-tubular membrane excitability, which can be very different from that of the outer cell membrane, as well as the interaction between the CaV1.1 Ca2+ channel, the voltage sensor, and the RyR1 channel, the sarcoplasmic reticulum Ca2+ release channel. The mechanically skinned fibre preparation has an intact t-tubular network allowing a better understanding of events occurring in t-tubules. One major advantage is the capacity to electronically stimulate t-tubules to trigger action potentials while measuring force to determine the extent of t-tubular membrane excitability. The effect of prolonged depolarization can also be determined by lowering the K+ concentration of the bathing solution, which mimics the intracellular environment. Although any experimental manipulations can also affect the sarcoplasmic reticulum Ca2+ release and the force generated by the sarcomere, these effects can be verified by exposing skinned fibres to caffeine or Mg2+ for an effect on Ca2+ release as well as measuring the pCa–force relationship for any effect at the sarcomere level. A limitation is the ionic t-tubular content itself, which can no longer be controlled as it depends on the activity of various ion channels and transporters. As a consequence, one can only estimate the t-tubular membrane potential from the bathing solution K+ concentration and a speculated t-tubular K+ concentration. Mechanically skinned fibres have increased our capacity to better understand potential intracellular signalling pathways that control t-tubular excitability. For example, exposing skinned fibres to high ATP and phosphocreatine levels while inhibiting mitochondrial ATP production reduces t-tubular excitability (Ørtenblad & Stephenson, 2003). The finding by Ørtenblad & Stephenson has two major implications. First, it suggests that mitochondria release signalling molecules that reduce t-tubular excitability when they cannot generate sufficient ATP to perhaps prevent damaging ATP depletion. Second, muscle fibre has mechanisms to anticipate potential ATP deficiency. In an another study, an inhibition of glycogenolysis in mechanically skinned fibres exposed to a high ATP concentration (in the bathing solution) reduced t-tubular membrane excitability by diminishing Na+,K+-ATPase pump activity (Jensen et al. 2020). Whether this effect is part of the anticipation mechanism or a reduced ATP level near the t-tubular membrane as glycolytic rate decreases remains to be determined. In a study published in this issue of The Journal of Physiology, mechanically skinned fibres were used to document t-tubular functionality following fatigue (Watanabe & Wada, 2020). The major finding of the study was of a longer repriming period under partially depolarized t-tubular membrane, i.e. longer time interval necessary to generate double action potentials. The authors then provided evidence that this longer repriming period was in part due to lower Na+,K+-ATPase pump activity. Interestingly, the decreased pump activity was related to greater pump S-glutathionylation, and not to a decreased phospholemman phosphorylation state, which was in fact higher after than before fatigue. It is important to understand that only long lasting changes of t-tubular functionality following fatigue can be studied using this approach. This is because intact muscle fibres were first fatigued before mechanically skinned fibres were dissected. Although one fibre was dissected within 4 min, up to three fibres were tested over a 60 min period. So, on the one hand, this approach does not allow the determination of events that rapidly recover after fatigue such as KATP and ClC-1 Cl− channel activities, which return to pre-fatigue level within 1 min, at least in intact muscle fibres (Pedersen et al. 2009). On the other hand, a major implication of the Watanabe and Wada study is that while force recovery following fatigue is rapid, functional recovery of some proteins such as the Na+,K+-pump may take longer. Thus, the mechanically skinned fibre preparation provides a new and better approach to study long lasting events in intact t-tubules. In all the above studies, the bathing K+ concentration is manipulated to partially depolarize the membrane, the extent of which can only be assumed. In the study of Watanabe and Wada, it was assumed that the t-tubular membrane potential was just a little more negative than the potential at which all Na+ channels are inactivated so that any hyperpolarization following an increased Na+,K+-pump activity results in less Na+ channel inactivation and a shorter repriming period, which is exactly what the authors were able to show. Future studies can be strengthened with the use of voltage-sensitive fluorescent indicators to obtain better estimations of any changes in t-tubular membrane potential when the Na+,K+-ATPase pump activity is modulated. Moreover, such indicators can be useful to study not only the property of the Na+,K+-pump but also that of ion channels by measuring changes in membrane potential when a channel agonist and antagonist are used. It would provide answers to questions such as whether mitochondrial signalling molecules and glycogenolysis inhibition reducing t-tubular excitability are involved in the activation of KATP and ClC-1 channels. Finally, action potential measurement has become possible with new and better performing fluorescent indicators, such as the di-propyl (di-3-ANEPPDHQ) and the di-butyl (di-4-ANEPPDHQ) forms of di-n-ANEPPDHQ (Obaid et al. 2004). No competing interests declared. Sole author. The author is funded by the Natural Sciences and Engineering Research Council of Canada: RGPIN-2020-04318.

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