Edited by: Julio L. Vergara, University of California, Los Angeles, USA
Reviewed by: David Grant Allen, University of Sydney, Australia; Laszlo Csernoch, University of Debrecen, Hungary
*Correspondence: Nicolas Place
This article was submitted to Striated Muscle Physiology, a section of the journal Frontiers in Physiology
†These authors have contributed equally to this work.
‡Share last authorship.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
The interpolated twitch technique (ITT) is the gold standard to assess voluntary activation and central fatigue. Yet, its validity has been questioned. Here we studied how peripheral fatigue can affect the ITT. Repeated contractions at submaximal frequencies were produced by supramaximal electrical stimulations of the human
The interpolated twitch technique (ITT) developed by Merton (
Place et al. (
In the present study we addressed two possible reasons to the above described conflicting results. First, the mouse FDB fibers were fatigued by repeated brief contractions, whereas the human
For human experiments, all participants gave their written informed consent before participation. The experimental protocol was approved by the Research Ethics Committees of the Geneva (13–107) and Vaud cantons (128/14) and were in agreement with the Declaration of Helsinki. Twenty-four healthy subjects participated in the study (8 women and 16 men, 28 ± 6 years old).
All animal experiments complied with the Swedish Animal Welfare Act, the Swedish Welfare Ordinance, and applicable regulations and recommendations from Swedish authorities. The study was approved by the Stockholm North Ethical Committee on Animal Experiments. Five 6–8 week old male Wistar rats were killed by placing them in a chamber filled with CO2.
Subjects sat on a chair that was adjustable for height, with their right forearm resting in a custom-made mold and the elbow and shoulder angles set to 90° in the sagittal axis. Two straps tightly secured the forearm (10 cm above the wrist and 5 cm below the elbow crease) to the ergometer. The thumb was adjusted to an angle allowing optimal force development and its first phalanx positioned on a support connected to the strain gauge (Z8 500 N, sensitivity 2 mV/V and 0.0083 V/N; HBM, Darmstadt, Germany). Force signals were recorded at 1 kHz using an analog-digital conversion system (MP150; BIOPAC, Goleta, CA, USA).
Transcutaneous electrical stimulation of the ulnar nerve was delivered by a high-voltage (400 V maximum) constant-current stimulator (DS7AH; Digitimer, Hertfordshire, UK) driven by a stimulation train generator (MP150; BIOPAC, Goleta, CA). The cathode and anode (4-mm plug bar-handle stimulator, SPES Medica, Genova, Italy) were located over the ulnar nerve anteriorly and just proximal to the wrist (Neyroud et al.,
The surface electromyographic (EMG) activity of the
Initially 1-s current trains (separated by ~10 s) were evoked, at 10, 15, 20, 30, 50, 80, and 100 Hz, in a counterbalanced order between subjects, to determine the force-frequency relationship in the rested state. Thereafter one of three different fatiguing protocols (
In the initial set of experiments (
In the next set of experiments (
In the final set of experiments (
For all parameters, the different values obtained throughout the course of the fatiguing task are expressed as a percentage of their value obtained during the first tetanus.
Every third evoked contraction of the fatiguing task was considered for analysis. For these contractions, amplitudes of potentiated and superimposed twitch forces as well as the force level just before the superimposed twitch (referred to as tetanic force from now on) were measured. The half-relaxation time (HRT) was measured in the first and last fatiguing contractions as the time from the end of stimulation until force had declined to 50% of the tetanic force. For the force-frequency relationship, the mean force over a 0.5-s window was measured at each frequency and expressed as a percentage of the force produced by the 100-Hz stimulation train.
The M waves associated with the superimposed electrical stimulus (superimposed M-wave) were analyzed. However, during fatiguing stimulation, the high stimulation frequency led to a truncated M-wave in between two stimulation artifacts in some participants and therefore peak-to-peak M-wave amplitude and duration could not be consistently measured. Therefore, the M-wave amplitude was quantified as the amplitude of the first peak of the M-wave (referred to as M-wave amplitude from now on, see Rodriguez-Falces and Place,
Whole
In another set of experiments designed to assess the effect of contractile slowing on ITT, force-frequency relationships were obtained at two temperatures (23 and 18°C). Three second duration tetani were evoked at 1-min intervals at 10, 15, 20, 30, 40, and 50 Hz at 18°C, and also at 70 and 100 Hz at 23°C. At each frequency, an additional electrical stimulus was delivered 2.5 s into the contraction with 10 ms separating this additional pulse from the previous regular pulse. As for the fatiguing experiments, an electrical doublet pulse with a 10 ms interpulse interval was delivered 1 s after each tetanus (i.e., the potentiated twitch). Peak forces were measured for tetani, and for the superimposed and potentiated twitches. The superimposed twitch force was measured as the force prior to the additional electrical stimulus up to peak force following the superimposed stimulus. HRT was measured as the time from the end of stimulation until force had declined to 50% of the tetanic force.
For human experiments, depending on the outcome of the Shapiro-Wilk normality test, one-way or Friedman repeated measures ANOVAs [time (tetanus 1, 4, 7, 10, 13, 16, 19, 22, 25, and 28)] were performed for all parameters. When significant differences were found, Dunnett's
Fourteen participants took part in
Superimposed twitch, N | 3.0 ± 1.3 | 3.3 ± 1.3 | 4.8 ± 1.3 |
M-wave amplitude, mV | 1.6 ± 1.5 | 1.5 ± 1.5 | 3.9 ± 1.5 |
M-wave latency, ms | 6.7 ± 1.1 | 6.5 ± 0.5 | 6.6 ± 0.4 |
Tetanic force, N | 59.7 ± 17.8 | 62.2 ± 18.8 | 52.9 ± 13.8 |
Tetanic HRT, ms | 88 ± 13 | 82 ± 7 | 81 ± 9 |
Potentiated twitch, N | 9.2 ± 2.7 | 10.7 ± 2.9 | 10.5 ± 3.0 |
Typical original force and EMG recordings from the start and end of the fatiguing stimulation in
As action potentials were not preserved in
In
Figure
Rat
A marked difference between the human and rat muscle experiments was a greater fatigue-induced slowing of tetanic relaxation in the human
In the present study, we used both an
In
Earlier studies have shown action potential failure during continuous electrical stimulation (e.g., Bigland-Ritchie et al.,
It is possible that the 30-Hz stimulation used in
We previously showed a relative increase in the superimposed twitch force during fatigue induced by repeated tetanic stimulation of isolated mouse FDB fibers (Place et al.,
A marked difference between the present findings in human and rat muscle is the greater fatigue-induced slowing of tetanic relaxation in the human muscle, hence leading to increased fusion that leaves less room for a force increase. Accordingly, cooling of rat
Fatigue-induced slowing of relaxation can in principle be due to slowed removal of Ca2+ from the myoplasm and/or slowing of the subsequent myofibrillar inactivation, which involves Ca2+ dissociation from troponin C followed by cross-bridge detachment (Gordon et al.,
The usage of ITT to assess voluntary activation during maximal voluntary contraction involves complex interactions between peripheral fatigue factors and changes in the pattern of motor unit activation during fatigue. For instance, all motor units are readily activated by supramaximal electrical nerve stimulation, whereas voluntarily later-recruited motor units have been shown to fire at submaximal frequencies even during maximal efforts (Contessa and De Luca,
Measurement of the superimposed twitch force is an essential component when ITT is used to assess the level of voluntary activation. Our results show that peripheral fatigue factors particularly affect the superimposed twitch force, including impaired membrane excitability, decreased myofibrillar force, and fatigue-induced contractile slowing.
DN, AC, BK, HW, and NP contributed to the conception and design of the study. DN, AC, and NP were responsible for data collection. DN, AC, NB, BK, HW, and NP participated in the analysis and interpretation of the data. All authors were involved in writing the manuscript and approved the final version. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Human experiments were performed in the Institute of Movement Sciences and Sports Medicine of Geneva University, Switzerland and in the Institute of Sport Sciences of the University of Lausanne, Switzerland. All animal experiments were performed at the Cellular Muscle Function Laboratory in the Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
AC and HW acknowledge funding from the Swedish National Centre for Sports Research, and the Swedish Research Council.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank all the participants who took part in the human experiments as well as Joseph Bruton and Barbara Uva for their help with data collection and analysis.