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Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females
Journal of Biomechanics ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females Hanna Drzymała-Celichowska a,b,n, Rositsa Raikova c, Piotr Krutki a a
Department of Neurobiology, University School of Physical Education in Poznań, 27/39 Królowej Jadwigi Street, 61-871 Poznań, Poland Division of Biochemistry, University School of Physical Education in Poznań, Poland c Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria b
art ic l e i nf o
a b s t r a c t
Article history: Accepted 18 July 2015
Mathematical decomposition of tetanic contractions of slow motor units (MUs) of the rat heterogeneous medial gastrocnemius muscle revealed immense variability of twitch-shape responses to successive pulses, contrary to results obtained for fast MUs. The aim of this study in rat soleus muscle, almost exclusively composed of slow MUs, was to reveal whether such variability of twitch-shape decomposed components was a common property of slow MUs in the two studied muscles, and whether ranges of the force amplitude or time parameters of these decomposed twitches showed sex differences. Unfused tetanic contractions evoked by stimulation at variable interpulse intervals were analyzed for 10 MUs of males and 10 MUs of females. Significantly higher variability between parameters of the decomposed responses was observed for male soleus MUs, as the mean ratio of forces of the strongest decomposed twitch and the first (the weakest) decomposed twitch amounted to 3.8 for males and 2.8 for females. The ratios of the contraction times of the longest decomposed to the first twitch were much more similar between male and female MUs, 2.6 and 2.9, respectively. Consequently, the mean ratio of the force–time area for the strongest decomposed to the first twitch was much bigger in male than female MUs (7.35 vs. 5.07, respectively). Our observations indicate that high variability of responses to successive stimuli is a general property of slow MUs in different rat muscles, but the mechanisms of summation of individual twitches into tetanic contractions of MUs are not identical for male and female rats. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Slow motor units Sex differences Random stimulation pattern Soleus Rat
1. Introduction During voluntary activity, motoneurons generate trains of pulses at variable intervals, which substantially influence the development and profiles of tetanic forces of motor units (MUs). The discharge patterns of MUs have been most frequently studied by the decomposition of electromyograms into trains of MU action potentials (Boe et al., 2005; Moritz et al., 2005). On the other hand, the mathematical decomposition of unfused tetanic contractions of MUs into twitch-shape responses to successive pulses can provide information concerning contractile effects induced by individual action potentials. This method, applied to tetani evoked by stimulation of the medial gastrocnemius MUs at variable interpulse intervals (IPIs), resembling motoneuronal firing patterns observed during voluntary contractions, has revealed substantial variability of n Corresponding author at: Department of Neurobiology University School of Physical Education in Poznań, 27/39 Królowej Jadwigi Street, 61-871 Poznań, Poland. Tel.: þ48 61 8355435; fax: þ 48 61 8355444. E-mail address: [email protected] (H. Drzymała-Celichowska).
individual responses to successive pulses (Raikova et al., 2008). The decomposed twitches of fast MUs have displayed up to 2.2 times higher forces, and up to 2.5 times longer contraction times than the single twitch. For slow MUs, the forces of decomposed twitches have reached even 7 times higher values, while the twitch contraction and relaxation parameters have been up to 3.5 times longer than those of the single twitch (Celichowski et al., 2014). The above observations have only been made for MUs of one muscle so far, and it is unknown whether similar variability in amplitudes and duration of responses summating into a tetanic contraction could be obtained for MUs of another muscle, anatomically and physiologically different. Such an example is the soleus, a homogeneous, slow-twitch muscle, composed predominantly of slow MUs (Kugelberg, 1973; Burke et al., 1974). Although the soleus and medial gastrocnemius muscles contain slow MUs, it has been shown that basic contractile properties of slow MUs of these two muscles are considerably different, as they are active in different motor tasks (Burke and Tsairis, 1973; Burke et al., 1974; Kanda and Hashizume, 1992; Drzymała-Celichowska and Krutki, 2015). Moreover, previous studies of rat soleus MUs
http://dx.doi.org/10.1016/j.jbiomech.2015.07.019 0021-9290/& 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Drzymała-Celichowska, H., et al., Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females. Journal of Biomechanics (2015), http://dx.doi.org/10.1016/j.jbiomech.2015.07.019i
H. Drzymała-Celichowska et al. / Journal of Biomechanics ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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have revealed that this muscle is also sexually dimorphic, and properties of its MUs are different for males and females (Drzymała-Celichowska and Krutki, 2015). MUs of the male soleus have shorter contraction times and generate higher tetanic forces, whereas the twitch forces are similar for animals of both sexes; consequently, the twitch-to-tetanus force ratios are considerably (about twice) higher for females. It is expected that results of a decomposition of unfused tetani of the slow soleus MUs will not be identical to those previously observed for the medial gastrocnemius muscle. Moreover, significant sex differences will be found, in line with previous observations of sexual dimorphism in MU properties. The study is important for understanding processes of development of MU tetanic forces, and the specific role of slow MUs in the activity of skeletal muscles. Therefore, the principal purpose of this study was to mathematically decompose several unfused tetanic contractions of the soleus MUs, evoked by trains of stimuli at variable IPIs. We aimed at analyzing the contraction and relaxation times and the force and force–time area of all decomposed twitch-like responses to successive stimuli, separately for MUs of male and female soleus muscles, which had not been done so far. We compare and discuss the results in relation to muscle structure and function, in comparison with previous observations concerning slow MUs of the medial gastrocnemius and especially with respect to sex differences. 2. Materials and methods 2.1. Electrophysiological experiments, MU isolation, and recordings The experiments were performed on adult Wistar rats (three males and three females). The body weight was 470–520 g for male and 260–300 g for female animals. Rats were anesthetized with pentobarbital sodium (Morbital, Biowet Puławy, 60 mg kg 1, i.p., supplemented after 2 h with additional doses of 10 mg kg 1 h 1), and the depth of anesthesia was controlled by monitoring pinna and limb withdrawal reflexes. After the experiments, the animals were killed by a lethal dose of the anesthetic (180 mg kg 1 i.p.). All experimental procedures were approved by the Local Ethics Committee and followed the European Union guidelines of animal care as well as principles of the Polish Law on The Protection of Animals. Experimental procedures were identical for male and female animals. During surgery, the distal part of the soleus muscle was partly isolated from surrounding tissues; the innervation and blood supply to the examined muscle were left intact, whereas other muscles were denervated by cutting remaining branches of the sciatic nerve. The laminectomy was performed over L2–S1 segments. Dorsal and ventral roots were cut proximally to the spinal cord. The animals were immobilized with steel clamps on the tibia, the sacral bone, and the L1 vertebra. The operated hind limb, isolated spinal cord, and ventral and dorsal roots of spinal nerves were covered with warm paraffin oil. The oil and animal core temperature were kept at a constant level (377 1 °C) by an automatic heating system. The functional isolation of a single MU was achieved by splitting the L5 ventral roots into thin filaments, which were electrically stimulated with rectangular electrical pulses (duration of 0.1 ms, amplitude of up to 0.5 V), generated by the dual-channel square pulse stimulator (Grass Instrument Company, model S88). The isolation of an MU was confirmed when the action potential in a muscle and the twitch force were of the “all-or-none” type and did not change in shape and size with an increase in the stimulus strength. The MU action potentials were recorded with two non-insulated silver wire electrodes inserted into the muscle belly, perpendicularly to its long axis. The force was measured under isometric conditions by a custom made force transducer (deflection sensitivity of 100 μm per 100 mN). The muscle was stretched and kept at a passive force of 40 mN, at which the majority of its MUs had maximum twitch amplitudes. The following experimental protocol was performed: 1. 5 single twitches were evoked (5 stimuli at 1 Hz) and the averaged twitch was estimated.
stimulation frequencies, two tetanic contractions were recorded. The following IPIs were applied: 70, 80, and 100 ms (i.e., the mean frequencies were 14.2, 12.5, and 10 Hz, respectively). For each of the random stimulation patterns, IPIs had a normal distribution within a variability range of 50–150% of the mean IPI, i.e., the respective ranges of IPIs were 35–105 ms, 40–120 ms and 50–150 ms. 5. After completing the above procedure, a standard fatigue test was performed: stimulation with trains of 14 pulses at 40 Hz, repeated each second within 3 min (Burke and Tsairis, 1973). Ten-second intervals were applied between all successive steps of the above protocol and between all tetanic contractions evoked within point 4 of the protocol. For each studied MU, the following parameters were calculated for its single twitch recordings: the contraction time (from the beginning of twitch to the force peak); the half-relaxation time (from the force peak to the moment when the force decreased to half of the peak value); and the peak twitch force. For the 150 Hz tetanus recordings, the maximum MU force was determined. For each unfused tetanus recorded at constant IPIs, the fusion index was calculated, as a ratio of the minimum force when the response to the last stimulus began to the peak force of the last response (Bakels and Kernell, 1995; Celichowski and Grottel, 1995); the relative force level of such a tetanus was determined as a percentage of the maximum tetanus force. Among 67 MUs recorded during the experiments, 10 units of male and 10 of female soleus muscle were chosen for the mathematical decomposition. The selection criteria were a high signal-to-noise ratio and a lack of artifacts in force recordings (sporadically observed, due to respiratory movements or artifacts from the non denervated tail and hip muscles). The contractile properties of the selected MUs were within ranges of contraction force and time parameters obtained earlier for a large population of slow MUs in the soleus muscle of males and females. Moreover, for reliable comparison of results of decomposition, it was important to obtain similar relative force levels and similar fusion degrees of tetanic contractions for all studied MUs. Therefore, among four tetani recorded for each MU at different frequencies at constant IPIs, we chose those with force levels ranging from 46% to 70% of the maximum force, and with fusion indices in the range 0.78–0.94. Subsequently, the tetanus evoked at the respective mean frequency but in a random stimulation pattern was taken for the decomposition. 2.2. Decomposition of the tetanic contractions The mathematical method for decomposition of an unfused MU tetanic force curve into successive twitch-shape contractions, which are mechanical responses to a train of electrical pulses, was described in detail previously (Raikova et al., 2007). To model the single twitch as well as the decomposed twitch-shape responses, a six-parameter analytical function was applied. These six parameters were: the lead time (Tlead); the half-contraction time (Thc); the contraction time (Tc); the half-relaxation time (Thr); the duration of the twitch (Ttw); and the force amplitude (Fmax) – see the equations in Raikova et al. (2008). In this study, the algorithm and the computer program were ameliorated as described below: 1. From the five individual twitches recorded during the first step of the experiments, the most noiseless was chosen and the six parameters for this MU twitch were calculated so that the error between the experimental force and the model was minimal. This automatic process was followed by a visual inspection and manual adjustment of the parameters if necessary. 2. This model was compared with the visible part of the first contraction into the tetanus and, if necessary, some of the parameters were manually adjusted (Fig. 1a) to model the curve between the first and second pulses as precisely as possible.
3. The model of the first contraction was mathematically subtracted from the tetanic curve, and the contraction between the second and third pulses, i.e., the twitch response to the second pulse, was visible (green line in Fig. 1b). The basic twitch parameters for this second contraction (the amplitude, half-contraction time, and contraction time) were automatically calculated. To obtain the remaining three parameters, their values from the first modeled contraction were assigned initially and then were manually corrected to match the visible part between the second and third pulses, and to adjust the second modeled contraction (blue line in Fig. 1b). The force curve, obtained by subtraction of the first and the second models from the recorded curve (the red line in Fig. 1b), was visible. Then, the twitch parameters could be modified for the next, third contraction (i.e., the red line after the third pulse). This process was repeated till the last, 41st pulse (Fig. 1c).
2. An unfused tetanus was evoked by a 500 ms train of stimuli at 40 Hz. 3. The maximum tetanus was evoked by a 200 ms train of stimuli at 150 Hz.
2.3. Statistical analysis
4. Tetanic contractions were evoked by trains of 41 electrical pulses at three frequencies, first at constant and then at random IPIs (prepared with the random number generator in MATLAB). This means that for each of the three applied
All data are expressed as means 7 standard deviations (SDs), and the minimum and maximum values are given. The normality of distribution of interval scale data
Please cite this article as: Drzymała-Celichowska, H., et al., Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females. Journal of Biomechanics (2015), http://dx.doi.org/10.1016/j.jbiomech.2015.07.019i
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Fig. 1. Description of the method for decomposition of a motor unit (MU) tetanic contraction into twitch-shape responses to successive pulses. (a) The first contraction (green line between the first and second pulses) was modeled by the six-parameter analytical function (blue line). (b) The first modeled contraction was subtracted from the tetanic curve to reveal the second contraction (green line between the second and third pulses), which was then modeled (blue line). The red curve was obtained by subtraction of the first two modeled contractions from the tetanic curve. (c) The green line is the curve obtained by subtraction of the first two modeled contractions from the tetanic curve; the blue line is the model of the third contraction; and the red line is the curve obtained by subtraction of the first three modeled contractions from the experimental tetanic curve. Arrows show the moments of application of the successive pulses (there was lead time between each pulse and the mechanical response of the MU).
was tested with the Shapiro–Wilk test. In all cases, the distribution was normal and the homogeneity of variance was noted. Therefore, in order to compare values between the studied groups of MUs in males and females, statistical evaluation with Student's t-test was performed. All above calculations were carried out with Statistica, Stat Soft 10.0 software. Moreover, the effect size as well as the power of the test were calculated using G*Power software. For all significant differences the power of the test was above 0.80.
3. Results Ten MUs of male and 10 MUs of female rats were analyzed in this study. Two examples of representative units, with similar tetanus forces and fusion indices, are presented in Fig. 2, which also illustrates results of the decomposition of tetanic curves into series of responses to successive stimuli. There was immense diversity of force and time parameters between the decomposed twitch responses in each MU (Fig. 2c and d). In all cases, the first twitch had the smallest amplitude and the shortest contraction time (Fig. 3a and e), while the maximal amplitude of decomposed twitches (Fmax) was up to 4.71 times higher, and the maximal contraction time (Tc max) was up to 3.39 times longer than respective parameters of the first twitch (Table 1). A similar observation was made for the half-relaxation time (Thr max) in 9 out of 10 male MUs and in 9 out of 10 female MUs (Table 1 and Fig. 3c). Consequently, the calculated maximum force–time area per one pulse (FTA) was usually (in 9 out of 10 female and in all male MUs) the smallest for the first decomposed twitch, while the maximum FTA amounted up to 11.59 times the FTA of the first twitch (Table 1 and Fig. 3h). There was a statistically significant difference (Po0.05) between the results obtained for male and female soleus MUs with respect to the Fmax and FTA (Table 1). For the male MUs, higher variability of amplitudes of the decomposed twitches, expressed by the ratio of force of the strongest to the weakest decomposed twitch, was observed in comparison with female MUs (Table 1). However, the variability in the contraction and half-relaxation times (Thr) showed no difference between male and female MUs (Table 1).
4. Discussion The present study is the first on the decomposition of tetanic contractions of rat soleus MUs. These contractions were evoked by stimulation with variable, random IPIs to resemble discharge patterns observed during voluntary MU activity. Previously, the decomposition of slow MU contractions was performed in
experiments on the rat medial gastrocnemius (Celichowski et al., 2014). Therefore, some of the results obtained in this study could be expected, and indeed several similarities were observed, namely, considerable diversity of either force and time parameters was common for the decomposed twitch-shape responses in each MU, and the first decomposed twitch was the weakest and the shortest. On the other hand, this variability of decomposed twitch properties was stronger for slow MUs in the medial gastrocnemius muscle in comparison with the present results in the soleus muscle. For the medial gastrocnemius, the ratio of the force of the strongest decomposed twitch to the first twitch force was up to 6.9, the ratio for the contraction time was up to 3.5, and the ratio for the force–time area was up to 14.2 (Celichowski et al., 2014). This indicates that slow-type MUs in different muscles are not uniform with respect to mechanisms of summation into tetanic contractions, though they evidently have different properties to fast-type MUs in which this diversity of twitch force and time parameters is much smaller (Raikova et al., 2010; Celichowski et al., 2014). One of the most obvious differences between slow MUs of these two muscles is that the values of the twitch contraction and relaxation times as well as of the twitch force are substantially higher in soleus MUs than in medial gastrocnemius MUs (Drzymała-Celichowska and Krutki, 2015). These differences should be related to differences in the innervation ratio, muscle fiber diameter and density within a MU (Mierzejewska-Krzyżowska et al., 2011) as well as in the architecture of the two muscles – the soleus is a pennate muscle with a smaller pennation angle and shorter muscle fibers (Eng et al., 2008). Moreover, one should consider the different functions of slow MUs in the two muscles due to the different muscle fiber composition. The soleus is almost entirely composed of slow MUs, while the medial gastrocnemius has about 14% and 23% of slow MUs in males and females, respectively, so its slow units generate only 5% and 10% of the total muscle force, respectively (Celichowski and DrzymałaCelichowska, 2007). Despite the above-mentioned differences between muscles, considerable variability of responses to successive stimuli is a general property of slow MUs, which contrasts with the much smaller variability observed for fast units. This points to a common feature of slow muscle fibers – their capacity to significantly increase the efficiency of responses to successive stimuli during activation. It is worth noticing that this high effectiveness of force summation during development of tetanic contractions is also manifest in slow MUs of different muscles by relatively low twitch-to-tetanus ratios, considerably lower than values observed for fast MUs (Stephens and Stuart, 1975; Gardiner and Olha, 1987; Chamberlain and Lewis, 1989). The twitch-to-
Please cite this article as: Drzymała-Celichowska, H., et al., Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females. Journal of Biomechanics (2015), http://dx.doi.org/10.1016/j.jbiomech.2015.07.019i
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Fig. 2. Results of the decomposition of tetanic curves for two slow soleus motor units (MUs), from the female group (left column) and from the male group (right column). The first row (a and b) shows the experimentally recorded tetanic forces obtained following stimulation at the same mean frequencies, but with constant (blue) or random (red) interpulse intervals (IPIs). The MU of the female soleus (a) was stimulated at 14.2 Hz (mean IPI of 70 ms), while the MU of the male muscle (b) was stimulated at 10 Hz (mean IPI of 100 ms). The relative force levels were comparable for both MU and amounted to 64% and 59% of the maximum tetanic force, respectively. The fusion indices of constant stimulation unfused tetani were also similar for both presented records: 0.91 and 0.87, respectively. The forces shown in green (a, b) were calculated by summation of equal twitches (identical to the first decomposed twitch), by using the respective random stimulation pattern. They illustrate that the force development was a non-linear process. The second row (c and d) shows decomposed twitch-shape responses to consecutive stimuli, which have been superimposed. All pulses were delivered at zero time. In the third row (e and f), the same decomposed contractions are presented according to their time position. In c–f, the first (the weakest) decomposed twitches are distinguished by thick blue lines, and the strongest decomposed twitches by thick black lines.
tetanus ratio is a parameter characterizing the range between a single twitch (the minimum force) and the fused tetanic contraction (the maximum force). The force of subfused contractions may vary within this range. This parameter is strictly related to the rate coding, one of the principal mechanisms of regulation of the MU force during motor activity. The striking result of this study was the difference between male and female rats with respect to several properties of decomposed twitches. The ratios of values of the force and the force–time area for the strongest decomposed twitch to the weakest one were significantly higher for male MUs. The above results well reflect sex differences in the twitch-to-tetanus ratios between male and female soleus MUs, as this parameter has been found to be almost twice lower for males in relation to females
(0.11 and 0.19, respectively), while no sex differences have been observed in twitch forces between male and female soleus MUs (Drzymała-Celichowska and Krutki, 2015). This difference suggests that in male soleus MUs, the rate coding plays a bigger role in force regulation processes than in females. Indeed, it has been observed in our previous study that the force–frequency relationships in their steep parts (attributed to unfused tetanic contractions) have higher slopes in males, indicating ability for a higher force increase in response to 1 Hz increase in the stimulation frequency (Drzymała-Celichowska and Krutki, 2015). It is possible that the presented discrepancies between male and female animals are influenced by sex differences in the muscle architecture. In the literature data for male rats are available (Eng et al., 2008), but there are no respective data for female rats. For the humans soleus
Please cite this article as: Drzymała-Celichowska, H., et al., Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females. Journal of Biomechanics (2015), http://dx.doi.org/10.1016/j.jbiomech.2015.07.019i
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Fig. 3. Distribution of four parameters of all 41 decomposed twitches for 10 female soleus motor units (MUs) (blue plots) and 10 male soleus MUs (red plots) analyzed in this study. The contraction time (Tc – a and b), half-relaxation time (Thr – c and d), maximal twitch amplitude (Fmax – e and f), and area under the force curve (FTA – g and h) are presented as absolute (left column) and normalized values (right column). The normalization was performed according to the respective values of the first decomposed twitch. The circles in a, c, e, and g mark the values of respective parameters calculated for the first contraction in each MU.
the muscle fibers are in average longer and their pennation angle is lower in females (Chow et al., 2000). Anatomy and physiological function of a predominantly slow soleus muscle is similar for various animals and from this reason one might expect that the presented results are not unique for rat soleus, but are characteristic for this muscle of other mammals, including humans. On the other hand, there is sparse knowledge on sexual dimorphism of MU contractile properties or biomechanical structure of the soleus muscle in various species, and none of the previous studies on decomposition and modeling of tetanic contractions have taken into consideration differences in force summation between
male and female muscles. The applied mathematical method also has limitations – the decomposition can be made only for unfused tetanic contractions, when the beginning of relaxation following a response to each stimulus is visible. Such unfused tetanic contractions can be evoked for isolated MUs only, and for humans this method was only used in experiments on small distal muscles of the limbs (Thomas et al., 2005; Häger-Ross et al., 2006). From these reasons one cannot simply generalize the obtained results to humans, neither with respect to high variability of responses to individual stimuli nor to sex differences in the process of force generation for slow soleus MUs.
Please cite this article as: Drzymała-Celichowska, H., et al., Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females. Journal of Biomechanics (2015), http://dx.doi.org/10.1016/j.jbiomech.2015.07.019i
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Table 1 The ratio (R) of the highest values noted for the decomposed twitches to the values of the single twitch. RFmax is calculated by obtaining the maximal value of Fmax (i)/Fmax (1) (i¼ 1,2,..,41), where Fmax (1) is the force amplitude of first decomposed twitch. RTc is calculated in the same way but using the contraction time parameters, RThr – the half-relaxation time, RFTA – the force–time area per one pulse. The data are presented as means7 standard deviations and ranges. *** – Difference between mean values for males and females significant at Po 0.001, ** – difference significant at Po 0.01, NS – difference not significant, Student's t-test. Additionally, the Cohen's d (effect size) and power of the test are given below.
Male n ¼10 Female n ¼10 Cohen’s d Power of the test
RFmax
RTc
RThr
RFTA
3.83 7 0.52 3.01–4.71 2.08 7 0.26 2.45–3.40 *** 2.471 0.999
2.687 0.45 2.16–3.39 2.917 0.24 2.60–3.26 NS 0.199 0.071
2.09 70.22 1.84–2.61 2.177 0.17 1.88–2.41 NS 0.398 0.134
7.357 1.85 5.12–11.59 5.077 0.68 4.37–6.66 ** 1.631 0.931
Despite the above reservations, a method of mathematical decomposition of tetanic contractions appears to be a useful and an effective tool that may reveal differences in mechanisms of MU force development between different MU types, the same MU types in different muscles or the same muscles in different sexes. This study in the soleus muscle confirmed that high variability of the mechanical responses to subsequent motoneuronal action potentials (delivered at a random stimulation pattern) is a characteristic property of slow MUs. On the other hand, several morphological, biomechanical and possibly biochemical factors influencing MU force production lead to significant differences in effects of decomposition of slow MU contractions between different muscles (medial gastrocnemius and soleus) or between males and females. All the above should be taken into consideration while interpreting results of studies based on motoneuronal firing patterns or a decomposition of electromyograms during voluntary movements.
Conflict of interest statement No conflicts of interest, financial or otherwise, are declared by the authors.
Acknowledgments This study was partially supported by Bilateral Agreement Between Polish and Bulgarian Academy of Sciences. Authors thank Maciej Tomczak PhD for statistical analysis of the data.
References Bakels, R., Kernell, D., 1995. Measures of ‘‘fastness’’: force profiles of twitches and partly fused contractions in rat medial gastrocnemius and tibialis anterior muscle units. Pflüg. Arch. 431, 230–236. Boe, S.G., Stashuk, D.W., Brown, W.F., Doherty, T.J., 2005. Decomposition-based quantitative electromyography: effect of force on motor unit potentials and motor unit number estimates. Muscle Nerve 31, 365–373. Burke, R.E., Tsairis, P., 1973. Anatomy and innervation ratios in motor units of cat gastrocnemius. J. Physiol. 234, 749–765. Burke, R.E., Levine, D.N., Salcman, M., Tsairis, P., 1974. Motor units in cat soleus muscle: physiological, histochemical and morphological characteristics. J. Physiol. 238, 503–514. Celichowski, J., Grottel, K., 1995. The relationship between fusion index and stimulation frequency in tetani of motor units in rat medial gastrocnemius. Arch. Ital. Biol. 133, 81–87. Celichowski, J., Drzymała-Celichowska, H., 2007. The number of motor units in the medial gastrocnemius muscle of male and female rats. J. Physiol. Pharmacol. 58, 821–828. Celichowski, J., Raikova, R., Aladjov, H., Krutki, P., 2014. Dynamic changes of twitchlike responses to successive stimuli studied by decomposition of motor unit tetanic contactions in rat medial gastrocnemius. J. Neurophysiol. 112 (12), 3116–3124. Chamberlain, S., Lewis, D.M., 1989. Contractile characteristics and innervation ratio of rat soleus motor units. J. Physiol. 412, 1–21. Chow, R.S., Medri, M.K., Martin, D.C., Leekam, R.N., Agur, A.M., McKee, N.H., 2000. Sonographic studies of human soleus and gastrocnemius muscle architecture: gender variability. Eur. J. Appl. Physiol. 82, 236–244. Drzymała-Celichowska, H., Krutki, P., 2015. Slow motor units in female rat soleus are slower and weaker than their male counterparts. J. Muscle Res. Cell Motil. 36, 287–295. Eng, C.M., Smallwood, L.H., Rainiero, M.P., Lahey, M., Ward, S.R., Lieber, R.L., 2008. Scaling of muscle architecture and fiber types in the rat hindlimb. J. Exp. Biol. 211, 2336–2345. Gardiner, P.F., Olha, A.E., 1987. Contractile and electromyographic characteristic of rat plantaris motor unit types during fatigue in situ. J. Physiol. 385, 13–34. Häger-Ross, C.K., Klein, C.S., Thomas, C.K., 2006. Twitch and tetanic properties of human thenar motor units paralyzed by chronic spinal cord injury. J. Neurophysiol. 96 (1), 165–174. Kanda, K., Hashizume, K., 1992. Factors causing difference in force output among motor units in the rat medial gastrocnemius muscle. J. Physiol. 448, 677–695. Kugelberg, E., 1973. Histochemical composition, contraction speed and fatiguability of rat soleus motor units. J. Neurol. Sci. 20, 177–198. Mierzejewska-Krzyżowska, B., Drzymała-Celichowska, H., Celichowski, J., 2011. Gender differences in the morphometric properties of muscle fibers and the innervation ratio of motor units in the rat medial gastrocnemius muscle. Anat. Histol. Embryol. 40, 249–255. Moritz, C.T., Barry, B.K., Pascoe, M.A., Enoka, R.M., 2005. Discharge rate variability influences the variation in force fluctuation across the working range of a hand muscle. J. Neurophysiol. 93, 2449–2459. Raikova, R., Celichowski, J., Pogrzebna, M., Aladjov, H., Krutki, P., 2007. Modeling of summation of individual twitches into unfused tetanus for various types of rat motor units. J. Electromyogr. Kinesiol. 17, 121–130. Raikova, R., Pogrzebna, M., Drzymała, H., Celichowski, J., Aladjov, H., 2008. Variability of successive contractions subtracted from unfused tetanus of fast and slow motor units. J. Electromyogr. Kinesiol. 8, 741–751. Raikova, R., Rusev, R., Drzymała-Celichowska, H., Krutki, P., Aladjov, H., Celichowski, J., 2010. Experimentally verified mathematical approach for the prediction of force developed by motor units at variable frequency stimulation patterns. J. Biomech. 28, 1546–1552. Stephens, J.A., Stuart, D.G., 1975. The motor units of cat medial gastrocnemius. Twitch potentiation and twitch-tetanus ratio. Pflüg. Arch. 365 (4), 359–372. Thomas, C.K., Johansson, R.S., Bigland-Ritchie, B., 2005. EMG changes in human thenar motor units with force potentiation and fatigue. J. Neurophysiol. 95 (3), 1518–1526.
Please cite this article as: Drzymała-Celichowska, H., et al., Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females. Journal of Biomechanics (2015), http://dx.doi.org/10.1016/j.jbiomech.2015.07.019i
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Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females Hanna Drzymała-Celichowska a,b,n, Rositsa Raikova c, Piotr Krutki a a
Department of Neurobiology, University School of Physical Education in Poznań, 27/39 Królowej Jadwigi Street, 61-871 Poznań, Poland Division of Biochemistry, University School of Physical Education in Poznań, Poland c Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria b
art ic l e i nf o
a b s t r a c t
Article history: Accepted 18 July 2015
Mathematical decomposition of tetanic contractions of slow motor units (MUs) of the rat heterogeneous medial gastrocnemius muscle revealed immense variability of twitch-shape responses to successive pulses, contrary to results obtained for fast MUs. The aim of this study in rat soleus muscle, almost exclusively composed of slow MUs, was to reveal whether such variability of twitch-shape decomposed components was a common property of slow MUs in the two studied muscles, and whether ranges of the force amplitude or time parameters of these decomposed twitches showed sex differences. Unfused tetanic contractions evoked by stimulation at variable interpulse intervals were analyzed for 10 MUs of males and 10 MUs of females. Significantly higher variability between parameters of the decomposed responses was observed for male soleus MUs, as the mean ratio of forces of the strongest decomposed twitch and the first (the weakest) decomposed twitch amounted to 3.8 for males and 2.8 for females. The ratios of the contraction times of the longest decomposed to the first twitch were much more similar between male and female MUs, 2.6 and 2.9, respectively. Consequently, the mean ratio of the force–time area for the strongest decomposed to the first twitch was much bigger in male than female MUs (7.35 vs. 5.07, respectively). Our observations indicate that high variability of responses to successive stimuli is a general property of slow MUs in different rat muscles, but the mechanisms of summation of individual twitches into tetanic contractions of MUs are not identical for male and female rats. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Slow motor units Sex differences Random stimulation pattern Soleus Rat
1. Introduction During voluntary activity, motoneurons generate trains of pulses at variable intervals, which substantially influence the development and profiles of tetanic forces of motor units (MUs). The discharge patterns of MUs have been most frequently studied by the decomposition of electromyograms into trains of MU action potentials (Boe et al., 2005; Moritz et al., 2005). On the other hand, the mathematical decomposition of unfused tetanic contractions of MUs into twitch-shape responses to successive pulses can provide information concerning contractile effects induced by individual action potentials. This method, applied to tetani evoked by stimulation of the medial gastrocnemius MUs at variable interpulse intervals (IPIs), resembling motoneuronal firing patterns observed during voluntary contractions, has revealed substantial variability of n Corresponding author at: Department of Neurobiology University School of Physical Education in Poznań, 27/39 Królowej Jadwigi Street, 61-871 Poznań, Poland. Tel.: þ48 61 8355435; fax: þ 48 61 8355444. E-mail address: [email protected] (H. Drzymała-Celichowska).
individual responses to successive pulses (Raikova et al., 2008). The decomposed twitches of fast MUs have displayed up to 2.2 times higher forces, and up to 2.5 times longer contraction times than the single twitch. For slow MUs, the forces of decomposed twitches have reached even 7 times higher values, while the twitch contraction and relaxation parameters have been up to 3.5 times longer than those of the single twitch (Celichowski et al., 2014). The above observations have only been made for MUs of one muscle so far, and it is unknown whether similar variability in amplitudes and duration of responses summating into a tetanic contraction could be obtained for MUs of another muscle, anatomically and physiologically different. Such an example is the soleus, a homogeneous, slow-twitch muscle, composed predominantly of slow MUs (Kugelberg, 1973; Burke et al., 1974). Although the soleus and medial gastrocnemius muscles contain slow MUs, it has been shown that basic contractile properties of slow MUs of these two muscles are considerably different, as they are active in different motor tasks (Burke and Tsairis, 1973; Burke et al., 1974; Kanda and Hashizume, 1992; Drzymała-Celichowska and Krutki, 2015). Moreover, previous studies of rat soleus MUs
http://dx.doi.org/10.1016/j.jbiomech.2015.07.019 0021-9290/& 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Drzymała-Celichowska, H., et al., Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females. Journal of Biomechanics (2015), http://dx.doi.org/10.1016/j.jbiomech.2015.07.019i
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have revealed that this muscle is also sexually dimorphic, and properties of its MUs are different for males and females (Drzymała-Celichowska and Krutki, 2015). MUs of the male soleus have shorter contraction times and generate higher tetanic forces, whereas the twitch forces are similar for animals of both sexes; consequently, the twitch-to-tetanus force ratios are considerably (about twice) higher for females. It is expected that results of a decomposition of unfused tetani of the slow soleus MUs will not be identical to those previously observed for the medial gastrocnemius muscle. Moreover, significant sex differences will be found, in line with previous observations of sexual dimorphism in MU properties. The study is important for understanding processes of development of MU tetanic forces, and the specific role of slow MUs in the activity of skeletal muscles. Therefore, the principal purpose of this study was to mathematically decompose several unfused tetanic contractions of the soleus MUs, evoked by trains of stimuli at variable IPIs. We aimed at analyzing the contraction and relaxation times and the force and force–time area of all decomposed twitch-like responses to successive stimuli, separately for MUs of male and female soleus muscles, which had not been done so far. We compare and discuss the results in relation to muscle structure and function, in comparison with previous observations concerning slow MUs of the medial gastrocnemius and especially with respect to sex differences. 2. Materials and methods 2.1. Electrophysiological experiments, MU isolation, and recordings The experiments were performed on adult Wistar rats (three males and three females). The body weight was 470–520 g for male and 260–300 g for female animals. Rats were anesthetized with pentobarbital sodium (Morbital, Biowet Puławy, 60 mg kg 1, i.p., supplemented after 2 h with additional doses of 10 mg kg 1 h 1), and the depth of anesthesia was controlled by monitoring pinna and limb withdrawal reflexes. After the experiments, the animals were killed by a lethal dose of the anesthetic (180 mg kg 1 i.p.). All experimental procedures were approved by the Local Ethics Committee and followed the European Union guidelines of animal care as well as principles of the Polish Law on The Protection of Animals. Experimental procedures were identical for male and female animals. During surgery, the distal part of the soleus muscle was partly isolated from surrounding tissues; the innervation and blood supply to the examined muscle were left intact, whereas other muscles were denervated by cutting remaining branches of the sciatic nerve. The laminectomy was performed over L2–S1 segments. Dorsal and ventral roots were cut proximally to the spinal cord. The animals were immobilized with steel clamps on the tibia, the sacral bone, and the L1 vertebra. The operated hind limb, isolated spinal cord, and ventral and dorsal roots of spinal nerves were covered with warm paraffin oil. The oil and animal core temperature were kept at a constant level (377 1 °C) by an automatic heating system. The functional isolation of a single MU was achieved by splitting the L5 ventral roots into thin filaments, which were electrically stimulated with rectangular electrical pulses (duration of 0.1 ms, amplitude of up to 0.5 V), generated by the dual-channel square pulse stimulator (Grass Instrument Company, model S88). The isolation of an MU was confirmed when the action potential in a muscle and the twitch force were of the “all-or-none” type and did not change in shape and size with an increase in the stimulus strength. The MU action potentials were recorded with two non-insulated silver wire electrodes inserted into the muscle belly, perpendicularly to its long axis. The force was measured under isometric conditions by a custom made force transducer (deflection sensitivity of 100 μm per 100 mN). The muscle was stretched and kept at a passive force of 40 mN, at which the majority of its MUs had maximum twitch amplitudes. The following experimental protocol was performed: 1. 5 single twitches were evoked (5 stimuli at 1 Hz) and the averaged twitch was estimated.
stimulation frequencies, two tetanic contractions were recorded. The following IPIs were applied: 70, 80, and 100 ms (i.e., the mean frequencies were 14.2, 12.5, and 10 Hz, respectively). For each of the random stimulation patterns, IPIs had a normal distribution within a variability range of 50–150% of the mean IPI, i.e., the respective ranges of IPIs were 35–105 ms, 40–120 ms and 50–150 ms. 5. After completing the above procedure, a standard fatigue test was performed: stimulation with trains of 14 pulses at 40 Hz, repeated each second within 3 min (Burke and Tsairis, 1973). Ten-second intervals were applied between all successive steps of the above protocol and between all tetanic contractions evoked within point 4 of the protocol. For each studied MU, the following parameters were calculated for its single twitch recordings: the contraction time (from the beginning of twitch to the force peak); the half-relaxation time (from the force peak to the moment when the force decreased to half of the peak value); and the peak twitch force. For the 150 Hz tetanus recordings, the maximum MU force was determined. For each unfused tetanus recorded at constant IPIs, the fusion index was calculated, as a ratio of the minimum force when the response to the last stimulus began to the peak force of the last response (Bakels and Kernell, 1995; Celichowski and Grottel, 1995); the relative force level of such a tetanus was determined as a percentage of the maximum tetanus force. Among 67 MUs recorded during the experiments, 10 units of male and 10 of female soleus muscle were chosen for the mathematical decomposition. The selection criteria were a high signal-to-noise ratio and a lack of artifacts in force recordings (sporadically observed, due to respiratory movements or artifacts from the non denervated tail and hip muscles). The contractile properties of the selected MUs were within ranges of contraction force and time parameters obtained earlier for a large population of slow MUs in the soleus muscle of males and females. Moreover, for reliable comparison of results of decomposition, it was important to obtain similar relative force levels and similar fusion degrees of tetanic contractions for all studied MUs. Therefore, among four tetani recorded for each MU at different frequencies at constant IPIs, we chose those with force levels ranging from 46% to 70% of the maximum force, and with fusion indices in the range 0.78–0.94. Subsequently, the tetanus evoked at the respective mean frequency but in a random stimulation pattern was taken for the decomposition. 2.2. Decomposition of the tetanic contractions The mathematical method for decomposition of an unfused MU tetanic force curve into successive twitch-shape contractions, which are mechanical responses to a train of electrical pulses, was described in detail previously (Raikova et al., 2007). To model the single twitch as well as the decomposed twitch-shape responses, a six-parameter analytical function was applied. These six parameters were: the lead time (Tlead); the half-contraction time (Thc); the contraction time (Tc); the half-relaxation time (Thr); the duration of the twitch (Ttw); and the force amplitude (Fmax) – see the equations in Raikova et al. (2008). In this study, the algorithm and the computer program were ameliorated as described below: 1. From the five individual twitches recorded during the first step of the experiments, the most noiseless was chosen and the six parameters for this MU twitch were calculated so that the error between the experimental force and the model was minimal. This automatic process was followed by a visual inspection and manual adjustment of the parameters if necessary. 2. This model was compared with the visible part of the first contraction into the tetanus and, if necessary, some of the parameters were manually adjusted (Fig. 1a) to model the curve between the first and second pulses as precisely as possible.
3. The model of the first contraction was mathematically subtracted from the tetanic curve, and the contraction between the second and third pulses, i.e., the twitch response to the second pulse, was visible (green line in Fig. 1b). The basic twitch parameters for this second contraction (the amplitude, half-contraction time, and contraction time) were automatically calculated. To obtain the remaining three parameters, their values from the first modeled contraction were assigned initially and then were manually corrected to match the visible part between the second and third pulses, and to adjust the second modeled contraction (blue line in Fig. 1b). The force curve, obtained by subtraction of the first and the second models from the recorded curve (the red line in Fig. 1b), was visible. Then, the twitch parameters could be modified for the next, third contraction (i.e., the red line after the third pulse). This process was repeated till the last, 41st pulse (Fig. 1c).
2. An unfused tetanus was evoked by a 500 ms train of stimuli at 40 Hz. 3. The maximum tetanus was evoked by a 200 ms train of stimuli at 150 Hz.
2.3. Statistical analysis
4. Tetanic contractions were evoked by trains of 41 electrical pulses at three frequencies, first at constant and then at random IPIs (prepared with the random number generator in MATLAB). This means that for each of the three applied
All data are expressed as means 7 standard deviations (SDs), and the minimum and maximum values are given. The normality of distribution of interval scale data
Please cite this article as: Drzymała-Celichowska, H., et al., Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females. Journal of Biomechanics (2015), http://dx.doi.org/10.1016/j.jbiomech.2015.07.019i
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Fig. 1. Description of the method for decomposition of a motor unit (MU) tetanic contraction into twitch-shape responses to successive pulses. (a) The first contraction (green line between the first and second pulses) was modeled by the six-parameter analytical function (blue line). (b) The first modeled contraction was subtracted from the tetanic curve to reveal the second contraction (green line between the second and third pulses), which was then modeled (blue line). The red curve was obtained by subtraction of the first two modeled contractions from the tetanic curve. (c) The green line is the curve obtained by subtraction of the first two modeled contractions from the tetanic curve; the blue line is the model of the third contraction; and the red line is the curve obtained by subtraction of the first three modeled contractions from the experimental tetanic curve. Arrows show the moments of application of the successive pulses (there was lead time between each pulse and the mechanical response of the MU).
was tested with the Shapiro–Wilk test. In all cases, the distribution was normal and the homogeneity of variance was noted. Therefore, in order to compare values between the studied groups of MUs in males and females, statistical evaluation with Student's t-test was performed. All above calculations were carried out with Statistica, Stat Soft 10.0 software. Moreover, the effect size as well as the power of the test were calculated using G*Power software. For all significant differences the power of the test was above 0.80.
3. Results Ten MUs of male and 10 MUs of female rats were analyzed in this study. Two examples of representative units, with similar tetanus forces and fusion indices, are presented in Fig. 2, which also illustrates results of the decomposition of tetanic curves into series of responses to successive stimuli. There was immense diversity of force and time parameters between the decomposed twitch responses in each MU (Fig. 2c and d). In all cases, the first twitch had the smallest amplitude and the shortest contraction time (Fig. 3a and e), while the maximal amplitude of decomposed twitches (Fmax) was up to 4.71 times higher, and the maximal contraction time (Tc max) was up to 3.39 times longer than respective parameters of the first twitch (Table 1). A similar observation was made for the half-relaxation time (Thr max) in 9 out of 10 male MUs and in 9 out of 10 female MUs (Table 1 and Fig. 3c). Consequently, the calculated maximum force–time area per one pulse (FTA) was usually (in 9 out of 10 female and in all male MUs) the smallest for the first decomposed twitch, while the maximum FTA amounted up to 11.59 times the FTA of the first twitch (Table 1 and Fig. 3h). There was a statistically significant difference (Po0.05) between the results obtained for male and female soleus MUs with respect to the Fmax and FTA (Table 1). For the male MUs, higher variability of amplitudes of the decomposed twitches, expressed by the ratio of force of the strongest to the weakest decomposed twitch, was observed in comparison with female MUs (Table 1). However, the variability in the contraction and half-relaxation times (Thr) showed no difference between male and female MUs (Table 1).
4. Discussion The present study is the first on the decomposition of tetanic contractions of rat soleus MUs. These contractions were evoked by stimulation with variable, random IPIs to resemble discharge patterns observed during voluntary MU activity. Previously, the decomposition of slow MU contractions was performed in
experiments on the rat medial gastrocnemius (Celichowski et al., 2014). Therefore, some of the results obtained in this study could be expected, and indeed several similarities were observed, namely, considerable diversity of either force and time parameters was common for the decomposed twitch-shape responses in each MU, and the first decomposed twitch was the weakest and the shortest. On the other hand, this variability of decomposed twitch properties was stronger for slow MUs in the medial gastrocnemius muscle in comparison with the present results in the soleus muscle. For the medial gastrocnemius, the ratio of the force of the strongest decomposed twitch to the first twitch force was up to 6.9, the ratio for the contraction time was up to 3.5, and the ratio for the force–time area was up to 14.2 (Celichowski et al., 2014). This indicates that slow-type MUs in different muscles are not uniform with respect to mechanisms of summation into tetanic contractions, though they evidently have different properties to fast-type MUs in which this diversity of twitch force and time parameters is much smaller (Raikova et al., 2010; Celichowski et al., 2014). One of the most obvious differences between slow MUs of these two muscles is that the values of the twitch contraction and relaxation times as well as of the twitch force are substantially higher in soleus MUs than in medial gastrocnemius MUs (Drzymała-Celichowska and Krutki, 2015). These differences should be related to differences in the innervation ratio, muscle fiber diameter and density within a MU (Mierzejewska-Krzyżowska et al., 2011) as well as in the architecture of the two muscles – the soleus is a pennate muscle with a smaller pennation angle and shorter muscle fibers (Eng et al., 2008). Moreover, one should consider the different functions of slow MUs in the two muscles due to the different muscle fiber composition. The soleus is almost entirely composed of slow MUs, while the medial gastrocnemius has about 14% and 23% of slow MUs in males and females, respectively, so its slow units generate only 5% and 10% of the total muscle force, respectively (Celichowski and DrzymałaCelichowska, 2007). Despite the above-mentioned differences between muscles, considerable variability of responses to successive stimuli is a general property of slow MUs, which contrasts with the much smaller variability observed for fast units. This points to a common feature of slow muscle fibers – their capacity to significantly increase the efficiency of responses to successive stimuli during activation. It is worth noticing that this high effectiveness of force summation during development of tetanic contractions is also manifest in slow MUs of different muscles by relatively low twitch-to-tetanus ratios, considerably lower than values observed for fast MUs (Stephens and Stuart, 1975; Gardiner and Olha, 1987; Chamberlain and Lewis, 1989). The twitch-to-
Please cite this article as: Drzymała-Celichowska, H., et al., Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females. Journal of Biomechanics (2015), http://dx.doi.org/10.1016/j.jbiomech.2015.07.019i
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Fig. 2. Results of the decomposition of tetanic curves for two slow soleus motor units (MUs), from the female group (left column) and from the male group (right column). The first row (a and b) shows the experimentally recorded tetanic forces obtained following stimulation at the same mean frequencies, but with constant (blue) or random (red) interpulse intervals (IPIs). The MU of the female soleus (a) was stimulated at 14.2 Hz (mean IPI of 70 ms), while the MU of the male muscle (b) was stimulated at 10 Hz (mean IPI of 100 ms). The relative force levels were comparable for both MU and amounted to 64% and 59% of the maximum tetanic force, respectively. The fusion indices of constant stimulation unfused tetani were also similar for both presented records: 0.91 and 0.87, respectively. The forces shown in green (a, b) were calculated by summation of equal twitches (identical to the first decomposed twitch), by using the respective random stimulation pattern. They illustrate that the force development was a non-linear process. The second row (c and d) shows decomposed twitch-shape responses to consecutive stimuli, which have been superimposed. All pulses were delivered at zero time. In the third row (e and f), the same decomposed contractions are presented according to their time position. In c–f, the first (the weakest) decomposed twitches are distinguished by thick blue lines, and the strongest decomposed twitches by thick black lines.
tetanus ratio is a parameter characterizing the range between a single twitch (the minimum force) and the fused tetanic contraction (the maximum force). The force of subfused contractions may vary within this range. This parameter is strictly related to the rate coding, one of the principal mechanisms of regulation of the MU force during motor activity. The striking result of this study was the difference between male and female rats with respect to several properties of decomposed twitches. The ratios of values of the force and the force–time area for the strongest decomposed twitch to the weakest one were significantly higher for male MUs. The above results well reflect sex differences in the twitch-to-tetanus ratios between male and female soleus MUs, as this parameter has been found to be almost twice lower for males in relation to females
(0.11 and 0.19, respectively), while no sex differences have been observed in twitch forces between male and female soleus MUs (Drzymała-Celichowska and Krutki, 2015). This difference suggests that in male soleus MUs, the rate coding plays a bigger role in force regulation processes than in females. Indeed, it has been observed in our previous study that the force–frequency relationships in their steep parts (attributed to unfused tetanic contractions) have higher slopes in males, indicating ability for a higher force increase in response to 1 Hz increase in the stimulation frequency (Drzymała-Celichowska and Krutki, 2015). It is possible that the presented discrepancies between male and female animals are influenced by sex differences in the muscle architecture. In the literature data for male rats are available (Eng et al., 2008), but there are no respective data for female rats. For the humans soleus
Please cite this article as: Drzymała-Celichowska, H., et al., Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females. Journal of Biomechanics (2015), http://dx.doi.org/10.1016/j.jbiomech.2015.07.019i
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Fig. 3. Distribution of four parameters of all 41 decomposed twitches for 10 female soleus motor units (MUs) (blue plots) and 10 male soleus MUs (red plots) analyzed in this study. The contraction time (Tc – a and b), half-relaxation time (Thr – c and d), maximal twitch amplitude (Fmax – e and f), and area under the force curve (FTA – g and h) are presented as absolute (left column) and normalized values (right column). The normalization was performed according to the respective values of the first decomposed twitch. The circles in a, c, e, and g mark the values of respective parameters calculated for the first contraction in each MU.
the muscle fibers are in average longer and their pennation angle is lower in females (Chow et al., 2000). Anatomy and physiological function of a predominantly slow soleus muscle is similar for various animals and from this reason one might expect that the presented results are not unique for rat soleus, but are characteristic for this muscle of other mammals, including humans. On the other hand, there is sparse knowledge on sexual dimorphism of MU contractile properties or biomechanical structure of the soleus muscle in various species, and none of the previous studies on decomposition and modeling of tetanic contractions have taken into consideration differences in force summation between
male and female muscles. The applied mathematical method also has limitations – the decomposition can be made only for unfused tetanic contractions, when the beginning of relaxation following a response to each stimulus is visible. Such unfused tetanic contractions can be evoked for isolated MUs only, and for humans this method was only used in experiments on small distal muscles of the limbs (Thomas et al., 2005; Häger-Ross et al., 2006). From these reasons one cannot simply generalize the obtained results to humans, neither with respect to high variability of responses to individual stimuli nor to sex differences in the process of force generation for slow soleus MUs.
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Table 1 The ratio (R) of the highest values noted for the decomposed twitches to the values of the single twitch. RFmax is calculated by obtaining the maximal value of Fmax (i)/Fmax (1) (i¼ 1,2,..,41), where Fmax (1) is the force amplitude of first decomposed twitch. RTc is calculated in the same way but using the contraction time parameters, RThr – the half-relaxation time, RFTA – the force–time area per one pulse. The data are presented as means7 standard deviations and ranges. *** – Difference between mean values for males and females significant at Po 0.001, ** – difference significant at Po 0.01, NS – difference not significant, Student's t-test. Additionally, the Cohen's d (effect size) and power of the test are given below.
Male n ¼10 Female n ¼10 Cohen’s d Power of the test
RFmax
RTc
RThr
RFTA
3.83 7 0.52 3.01–4.71 2.08 7 0.26 2.45–3.40 *** 2.471 0.999
2.687 0.45 2.16–3.39 2.917 0.24 2.60–3.26 NS 0.199 0.071
2.09 70.22 1.84–2.61 2.177 0.17 1.88–2.41 NS 0.398 0.134
7.357 1.85 5.12–11.59 5.077 0.68 4.37–6.66 ** 1.631 0.931
Despite the above reservations, a method of mathematical decomposition of tetanic contractions appears to be a useful and an effective tool that may reveal differences in mechanisms of MU force development between different MU types, the same MU types in different muscles or the same muscles in different sexes. This study in the soleus muscle confirmed that high variability of the mechanical responses to subsequent motoneuronal action potentials (delivered at a random stimulation pattern) is a characteristic property of slow MUs. On the other hand, several morphological, biomechanical and possibly biochemical factors influencing MU force production lead to significant differences in effects of decomposition of slow MU contractions between different muscles (medial gastrocnemius and soleus) or between males and females. All the above should be taken into consideration while interpreting results of studies based on motoneuronal firing patterns or a decomposition of electromyograms during voluntary movements.
Conflict of interest statement No conflicts of interest, financial or otherwise, are declared by the authors.
Acknowledgments This study was partially supported by Bilateral Agreement Between Polish and Bulgarian Academy of Sciences. Authors thank Maciej Tomczak PhD for statistical analysis of the data.
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Please cite this article as: Drzymała-Celichowska, H., et al., Decomposition of motor unit tetanic contractions of rat soleus muscle: Differences between males and females. Journal of Biomechanics (2015), http://dx.doi.org/10.1016/j.jbiomech.2015.07.019i