Contraction-induced enhancement of relaxation during high force contractions of mouse lumbrical muscle at 37°C

The rate of force decline during muscle relaxation can become faster when a muscle is activated repeatedly. This effect is readily seen during repeated twitch and other submaximal tetanic contractions (Close and Hoh, 1968; Krarup, 1981; Smith et al., 2014; Vandenboom et al., 1993, 1997), as well as during the course of a prolonged unfused tetanic contraction (Burke et al., 1973; Smith et al., 2016). Reports are mixed regarding whether relaxation from high force tetanic contractions can be accelerated with repeated contractions (Nocella et al., 2011; Vandenboom et al., 1993, 1997). Mechanistically, we have demonstrated that the accelerated relaxation cannot be explained by faster calcium removal from the cytosol (Smith et al., 2013, 2014). Instead, this phenomenon might be due to a contraction-induced accumulation of inorganic phosphate (P_i) (Nocella et al., 2011; Smith et al., 2016). While this theory is consistent with the enhancements in relaxation rates seen with increasing levels of P_i concentration in maximally activated mammalian skinned fibre preparations (Luo et al., 2002; Tesi et al., 2002), there is no direct evidence to support a causal relationship.

The force declines during relaxation from high force contractions are characterized by a slow, almost linear tension decline followed by a fast exponential tension decline. The transition between these two relaxation phases is referred to as the relaxation shoulder (Cleworth and Edman, 1972; Huxley and Simmons, 1970). Distinct relaxation phases are frequently absent during relaxation from low force contractions. In skinned mammalian muscle preparations, an increase in P_i concentration has been shown to affect the slow linear phase of relaxation from high force contractions by increasing the rate of tension decline and abbreviating its duration at 5°C (Tesi et al., 2002) and 15°C (Luo et al., 2002; Tesi et al., 2002). The effects of P_i on the fast phase of relaxation in mammalian skeletal muscle may be temperature dependent, with no effect seen at 5°C (Tesi et al., 2002) and an increased rate of relaxation at 15°C when P_i is elevated (Luo et al., 2002; Tesi et al., 2002). The effects of P_i on high force contractions in intact muscle preparations can be examined by comparing a well-rested muscle with a muscle in the early stages of contraction-induced fatigue, where P_i changes are large and Ca²⁺ and pH effects are negligible (discussed in Nocella et al., 2017). The lone study to demonstrate that relaxation from high force contractions in intact muscle is enhanced with contractile activity (Nocella et al., 2011) did not distinguish between the slow and fast relaxation phases, and was performed at room temperature. How P_i influences the fast and slow phases of relaxation has not been studied in intact muscle, and the effects of P_i on relaxation from high force contractions at physiological temperatures are not known. The temperature effect is important to distinguish because the force-depressing effects of P_i are greatly diminished as temperature is increased (Coupland et al., 2001; Debold et al., 2004), and the effects of P_i on relaxation may exhibit a similar temperature dependency. The purpose of the present study was to determine the effects of contractile activity on the fast and slow phases of relaxation from high force contractions in mouse lumbrical muscle at physiological temperature. It was hypothesized that the rate of relaxation would be increased following contractile activity for both the fast and slow phases of relaxation, and that contractile activity would shorten the duration of the slow phase of relaxation.

Experimental protocols were approved by the University of Waterloo Animal Care Committee, and all experiments were performed in the laboratory of A.R.T. at the University of Waterloo. Lumbrical muscles were isolated from the second digit of the hind feet of 3- to 6-month-old male C57BL/6 mice (n=4) as described previously (Smith et al., 2013, 2014). Dissection was performed in ice-cold Tyrode's dissecting solution containing (in mmol l⁻¹): 136.5 NaCl, 5.0 KCl, 11.9 NaHCO₃, 1.8 CaCl₂, 0.40 NaH₂PO₄, 0.10 EDTA and 0.50 MgCl₂, pH 7.5. Muscles were mounted between a model 322C high-speed length controller (Aurora Scientific, Aurora, ON, Canada) and a model 400A force transducer (Aurora Scientific), affixed using four surgeon's knots on each tendon with 10-0 monofilament nylon thread. Muscles were immersed in a 37°C bath containing oxygenated (95% O₂, 5% CO₂) Tyrode's experimental solution containing (in mmol l⁻¹): 121.0 NaCl, 5.0 KCl, 24.0 NaHCO₃, 1.8 CaCl₂, 0.40 NaH₂PO₄, 5.50 glucose, 0.10 EDTA and 0.50 MgCl₂, pH 7.3. The lumbrical muscle was chosen because it does not exhibit myosin phosphorylation-mediated force potentiation (Smith et al., 2013). In addition to potentiating submaximal force, phosphorylation of the myosin regulatory light chain may slow the linear phase of relaxation from high force or maximal contractions (Brown and Loeb, 1999; Gittings et al., 2011; Patel et al., 1998). Thus, stimulation-induced elevations in myosin phosphorylation may have prevented an enhanced relaxation from high force from being observed in previous studies using mouse extensor digitorum longus muscle (Vandenboom et al., 1993, 1997).

At the onset of each experiment, muscles were allowed to rest for 20 min. Muscles were then stimulated via flanking platinum plate field stimulus electrodes using a model 701C stimulator (Aurora Scientific) at supramaximal voltage. Muscles were slowly stretched from just slack length and periodically stimulated until optimum length for twitch tension was found. Next, a tetanic contraction was evoked at 200 Hz for 300 ms to take up any slack in the mechanical system. Muscles then rested for 5 min, optimum length was reaffirmed, and the muscles rested again for 5 min before beginning experimental testing. Because the changes in relaxation rate occur quickly, and relaxation rate can slow when stimulation is applied for a long period of time (Smith et al., 2014), our test contractions were evoked at 500 Hz for 20 ms (pulse width 0.2 ms) to increase force rapidly and create a very short contraction. These test contractions were applied 30 s prior to (pre) and 7 s after (post) a 2.5 s 20 Hz intervening contraction (pulse width 0.2 ms). The 2.5 s 20 Hz stimulation protocol and 7 s wait period were chosen because this combination has previously been shown to maximize the increases in relaxation rate of twitch contractions in mouse lumbrical muscle at 37°C (Smith et al., 2013). A set of control experiments was performed on these muscles in which twitch contractions were evoked 30 s prior to and 7 s after a 2.5 s 20 Hz intervening contraction 39.5 s apart with no intervening contraction. Analogue tension signals were digitized and collected at 10 kHz using ASI 600A software (Aurora Scientific) and stored for later analysis.

Measures of interest pertaining to the tension rise included the peak tension (P₀), time to P₀ and peak rate of tension production. Measures of interest during relaxation included the rate of tension decline during the fast and slow phases of relaxation, and the duration of the slow phase of relaxation. The rates were measured relative to P₀ in each condition as done in Tesi et al. (2002). P₀ was measured as the highest tension achieved during the contraction minus the baseline tension of the relaxed muscle prior to contraction. The time to P₀ was measured as the time between the onset of stimulation and P₀. The peak rate of tension production and peak rate of relaxation in the fast phase were determined from the first derivative of the tension–time record. The rate of relaxation in the slow phase was taken between 98% P₀ and 88% P₀. The duration of the slow phase of relaxation was measured between P₀ and the relaxation shoulder. The relaxation shoulder could be consistently identified as a local minimum in the second derivative of the tension–time record following the application of a low-pass digital filter with a cut-off frequency of 50 Hz. Pre–post differences were assessed using one-tailed paired Student's t-tests, with differences considered significant at α=0.05.

Representative traces of pre and post tetanic contractions are shown in Fig. 1. Relative to pre contractions, post contractions exhibited a small decrease in P₀ but also an increased peak rate of tension development and lower time to P₀ (all P<0.05; see Fig. 2). This combination of results is consistent with results from studies on intact mouse fibers examining the early effects of fatigue (Allen et al., 2008; Jones, 1996; Westerblad and Allen, 1991; Westerblad and Lännergren, 1991), with the small decline in P₀ being attributable to a metabolically mediated reduction in actomyosin complex function (Cooke and Pate, 1985; Coupland et al., 2001; Debold et al., 2004; Edman and Lou, 1990, 1992; Nocella et al., 2011, 2017). The faster rate of tension development is consistent with findings from earlier studies examining mouse lumbrical (Smith et al., 2013, 2014) and mouse flexor digitorum brevis bundles (Nocella et al., 2011, 2017). This effect may be due to the demonstrated effects of P_i on cross-bridge kinetics in permeabilized muscle preparations (Hibberd et al., 1985; Millar and Homsher, 1990; Takagi et al., 2004; Tesi et al., 2000). The faster time to P₀ may be the product of both the decrease in P₀ and the faster rate of tension development and is consistent with earlier studies examining contractions with enhanced relaxation properties (Close and Hoh, 1968; Krarup, 1981; Nocella et al., 2011; Smith et al., 2013, 2014).

In accordance with our hypothesis, the duration of the slow relaxation phase was lower at post than at pre contraction, and the rates of relaxation during both the fast and slow phases of relaxation were increased (all P<0.05; see Fig. 2). Our finding that contractile activity enhances relaxation rates from high force contractions is consistent with the findings of Nocella et al. (2011) but not Vandenboom et al. (1993, 1997). Our findings are also generally consistent with the effects of P_i on the phases of relaxation in highly activated skinned muscle preparations at 15°C (Luo et al., 2002; Tesi et al., 2002). However, we report magnitudes of change in relaxation parameters much lower than the effects seen in previous studies (Luo et al., 2002; Nocella et al., 2011; Tesi et al., 2002), which is likely to be largely due to the lower temperatures used in the earlier studies, though the differences in muscle, animal, preparation type and experimental protocol would certainly also impact the results. How temperature impacts the P_i-mediated changes in relaxation parameters has not been systematically tested. However, repeated twitch contractions show greater reductions in 50% relaxation time as temperature is decreased (Krarup, 1981; Smith et al., 2014), suggesting a temperature dependency of the effects of P_i on relaxation. However, low force contractions have temperature-dependent contraction-induced changes in activating calcium levels (Smith et al., 2014), which could confound the interpretations. Future studies should examine in detail how P_i influences relaxation at different temperatures.

Similar to our previous work (Smith et al., 2013), we found that twitches evoked 7 s after the intervening contraction had 22% higher peak force, 36% faster rates of force production, 37% faster rates of relaxation and 11% lower full duration at 50% maximum (all P<0.01) than twitches evoked 30 s prior to the intervening contraction (not depicted). Fast and slow phases of relaxation were not apparent in the twitch contractions. Twitch contractions evoked 39.5 s apart with no intervening contraction were not significantly different from each other. No significant differences were observed in the contractile characteristics of the intervening contractions that were preceded by a tetanus versus those preceded by a twitch contraction (Fig. S1, Table S1), suggesting that the differences in contractile characteristics we observed were caused by the intervening contraction and not the initial contraction.

In summary, we have shown that the contraction-induced enhancement of relaxation from high force contraction influences both the fast and slow phases of relaxation in mouse lumbrical muscle at 37°C. The changes in relaxation parameters we observed are similar to the known effects of P_i on muscle relaxation, and are likely to be caused by contraction-induced P_i accumulation. However, direct causal evidence linking changes in P_i to the changes in relaxation parameters in intact muscle is lacking and the precise influence of P_i on cross-bridge cycling is not fully clear, particularly in intact muscle preparations.