Attenuated Fatigue in Slow Twitch Skeletal Muscle during Isotonic Exercise in Rats with Chronic Heart Failure

During isometric contractions, slow twitch soleus muscles (SOL) from rats with chronic heart failure (chf) are more fatigable than those of sham animals. However, a muscle normally shortens during activity and fatigue development is highly task dependent. Therefore, we examined the development of skeletal muscle fatigue during shortening (isotonic) contractions in chf and sham-operated rats. Six weeks following coronary artery ligation, infarcted animals were classified as failing (chf) if left ventricle end diastolic pressure was >15mmHg. During isoflurane anaesthesia, SOL with intact blood supply was stimulated (1s on 1s off) at 30Hz for 15 min and allowed to shorten isotonically against a constant afterload. Muscle temperature was maintained at 37°C. In resting muscle, maximum isometric force (Fmax) and the concentrations of ATP and CrP were not different in the two groups. During stimulation, Fmax and the concentrations declined in parallel sham and chf. Fatigue, which was evident as reduced shortening during stimulation, was also not different in the two groups. The isometric force decline was fitted to a bi-exponential decay equation. Both time constants increased transiently and returned to initial values after approximately 200 s of the fatigue protocol. This resulted in a transient rise in baseline tension between stimulations, although this effect which was less prominent in chf than sham. Myosin light chain 2s phosphorylation declined in both groups after 100 s of isotonic contractions, and remained at this level throughout 15 min of stimulation. In spite of higher energy demand during isotonic than isometric contractions, both shortening capacity and rate of isometric force decline were as well or better preserved in fatigued SOL from chf rats than in sham. This observation is in striking contrast to previous reports which have employed isometric contractions to induce fatigue

Multiple Causes of Fatigue during Shortening Contractions in Rat Slow Twitch Skeletal Muscle

Fatigue in muscles that shorten might have other causes than fatigue during isometric contractions, since both cross-bridge cycling and energy demand are different in the two exercise modes. While isometric contractions are extensively studied, the causes of fatigue in shortening contractions are poorly mapped. Here, we investigate fatigue mechanisms during shortening contractions in slow twitch skeletal muscle in near physiological conditions. Fatigue was induced in rat soleus muscles with maintained blood supply by in situ shortening contractions at 37°C. Muscles were stimulated repeatedly (1 s on/off at 30 Hz) for 15 min against a constant load, allowing the muscle to shorten and perform work. Fatigue and subsequent recovery was examined at 20 s, 100 s and 15 min exercise. The effects of prior exercise were investigated in a second exercise bout. Fatigue developed in three distinct phases. During the first 20 s the regulatory protein Myosin Light Chain-2 (slow isoform, MLC-2s) was rapidly dephosphorylated in parallel with reduced rate of force development and reduced shortening. In the second phase there was degradation of high-energy phosphates and accumulation of lactate, and these changes were related to slowing of muscle relengthening and relaxation, culminating at 100 s exercise. Slowing of relaxation was also associated with increased leak of calcium from the SR. During the third phase of exercise there was restoration of high-energy phosphates and elimination of lactate, and the slowing of relaxation disappeared, whereas dephosphorylation of MLC-2s and reduced shortening prevailed. Prior exercise improved relaxation parameters in a subsequent exercise bout, and we propose that this effect is a result of less accumulation of lactate due to more rapid onset of oxidative metabolism. The correlation between dephosphorylation of MLC-2s and reduced shortening was confirmed in various experimental settings, and we suggest MLC-2s as an important regulator of muscle shortening. Copyright 2013 Hortemo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License

Time course of fatigue and recovery during 20 s and 100 s exercise.

<p>The panels show development of contraction (panels <i>A</i>, <i>B</i> and <i>C</i>) and relaxation (panels <i>D</i>, <i>E</i> and <i>F</i>) parameters during fatigue development (black symbols, solid line) and recovery (white symbols, dotted line). Exercise times are given at bottom x-axis and recovery times at top x-axis. <i>A)</i> Maximal rate of isometric force development, dF/dt. <i>B</i>) Maximal isotonic shortening velocity, dL/dt. C) Maximal shortening, S_max. D) Maximal isotonic relengthening velocity, −dL/dt. E) Maximal isometric relaxation rate, −dF/dt. <i>F</i>) Time to resting length, TTL₀ (squares) and tau2 (circles). Symbols are averages ± SEM. * p<0.05 <i>vs.</i> initial value. † p<0.05 <i>vs.</i> 20 s. <i>N</i> start<i>24</i>; 20 s = <i>12</i>; 100 s = <i>12</i>; 20 s +2.5 min recovery = <i>6</i>; 100 s +15 min recovery = <i>6</i>.</p

Contractile performance in the 1^st bout (open bars) <i>vs.</i> the 2^nd bout (black bars).

<p>Contraction (panels <i>A</i>, <i>B</i> and <i>C</i>) and relaxation (panels <i>D</i>, <i>E</i> and <i>F</i>) parameters at start (0 s), at 100 s and at 900 s of shortening contractions. <i>A</i>) Maximal rate of isometric force development, dF/dt. <i>B</i>) Maximal isotonic shortening velocity, dL/dt. <i>C</i>) Maximal shortening, S_max. <i>D</i>) Maximal isotonic relengthening velocity, −dL/dt. <i>E</i>) Maximal isometric relaxation rate, −dF/dt. <i>F</i>) Tau2 values. Bars are averages ± SEM. * p<0.05 <i>vs.</i> initial value. # p<0.05 <i>vs.</i> corresponding 1^st bout value. † p<0.05 <i>vs.</i> 100 s. <i>N</i> start 1^st = <i>20</i>; 100 s 1^st = <i>12</i>; 15 min 1^st = <i>6</i>; start 2^nd = <i>12</i>; 100 s 2^nd = <i>20</i>; 15 min 2^nd = <i>6</i>.</p

Isometric relaxation and lactate.

<p>Isometric relaxation rate (−dF/dt) was strongly correlated to muscle lactate throughout the exercise protocols. Data are obtained from all measured time points in the 1^st bout (black), after recovery (grey) and in the 2^nd bout (white), presented as a linear regression (r² = 0.81, p<0,01) based on group means ± SEM.</p

Effects of afterload and stimulation frequency on dephosphorylation of MLC-2s.

<p>Panel <i>A</i> and <i>B</i>: The effects of altered afterload of the exercising muscle. Stimulation frequency was 30 Hz as in the standard protocols. <i>A</i>) Shortening (S_max) at start was strongly correlated to the pre-set afterload (r² = 0.99). <i>B</i>) The phosphorylation level of MLC-2s (white bars) relative to resting control (100%) and the corresponding S_max (black bars) at 15 min exercise. Afterload was set to 10, 20 or 33% of F_max. Panel <i>C</i> and <i>D:</i> The effects of altered muscle stimulation frequency. Afterload was 33% of F_max as in the standard protocols. <i>C</i>) S_max (black circles) and force development (grey squares) in the unfatigued muscle at various stimulation frequencies. <i>D</i>) The phosphorylation level of MLC-2s (white bars) and S_max (black bars) at 15 min exercise with stimulation frequency 40, 30 or 20 Hz. Symbols are averages ± SEM. *p<0.05 <i>vs.</i> 33% afterload. †p<0.05 <i>vs.</i> 30 Hz. <i>N</i> 10% afterload = <i>6</i>; 20% afterload = <i>5</i>; 33% afterload = <i>8</i>; 20 Hz = <i>6</i>; 40 Hz = <i>6</i>.</p

Metabolites in soleus muscle at rest (Ctr) and at different exercise and recovery times.

<p>Values are in mmol kg wet weight^-1, average ± SEM.</p>*<p>p<0.05 <i>vs.</i> control,</p>†<p>p<0.05 <i>vs.</i> 20 s exercise,</p>#<p>p<0.05 <i>vs</i>. 100 s exercise in the 1^st bout. <i>Rec</i>. recovery.</p>1<p>i.e. at start of the 2^nd bout.</p

Definitions of calculated contractile parameters.

<p>Definitions of calculated contractile parameters.</p

Overview of the three experimental protocols.

<p>Intermittent stimulation at 30 Hz for 1 s every 2 s. <i>A</i>) 20 s exercise (stimulation) followed by 2.5 min rest. <i>B</i>) 100 s exercise followed by 15 min rest. <i>C</i>) 15 min exercise (1^st bout) followed by 15 min rest before initiating another 15 min exercise (2^nd bout). In all protocols, muscles were harvested (arrows) at start and at end of exercise, as well as after rest. In <i>C</i>, there was an additional harvesting point at 100 s both in the 1^st and 2^nd bout.</p

Dephosphorylation of MLC-2s.

<p><i>A</i>) Immunoblot of the extensor digitorum longus (EDL) and soleus (SOL) muscle probed with monoclonal MLC-2 antibody. <i>B</i>) Gel showing myofibrillar proteins from SOL stained with ProQ Diamond for phosphorylated proteins. <i>C</i>) The same gel as in <i>B</i> stained with Sypro Ruby for total proteins. The phosphorylation level of MLC-2s was normalized to protein content of MLC-2s in individual muscles by dividing the staining intensity reflecting phosphoryration level of the MLC-2s (ProQ Diamond) by the staining intensity of the MLC-2s protein band (Sypro Ruby). This normalized phosphorylation level was then calculated relative to the resting, contralateral control muscle. <i>D</i>) Immunoblot of SOL probed with MLC-2 pSer18 confirmed the same phosphorylation pattern as seen with ProQ Diamond gel stain. <i>E</i>) MLC-2s phosphorylation in exercised SOL after different exercise durations (black bars) and after respective recovery periods (grey bars) in the 1^st bout, relative to the resting control muscle (100%, white bar). Bars are averages ± SEM. <i>F</i>) MLC-2s phosphorylation plotted against maximal shortening at all measured time points in the 1^st bout (black), after recovery (grey) and in the 2^nd bout (white). Symbols are group means ± SEM. *p<0.05 <i>vs.</i> control. <i>N,</i> 20 s = <i>6</i>; 20 s+ recovery = <i>7</i>; 100 s = <i>15</i>; 100 s+recovery = <i>6</i>; 15 min = <i>8</i>; 15 min+recovery = <i>6</i>. 100 s 2^nd bout = <i>6</i>; 15 min 2^nd bout = <i>7</i>.</p