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

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

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

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

Expanded view of representative tracings at selected time points in the 1^st and 2^nd bout.

<p>Shortening (upper panels) and force (lower panels) from a representative tracing at different time points in the 1^st (black) and 2^nd (grey) exercise bout. <i>A</i>) The initial exercise cycle. <i>B</i>) At 100 s of exercise. <i>C</i>) At 15 min exercise.</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

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

Definitions of calculated contractile parameters.

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

SR calcium leak.

<p><i>A)</i> SR calcium leak after 20 s and 100 s exercise and after recovery from 20 s and 100 s exercise (2.5 min and 15 min recovery, respectively). <i>B)</i> Individual SR calcium leak values from start, 20 s and 100 s time points plotted against corresponding tau2 values (r² = 0.55, p<0.01). Bars are means ± SEM (<i>N</i> Ctr = <i>8</i>; 20 s = <i>4</i>; 20+ rec = <i>4</i>; 100 s = <i>6</i>; 100 s+rec = <i>4</i>).*p<0.05 <i>vs.</i> control.</p