Table1_Moderate muscle cooling induced by single and intermittent/prolonged cold-water immersions differently affects muscle contractile function in young males.docx

Background: We investigated the impact of moderate muscle cooling induced by single and intermittent/prolonged cold-water immersions (CWI) on muscle force and contractility in unfatigued state and during the development of fatigue resulting from electrically induced contractions.Methods: Twelve young males participated in this study consisting of two phases [single phase (SP) followed by intermittent/prolonged phase (IPP)], with both phases including two conditions (i.e., four trials in total) performed randomly: control passive sitting (CON) and cold-water immersions (10°C). SP-CWI included one 45 min-bath (from 15 to 60 min). IPP-CWI included three baths (45 min-bath from 15 to 60 min, and 15 min-baths from 165 to 180 min and from 255 to 270 min), with participants sitting at room temperature the rest of the time until 300 min. Blood pressure and intramuscular (Tmu) temperature were assessed, and neuromuscular testing was performed at baseline and 60 min after baseline during SP, and at baseline, 60, 90, 150 and 300 min after baseline during IPP. A fatiguing protocol (100 electrical stimulations) was performed after the last neuromuscular testing of each trial.Results: In unfatigued state, SP-CWI and IPP-CWI reduced electrically induced torque at 100 Hz (P100) but not at 20 Hz (P20), and increased P20/P100 ratio. The changes from baseline for P100 and P20/P100 ratio were lower in IPP-CWI than SP-CWI. Both cold-water immersion conditions slowed down muscle contraction and relaxation, and reduced maximal isokinetic contraction torque, but the changes from baseline were lower after IPP-CWI than SP-CWI. cold-water immersions did not impair maximal voluntary isometric contraction. During the fatiguing protocol, torque fatigue index and the changes in muscle contractile properties were larger after IPP-CWI than SP-CWI, but were in the same range as after CON conditions. The differences of muscle contractile function between SP-CWI and IPP-CWI were accompanied by a lower reduction of superficial Tmu and a smaller increase in systolic blood pressure after IPP-CWI than SP-CWI.Conclusion: IPP-CWI induces a less pronounced fast-to-slow contractile transition compared to SP-CWI, and this may result from the reduced vasoconstriction response and enhanced blood perfusion of the superficial muscle vessels, which could ultimately limit the reduction of superficial Tmu.</p

Image1_Moderate muscle cooling induced by single and intermittent/prolonged cold-water immersions differently affects muscle contractile function in young males.tif

Background: We investigated the impact of moderate muscle cooling induced by single and intermittent/prolonged cold-water immersions (CWI) on muscle force and contractility in unfatigued state and during the development of fatigue resulting from electrically induced contractions.Methods: Twelve young males participated in this study consisting of two phases [single phase (SP) followed by intermittent/prolonged phase (IPP)], with both phases including two conditions (i.e., four trials in total) performed randomly: control passive sitting (CON) and cold-water immersions (10°C). SP-CWI included one 45 min-bath (from 15 to 60 min). IPP-CWI included three baths (45 min-bath from 15 to 60 min, and 15 min-baths from 165 to 180 min and from 255 to 270 min), with participants sitting at room temperature the rest of the time until 300 min. Blood pressure and intramuscular (Tmu) temperature were assessed, and neuromuscular testing was performed at baseline and 60 min after baseline during SP, and at baseline, 60, 90, 150 and 300 min after baseline during IPP. A fatiguing protocol (100 electrical stimulations) was performed after the last neuromuscular testing of each trial.Results: In unfatigued state, SP-CWI and IPP-CWI reduced electrically induced torque at 100 Hz (P100) but not at 20 Hz (P20), and increased P20/P100 ratio. The changes from baseline for P100 and P20/P100 ratio were lower in IPP-CWI than SP-CWI. Both cold-water immersion conditions slowed down muscle contraction and relaxation, and reduced maximal isokinetic contraction torque, but the changes from baseline were lower after IPP-CWI than SP-CWI. cold-water immersions did not impair maximal voluntary isometric contraction. During the fatiguing protocol, torque fatigue index and the changes in muscle contractile properties were larger after IPP-CWI than SP-CWI, but were in the same range as after CON conditions. The differences of muscle contractile function between SP-CWI and IPP-CWI were accompanied by a lower reduction of superficial Tmu and a smaller increase in systolic blood pressure after IPP-CWI than SP-CWI.Conclusion: IPP-CWI induces a less pronounced fast-to-slow contractile transition compared to SP-CWI, and this may result from the reduced vasoconstriction response and enhanced blood perfusion of the superficial muscle vessels, which could ultimately limit the reduction of superficial Tmu.</p

Image1_Passive heating-induced changes in muscle contractile function are not further augmented by prolonged exposure in young males experiencing moderate thermal stress.tif

Background: We investigated the impact of 1) passive heating (PH) induced by single and intermittent/prolonged hot-water immersion (HWI) and 2) the duration of PH, on muscle contractile function under the unfatigued state, and during the development of muscle fatigue.Methods: Twelve young males volunteered for this study consisting of two phases: single phase (SP) followed by intermittent/prolonged phase (IPP), with both phases including two conditions (i.e., four trials in total) performed randomly: control passive sitting (CON) and HWI (44–45°C; water up to the waist level). SP-HWI included one continuous 45-min bath (from 15 to 60 min). IPP-HWI included an initial 45-min bath (from 15 to 60 min) followed by eight additional 15-min baths interspaced with 15-min breaks at room temperature between 75 and 300 min. Intramuscular (Tmu; measured in the vastus lateralis muscle) and rectal (Trec) temperatures were determined. Neuromuscular testing (performed in the knee extensors and flexors) was performed at baseline and 60 min later during SP, and at baseline, 60, 90, 150 and 300 min after baseline during IPP. A fatiguing protocol (100 electrical stimulations of the knee extensors) was performed after the last neuromuscular testing of each trial.Results: HWI increased Tmu and Trec to 38°C–38.5°C (p Conclusion: PH induces changes in muscle contractile function which are not augmented by prolonged exposure when thermal stress is moderate.</p

Participant characteristics by group.

<p>All values are mean ± SD. With the exception of age, all measures are significantly different by ANOVA (p < 0.05).</p><p>* significantly different from CON by t-test;</p><p>^† significantly different from STR by t-test (p < 0.05). VO₂ max (mL·kg^-0.75·min^-1) has been included for comparison to other studies.</p><p>Participant characteristics by group.</p

Cell signalling proteins in Con shRNA and Cs shRNA myotubes.

<p>The protein levels were was assayed by immunoblotting and the blots were analyzed using ImageJ software. The ratios of phosphorylated to total protein were calculated for AMPK (A) and ACC (B), MAPK p38 (C) and mTOR (D) for Con shRNA cells (n = 8) and Cs shRNA (n = 8) cells. Values are means ± SEM.</p

Cellular metabolism in Con shRNA and Cs shRNA myotubes.

<p>A, Oxygen consumption rate and (B) proton production rate were assessed using Seahorse Bioscience XF24-3 Extracellular Flux Analyser; C, Basal respiration, maximal respiration and spare respiratory capacity; D, Proton leak, aerobic ATP production and non-mitochondrial respiration. Values are means ± SEM (n = 10 each); *P < 0.05, ** <i>p</i> < 0.01 different between Con shRNA and Cs shRNA myotubes.</p

Palmitate metabolism in C2C12 muscle cells.

<p>The cells were treated with Con shRNA or Cs shRNA and then incubated in the differentiation media containing 5.5 mM glucose (G) and/or 0.8 mM palmitate for 2 h (P). A, palmitate oxidation was assessed by adding [1-¹⁴C]palmitate to the media and measuring its incorporation into CO₂ (n = 6); B, palmitate incorporation was measured as from the amount of [1-¹⁴C]palmitate cell lysates generated from the cells after the experiments with palmitate oxidation (n = 6). Results are means ± SEM; * <i>p</i> < 0.05 between Cs shRNA and Con shRNA cells.</p

Relative expression levels of miR-222 (A), -21 (B), -146a (C), and -221 (D) in STR, CON and END.

<p>Box plots depict the range (upper and lower whiskers), median (centre line) and interquartile range (edge of boxes). * significantly different between all groups (One way ANOVA; p < 0.05); † significantly different from STR (t-test; p < 0.05).</p

Citrate synthase (CS) activity in mouse tissues.

<p>Data are shown for the heart (A), liver (B) and gastrocnemius (C) muscle of BALB, C57BL/6J (B6), offspring of the cross between B6 and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6/B6.A) mice, congenic B6.A and A/J mice, respectively. CS activity and protein levels were measured using spectrophotometric assays. Data are shown as mean ± S.E. Numbers of samples are indicated below in brackets. ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 are different to BALB and B6 strains, respectively; ## <i>p</i> < 0.01 different to B6/B6.A mice, respectively.</p

Citrate synthase (CS) activity, mitochondrial markers and proliferation of Con shRNA and Cs shRNA cells.

<p>A, citrate synthase (CS) activity; B, CS protein levels; C, HAD activity; D, cytochrome C oxidase (COX) activity; E, proliferation rate as reflected in cell impedance index of growing cells. CS and HAD activity (n = 9 each) was measured using a spectrophotometric assays. COX activity (n = 6) was assessed using COX assay kit; F, Citrate accumulation was measured using the standard assay kit. Values for CS, HAD, COX, and citrate are shown as mean ± SEM. Cell impedance index was assessed in cells incubated in growth media and differentiation media (n = 4). Cells were grown in 6 well plates and the media was changed every day.</p