Ajpcell 00008 2019

1 Mild hypothermia affects the morphology and impairs glutamine-
2 induced anabolic response in human primary myotubes

3
4 Robert Rantala1 and Thomas Chaillou1

5
1
6 Department of Health Sciences, Örebro University, Örebro, Sweden
7
8
9 Running head: Effect of mild hypothermia on human myotube growth
10
11 Address for correspondence:
12 Thomas Chaillou,
13 Örebro University, Department of Health Sciences, Örebro, Sweden
14 E-mail: thomas.chaillou@oru.se
15
16
17
Downloaded from www.physiology.org/journal/ajpcell at Univ of Louisiana Lafayette (130.070.008.131) on March 28, 2019.
18 ABSTRACT
19 The specific impact of reduced temperature on skeletal muscle adaptation has been
20 poorly investigated. Cold water immersion, one situation leading to decreased
21 skeletal muscle temperature, is commonly proposed to reduce the perception of
22 fatigue and muscle soreness after strenuous exercise. In contrast, it may impair long-
23 term benefits of resistance exercise training on muscle strength and hypertrophy. To
24 date, the physiological factors responsible for this blunted muscle adaptation remain
25 unclear. Here, we used a cell culture model of human primary myotubes to
26 specifically investigate the intrinsic behavior of muscle cells during mild hypothermia
27 (MH). Newly formed myotubes were exposed to either 37°C or 32°C to evaluate the
28 effect of MH on myotube size and morphology, protein synthesis and anabolic
29 signaling. We also compared the glutamine (GLUT)-induced hypertrophic response
30 between myotubes incubated at 32°C or 37°C. We showed that 48 h exposure to MH
31 altered the cellular morphology (greater myotube area, shorter myosegments,
32 myotubes with irregular shape), and impaired GLUT-induced myotube hypertrophy.
33 Moreover, MH specifically reduced protein synthesis at 8 h. This result may be
34 explained by an altered regulation of ribosome biogenesis, as evidenced by a lower
35 expression of 45S pre-rRNA and MYC protein, and a lower total RNA concentration.
36 Furthermore, MH blunted GLUT-induced increase in protein synthesis at 8 h, a
37 finding consistent with an impaired activation of the mechanistic target of rapamycin
38 (mTOR) pathway. In conclusion, this study demonstrates that MH impairs the
39 morphology of human myotubes and alters the hypertrophic response to GLUT.
40
41
42 Key words: mTOR signaling pathway, skeletal muscle hypertrophy, reduced
43 temperature, protein synthesis, ribosome biogenesis
44
45 List of abbreviations
46 2-way RM ANOVA: two-way repeated measures analysis of variance
47 45S pre-rRNA: 45S pre-ribosomal RNA
48 4E-BP1: eukaryotic initiation factor-4E-binding protein-1
49 ACTA1: actin alpha-1
50 COL6A1: collagen, type VI, alpha 1
51 CT: control
52 DAPI: 4’, 6-diamidino-2-phenylindole
53 DES: desmin
54 DMD: dystrophin
55 DMEM: Dulbecco’s Modified Eagle Medium
56 FBS: fetal bovine serum
57 GLUT: glutamine
58 LAMB2: laminin b2
59 MH: mild hypothermia
60 mTOR: mechanistic target of rapamycin
61 MYH1: myosin heavy chain 1
64 NEB: nebulin
65 p70S6K: p70S6 kinase
66 qPCR: quantitative polymerase chain reaction
67 RE: resistance exercise
68 RPLP0: ribosomal protein lateral stalk subunit P0
69 SUnSET: surface sensing of translation
70 TEMP: temperature
71 TTN: titin
72
73
74 INTRODUCTION
75 Homothermal living beings such as humans are able to maintain their core
76 temperature nearly constant in spite of changes in environmental temperatures. Most
77 thermal responses to maintain core temperature in mammal organisms are mediated
78 by central adaptations, including sympathetic output, endocrine secretions,
79 behavioral changes and cardiovascular adjustments (6, 17, 23). In contrast, the
80 temperature of tissues distant from the core body such as peripheral skeletal muscles
81 can considerably vary in response to cold stress (8, 21).
82
83 The impact of reduced temperature on skeletal muscle adaptation is currently not
84 well-established. Cold water immersion is one situation that leads to reduced skeletal
85 muscle temperature in humans (16, 21, 22). This method of cryotherapy has recently
86 attracted increasing attention in recreational and elite athletes due to its potential to
87 improve recovery and reduce fatigue following training and competition (5, 28). It is
88 usually utilized directly after intense exercise, and typically consists in immersing the
89 whole body or a part of it for at least 10 min in water cooler than 15°C (41). This
90 method has been proposed to reduce the perception of fatigue, muscle soreness and
91 edema, as well as to limit inflammatory response and exercise-induced muscle
92 damage following strenuous exercise (4, 15). Despite its popularity among athletes,
93 an increasing number of studies indicates that chronic use of cryotherapy could
94 impair long-term benefits of resistance exercise (RE) training on muscle strength and
95 hypertrophy in human (12, 29, 44). Recently, an acute session of cold water
96 immersion (10 min at 10°C) has been shown to attenuate the increased
97 phosphorylation of p70S6 kinase (p70S6K) after RE (29), providing the first evidence
98 of an impaired activation of the mechanistic target of rapamycin (mTOR) signaling
99 pathway, an anabolic pathway controlling skeletal muscle protein synthesis and
100 hypertrophy (31). In addition, cold water immersion appeared to limit the activation of
101 several transcriptional factors and upstream signaling events associated with
102 ribosome biogenesis after a single bout of RE (11). Ribosome biogenesis constitutes
103 a cellular process recently proposed to regulate protein synthesis and skeletal
104 muscle mass through its control of translational capacity (7, 9, 10, 40, 42). To date,
105 the effect of cryotherapy on the rate of protein synthesis has never been investigated
106 in human skeletal muscles.
107
108 The physiological and/or biological factors responsible for the blunted anabolic
109 response to RE following cryotherapy remain elusive. It has been hypothesized that
110 decreased muscle blood flow consecutive to cold water immersion may impair the
111 delivery of amino acids to skeletal muscle cells, thereby leading to an attenuated
112 activation of anabolic signaling pathways and a reduced rate of muscle protein
113 synthesis after RE (29). Considering that the human organism optimally functions in a
114 rather narrow temperature range, and that the rate of enzymatic processes is
115 generally decreased at lower temperature (37), another hypothesis could be that
116 reducing intramuscular temperature per se could impair skeletal muscle anabolic
117 response.
118
119 In-vitro exploration of primary human myogenic cells provides a relevant approach to
120 study the intrinsic behavior of muscle cells in response to locally reduced
121 temperature, while excluding potential interferences from systemic and central
122 factors. It has been shown that low temperature (30°C) inhibits myogenic
123 differentiation and myotube growth in murine C2C12 cell line (32-34). However, the
124 anabolic response of differentiated human myotubes exposed to mild hypothermia
125 (MH) and subjected to a hypertrophic stimulus has never been investigated. This
126 study was not designed to mimic the physiological conditions observed in response
127 to cold water immersion, which is technically unrealistic in a cell culture model, but
128 rather to evaluate the intrinsic behavior of muscle cells during exposure to mild
129 hypothermia. The aims of this study were to 1) evaluate the effect of MH on human
130 myotube size and morphology, protein synthesis and anabolic signaling, and 2)
131 compare the hypertrophic response to the amino acid glutamine between human
132 myotubes exposed at either 32°C or 37°C. We hypothesized that MH would reduce
133 myotube size and protein synthesis, alter cellular morphology and impair the
134 hypertrophic response to glutamine.
135
136 MATERIAL AND METHODS
137 Ethics approval
138 Informed consent from the subjects was obtained before participation in the study,
139 and all procedures were performed in accordance to the declaration of Helsinki.
140 Ethical approval was given by the regional Ethical review Board in Uppsala (DNR
141 2015/489).
142
143 Isolation and purification of human myoblasts
144 Muscle biopsies were obtained from the mid-portion of the vastus lateralis from 8
145 healthy participants (4 females and 4 males; 32.0 ± 6.6 years old) by a trained
146 medical doctor. Explants were trapped inside a layer (1 ml) containing 6 mg of
147 Matrigel (#354263; Corning B.V, The Netherlands) and migration medium. Migration
148 medium consists in Dulbecco’s Modified Eagle Medium (DMEM; D5671, Merck,
149 Sweden), supplemented with 20% fetal bovine serum (FBS; Hyclone, France), L-
150 glutamine (0,5g/l; G8540, Merck), 0.5% Ultroser G (#15950-017, Pall Corporation,
151 France), 10 mM Hepes (H0887, Merck) and 50 µg/ml gentamycin (G1397, Merck).
152 After 6-12 days, the migrated cells were enzymatically harvested using dispase
153 (#354235, Corning B.V.) and sub-cultured in proliferation medium (i.e. migration
154 medium without Hepes). The harvested cells were purified with magnetic activated
155 cell sorter microbeads (Miltenyi Biotec, Sweden) coupled to an antibody against
156 CD56 (BD 559043, BD Biosciences, France) (36, 38). As previously shown by our
157 research group, this method allows to obtain highly purified myoblasts (>99%) (38).
158 At purification stage, all myoblasts were considered to be at passage number 0. All
159 experiments were performed on cells from passage number 4 to 7.
160
161 Cell culture experiments
162 Myoblasts were seeded at either 5 x 104 cells per dish into collagen type I-coated six-
163 well plate (Corning B.V.) for immunocytochemistry analysis, puromycin incorporation,
164 and gene expression analysis, or 2.5 x 105 cells per 100 mm collagen-coated Petri
165 dish (Corning B.V.) for immunoblotting (mTOR pathway and MYC). The density of
166 cells was similar in both types of dish (~5,000 cells/cm2). Myoblasts were grown in
167 proliferation medium in a humid air incubator containing 5% CO2 at 37°C. At 70-80%
168 confluence, proliferation medium was replaced with differentiation medium containing
169 DMEM (D5546, Merck), 2% FBS and 50 µg/ml gentamycin to induce myogenic
170 differentiation. It is noteworthy that this DMEM did not contain any amino acids. After
171 48 h of differentiation, fused myotubes were subjected to one of the four following
172 conditions to assess the effect of temperature (37°C or 32°C) and its combination
173 with the amino acid glutamine [control condition (CT) or glutamine condition (GLUT)]:
174 37°C CT, 37°C GLUT, 32°C CT and 32°C GLUT. For these experiments, myotubes
175 were incubated in normal differentiation medium or differentiation medium
176 supplemented with the amino acid L-glutamine (0,5g/l; G8540, Merk). We chose this
177 amino acid based on preliminary experiments showing that it acts as a strong
178 anabolic stimulus inducing myotube hypertrophy. Glutamine constitutes the main
179 source of amino acids within the entire free amino acid pool (more than 60%) (3), and
180 thus represents the main bulk of intramuscular free amino acids. In addition,
181 glutamine appears to be a strong anabolic factor promoting the activation of mTOR
182 signaling pathway in vitro (26). A temperature reduction of 5°C was selected in order
183 to induce a mild cold stimulus. This moderate reduction of temperature has been
184 observed in skeletal muscles such as vastus lateralis muscles following cold water
185 immersion (16, 21).
186
187 Cell morphological changes were assessed in myotubes maintained in their
188 respective condition for 48 h. This time point was chosen in order to evaluate the
189 prolonged effect of mild hypothermia and GLUT supplementation on growing
190 myotubes. The molecular response to reduced temperature and GLUT
191 supplementation was determined at 1 h (short duration) and 8 h (moderate duration).
192 These time points were chosen to explore some potential molecular factors
193 responsible for the morphological changes. All the experiments were performed in
194 already formed myotubes in order to focus on myotube growth and limit potential
195 effects of reduced temperature and GLUT on myoblast fusion during the early phase
196 of differentiation.
197
198 Immunohistochemistry
199 Myotubes were fixed in 2% paraformaldehyde and blocked overnight in 0.5% bovine
200 serum albumin (BSA). They were subsequently permeabilized in 0.25% triton X-100,
201 and incubated for 90 minutes at room temperature with an antibody against troponin-
202 T (1:100; mouse monoclonal, T6277, Merck). Following this step, myotubes were
203 incubated for 1 h at room temperature with fluorescent labelled Alexa 488 (1:100;
204 goat anti-mouse, A11001, Invitrogen, USA) secondary antibody. 4',6-diamidino-2-
205 phenylindole (DAPI) (Invitrogen) was used to stain the nuclei. The cells were
206 visualized using a fluorescent microscope (Zeiss Axiovert; Carl Zeiss AG, Germany),
207 and image capturing was performed through the ZEN 2.3 blue edition microscope
208 software (Carl Zeiss AG).
209
210 Analysis of myotube size
211 Myotube size was assessed from the analysis of myotube diameter, length and area.
212 For these analyses, 6 non-overlapping microscopic fields were captured per well (one
213 well per cell culture condition) with a 10x objective. These experiments were carried
214 out blinded by a single scientist. To evaluate myotube diameter, more than 100
215 myotubes in average per condition were included in the analysis. Myotube diameter
216 was determined by calculating the average of three measurements taken along the
217 length of the fusiform portions of the myotubes (the trunks of the myotubes were not
218 included in the analysis). Myotube length was assessed in at least 60 myotubes per
219 condition, by measuring the length of the longest straight axis that could be
220 incorporated within a myotube. Myotube diameter and myotube length were analyzed
221 using ImageJ Software (US National Institutes of Health, USA).
222
223 Myotube area was measured as previously described by Agley and colleagues (1)
224 using Adobe Photoshop version 19.0 (Adobe Systems, USA). Briefly, a color range
225 mask consisting of a representative panel of color tones that were specifically
226 expressed by troponin-T labeled myotubes colors was created to automatically
227 delineate the myotubes. In addition, incoherent edges were manually trimmed using
228 the lasso tool to assure a tight selection. Number of pixels contained within the
229 myotubes were recorded and converted into square micrometers.
230
231 Protein synthesis rate
232 Relative protein synthesis was assessed using the surface sensing of translation
233 (SUnSET) method (13). This method requires the incubation of cells with puromycin
234 (a tyrosol-transfer RNA analog) and subsequent detection of its incorporation with
235 immunoblotting. Following 1 h or 8 h of treatment, puromycin (final concentration:
236 1µg/ml; P9620, Merck) was added to the medium, and the cells were further
237 incubated in their respective treatment condition for an additional 30 min. The cells
238 were then harvested in homogenization buffer for protein extraction.
239
240 Immunoblotting
241 Myotubes were washed in cold PBS and scraped with ice-cold homogenization buffer
242 [50 mM Tris–HCL, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 50 mM
243 NaF, 0.5 mM sodium orthovanadate, 0.5% triton X-100, and a protease inhibitor
244 tablet (Roche Applied Science, Germany)]. After mechanical disruption and
245 sonication, the cell lysates were incubated 30 min on ice before centrifugation. For
246 the measurement of protein synthesis using the SUnSET method, protein
247 homogenates were centrifuged at 500 g for 10 min at 4°C. For the analysis of mTOR
248 pathway and MYC, homogenates were centrifuged at 10,000 g for 10 min at 4°C.
249 Total protein concentration was determined using the BCA protein assay (Thermo
250 Scientific, USA). Equal amounts of total proteins (5-10 µg) from each cell lysate
251 sample prepared in laemmli buffer were heated at 95°C for 5 min. Subsequently, the
252 samples were separated on 8-12% SDS-polyacrylamide gels for 15 min at 50 V,
253 followed by 45-60 min at 200 V. A standardized amount of protein prepared from a
254 pooled sample was also loaded into each gel to serve as an internal standard for
255 comparison between blots. Then, separated proteins were transferred onto
256 nitrocellulose (i.e. for the analysis of mTOR pathway and MYC) or PVDF (i.e. for the
257 SUnSET method) membranes for 60 min at 100 V. The membranes were incubated
258 for 1 h at room temperature with a blocking solution containing 5% non-fat dry milk.
259 The membranes were then incubated for 2 h at room temperature with primary
260 antibodies against puromycin (1:5,000; MABE343, Millipore, USA), MYC (1:1000,
261 ab32072, Abcam, United Kingdom), phosphorylated mTOR (p-mTOR, Ser2448;
262 1:1,000, #5536, Cell Signaling technology, The Netherlands), phosphorylated
263 p70S6K (p-p70S6K, Thr389; 1:1000, #9234, Cell Signaling technology),
264 phosphorylated eukaryotic initiation factor-4E-binding protein-1 (p-4E-BP1, Thr37/46;
265 1:1000, #9459, Cell Signaling technology), and β-actin (1:5,000, #4967, Cell
266 Signaling technology). Subsequently, the membranes were probed with anti-rabbit
267 (1:3,000, #7074) or anti-mouse (1:3,000, #7076) secondary horseradish peroxidase-
268 conjugated antibodies from Cell Signaling technology for 1 h at room temperature.
269 The membranes were treated with chemiluminescent horseradish peroxidase
270 substrate (#926-95000, LI-COR Biosciences, USA), and the bands were visualized
271 using C-DiGit Blot Scanner (LI-COR Biosciences). The membranes were analyzed
272 using UN-SCAN-IT (Silk Scientific, USA). β-actin was used to normalize the levels of
273 phosphorylated proteins and MYC. Coomassie staining was used as a loading
274 control for the assessment of puromycin incorporation.
275
276 Gene expression analysis
277 Total RNA from myotubes was isolated using NucleoSpin® RNA Isolation Kit
278 (Macherey-Nagel, Germany), according to the manufacturer’s directions. Purity and
279 RNA concentration were determined by measuring the absorbance and assessing
280 the ratio at 260/280 nm and 260/230 nm using a photospectometer (NanoDrop 2000;
281 Thermo Scientific). 700 ng of total RNA was reverse transcribed using oligo(dT)
282 primers, random primer mix, 5x iScript select reaction mix, and iScript reverse
283 transcriptase according to manufacturer’s instructions (iScript™ Select cDNA
284 synthesis Kit, Bio-Rad, Sweden). Quantitative polymerase chain reaction (qPCR) was
285 performed with samples loaded in duplicate, by using 5 µl of diluted cDNA (1/20
286 dilution from stock cDNA mixture), 10 µl of iTaq Universal SYBR green super mix
287 (Bio-Rad) and 1 µl (Bio-Rad) or 0.8 µl (Qiagen, Germany) of gene-specific primers to
288 a final volume of 20 µl (list of primers presented in Table 1). qPCR was performed
289 using a Rotor Gene Q (Qiagen) for 40 cycles (95°C for 5 s and 60°C for 30 s)
290 followed by melting curve analysis. Threshold cycles (Ct) were determined with the
291 Rotor-Gene Q software (Qiagen) and qPCR efficiency was estimated for each gene
292 by using standard curves obtained from serial dilutions of a pooled sample. qPCR
293 data were obtained using relative quantification with the reference gene RPLP0
294 (ribosomal protein lateral stalk subunit P0), after correction for qPCR efficiencies
295 (27). We chose this reference gene because RPLP0 mRNA levels were not affected
296 by our experimental conditions (data not shown).
297
298 Statistical analysis
299 Data were obtained from myotubes derived from the muscle biopsies of 8 participants
300 (8 biological replicates) and are expressed as mean ± SD. For each specific
301 experiment, cells from each biological replicate were simultaneously seeded in 4
302 wells or dishes (i.e. 4 conditions: 37°C CT, 37°C GLUT, 32°C CT and 32°C GLUT) to
303 investigate the effects of temperature and GLUT. Shapiro-Wilk tests were used to
304 check the normality assumption and skewed data were log transformed. In our
305 experimental design (each biological replicate exposed to 4 conditions), two-way
306 repeated measures analysis of variance (2-way RM ANOVA) was used to assess the
307 effect of GLUT, temperature (TEMP) and GLUT x TEMP interaction. When an
308 interaction was observed, a Fisher’s LSD test was used to compare the CT and
309 GLUT conditions. For the analysis of mTOR pathway (Fig.5), 2-way RM ANOVA was
310 used to assess the effect of TEMP, time, and TEMP x time interaction. The alpha
10
311 level of significance was set to P < 0.05. All statistical analyses were performed using
312 Graph-Pad Prism 7.05 (GraphPad Software, USA).
313
314 RESULTS
315 Myotube morphology and size
316 To investigate the direct effects of MH on myotube morphology and hypertrophy in
317 response to GLUT, differentiated human myotubes were incubated in their respective
318 conditions for 48 h. As illustrated in Fig. 1A, myotube morphology was markedly
319 affected at 32°C. Myotubes exposed to MH contained numerous myosegments of
320 short length, and appeared to be less fusiform and more irregular in their shape, as
321 compared to the myotubes maintained at 37°C. In addition, anti-troponin-T staining
322 was more diffuse and less uniform in myotubes grown at 32°C. A high proportion of
323 short myotubes were observed in the 32°C CT condition (length between 200 and
324 400 µm, Fig 1B), while the long myotubes (> 800 µm) were almost absent in this
325 group. Myotube area was larger at 32°C than 37°C (TEMP effect: P < 0.01; Fig. 1C),
326 while myotube diameter was not affected by MH (Fig. 1D). GLUT induced myotube
327 hypertrophy, as evidenced by an increased myotube area and diameter (GLUT
328 effect: P < 0.05; Fig. 1C-1D). However, the increase in myotube area was greater at
329 37°C than 32°C (+ 40%, P < 0.01 and + 4%, NS, respectively; TEMP x GLUT
330 interaction: P < 0.001). In addition, the proportion of long myotubes (> 800 µm),
331 which was modest in the 37°C GLUT condition, was very low in the 32°C GLUT
332 condition (6% and 2%, respectively; Fig. 1B). Altogether, these results indicated that
333 MH led to important changes in myotube morphology and altered the hypertrophic
334 response to GLUT.
335
336 Expression of genes encoding structural proteins
337 To obtain a more comprehensive understanding of the changes observed in myotube
338 morphology, several genes encoding structural proteins were analyzed using qPCR.
339 This analysis was performed in differentiated myotubes incubated in their respective
340 conditions for 8 h. 5 genes encoding sarcomeric proteins (ACTA1, MYH7, MYH1,
341 MYH2 and TTN) are presented in Fig. 2, and 5 genes encoding proteins expressed in
342 either the cytoskeleton (NEB, DMD, DES) or the extracellular matrix (ECM) (LAMB2,
343 and COL6A1) are shown in Fig. 3. MH markedly decreased the mRNA levels of 3
344 genes encoding myosin heavy chain isoforms (MYH7, MYH1, and MYH2; TEMP
11
345 effect: P < 0.001; Fig. 2A-2B-2C) while it only slightly decreased the mRNA levels of
346 the ECM gene LAMB2 (TEMP effect: P < 0.05; Fig. 3E). A TEMP x GLUT interaction
347 was found for the 3 contractile genes MYH1 (P < 0.05; Fig. 2A), ACTA1 (P < 0.01;
348 Fig. 2D) and TTN (P < 0.05; Fig. 2E), as well as for the 2 ECM genes COL6A1 (P <
349 0.05; Fig. 2D) and LAMB2 (P < 0.05; Fig. 2E). Fisher’s LSD tests indicated that the
350 GLUT-induced increase in mRNA levels was only significant at 37°C for these genes
351 (except for LAMB2 expression that remained unchanged).
352
353 Protein synthesis and mTOR signaling pathway
354 We evaluated the effect of MH on the rate of protein synthesis in human myotubes
355 incubated in CT or GLUT conditions for 1 h and 8 h, by quantifying the level of
356 puromycin incorporation (Fig. 4). MH decreased puromycin incorporation at 8 h
357 (TEMP effect: P < 0.01; Fig. 4B), but not at 1 h (Fig. 4A). Puromycin incorporation
358 was increased by GLUT at 1 h at both 37°C (+53%) and 32°C (+23%). However, the
359 magnitude of these changes was not significantly different. After 8 h, puromycin
360 incorporation was 17% higher in 37°C GLUT condition than 37°C CT condition, while
361 GLUT did not influence puromycin incorporation at 32°C. These results indicated that
362 MH impaired protein synthesis and GLUT-induced increase in protein synthesis at 8
363 h in human myotubes.
364 Then, immunoblotting was used to study the anabolic signaling pathway mTOR in
365 myotubes incubated in CT or GLUT conditions for 1 h and 8 h (Fig. 5). Since 2-way
366 RM ANOVA did not reveal any effect of temperature on the phosphorylation levels of
367 the 3 components of the mTOR pathway (i.e. mTOR, p70S6K and 4E-BP1), we only
368 presented the GLUT-induced changes in phosphorylation levels. GLUT-induced
369 increases in phosphorylation levels of mTOR (Fig.5A), and 4E-BP1 (Fig. 5B) were
370 absent at 32°C (TEMP effect: P < 0.05). In addition, GLUT-induced increases in
371 p70S6K phosphorylation were more pronounced at 37°C than at 32°C (Fig. 5C;
372 TEMP effect: P < 0.05). These data indicated that MH impaired GLUT-induced
373 activation of mTOR signaling pathway in human myotubes.
374
375 Markers of ribosome biogenesis
376 In an effort to better understand the reduced protein synthesis and the blunted
377 GLUT-induced stimulation of protein synthesis observed at 32°C after 8 h, we
378 investigated several markers of ribosome biogenesis in human myotubes at this time
12
379 point (Fig. 6). Total RNA concentration was used as a proxy to evaluate ribosome
380 content since over 80% of all the RNAs in cells is made up of ribosomal RNA (24).
381 45S pre-rRNA levels were determined to assess rDNA transcription via RNA
382 polymerase I and MYC protein levels were analyzed since this protein is a central
383 regulator of ribosome biogenesis (39).
384 MH decreased total RNA concentration (TEMP effect: P < 0.01; Fig. 6A), 45S pre-
385 rRNA levels (TEMP effect: P < 0.05; Fig. 6B) and MYC protein levels (TEMP effect: P
386 < 0.01; Fig. 6C). A TEMP x GLUT interaction was found for total RNA concentration
387 (P < 0.01), 45S pre-rRNA levels (P < 0.05) and MYC protein levels (P < 0.05). GLUT-
388 induced increases in 45S pre-rRNA levels and MYC protein levels were only
389 significant at 37°C, while GLUT-induced changes in total RNA concentration
390 remained marginal at both temperatures.
391
392 DISCUSSION
393 The aims of the present study were to 1) evaluate the effect of MH on human
394 myotube size and morphology, protein synthesis and anabolic signaling, and 2)
395 compare the GLUT-induced hypertrophic response between human myotubes
396 exposed to either 32°C or 37°C. We showed that MH altered myotube morphology
397 and impaired myotube hypertrophy in response to GLUT. MH specifically reduced
398 protein synthesis at 8 h, a result associated with a lower expression of 45S pre-rRNA
399 and MYC protein, and a lower total RNA concentration. Protein synthesis was
400 increased by GLUT at 1 h at both 37°C (+53%) and 32°C (+23%), but the magnitude
401 of these changes was not significantly different. Furthermore, GLUT-induced
402 increase in protein synthesis was only observed in myotubes exposed to 37°C at 8 h.
403 This finding was consistent with a blunted GLUT-mediated activation of the mTOR
404 pathway observed at 32°C.
405
406 A 3°C reduction of culture temperature (i.e. 35°C vs. 38°C) was previously shown to
407 delay the fusion of C2C12 myoblasts into multinucleated myotubes, while a more
408 pronounced reduction of temperature (i.e. 30°C vs. 38°C) completely prevented
409 myotube formation (32). Myogenic fusion of C2C12 myoblasts was also delayed at
410 32°C compared with 37°C, as evidenced by similar stages of differentiation after 120
411 h and 72 h, respectively (19). The originality of the current study was to first
412 differentiate human myoblasts into myotubes during 48 h at 37°C, and then study the
13
413 growth phase of newly formed myotubes during the following 48 h at either 37°C or
414 32°C. We found that myotubes incubated at 32°C contained short myosegments and
415 exhibited irregular outlines and shapes compared with myotubes grown at 37°C.
416 Surprisingly, MH increased the total area covered by myotubes, while our analysis of
417 myotube diameter did not reveal any differences between the temperature conditions.
418 In contrast to the study of Shima et al. that did not observe any changes in the
419 appearance of C2C12 myotubes at lower temperature (i.e. 35°C vs. 38°C) (32), our
420 results indicate that MH alters the morphology and architecture of human myotubes.
421 Severe cold stress (4 and 10°C) has been shown to induce marked changes in the
422 structural architecture of several non-muscle cell lines, while cellular structure was
423 not substantially modified during mild cold stress (27°C and 32°C) (30). Altogether,
424 the cellular architecture of human myotubes seems highly sensitive to mild cold
425 stress. We also observed that three genes encoding myosin heavy chain isoforms
426 (MYH7, MYH1 and MYH2) were downregulated in myotubes grown at 32°C.
427 Meanwhile, except for LAMB2 gene, the levels of cytoskeleton and ECM-related
428 genes were not significantly affected by reduced temperature. An increased
429 expression of genes involved in myofibrillogenesis (such as Myh1 and Myh2) was
430 also observed in C2C12 myotubes exposed to moderate hyperthermia during 120 h
431 (i.e. 39°C vs. 37°C), while a more severe hyperthermia (41°C) decreased (for Myh1)
432 or had no effect (for Myh2) on the expression of these sarcomeric genes (14).
433 Collectively, moderate changes in temperature appear to influence myofibrillogenesis
434 in myotubes, and our results suggest that MH impairs this cellular process, which
435 may partly explain the abnormal morphology and appearance of human myotubes.
436
437 Recently, it was demonstrated that cold water immersion attenuates long-term gains
438 in quadriceps muscle mass in response to strength training (29). Unfortunately, this
439 work did not investigate the physiological factors that could potentially lead to this
440 blunted hypertrophic response, such as low blood perfusion, inflammatory response,
441 changes in endocrine secretions or reduced intramuscular temperature (5). In the
442 present study, human myotubes exposed to control condition or MH were
443 supplemented with GLUT in order to induce hypertrophy. It is obvious that our cell
444 culture model is unable to properly mimic the physiological conditions observed in
445 response to cold water immersion. However, it constitutes a relevant approach to
446 isolate the specific effect of mild hypothermia on muscle hypertrophic response.
14
447 Based on the changes of myotube area and myotube length, we found that GLUT-
448 induced myotube hypertrophy was altered at 32°C compared with 37°C. It is
449 noteworthy that myotube diameter was only increased by ~10% in response to
450 GLUT, a small change certainly explained by the criteria used to perform this analysis
451 (i.e. the exclusion of the central region of myotubes exhibiting a thick and non-
452 fusiform shape, which appears to extensively grow in response to GLUT at 37°C, see
453 Fig.1A). It is therefore not surprising that reduced temperature did not impair GLUT-
454 induced increase in myotube diameter. Another interesting result was that GLUT
455 supplementation upregulated three sarcomeric genes (MYH1, ACTA1 and TTN) in
456 myotubes grown at 37°C, but not at 32°C, suggesting that MH could blunt the GLUT-
457 mediated stimulation of myofibrillogenesis. Thus, our findings provide the first
458 evidence that reduced temperature per se impairs hypertrophy of skeletal muscle
459 cells in response to an anabolic stimulus.
460
461 Non-muscle cell lines exposed to severe hypothermia (20°C) displayed a dramatic
462 decrease in protein synthesis, while MH (32°C) during 6 or 30 h did not substantially
463 affect protein synthesis (30). To our knowledge, this current work offers the first
464 evidence that MH impairs protein synthesis and the increase in protein synthesis in
465 response to a nutrient stimulus (glutamine) in human skeletal muscle cells. These
466 changes were significant at 8 h, but not at 1 h, suggesting that the lower anabolic
467 response to MH observed in both experimental conditions (CT and GLUT) is probably
468 not immediate and seems to require several hours. However, our results do not
469 exclude the possibility that a short exposure to reduced temperature is sufficient to
470 impair protein synthesis several hours later, even when muscle temperature is fully
471 recovered. Two recent studies reported that temperature decreased by approximately
472 5°C during the post-exercise period in the vastus lateralis muscles exposed to cold
473 water immersion (16, 21). This change was maintained during at least 3 h following
474 cold water immersion (16). It is important to highlight that muscle temperature and
475 the temperature changes observed post-immersion vary within the vastus lateralis
476 muscle depending on the measurement site (i.e. how deep the needle thermistor is
477 inserted into the muscle) (21). Although our results cannot be extrapolated to in vivo
478 experiments, it appears safe to conclude that MH alters baseline protein synthesis in
479 skeletal muscle cells, and impairs the stimulation of protein synthesis in response to
480 an anabolic stimulus such as GLUT.
15
481
482 In an effort to understand the mechanism behind the altered anabolic response to
483 mild cold stress, we first evaluated the mTOR signaling pathway. We observed that
484 the phosphorylation levels of mTOR, p70S6K and 4E-BP1 were not directly affected
485 by reduced temperature, suggesting that this signaling pathway is not the main factor
486 responsible for the altered rate of protein synthesis at 8 h in the control condition.
487 However, MH prevented GLUT-induced increases in the phosphorylation levels of
488 mTOR and 4E-BP1, while it attenuated GLUT-induced increase in p70S6K
489 phosphorylation. These findings support the idea that blunted protein synthetic
490 response to GLUT at reduced temperature is partly explained by the altered
491 activation of the mTOR signaling pathway. To our knowledge, the mTOR signaling
492 pathway in conditions of reduced muscle temperature in humans has only been
493 investigated during cold water immersion. Roberts and colleagues found that this
494 cryotherapy method attenuated the increased p70S6K phosphorylation following an
495 acute session of RE, but it did not affect 4E-BP1 phosphorylation (29). In this latter
496 study, RE-induced increase in p70S6K phosphorylation was observed at 2 h and 24
497 h after exercise in the control recovery trial, whereas RE-induced increase in 4E-BP1
498 phosphorylation was only transiently found at 2 h. An early and transient increase in
499 4E-BP1 phosphorylation and a more prolonged increased phosphorylation of p70S6K
500 during post-exercise recovery have been confirmed by others (2). This finding is also
501 consistent with our results, since GLUT-induced increases in p70S6K and 4E-BP1
502 phosphorylation were observed during 8 h, and only during 1 h, respectively. We
503 propose that the temporal changes of 4E-BP1 and p70S6K activities are not identical
504 in response to anabolic stimuli, but both proteins may be involved in the defective
505 GLUT-mediated stimulation in protein synthesis in response to MH at 8 h.
506
507 Changes in ribosome content have been recently proposed to affect translational
508 capacity, thereby modulating protein synthesis (7, 9, 10, 40, 42). Cold water
509 immersion was shown to prevent RE-induced increase in MYC protein levels (a
510 central regulator of ribosome biogenesis) and 45S pre-rRNA expression (a marker of
511 rDNA transcription) (11). This recovery strategy could suppress ribosome biogenesis
512 after RE, which could potentially contribute to the impaired hypertrophic response
513 observed after strength training (29). In our model, we observed that MH reduced the
514 levels of MYC and 45S pre-rRNA, as well as total RNA concentration (a marker of
16
515 ribosome content) at 8 h. This potential reduction of ribosome biogenesis could
516 contribute to the reduced protein synthesis observed at 8 h. Furthermore, we found
517 that 45S pre-rRNA and MYC protein levels increased at 8 h in response to GLUT
518 supplementation, but only at 37°C, suggesting that the stimulation of ribosome
519 biogenesis by GLUT is already blunted by mild temperature stress at 8 h. However,
520 the fact that total RNA concentration is only marginally increased by GLUT at 37°C
521 suggests that translational capacity is likely not substantially increased by GLUT at 8
522 h yet, and may not play a major role in the increased protein synthesis at this time
523 point. Since rDNA transcription is an early event that precedes the accumulation of
524 rRNA during muscle hypertrophy (25, 43), total RNA concentration (and thus rRNA
525 concentration) may increase after a more prolonged incubation time with GLUT.
526 Although it remains to be examined, we hypothesize that MH may blunt long-term
527 GLUT-mediated increase in total RNA concentration, which might ultimately prevent
528 the increased translational capacity and hypertrophic response to GLUT.
529
530 In this study, our experiments were performed on 8 human myogenic cell cultures
531 obtained from 8 individual muscle biopsies (i.e. biologically independent samples). An
532 identical number of myoblasts from each biological sample (myoblasts from the same
533 original dish) were seeded in 4 wells or dishes (i.e. 4 conditions: 37°C CT, 37°C
534 GLUT, 32°C CT and 32°C GLUT) to investigate the effects of temperature and
535 glutamine. 2-way RM ANOVA were used to evaluate the effect of the two treatments
536 (reduced temperature and glutamine supplementation) on each biological sample.
537 This type of statistical analysis (repeated measures or comparison of paired samples)
538 is commonly performed in studies using independent biological samples of human
539 myogenic cells (18, 20, 35, 36) exposed to one or several treatments. In addition, we
540 believed that the use of 8 biological independent samples was relevant to provide
541 biological replication. For this reason, we did not use technical replicates in our study.
542
543 In conclusion, we demonstrate that MH impairs the morphology of human myotubes

544 and alters myotube hypertrophy in response to GLUT supplementation. This study
545 provides the first evidence that reduced temperature per se could reduce protein
546 synthesis in myotubes grown in control condition, and impair GLUT-induced protein
547 synthesis stimulation. This latter result was associated with an altered activation of
17
548 mTOR signaling pathway. Our findings also suggest that MH impairs ribosome
549 biogenesis, which may contribute to the reduced protein synthesis observed in
550 control condition at 8 h, and may potentially affect long-term GLUT-mediated
551 anabolic response. This in vitro study provides new and important insights into how
552 reduced temperature per se could attenuate anabolic response in human muscle
553 cells. It provides additional evidence suggesting that reduced muscle temperature is
554 probably detrimental to muscle hypertrophic response to anabolic stimuli.
555
556 GRANTS
557 No funding was received.
558
559 ACKNOWLEDGMENTS
560 The authors thank I. Sanna for his technical assistance.
561
562 DISCLOSURES
563 No conflicts of interest, financial or otherwise, are declared by the author.
564
18
565 Figure legends
566 Figure 1. Effect of reduced temperature on myotube size and morphology at 48 h. A)
567 Representative images showing troponin T-labelled myotubes. B) Relative frequency
568 of myotube length. C) Myotube area. D) Myotube diameter.
569 Scale bar (A): 100 µm; CT: control; GLUT: glutamine.
570 Histograms represent mean ± SD from myotubes derived from the muscle biopsies of
571 8 participants (8 biological replicates). ** P < 0.01
572
573 Figure 2. mRNA levels of genes encoding sarcomeric proteins (A: MHY1; B: MYH2;
574 C: MYH7; D: ACTA1; E: TTN) at 8 h.
575 MHY1: myosin heavy chain 1; MYH2: myosin heavy chain 2; MYH7: myosin heavy
576 chain 7; ACTA1: actin alpha-1; TTN: titin; CT: control; GLUT: glutamine.
578 8 participants (8 biological replicates). *** P < 0.001; ** P < 0.01
579
580 Figure 3. mRNA levels of genes encoding proteins expressed in the cytoskeleton (A:
581 NEB; B: DMD; C: DES) or the extracellular matrix (D: COL6A1; E: LAMB2) at 8 h.
582 NEB: nebulin; DMD: dystrophin; DES: desmin; COL6A1: collagen, type VI, alpha 1;
583 LAMB2: laminin b2; CT: control; GLUT: glutamine.
585 8 participants (8 biological replicates). * P < 0.05
586
587 Figure 4. Protein synthesis rate assessed by puromycin incorporation in human
588 myotubes after 1 h (A) and 8 h (B). C) Representative image of immunoblot analysis
589 for puromycin followed by Coomassie blue staining.
590 CT: control; GLUT: glutamine.
592 8 participants (8 biological replicates). * P < 0.05
593
594 Figure 5. Changes in phosphorylation levels of mechanistic target of rapamycin
595 (mTOR) (A), eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) (B), and
596 p70S6 kinase (p70S6K) (C) in response to glutamine supplementation after 1 h and 8
597 h. D) Representative images of immunoblot analysis (phosphorylated mTOR,
598 p70S6K, 4E-BP1 and beta-actin)
601 8 participants (8 biological replicates).
19
602 Figure 6. Total RNA concentration (A), levels of 45S pre-rRNA (B), and levels of
603 MYC protein (C) at 8 h. D) Representative images of immunoblot analysis (MYC and
604 beta-actin)
607 8 participants (8 biological replicates). * P < 0.05; ** P < 0.01
608
20
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23
CT GLUT
40
37 C CT
37 C GLUT
37°C
30 32 C CT
32 C GLUT
20
10
32°C
Myotube length (μm)
Myotube diameter (μm)
MYH7 mRNA levels
MYH1 mRNA levels
MYH2 mRNA levels
(Log-transformed)
(Log-transformed)
(Log-transformed)
TEMP x GLUT interaction: P < 0.05
0.5 **
0.0
-0.5
37°C 32°C
DMD mRNA levels
TEMP effect: P < 0.05
2.0
COL6A1 mRNA levels
1.5
1.0
0.5
0.0
37°C 32°C
A C
Puromycin incorporation
250 kDa
Puromycin
50 kDa
250 kDa
Coomassie
50 kDa
B TEMP effect: P < 0.01

32°C CT
37°C GLUT
37°C CT
32°C GLUT
32°C CT
37°C CT
32°C GLUT
37°C GLUT
8h
2.0 *
1.5
1h 8h
1.0
0.5
0.0
37°C 32°C
Fig.4
A B
GLUT-induced changes in p-4E-BP1

GLUT-induced changes in p-mTOR TEMP effect: P < 0.05 TEMP effect: P < 0.05
0.4 0.4
37°C
32°C
0.2 0.2
(Log2 fold-change)
(Log2 fold-change)
0.0 0.0
-0.2 -0.2
-0.4 -0.4
1h 8h 1h 8h
C D
TEMP effect: P < 0.05 p-mTOR 250 kDa
1.0 75 kDa
p-p70S6K
20 kDa
0.8
p-4EBP1
15 kDa
0.6
50 kDa
-actin
0.4
32°C CT
37°C CT
32°C GLUT
32°C CT
37°C GLUT
37°C CT
32°C GLUT
37°C GLUT
0.2
0.0
1h 8h
1h 8h
Fig.5
A B TEMP effect: P < 0.05
*
Total RNA concentration
2.0
1.5
(ng/μl)
1.0
0.5
0.0
37°C 32°C
C D
75 kDa
MYC
-actin
MYC protein levels
37 kDa
37°C CT
37°C GLUT
32°C CT
32°C GLUT
(Log-transformed)
Fig.6
Table 1. List of primers used for qPCR-analysis
Target Description Catalogue number
ACTA1 Actin alpha-1 qHsaCED0005405
MYH7 Myosin heavy chain 7 qHsaCID0011217
TTN Titin qHsaCID0020293
NEB Nebulin qHsaCID0011772
LAMB2 Laminin b2 qHsaCED0056976
DMD Dystrophin qHsaCID0010707
DES Desmin qHsaCED0047087
COL6A1 Collagen, type VI, alpha 1 qHsaCED0045048
45S pre-rRNA 45S pre-ribosomal RNA PPH82089A
RPLP0 Ribosomal protein lateral stalk subunit P0 PPH21138F

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1 Mild hypothermia affects the morphology and impairs glutamine-

2 induced anabolic response in human primary myotubes

4 Robert Rantala1 and Thomas Chaillou1

543 In conclusion, we demonstrate that MH impairs the morphology of human myotubes

Myotube length (μm)

Myotube diameter (μm)

MYH2 mRNA levels

TEMP x GLUT interaction: P < 0.05

B TEMP effect: P < 0.01

GLUT-induced changes in p-4E-BP1

TEMP effect: P < 0.05 p-mTOR 250 kDa

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