Influence of typology on heterogeneous response of two different muscles to hypoxia and 1
pulmonary inflammation. 2
Chabert Clovis1,2, Pison Christophe1,2,3, Schlattner Uwe1,2, and Dubouchaud Hervé1,2
3
4
1 INSERM, U1055, Laboratoire de Bioénergétique Fondamentale et Appliquée, SFR SEM, 5
UFRAPS, F-38000 Grenoble, France 6
2 Université Grenoble Alpes, Laboratoire de Bioénergétique Fondamentale et Appliquée, F- 7
38000 Grenoble, France 8
3 Clinique Universitaire de Pneumologie, Pôle Thorax et Vaisseaux, Centre Hospitalier 9
Universitaire de Grenoble, F-38000 Grenoble, France 10
11
Address for correspondence: 12
H. Dubouchaud, 13
Laboratoire de Bioénergétique Fondamentale et Appliquée, INSERM U1055, Université 14
Grenoble Alpes, F-38000 Grenoble, France. 15
Tel. +33 476 635 736; Fax +33 476 514 218; email: herve.dubouchaud@univ-grenoble-alpes.fr 16
Abstract 18
Rationale 19
The Chronic Obstructive Pulmonary Disease (COPD) is characterized by a chronic 20
pulmonary inflammation (PI) and a chronic hypoxia (CH). The COPD involves several 21
comorbidities, including impairment of the muscle tissue that is linked to the survival 22
probability of the patients. To limit this muscle alteration, retraining programs are usually 23
proposed. However the benefits are heterogeneous between patients and muscles. The 24
underlying mechanisms implicated in that altered response of the COPD muscle are still 25
unclear. 26
Objective 27
The aim of our work was to study the specificities of muscle adaptations to a 28
hypertrophic stimulus induced by surgical ablation of agonist muscles in the presence of PI 29
and CH in a slow oxidative (soleus) and mixed (plantaris) muscle. 30
Methods and Results 31
Animals were exposed to a chronic hypoxia associated or not with a pulmonary 32
inflammation induced by bi-weekly instillations of lipopolysaccharides. The muscle 33
hypertrophy was triggered by a bilateral ablation of the soleus or the plantaris agonists, one 34
week before sacrifice. Our results show a specific sensitivity of the plantaris to chronic hypoxia 35
which decreased the hypertrophy percentage (from +56 % to +14 %, p<0.05), without effect 36
of pulmonary inflammation. At the opposite, soleus hypertrophy is totally inhibited by 37
pulmonary inflammation (from +24 % to -6 %, p<0.05), and unchanged by chronic hypoxia. 38
This specific sensitivity of plantaris and soleus was associated with modifications in proteins 39
involved in the fiber typing and the regulation of the proteolysis/synthesis balance. 40
Conclusions 41
Our results highlight the role played by the chronic hypoxia and the pulmonary 42
inflammation in the non-response of the muscle to a retraining program. Moreover, the 43
specific sensitivity of the plantaris and the soleus open new insight to understand the 44
heterogeneity of the muscle alteration which occurs during COPD. 45
Introduction 47
The Chronic Obstructive Pulmonary Disease (COPD) is a progressive pathology with a 48
pulmonary starting point to systemic repercussions which lead to manifold comorbidities 49
(Barnes and Celli, 2009). Evolution of the COPD is marked by a muscle wasting linked to 50
survival probability (Marquis et al., 2002), placing the maintenance of this tissue as a priority 51
for clinicians (Vestbo et al., 2013). The degree of muscle impairment is influenced by the 52
muscle type and localization - thorax, lower limb versus upper limb (Miranda et al., 2011) - 53
and by its metabolic orientation. Indeed, oxidative fibers from lower limb muscles show larger 54
decrease in number, area, and functionality than glycolytic fibers (Gouzi et al., 2013, Swallow 55
et al., 2007, Kim et al., 2008). This suggests that there is a link between the fiber type and the 56
consequences of the COPD on these muscle-specific alterations. However, the heterogeneous 57
typology of muscles do not allow to distinguish the specific impact of COPD on each fiber type. 58
To restore muscle functionality and mass in COPD, retraining programs or pulmonary 59
rehabilitation, became a major medical care with effective results. Nowadays, the most 60
efficient retraining programs are based on the combination of endurance Training (ET) with 61
Resistance Training (RT). The first one is aimed at restoring oxidative capacities linked to the 62
health-related quality of life, and the second one seeks to prevent muscle wasting through its 63
anabolic effects (Iepsen et al., 2015). Additionally, including a nutritional program increases 64
the benefits of such retraining programs (Pison et al., 2011). However, up to one third of 65
patients undergoing such multimodal rehabilitation programs does not show the classical 66
effects observed (Celli et al., 2008). Furthermore the 70 % considered as good responders 67
show lower responses compared to healthy subjects by a network biology approach to model 68
the relationship between muscle molecular and physiological response to training and 69
systemic inflammatory mediators (Turan et al., 2011). Furthermore, this lower response is 70
present even though a lung transplantation was performed before retraining program 71
(Guerrero et al., 2005). The origin of this non-response remains unclear (Troosters et al., 72
2001), but could be related to the level of Pulmonary Inflammation (PI) (Garcia-Rio et al., 2010) 73
and an underlying Chronic Hypoxia (CH) (Kent et al., 2011) that are two major stimuli seen in 74
the COPD. 75
Indeed, the role played by the PI in COPD is well established in the different 76
comorbidities (Wagner, 2008) and in the decrease of oxidative fibers (Remels et al., 2010). PI 77
is also reported to be cause (Thomsen et al., 2013) and consequence (Anzueto, 2010) of the 78
acute decompensation phases that rhythm the evolution of this pathology (Anzueto, 2010). 79
However, classical inflammatory parameters measured to evaluate the severity of this 80
pathology do not correlate with the nonresponse of COPD to a retraining program (Scott et 81
al., 2010). Recently, the role of initial body profile of the patient (Vogiatzis et al., 2010) or of a 82
systemic inflammation based on the overspill concept have been proposed to explain this 83
nonresponse (van Eeden et al., 2001, Salvi et al., 1999). The PI could overspill into the 84
circulation by the crossing from the lung compartment to the circulation through the 85
pulmonary interstitium and then from the blood to the muscle compartment. The nature and 86
the kinetics of the inflammatory signals could be modified and thus could complicate the 87
relation between PI and muscle wasting in COPD (van Eeden et al., 2001, Salvi et al., 1999). 88
Despite of a lack of consensus, CH also plays an important role in the prognosis of the 89
COPD. Analysis of 10 different studies shows an O2 arterial pressure (PaO2) below healthy
90
subjects (Crul et al., 2007, Petersen et al., 2007, Lemire et al., 2012, Rabinovich et al., 2003, 91
Vogiatzis et al., 2007, Itoh et al., 2004, Broekhuizen et al., 2005, Takabatake et al., 2000, 92
Karadag et al., 2008, Du et al., 2014). During exacerbations, the increase of inflammation alters 93
the ventilation/perfusion ratio, leading to a higher hypoxemia level in the patients (Barbera et 94
al., 1997). Hypoxemia, through the Hypoxia Inducible Factor-1 (HIF-1) [301], is associated to a 95
part of the comorbidities observed in COPD, such as pulmonary hypertension (Chaouat et al., 96
2008), muscle atrophy (Slot et al., 2014), and decrease in the Body Mass Index (BMI) (Raguso 97
and Luthy, 2011). According to these results, CH and PI seem to be relevant factors to explain 98
the decrease in endurance and in strength observed in COPD patients. Moreover, recent 99
studies pointed out a crosstalk between inflammation-hypoxia (Jiang et al., 2010b) and 100
hypoxia-inflammation (Liu et al., 2009, Takabatake et al., 2000) implicating at least two 101
different proteins, i) the Tnf- which has been shown to be correlated with muscle wasting 102
only in already cachectic patients (Eagan et al., 2012), ii) the Nuclear Factor Kappa-B (NF-kB) 103
(Jiang et al., 2010a, Langen et al., 2001). These studies suggest that COPD evolution is induced 104
by the combination of these 2 stimuli leading to cachexia in 20-40 % of patients (Schols, 2002, 105
Schols et al., 1998). 106
Interestingly, while oxidative fibers appear to be sensitive to inflammation (Remels et 107
al., 2010, Remels et al., 2015), glycolytic fibers differentiation is inhibited by chronic hypoxia 108
(de Theije et al., 2015). We can therefore hypothesize that the lack or the inappropriate 109
response of the muscle to exercise rehabilitation reported in COPD patients could be due to a 110
specific sensitivity of the different fibers to the CH and the PI. To understand the respective 111
role played by these two factors on the response of muscle to a retraining program, we studied 112
the muscle hypertrophy response to CH and PI using an overload surgery model in rat to mimic 113
exercise strength training based on previous works (Adams et al., 1999, Chale-Rush et al., 114
2009, Nakada et al., 2016). We compared the response of the soleus muscle, as mainly 115
composed of oxidative fibers and of the plantaris muscle, as a mixed muscle with an attempt 116
to characterize the signaling pathways involved. 117
118
Material and methods 119
Animals 120
Wistar male rats (n = 80; 4 month-old) were fed ad-libitum and housed in a 12h/12h 121
light/dark cycle. The study was performed in accordance with the French Ministry of Research 122
(2013-118) and the European Council Directive 2010/63/UE recommendations. Protocols 123
were carried out in the animal facility of the Biology Department of the University 124
(D3842110001) after approval by the local ethical committee affiliated to the French Ministry 125
of Research (168_LBFA-U1055 and 345_LBFA-U1055). 126
Experimental design 127
To determine the respective roles played by PI, CH and both of them on the 128
hypertrophic capacities of two different muscles, animals were allocated to one of the 5 129
following groups: N: Normoxia with a sham operation; Ns: Normoxia with surgery; NPIs: 130
Normoxia with Pulmonary Instillations and surgery; CHs: Chronic Hypoxia with surgery; and 131
CHPIs: Chronic Hypoxia with Pulmonary Instillations and surgery. All the groups with surgery 132
were performed twice, for the soleus and the plantaris muscles. 133
Lipopolysaccharides instillations 134
Pulmonary inflammation was induced in NPIs and CHPIs groups by instillations of 135
lipopolysaccharides (LPS) at 1 mL/kg of body weight (E.coli, serotype O55:B5; Sigma-Aldrich 136
Chemical Co., St Louis, Missouri, USA, 0.4 mg/mL in NaCl 0.9 %). These instillations were 137
performed bi-weekly during 4 weeks before the sacrifice as previously described by Wang et 138 al (Wang et al., 2009). 139 Chronic Hypoxia 140
During the last 2 weeks of the protocol, animals from CHs and CHPIs groups were 141
exposed to a normobaric hypoxia (FiO2: 10 %) generated by a Nitrogen Gas generator G-2-0-
142
1-0-E (Parker Hannifin, Contamine-sur-Arve, France). Every day, the hypoxia chamber was 143
opened 45-60 minutes to weight the animals, the food, and to perform LPS instillations. 144
Muscle overload surgery 145
One week before sacrifice, animals were anesthetized by intraperitoneal injection of 146
xylazine/ketamine (8 mg/kg and 100 mg/kg, respectively). Animals were subjected to a 147
bilateral ablation of agonist muscles of either the soleus or the plantaris, as previously 148
described (Morioka et al., 2008). Animals from Sham groups were also operated in the same 149
conditions, without the removal of muscles. All animals were monitored until their total 150
awakening and those from CH groups were replaced in the hypoxia chamber 24 h after 151
surgery. Paracetamol was delivered in the drinking bottle (300 mg/L) during the week post- 152
surgery. 153
Spontaneous physical actimetry measurements 154
Spontaneous Physical Activity (SPA) was evaluated using a video tracking system 155
developed in our laboratory (Chabert et al., 2016). Briefly, this system is based on the 156
automatic detection, picture-by-picture, of a blue dot placed on the animal using a Matlab 157
script (Mathworks, Natick, Massachusetts, United States). Animals were filmed during 24h in 158
their own environment (N or CH) between the third and fourth day after surgery. Results were 159
expressed in meters. 160
Tissue sampling 161
Animals were euthanized using an intraperitoneal injection of sodium pentobarbital 162
(50 mg/kg of body weight) followed by heart excision. Soleus and plantaris were dissected, 163
weighted and directly frozen in liquid nitrogen until further measurements. Muscle 164
hypertrophy was expressed as the hypertrophy percentage calculated by dividing the muscle 165
mass of the soleus or the plantaris by the corresponding mean muscle mass obtained from 166
the control group N. 167
Protein isolation and biochemical analysis 168
About 30 mg of frozen soleus or plantaris muscle were homogenized in 1 mL of 20 mM 169
HEPES, 2 mM EGTA, 50 mM -glycerophosphate, 3 mM benzamidine, 10 % glycerol, 1 % triton 170
X-100, 1 mM DTT, 1 mM Na3VO4, pH 7.4, added with a proteases inhibitor cocktail (cOmplete
171
tablets, Roche, Bâle, Switzerland). Homogenization were performed using a ball mill (MM301; 172
Retsch, Düsseldorf, Germany). Protein were separated by SDS-PAGE, using a mini-PROTEAN II 173
system (Biorad, Marne-La-Coquette, France), with 10 to 20 % acrylamide/bis-acrylamide 174
(37.5:1) gradient gels and subsequently transferred on nitrocellulose membrane. Appropriate 175
secondary antibodies coupled with HRP (anti-rabbit NA934, GE Healthcare™, Little Chalfont, 176
England) were incubated at room temperature for 1h. Reactions were revealed using 177
enhanced chemiluminescence and signals were acquired on a LAS4000 imaging system (GE 178
healthcare, Little Chalfont, UK). Band intensities were quantified using ImageJ software and 179
expressed in arbitrary units based on the signal generated by a standard sample loaded on 180
each gel. 181
Enzymatic analysis 182
Citrate synthase (CS) activity was measured on muscle homogenates according to the 183
kinetic procedure of Faloona et al. (Faloona and Srere, 1969) using a Specord 210 184
spectrophotometer (Analytik Jena, Jena, Germany) at 412 nm and at 25°C. Results were 185
expressed in µmol.min-1.g-1.
186
Myosin Heavy Chain distribution 187
The proportions of the 4 different isoforms of Myosin Heavy Chain (MHC) I, IIa, IIx and 188
IIb were determined by electrophoresis as previously described (Talmadge and Roy, 1993), 189
and revealed by silver staining (SilverQuest Silverstaining Kit, Novex, Levallois-Perret, France). 190
Results were expressed as percentage of the total MHC content. 191
RNA extraction and RT-qPCR 192
Total RNA were purified from 20-25 mg of soleus or plantaris muscle homogenized in 193
Qiazol (Qiagen, Hilden, Germany). Total RNA concentration and purity were assessed at 230, 194
260 and 280 nm using a nanophotometer (Classic, Implen, München, Germany). Reverse 195
transcription of 1 µg of total RNA was performed using 100 U of M-MLV reverse transcriptase 196
in a final volume of 20 µL using oligo-DTs as primers (Superscript III, Invitrogen, Carlsbad, 197
California, USA). Relative contents of target mRNA were determined using a LightCycler 198
(Roche Applied Science, Bâle, Switzerland) by real-time PCR with a Fast Start DNA Master SYBR 199
Green kit (Roche Applied Science, Bâle, Switzerland). Primers used were designed on 200
ProbeFinder 2.50 (Roche Applied Science) and are presented in table 1. The (1+E)CT method
201
(Livak and Schmittgen, 2001) was used to determine the fold change of the target mRNA 202
compared with the control group (N). Hypoxanthine phosphoribosyltransferase-1 (Hprt1) was 203
used as a housekeeping gene to normalize the mRNA values of each sample. 204
Table 1: Primers sequences of genes amplified by RT-PCr. 205
Hprt1: hypoxanthine phosphoribosyltransferase 1; MHC: Myosin Heavy Chain; VEGF: Vascular 206
Endothelial Growth Factor, Tfam: Mitochondrial transcription factor A; PGC1: Peroxisome 207
proliferator-activated receptor gamma coactivator 1-alpha; MuRF-1: Muscle Ring Factor-1; 208
MAFbx: Muscle Atrophy F-Box. 209
210
Statistical analysis 211
All the data are presented as mean ± SEM. When data were normally distributed, a 212
one-way or a two-way ANOVA was performed to determine the effects of environment 213
(normoxia vs. chronic hypoxia) and inflammation (without instillations vs. LPS instillations). If 214
significant, the omnibus test of variance was followed by a Holm-Sidak post-hoc. For non- 215
parametric data, a Tukey test was used to determine the nature of the effect. 216
217
Genes Primers sequences Amplicon
length
Annealing temp (°C) Hprt1 5'-ggtccattcctatgactgtagatttt-3' 5'-caatcaagacgttctttccagtt-3' 126 53 MuRF1 5'-agcattgtagaagcttccaagg-3' 5'-gtaaactcctcctcctcatctgtc-3' 146 56 VEGF 5'-gagagcaacgccactatgc-3' 5'-cgccttggcttgtcacat-3' 146 54 MAFbx 5'-gcttgtgcgatgttacccaagaa-3' 5'-tgaaagtgagacggagcagctct-3' 139 58 MHC-I 5'-ttgctctacccaaccctaaggatg-3' 5'-ttgtgtttctgcctgaaggtgc-3' 81 49 MHC-IIa 5'-ctcaggcttcaagatttggtgg-3' 5'-ttgtgcctctcttcggtcattc-3' 265 49 MHC-IIx 5'-ggaggaacaatccaacgtcaacc-3' 5'-ggtcactttcctgctttggatcg-3' 178 51 MHC-IIb 5'-cacgaagcgtgtcatcca -3' 5'-aggtttcgatatctgcggagg-3' 242 59 1
Results 218
Table 2: Morphological data and spontaneous physical activity of animals subjected to chronic 219
hypoxia and/or pulmonary inflammation associated to a muscular overload. 220
N: Normoxia Control; Ns: Normoxia + Surgery; NPIs: Normoxia + Surgery + Inflammation; CHs: 221
Hypoxia + Surgery; CHPIs: Hypoxia + Surgery + Inflammation. *: Global effect of inflammation 222
(Ns+CHs vs. NPIs+CHPIs, p<0.05); #: Global effect of hypoxia (Ns+NPIs vs. CHs+CHPIs, p<0.05); 223
$: diff. of N (p<0.05), £: CHs diff. of CHPIs (p<0.05). 224
225
The table 2 present the main characteristics of the animals subjected to the two 226
different stimuli. Body weight was significant lower in the animals subjected to CH (- 11 % of 227
normoxic groups, p<0.001), without change in animal size as suggested by the unchanged tibia 228
length. Muscle mass was higher in response to overload surgery in soleus of animals subjected 229
to normoxia or CH compared to the mean value of control groups (respectively + 26.4 mg and 230
+ 49.8 mg of N, p<0.05). However, overload effect is inhibited in the soleus muscle of animals 231
from PI (NPIs+CHPIs) groups when compared to non-treated (Ns+CHs) groups (- 51 mg, 232
p<0.001). The plantaris muscle mass is increased after the overload surgery (from + 56mg 233
(p<0.05) for CHs group to + 223 mg for Ns group (p<0.05) when compared to the control (N) 234
group. Interestingly plantaris mass is decreased in CHs and CHPIs group compared to normoxic 235
(Ns) group (-167 mg and -130 mg, respectively, p<0.05). Spontaneous Physical Activity (SPA) 236
decreased in groups with overloaded soleus (Ns and CHPIs) when compared to N group (-43 % 237
and -46 %, respectively p<0.05). Similarly, SPA was lower in groups with overloaded plantaris 238
(Ns, CHPIs and NPIs) compared with N group (-37 %, -40 %, and -47 %, respectively, p<0.05). 239
However, no differences of the SPA level between animals subjected to soleus or plantaris 240
overload surgery are detected. 241
242
The hypertrophy of the soleus and plantaris muscle challenged by CH, PI and an 243
overload surgery. 244
245
Figure 1: Effects of pulmonary inflammation and hypoxia exposure on the hypertrophic 246
capacity of soleus and plantaris muscle triggered by an overload surgery. Hypertrophic 247
capacity was determined by the ratio between muscle weight and the mean of the muscle 248
weight from control group. (n = 7-8; Mean ± SEM); N: Normoxia Control; Ns: Normoxia + 249
Surgery; NPIs: Normoxia + Surgery + Inflammation; CHs: Hypoxia + Surgery; CHPIs: Hypoxia + 250
Surgery + Inflammation. **: Global effect of inflammation (Ns+CHs vs. NPIs+CHPIs, p<0.001); 251
##: Global effect of hypoxia (Ns+NPIs vs. CHs+CHPIs, p<0.001). 252
As shown in Figure 1, in Ns group, the overload surgery of the soleus induced a 254
significant hypertrophy compared with N group (+23.8 %, p<0.05). Whilst CH doesn’t alter the 255
hypertrophic response to overload surgery for the soleus (+ 25.9 % of N group, p<0.05), the 256
presence of PI totally was associated with an inhibition of the muscle growth in both NPIs and 257
CHPIs groups (-5.9 % and -2.9 % of N group, ns). Hypertrophic response of the plantaris 258
showed a higher growth percentage (+ 55 % at the sacrifice) in the group Ns compared with 259
the N group. PI had no effect on the plantaris hypertrophy in both NPIs and CHPIs groups (- 260
5 % of Ns and +9 % of CHs, respectively, ns), while CH strongly inhibits it in groups CHs and 261
CHPIs (-42 % and -26 % of Ns, respectively, p<0.05). 262
The hypertrophic capacities of these two muscles show a very different profile of 263
sensitivity with a lower hypertrophic response of the soleus than the plantaris (p < 0.001), 264
associated to a higher sensitivity to PI, while plantaris appears insensitive to PI but strongly 265
affected by CH (Figure 1). 266
The typology of the muscles and their evolution induced by CH, PI under overload 268
surgery. 269
Figure 2: Effects of chronic hypoxia and pulmonary inflammation on the four myosin heavy 270
chain isoforms (MHC-I (A), IIa (B), IIx (C), and IIb (D)) distribution in soleus and in plantaris 271
muscles of animals subjected to a muscle overload surgery. (n = 6-8; Mean ± SEM); N: 272
Normoxia Control; Ns: Normoxia + Surgery; NPIs: Normoxia + Surgery + Inflammation; CHs: 273
Hypoxia + Surgery; CHPIs: Hypoxia + Surgery + Inflammation. £: CHPIs vs. NPIs (p<0.05). 274
275
To determine the influence of the CH and the PI on the typology of the different 276
muscles, we analyzed the MHC distribution in soleus and plantaris muscles and results are 277
reported on Figure 2. The MHC distribution in soleus was similar in the different groups, 278
independently of the presence of CH or PI. In plantaris, MHC-1, IIa and IIx distributions were 279
also similar between groups (respectively Figure 2A, B and C). However, MHC-IIb percentage 280
is significantly lower in the CHPIs group when compared to the NPIs group (-29 %, p<0.05, 281
Figure 2D). Additionally, soleus and plantaris muscles displayed a very different typology, with 282
a soleus compound by over 95 % of MHC-I, while plantaris is composed of all MHC type: I 283
(5.02 % in Ns group), IIa (23.6 % in Ns group), IIx (35.4 % in Ns group), and IIb (34.6 % in Ns 284
group). 285
286
Figure 3: Effects of chronic hypoxia and pulmonary inflammation on the mRNA level of MHC-I 287
(A), IIa (B), IIx (C), and IIb (D) in soleus and in plantaris of animals subjected to a muscle 288
overload surgery. Hprt1 (hypoxanthine phosphoribosyltransferase 1) was used as a 289
housekeeping gene; (n = 6-8; Mean ± SEM). N: Normoxia Control; Ns: Normoxia + Surgery; 290
NPIs: Normoxia + Surgery + Inflammation; CHs: Hypoxia + Surgery; CHPIs: Hypoxia + Surgery + 291
Inflammation. *: Global effect of inflammation (Ns+CHs vs. NPIs+CHPIs, p<0.05); #: Global 292
effect of hypoxia (Ns+NPIs vs. CHs+CHPIs; $: CHs diff. of Ns and CHPIs (p<0.05). 293
294
The transcription level of MHC-I, IIx and IIb mRNAs in soleus (Figure 3A, C and D) are 295
unaffected by CH or PI. However, there is a significant decrease in the MHC-IIa mRNA under 296
the PI condition (- 50 % for Ns+CHs vs. NPIs+CHPIs, p<0.05, Figure 3B). In plantaris, there were 297
no differences in the MHC-I, IIa and IIx mRNA levels between groups (Figure 3A, B and C). 298
However, MHC-IIb mRNA was higher in CHs group compared to Ns and CHPIs groups (+ 145 % 299
and + 82 %, respectively, p<0.05). 300
301
The oxidative metabolism modifications induced by CH and PI in animals subjected 302
to an overload surgery. 303
304
Figure 4: Effects of chronic hypoxia and pulmonary inflammation on the citrate synthase 305
activity (A) and the protein content of the very long-chain acyl-CoA dehydrogenase (B) in the 306
soleus and plantaris muscles of animals subjected to a muscle overload surgery. (n = 5-8; Mean 307
± SEM); VLCAD: Very long-chain acyl-CoA dehydrogenase; N: Normoxia Control; Ns: Normoxia 308
+ Surgery; NPIs: Normoxia + Surgery + Inflammation; CHs: Hypoxia + Surgery; CHPIs: Hypoxia 309
+ Surgery + Inflammation. 310
311
Neither CH nor PI induced changes in the CS or in the VLCAD activities whatever the 312
muscle (soleus or plantaris) (Figure 4A). 313
314
Figure 5: Effects of chronic hypoxia and pulmonary inflammation in the mRNA transcription 316
level of three protein involved in the oxidative metabolism: VEGF (A), PGC1 (B), and Tfam (C 317
in animals subjected to an overload surgery. Panel 5(D) shows the correlation between PGC1- 318
and Tfam in soleus and plantaris muscle. Hprt1 was used as a housekeeping gene; (n = 5-6; 319
Mean ± SEM); VEGF: Vascular Endothelial Growth Factor, Tfam: Mitochondrial transcription 320
factor A; PGC1-: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; N: 321
Normoxia Control; Ns: Normoxia + Surgery; NPIs: Normoxia + Surgery + Inflammation; CHs: 322
Hypoxia + Surgery; CHPIs: Hypoxia + Surgery + Inflammation. *: Global effect of inflammation 323
(Ns+CHs vs. NPIs+CHPIs, p<0.05); #: Global effect of hypoxia (Ns+NPIs vs. CHs+CHPIs, p<0.05); 324
$: diff. of all (p<0.05). 325
326
The level of VEGF mRNA (Figure 5A,) in soleus was lower in animals subjected to PI 327
(NPIs+CHPIs) compared with Ns+CHs (-39 %, p<0.05). It also appeared that the increase in 328
VEGF mRNA level in CHPIs group was 44 % lower than the increae in CHs group (p<0.05). In 329
plantaris muscle, VEGF mRNA level was also lower in NPIs+CHPIs when compared to Ns+CHs 330
(-43 %, p<0.05). This was associated with a lower VEGF mRNA level in CHPIs group compared