Transient neonatal exposure to hyperoxia, an experimental model of 1 preterm birth, leads to skeletal muscle atrophy and fiber type switching.

Individuals born preterm show reduced exercise capacity and increased risk for pulmonary 26 and cardiovascular diseases, but the impact of preterm birth on skeletal muscle, an inherently 27 critical part of cardiorespiratory fitness, remains unknown. We evaluated the impacts of 28 preterm birth-related conditions on the development, growth, and function of skeletal muscle 29 using a recognized preclinical rodent model in which newborn rats are exposed to 80% 30 oxygen from day 3 to 10 of life. We analyzed different hindlimb muscles of male and female 31 rats at 10 days (neonatal), 4 weeks (juvenile) and 16 weeks (young adults). Neonatal high 32 oxygen exposure increased the generation of reactive oxygen species and the signs of 33 inflammation in skeletal muscles, which was associated with muscle fiber atrophy, fiber type 34 shifting (reduced proportion of type I slow fibers and increased proportion of type IIb fast- 35 fatigable fibers), and impairment in muscle function. These effects were maintained until 36 adulthood. Fast-twitch muscles were more vulnerable to the effects of hyperoxia than slow- 37 twitch muscles. Male rats, which expressed lower antioxidant defenses, were more susceptible 38 than females to oxygen-induced myopathy. Overall, preterm birth-related conditions have 39 long-lasting effects on the composition, morphology, and function of skeletal muscles; and 40 these effects are sex-specific. Oxygen-induced changes in skeletal muscles could contribute to 41 the reduced exercise capacity and to increased risk of diseases of preterm born individuals. 42


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Improved neonatal care over the last 3 decades has allowed the survival of the vast majority 64 of infants born preterm (< 37 weeks). Consequently, about 10% of current children and young 65 adults were born preterm, including 1.5% very preterm (< 32 weeks) [1]. Preterm birth and 66 related conditions can alter the development of the pulmonary and cardiovascular system 67 [2,3], and adults born preterm show an increased risk of chronic lung and cardiovascular 68 diseases. These dysfunctions have traditionally been evoked to explain the reduced exercise 69 capacity in children and adults born preterm [4,5]. However, the impact of preterm birth on 70 skeletal muscles, an inherently critical part of exercise capacity and of cardiorespiratory 71 health, has been overlooked. 72 Skeletal muscles are mostly constituted of long cylindrical multinucleated cells, called 73 myofibers, which are responsible for muscle contraction. The formation of myofibers during 74 prenatal development is divided into primary myogenesis (until week 10 of gestation), during 75 which a small number of large primary myofibers emerge and serve as templates for the 76 formation of smaller myofibers during secondary myogenesis (between weeks 11-20 of 77 gestation) [6,7]. Thereafter, during the end of the second and the beginning of the third 78 trimester, at the time of preterm birth, the skeletal muscles undergo a critical phase of 79 maturation characterized myofiber growth, fiber typing determination (slow vs fast fibers), 80 and maturation of the excitation-contraction coupling system. Physiological fetal hypoxia 81 plays a critical role in the proper regulation of these processes [8]. In vitro experiments have 82 shown that the levels of oxygen have a decisive impact on the establishment of fiber typing 83 [9]. Upon preterm birth, infants are exposed to higher blood oxygen levels (versus relative 84 hypoxia in utero), which could interfere with muscle growth, fiber typing, and contractile 85 function. 86 Few studies have provided indirect evidence of the contribution of skeletal muscles to 87 exercise capacity in preterm born individuals. These studies suggest a reduced skeletal muscle 88 mass, with lower maximal power output, and altered strength and endurance in individuals 89 born preterm [4,5,10,11], that is proportional to the decrease in gestational age [12]. 90 Supporting the clinical observations, experimental rodent model of bronchopulmonary 91 dysplasia (BPD) showed that transient neonatal exposure to high oxygen levels is associated 92 with higher body weight without increase in muscle mass, lower muscle mitochondrial 93 biogenesis, lower oxidative activity and compensatory higher glycolytic enzyme expression 94 [13]. However, the impact of preterm birth-related conditions on skeletal muscle growth, 95 composition, fiber typing, and contractile function remains unknown. Considering that 96 skeletal muscle atrophy was shown to be associated with chronic obstructive pulmonary 97 diseases and contributes negatively to the patient's prognosis [14], and that very preterm birth 98 is associated with higher risk of BPD and reduced exercise capacity, it is essential to 99 investigate further the direct contribution of preterm birth-related conditions on skeletal 100 muscles. 101 In the context of preterm birth, newborns are exposed to high levels of oxygen, especially 102 compared to in utero oxygen tension. This relative "hyperoxic" environment is often 103 exacerbated by neonatal oxygen supplementation. The sudden rise in oxygen levels induces 104 an increase in reactive oxygen species (ROS) production while the antioxidant system is still 105 immature [15]. Elevated ROS production can activate canonical Nuclear Factor-kappa B (NF-106 B) signaling and the expression of downstream inflammatory genes, such as Tumor Necrosis 107 to high oxygen induces an oxidative stress and a chronic inflammatory state in skeletal 119 muscles, which is associated with reduced muscle mass and fiber size, shift in the proportion 120 of fiber types toward fast fatigable fibers, and impaired contractile properties of the motor 121 units. 122

Animals 125
Sprague-Dawley (Charles River, St-Constant, Québec, Canada) pups were maintained with a 126 dam in 80% O 2 (OI, oxygen-induced injury group) in an oxycycler (A82OCV, Biospherix) or 127 in room air (CTRL, control group) from day 3 to 10 of life. Dams in hyperoxia were 128 interchanged every 12 h with a dam from a litter maintained in room air to avoid maternal O 2 129 toxicity. Both males and females were studied. In total 8 litters of 12 rats were used (4 litters 130 per group: OI and CTRL) and no more than 4 animals per litter (2 males and 2 females, 131 chosen randomly) were used for each experimental procedure. The proximal and distal tendons of the EDL muscles were attached with a 3-0 silk suture. 214 Muscles were carefully dissected and placed in the 300A Test System organ bath (Aurora 215 Scientific Inc., Ontario, Canada) filled with buffered physiological salt solution (Krebs-Ringer 216 supplemented with glucose) continuously perfused with carbogen bubbling (5% CO 2 , 95% 217 O 2 ), and thermostatically maintained at 25°C. After calibration of the optimal muscle length, 218 muscles were stimulated (25 V, 500 ms) at increasing frequencies: 1, 25, 50, 80, 100, 150 Hz. 219 The muscle was allowed to rest for 3 min between two stimulations [29]. The specific muscle 220 force was calculated as follow: [maximal force × optimal fiber length (0.44 x muscle length) 221 × muscle density (1.06 g/cm 3 )]/muscle mass. At the end of the protocol, the length and weight 222 of the muscle were measured. The twitch-to-tetanus (Pt/P 0 ) ratio was calculated as the ratio of

Relevance of transient neonatal hyperoxia as a model to study skeletal muscle in 246
preterm birth conditions. 247 Neonatal rats were exposed to high concentrations of oxygen to mimic the sudden rise in pO 2 248 levels associated with preterm birth. Even though newborn rats are not preterm, the 249 maturational stage at birth for many organs including the eyes, brain, kidneys, lungs, heart, 250 and skeletal muscles correspond to those of a human infant born very preterm in the second 251  Figure 3A, B). The density of CD163+ 295 cells, a marker for a subset of resident/anti-inflammatory macrophages, was increased in the 296 TA of OI males and females at 4 weeks, and only for males at 16 weeks ( Figure 2G, J, K). 297 The proportion of anti-inflammatory macrophages relative to total macrophages 298 (CD163+/CD68+ cells) was significantly reduced in OI male rats at 16 weeks, but not in 299 females (Supplemental Figure 3E-H). Analysis of sex differences showed a higher level of 300 CD68+ and CD163+ macrophages in the TA of OI males vs OI females, suggesting a stronger 301 inflammatory response in males ( Figure 2G-K). 302 303

Impact of transient neonatal exposure to high oxygen on muscle atrophy 304
To assess muscle atrophy, we first measured the body weight and the Lee index (surrogate 305 marker for body mass index) and observed that they were similar between the groups for both 306 males and females throughout the experimental period (Supplemental Figure 4A-C). At 10 307 days (end of the hyperoxia exposure period), the ratio of both TA and soleus muscle weight to 308 body weight were not different between CTRL and OI groups for males and females ( Figure  309 3A and Supplemental Figure 4D). This ratio was significantly reduced in the TA of OI vs 310 CTRL males 4-and 16 weeks and in the EDL at 4 weeks, but not in the soleus muscle ( Figure  311 3B,C and Supplemental Figure 4D-H). In OI vs CTRL females, the TA, EDL, and soleus 312 weight to body weight ratio were similar at 4 weeks, but a significant reduction was observed 313 at 16 weeks only for the TA ( Figure 3B, C and Supplemental Figure 4D-H). Analysis of sex 314 differences showed a lower ratio of muscle weight to body weight for the TA of OI males 315 compared to OI females at 16 weeks ( Figure 3C, Supplemental Figure 4F). 316 Next, we assessed muscle fiber size as a direct measure of muscle atrophy. In OI vs 317 CTRL males, TA fiber size was similar at 10 days but was strongly reduced at 4-and 16 318 weeks ( Figure 3D-F). Soleus muscle fiber size was similar between groups at all ages studied 319 (Supplemental Figure 4I-K). In OI vs CTRL females, the fiber size of both the TA and soleus Considering the importance of oxygen levels in fiber typing, we next examined whether 333 transient neonatal exposure to high oxygen leads to long-lasting changes in muscle fiber 334 typing. We assessed the proportion of type I fibers (slow oxidative), type IIA fibers (fast 335 oxidative), IIX and IIB fibers (fast glycolytic) by co-immunofluorescence ( Figure 5A). In the 336 TA of OI vs CTRL males, the proportion of type I and IIA fibers was reduced at 4-and 16 337 weeks along with a concomitant increase in type IIB fibers ( Figure 5B, C). A small proportion 338 (3-4%) of hybrid IIA/IIB fibers was observed in both groups at 4 weeks. Similar changes 339 were observed in the TA of OI vs CTRL females at 4 weeks; however, this switch was less 340 pronounced in 16 weeks OI females, which only showed a significant reduction in the 341 proportion of type IIA fibers ( Figure 5D, E). This switch from slow to fast fibers was also 342 observed in the extensor digitorum longus (fast-twitch muscle), in which a reduction in the 343 proportion of type I fibers was associated with an increase in type IIB fibers at 4-and 16 344 weeks in OI vs CTRL male and female rats (Supplemental Figure 6A-D). In the soleus, high 345 oxygen exposure did not affect fiber typing in both males and females at any time point. 346 (Supplemental Figure 6E-H).  force did not significantly differ between groups for both males and females at 4-and 16 368 weeks (Figure 7 A, B, D, E). As expected, maximal contractile force was increased in CTRL 369 and OI male rats compare to CTRL and OI females at 16 weeks ( Figure 7D,E). Next, we 370 assessed the twitch-to-tetanus ratio (Pt/P 0 ratio), which compares the muscle force generated 371 by a single electrical stimulus to its maximal contractile force. This indicator of the 372 physiological properties of single motor units is directly correlated to fiber typing and 373 fatiguability (i.e. Pt/P 0 ratio is higher in fast fatigable muscles). In males, Pt/P 0 ratio was 374 significantly increased in OI vs CTRL rats at 4 weeks, but not at 16 weeks ( Figure 7C, F). In 375 females, Pt/P 0 was similar between groups at both 4-and 16 weeks ( Figure 7C, F). Analysis 376 of sex differences showed that the Pt/P 0 ratio is higher in OI males compared to OI females at 377 4 weeks. 378

Discussion 380
In this study, we showed, using a well-recognized animal model of BPD and preterm birth-381 related conditions, that transient neonatal exposure to high oxygen leads to long-term 382 alterations in the skeletal muscles. These changes are characterized by an increased 383 production of ROS and a chronic inflammatory state, alongside with signs of muscle atrophy, 384 fibrosis, fiber type shifting, and impairment in contractile properties of the motor unit. These 385 detrimental changes are observed in juvenile rats and are maintained until adulthood. Fast-386 twitch muscles were more vulnerable to the effects of hyperoxia than slow-twitch muscles. 387 Moreover, male rats are more susceptible than females to oxygen-induced myopathy. Overall, 388 these findings demonstrate that preterm birth-related conditions have long-lasting effects on 389 the composition, morphology, and function of skeletal muscle, which could contribute to the 390 development and prognosis of long-term cardiopulmonary consequences of prematurity 391 muscles are also strongly affected by this pathological condition. In the TA muscle of male 399 rats, we observed a strong rise in ROS production immediately after the high oxygen exposure 400 (at 10 days) that was still significantly higher than control at 4-and 16-weeks. A similar 401 increase was also observed in the fast-twitch EDL muscle at 4 weeks. However, this increase 402 in ROS production was milder in the soleus muscle of OI males, suggesting that the muscle 403 type may affect its capacity to maintain its redox balance. The soleus muscle is mainly 404 production and/or NF-kB activation, which promotes muscle atrophy. 453 Analysis of distinct types of muscle revealed that oxygen-induced muscle atrophy is 454 fiber-type specific. In the fast-twitch TA muscle that has lower antioxidant defenses there is a 455 stronger rise in ROS production, increased Atrogin-1 and MuRF-1 expression, and reduced 456 muscle mass and size, compared to the slow-type soleus muscle. Fast-fiber specific muscle 457 atrophy has also been observed in other acquired myopathies such as cancer cachexia, 458 diabetes, chronic heart failure, and sarcopenia Assessment of maximal muscle force did not reveal significant differences in the OI vs 470 CTRL rats; however, at 4 weeks of age the interindividual variability in the maximal force 471 was high and precluded any conclusions. This heterogeneity in the phenotype and the 472 prognosis is observed in preterm born individuals (i.e. some individuals being highly affected 473 while others have no apparent complications) [66]. It could be hypothesized that transient 474 neonatal exposure to hyperoxia induce a predisposition to reduced muscle force, which could 475 be exacerbated by additional deleterious conditions and/or aging. Nevertheless, the absence of 476 muscle force deficit was unexpected considering the strong evidence indicative of muscle 477 atrophy in OI male rats. Importantly, force generation requires a complex system that is not 478 solely driven by muscle size. Changes in the excitation-contraction coupling could 479 compensate for the loss of muscle mass. In this regard, further analysis of contractile 480 properties showed that the twitch-to-tetanus (Pt/P 0 ) ratio was increased in OI male rats. A 481 similar increase in the Pt/P 0 ratio was observed in a variety of conditions such as disuse and 482 aging [67][68][69]. This ratio represents a physiological property of motor units that is correlated 483 to fiber typing and fatigability (i.e. higher Pt/P 0 ratio in fast-fatigable muscle) [30]. The rise in 484 Pt/P 0 ratio is coherent with the switch that we observed in OI rats in favour of fast fiber type. 485 The higher content of fast fibers in OI rats could help to partially mitigate the loss of muscle 486 force induced by muscle atrophy considering that fast-twitch fibers are more powerful than 487 slow-twitch fibers; however, they are also more fatigable. Taken together, these findings 488 suggest that the reduced exercise capacity of individuals born preterm does not solely depend 489 on reduced cardiopulmonary capacity but also on impaired contractile properties of their 490 skeletal muscles [68,69]. 491 In summary, this study shows that a transient exposure to high oxygen levels in 492 newborn rats induces an imbalance in the redox status associated with chronic inflammation, 493 muscle atrophy, fiber type switching, and impaired muscle function. These alterations in 494 skeletal muscles closely mimic the ones observed in premature aging [70,71]   oxygen-induced injury (OI) male and female rats. Error bars represent means ± SEM; n=3-6 864 per group. Statistical analyses were performed using two-way ANOVA with Tukey's post-test 865 *p<0.05; **p<0.01; ***p<0.001 vs. group indicated. # CTRL males vs CTRL females; ‡ OI 866 males vs OI females. 867 868 Figure 8: Schematic overview of the mechanism proposed. Preterm newborns are exposed 869 to high levels of oxygen compared to in utero that induces oxidative stress and systemic 870 inflammation in a context of immature tissue development. Using a rodent model of neonatal 871 exposure to hyperoxia, we showed that preterm birth-related condition induced an imbalance 872 in the redox status in skeletal muscles associated with the activation of the key inflammatory 873 pathway NF-kB and chronic inflammatory cell recruitment. These changes were associated 874 with muscle atrophy, fibrosis, fiber type switching (favoring fast-fatigable fibers), and 875 impaired contractile properties. These changes develop overtime and persist until adulthood 876 and are more severe in males than females. 877 878