Effect of Chronic Intermittent Hypoxia (CIH) on Neuromuscular Junctions and Mitochondria in Slow- and Fast-Twitch Skeletal Muscle of Mice – Role of iNOS

Effect of Chronic Intermittent Hypoxia (CIH) on Neuromuscular Junctions and Mitochondria in Slow- and Fast-Twitch Skeletal Muscle of Mice – Role of iNOS | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Effect of Chronic Intermittent Hypoxia (CIH) on Neuromuscular Junctions and Mitochondria in Slow- and Fast-Twitch Skeletal Muscle of Mice – Role of iNOS Laura Isabel Bannow, Gabriel A. Bonaterra, Mirjam Bertoune, Sabrina Maus, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-102618/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Feb, 2022 Read the published version in Skeletal Muscle → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Obstructive sleep apnea (OSA) imposes vascular and metabolic risks through chronic intermittent hypoxia (CIH) and impairs skeletal muscle performance. As studies addressing limb muscles are rare, the reasons for the lower exercise capacity are unknown. We hypothesize that CIH-related morphological alterations in neuromuscular junctions (NMJ) and mitochondrial integrity might be the cause of functional disorders in skeletal muscles. Methods: Mice were kept under 6-weeks-CIH (alternating 7% and 21% O 2 -fractions every 30s, 8h/d, 5d/w) compared to normoxia (NOX). Analyses included neuromuscular junctions (NMJ) postsynaptic morphology and integrity, fiber cross-sectional area (CSA) and composition (ATPase), mitochondrial ultrastructure (transmission-electron-microscopy) and relevant transcripts (qRT-PCR). Beside wildtype (WT) we included inducible-nitric-oxide-synthase knockout mice (iNOS -/- ) to evaluate whether iNOS is protective or risk-mediating. Results: In WT soleus muscle, CIH vs. NOX reduced NMJ size (-37.0%, p<0.001) and length (-25.0%, p<0.05) together with fiber CSA of type IIa fibers (-14%, p<0.05) and increased centronucleated fiber fraction (p<0.001). Moreover, CIH vs. NOX increased the fraction of damaged mitochondria (1.8-fold, p<0.001). Compared to WT, iNOS -/- similarly decreased NMJ area and length with NOX (-55%, p<0.001 and -33%, p<0.05, respectively) or with CIH (-37%, p<0.05 and -29%, p<0.05), however, prompted no fiber atrophy. Moreover, increased fractions of damaged (2.1-fold, p6-fold, p10-fold. None of these morphological alterations with CIH- or iNOS -/- were detected in gastrocnemius muscle. Notably, iNOS expression was undetectable in WT muscle, unlike the liver, where it was massively decreased with CIH. Conclusion: CIH leads to NMJ and mitochondrial damage associated with fiber atrophy/centronucleation selectively in slow-twitch muscle of WT. This effect is largely mimicked by iNOS -/- at NOX (except for atrophy). Both conditions involve massive SOCS3 upregulation likely through denervation. In the absence of muscular iNOS expression in WT, this damage may arise from extramuscular, e.g. motoneuronal iNOS deficiency (through CIH or knockout) awaiting functional evaluation. Orthopedics denervation muscle atrophy mitochondria fiber type oxidative stress neuromuscular junction iNOS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Obstructive sleep apnea (OSA), which describes a repetitive collapse of the upper airways during sleep, causes recurrent episodes of hypopnea or even apnea resulting in chronic intermittent hypoxia (CIH). Over the last two decades, the prevalence of sleep apnea has increased by a double-digit figure [ 1 – 5 ]. In a population-based study among the group of 40- to 85-year-old adults, 49.7% of men and 23.4% of women suffered from a moderate to severe OSA defined as an apnea–hypopnea index (AHI) ≥ 15 [ 6 ]. Due to the growing rate of obesity that is suspected as one of the strongest causal triggers, a further increase of sleep associated disorders is expected [ 5 ]. Among various comorbidities, OSA patients often experience muscle fatigue and reduced physical performance [ 7 ] resulting in limited daily activities and quality of life. In fact, recent meta-analyses showed that maximum oxygen uptake (aerobic capacity) under cycle ergometer test conditions is significantly reduced in patients with (severe) OSAS [ 8 , 9 ] compared to healthy controls and may be improved by continuous-positive-airway-pressure (CPAP) therapy [ 10 ]. The limiting factors of O 2 -transport or muscle function responsible for an inverse relation between AHI and aerobic capacity have yet remained unclear, but may not include histomorphological muscle microvascularisation, which was found to be increases at least in the tibialis anterior muscle [ 11 ]. While histomorphological data on locomotor muscle with OSA are surprisingly scarce, several studies in striated upper airway muscles have indicated various important alterations such as fiber type grouping [ 12 , 13 ], decreases in fiber cross-sectional area [ 14 ], muscle fiber atrophy [ 13 ], centrally located nuclei [ 12 ] or abnormal mitochondrial distribution [ 15 ]. Thus, mechanisms underlying functional impairment of locomotor muscles have remained to be assessed. Notably, adequate muscle function is not only dependent on the integrity of muscle fibers themselves but also reliant on intact neuromuscular signal transmission via myelinated motor neuron, neuromuscular junction (NMJ) and postsynaptic sarcolemma [ 16 ]. NMJ integrity and plasticity is considered critical for muscular function: While several studies have demonstrated a relationship between increases in pre- and postsynaptic areas and enhanced neuromuscular activity as well as increased fatigue resistance [ 17 – 20 ], age-related changes in NMJ, such as fragmentation or lowered NMJ area, are considered to contribute to sarcopenia [ 21 , 22 ]. Moreover, investigations on mice lacking antioxidant enzymes (SOD −/− ) or overexpressing uncoupling proteins (UCP1) supported the hypothesis, that oxidative stress may trigger NMJ degenerations which occur in association with mitochondrial dysfunction [ 23 , 24 ]. Most relevant to OSA, a massive generation of reactive oxygen/nitrogen species (ROS/RNS) and related excessive oxidative and nitrosative stress has been attributed to repetitive nocturnal hypoxia-reoxygenation cycles in analogy to repeated ischemia and reperfusion injuries [ 25 , 26 ]. Indeed, evidence exists in OSA patients for increased production of superoxide, which was normalized under CPAP therapy [ 27 ], as well as for a decrease in anti-oxidative capacity [ 28 , 29 ]. Moreover, studies in OSA patients as well as in CIH animal models detected an overexpression of inducible nitric oxide synthase (iNOS) via inflammatory triggers involving NF-κB activation, especially in neuronal or cardiovascular tissues, e.g. activated macrophages [ 30 – 32 ]. iNOS may become a source of massive amounts of nitric oxide (NO, only limited by a lack of substrate or coenzymes) which as a highly reactive free radical forms peroxynitrite and other RNS compromising mitochondrial respiration, cell membrane integrity or insulin signaling [ 33 ]. iNOS expression in skeletal muscle is observed in (type 2 fibers) of obese/diabetic adult, but rarely in healthy young subjects, unless exercising, while muscular iNOS expression in rodents was mainly detectable and studied in rats [ 34 ]. The present study used CIH as compared to normoxic control (NOX) as model of OSA in wildtype (WT) mice to investigate the long-term effect of OSA on skeletal muscle histomorphological measures of NMJ integrity, fiber size and composition as well as on transition electron microscopy (TEM) parameters of ultrastructural mitochondria integrity. Moreover, besides WT, we included iNOS-deficient (iNOS −/− ) mice into this analysis in order to evaluate the putatively risk-mediating role of iNOS within the pro-oxidative or -inflammatory stress arising from CIH-exposure. To the best of our knowledge, this is the first report on CIH-induced muscle-specific damage to NMJ, fiber histomorphology or mitochondrial ultrastructure in WT mice locomotor muscle, which unexpectedly is not inhibited by iNOS deficiency, but strikingly mimicked already at NOX. Materials And Methods CIH - mouse model of OSA: The mouse model of OSA was based on the long-term exposure to CIH as described by Schulz et al. (Schulz et al. 2014). Male C57BL/6J-mice (WT, Charles River Deutschland GmbH, Sulzfeld, Germany) and iNOS-deficient mice (iNOS −/− ; strain B6.129P2-Nos2 tm1Lau /J) aged 8–10 weeks were exposed to a 6-week CIH profile (8 h/d 5 d/week) in a PC-controlled normobaric gas chamber which allowed alternating O 2 -concentration between 7% and 21% at cycles of 120 s corresponding to an AHI of 30 h − 1 , i.e. border between moderate to severe sleep apnea. NOX control conditions for age-matched WT and iNOS −/− mice were rendered by the same chamber system flushed with room air (21% O 2 ). CIH and control intervention were limited to daytime as mice are active at night. All animals were provided with standard diet and water ad libitum. All mice were weighed prior to intervention and immediately before euthanization. After 6 weeks of CIH-/control intervention, mice were euthanized and the triceps surae (gastrocnemius, soleus and plantaris muscles) as well as the vastus lateralis muscle carefully removed, immediately shock frozen in liquid nitrogen-cooled isopentane and stored at -80 °C. Transversal cryosections of 7 µm (microtom Hyrax C60, Carl Zeiss AG, Oberkochen, -20 °C) were obtained for (immuno)histochemistry. Tibia length was carefully determined by a caliper. Muscle fiber composition, size and centronucleation: Muscle fiber types (1, 2a, 2x) were identified via the acid-sensitive myofibrillar ATPase (Adenosine triphosphatase, Sigma-Aldrich Co. LLC, St. Louis, Missouri) staining at pH 4.55 as previously described (Friedmann-Bette et al. 2010) in randomly distributed two to three images taken at 200-fold magnification by the Zeiss Axio Imager.M2 microscope (Carl Zeiss AG; Oberkochen, Germany) after digitalization by the imaging system Axio-Cam HRc/AxioVision (Carl Zeiss GmbH). Type-specific fiber cross-sectional area (CSA) was determined by manually encircling each cross-section of at least 100 fibers using standard imaging software ImageJ (Scion Image, National Institutes of Health, Bethesda, USA). In addition, transverse cryosections were stained by hematoxylin and eosin to determine the percentage of centronucleated fibers. Postsynaptic NMJ morphology and integrity: NMJ analyses were based on α-bungarotoxin (BTX) staining in cryosections after fixation with 4% PFA/PBS (paraformaldehyde/phosphate buffered saline pH 7.4) for 10 min and blocking of endogenous peroxidase with 3% H 2 O 2 . Thereafter, PBS-washed sections were incubated overnight with biotinylated BTX (1:500; Invitrogen Eugene, USA) in a humidified chamber, PBS-washed, blocked with 2% bovine serum albumin (BSA)/PBS, PBS-washed again, and incubated with horseradish peroxidase (HRP)-conjugated Streptavidin (Jackson ImmunoResearch Laboratories. Inc., West Grove, USA). 3,3'-Diaminobenzidine (DAB) was used as a chromogen substrate. Nuclei were counterstained with Mayer’s Hematoxylin (Carl Roth GmbH, Germany). NMJ were identified in three consecutive, complete cross-sections per muscle from digital images (200-fold magnification) obtained by the Zeiss Axio Imager.M2 microscope (Carl Zeiss AG; Oberkochen, Germany) combined with Axio-Cam HRc/AxioVision (Carl Zeiss GmbH). The NMJ length and area were determined manually together with the corresponding myofiber CSA and perimeter using ImageJ software (Scion Image, National Institutes of Health). Single NMJ were considered as “fragmented” when the BTX-stained area was divided into several sections. Further calculations included NMJ area per myofiber CSA, NMJ length per myofiber perimeter and the percentage of fragmented NMJ (McLoon et al. 2016). On average, 21 ± 2 (16 ± 3) NMJ were analyzed per soleus or gastrocnemius muscle. Pre- and postsynaptic NMJ co-staining: To analyze the innervation status by double staining of NMJ pre- and postsynapse, every 6th of 60 serial 7 µm transverse sections of the vastus muscle (WT: n = 4 NOX, n = 6 CIH; iNOS −/− : n = 6 NOX, n = 6 CIH) was stained for immunofluorescence with biotin-XX-conjugated BTX (Invitrogen Eugene, USA) and RvAChT (vesicular acetylcholine transporter) antibody (Lee Eiden, Laborchargen-Nr.: bl. 6/97). In detail, after fixation for 10 min in 4% PFA/PBS, and blockage by 1% BSA/PBS for 30 min, sections were incubated overnight at 4 °C with biotinylated BTX (1:500) or vAChT 80259 (1:1000) in a humidified chamber. The next day, washing was followed by two hours incubation with Alexa Fluor® 488 labeled donkey anti-rabbit IgG ( 1:200, MoBiTec GmbH, Göttingen, Germany ) or Cy3-conjugated streptavidin (1:200, Dianova GmbH, Hamburg, Germany) respectively. Finally, slides were mounted using Immu-Mount™, (Fisher Scientific GmbH, Schwerte, Germany) and glass coverslips. Control sections were treated similarly but with either no primary antibody to exclude non-specific staining of secondary antibody. Confocal images were taken by the scanning laser microscope C2 (Nikon GmbH, Düsseldorf, Germany) using the software NIS-Elements AR 4.30.01 (Laboratory Imaging). Each NMJ was scanned in a 630-fold magnification at 250 Hz with an image size of 1024 × 1024 pixels. Subsequently, Fiji software (National Institute of Health, Bethesda, USA) was used for morphometrical analysis, determining the BTX and vAChT immunolabeled areas as well as their overlap area. Transmission electron microscopy (TEM): Mitochondrial ultrastructure of soleus and gastrocnemius muscle was evaluated using TEM as previously described [ 35 ]. Images were taken at 10.000-fold magnification and ten random pictures from both muscles of each mouse were analyzed using ImageJ software (Scion Image, National Institutes of Health, Bethesda, USA). Mean mitochondrial size was determined by manually encircling all identified mitochondria. Alterations on mitochondrial architecture were assessed by the categorization “normal” or “damaged” as follows: Mitochondria showing a loss of more than 50% of the cristae or a disruption of more than 50% of the outer membrane were assigned to “damaged” or otherwise to “normal”. The results were given as the percentage of the total number of mitochondria. Moreover, the percentage of swollen mitochondria and mitochondria containing multi-lamellar bodies was determined. Quantitative RT-PCR: The extraction of RNA was performed using peqGOLD Isolation Systems TriFast™ (PEQLAB Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer’s instructions. RNA quality (OD260nm / OD280nm = 1.7 to 2.0) and concentration were determined using the NanoDrop 2000c spectrophotometer (Thermo Scientific, Schwerte, Germany). RNA integrity was confirmed by lab-on-a-chip technology, using an RNA 6000 NanoChip kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Total RNA (0.7 µg) was then treated with 1 unit DNAse (Thermo Scientific, St. Leon-Rot, Germany; 30 min, 37 °C). Thereafter, reverse transcription of RNA was carried out with 500 ng oligo (dT) 12− 18 primer, 20 units of the Affinity Script multiple temperature cDNA synthesis (Agilent) and 24 units of Ribo Lock™ RNAse inhibitor (Fermentas; 1 h, 42 °C) and 4 mM dNTP-Mix. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed in duplicate using the QuantiTect/primerAssays from QIAGEN GmbH (Hilden, Germany). To confirm the primer specificity and the presence of a single amplicon, a melting curve (55–95 °C) of the amplified product was performed. For amplification and data analysis the Mx3005P™ QPCR System (Stratagene) was used. For each sample, the relative amount was calculated by linear regression analysis from their respective standard curves which was generated from a pool of cDNA. Among the transcripts Actb, Gapdh and Tbp, Tbp was identified by the NormFinder software as the most stable reference gene for RNA normalization of gene of interest (additional file 1). Statistics: Data are presented as means ± standard error of the mean (SEM). Differences between CIH and NOX or iNOS −/− and WT were detected by ANOVA and Student’s t-test (post-hoc) or, in case of not normally distributed data, by the Kruskal-Wallis ANOVA and Dunn's post-hoc test. A p < 0.05 was considered as statistically significant. For all statistical procedures SigmaPlot 14.0 software (Systat Software, Inc, Chicago, USA) was used. Results Body weight: Effect of CIH in WT In WT mice, 6 weeks of CIH compared to NOX significantly reduced the pre- to post-interventional gain in body weight, i.e., the weight gain between 2 and 3.5 months of age (starting from a 9.1% higher baseline) (Table 1 ). Effect of iNOS w/o CIH Under NOX conditions, iNOS −/− mice showed a significantly lower weight gain compared to WT (Table 1 ), notably, starting from by 10.7% (p < 0.001) higher pre-interventional body weight than WT mice. When exposed to CIH, iNOS −/− mice even showed a weight loss and thus differed significantly from NOX conditions compared to NOX (Table 1 ). Table 1 Weight (g) and weight changes (%) of WT and iNOS −/− mice pre- to post-intervention. Values are given as mean + SEM, n = 6–14 animals per group. * p < 0.05, significance between CIH and NOX; ## p < 0.01, ### p < 0.001, significance between WT and iNOS −/− . WT NOX (n = 11) WT CIH (n = 14) iNOS −/− NOX (n = 6) iNOS −/− CIH (n = 8) Pre-intervention weight (g) 21.9 ± 0.5 23.9 ± 0.5* 24.8 ± 0.7 ### 26.0 ± 0.9 Post-intervention weight (g) 26.4 ± 0.5 25.9 ± 0.3 25.5 ± 0.7 24.1 ± 0.3 Weight change (%) 21.1 ± 3.7 9.0 ± 2.0* 2.9 ± 3.0 ## -7.4 ± 0.8* Fiber morphology and fraction in soleus muscle: Effect of CIH in WT The mean CSA covering all fiber types in soleus muscle did not significantly differ between CIH intervention and NOX in WT (Fig. 1 A-B, E left ), though it correlated significantly and positively with the weight gain (r = 0.41; p = 0.047) in WT of both interventional groups. A fiber type-specific analysis showed, that CIH compared to NOX led to a significant decrease in fiber CSA exclusively of type 2a fibers by 13.5% (p < 0.05) (Fig. 1 E, right ). Thereby, CIH as compared to NOX resulted in an increase in type 1 fiber fraction (37% vs. 31%; p < 0.01) in association with a decrease in type 2a fiber fraction (44% vs. 53%; p < 0.001) at unchanged type 2x fiber fraction (Fig. 1 A-B, G). A parallel CSA-specific analyses of fiber distribution showed that CIH compared to NOX significantly increased the fraction of small fibers (CSA < 800µm²) in soleus muscle (Fig. 2 ). Moreover, there was a significantly higher fraction of centronucleated fibers with CIH compared to NOX (1.5% vs. 0.2%; p = 0.01) in WT soleus muscle, as counted in HE stained sections (Fig. 3 , left ). A significant inverse correlation was found between the fraction of centronucleated fibers and the CSA of the fiber population in WT undergoing CIH or NOX (r=-0.417; p = 0.043). Effect of iNOS w/o CIH : In both conditions, NOX and CIH, iNOS −/− compared to WT was without any significant effect on fiber CSA of the total fiber population or that of fiber type 1, 2a or 2x. In iNOS −/− mice, CIH as compared to NOX caused no significant changes in CSA of fibers in total or of type (Fig. 1 F), while CIH-related changes in fiber composition partly resembled those in WT (Fig. 1 A, C, H): CIH compared to NOX in iNOS −/− mice led to a decrease in type 2a fiber fraction (45% vs. 51%; p < 0.05) and an associated non-significant increase in type 1 fiber fraction (39% vs. 35%) at unchanged type 2x fiber fraction (Fig. 1 C, D, H). Concerning the centronucleation, no significant difference was detected between the two genotypes or between CIH and NOX in iNOS −/− mice (Fig. 3 ). Fiber morphology and fraction in gastrocnemius muscle: In contrast to soleus muscle, the gastrocnemius muscle mostly consisting of fiber type 2x revealed no significant effect of CIH vs. NOX and iNOS −/− vs. WT or their combination with regard to CSA (additional file 2) or percentage of central nuclei (additional file 3). NMJ morphology/integrity in soleus muscle: Effect of CIH in WT mice In soleus muscle of WT mice BTX-staining of NMJ showed, that CIH compared to NOX led to a significantly by 37% smaller postsynaptic NMJ area (Fig. 4 A-C, left ). When calculating NMJ area relative to fiber CSA, (Fig. 4 D, left ), the CIH compared to NOX significantly reduced postsynaptic NMJ size. An alternative normalizing of NMJ length to fiber perimeter similarly decreased NMJ size with CIH compared to NOX (Fig. 4 E, left ). No sigificant CIH-related change occurred regarding the fraction of fragmented NMJ in WT mice (Fig. 4 F, left , G). Effect of iNOS −/− w/o CIH In comparison to WT, iNOS −/− mice demonstrated a significantly by 55% diminished NMJ area under the condition of NOX (Fig. 4 C), with this effect being significant also upon normalization of postsynaptic NMJ area for fiber CSA (Fig. 4 D) or, alternatively, of postsynaptic NMJ length for fiber perimeter (Fig. 4 E). CIH compared to NOX intervention in iNOS −/− mice led to an additional decrease in NMJ area (in absolute terms) by trend (Fig. 4 C, right ), however, this effect reached significance when normalizing postsynaptic NMJ area for fiber CSA (Fig. 4 D, right ) or, alternatively, postsynaptic NMJ length for fiber perimeter (Fig. 4 E, right ). Notably, a strikingly higher percentage of NMJ fragmentation was observed selectively with iNOS −/− as compared to WT under both conditions (Fig. 4 F, right , H). NMJ morphology/integrity in gastrocnemius and vastus muscle: Gastrocnemius muscle In contrast to soleus muscle, gastrocnemius muscle in WT mice showed a significantly increased NMJ size with CIH as compared to NOX (72%, p < 0.05, additonal file 4). However, this difference was abolished when normalizing postsynaptic NMR area for fiber CSA. Moreover, unlike the soleus muscle, the gastrocnemius muscle in iNOS −/− mice revealed no significant alterations in postsynaptic NMJ area, and this was also true for both above-mentioned normalization of NMJ area or length for fiber CSA or perimeter, respectively. Vastus muscle For further evaluation of functional NMJ integrity, double fluorescent staining was used in vastus muscle to quantify the area of the NMJ presynaptic nerve terminal (vACHT-antibodies, Fig. 5 A, D), of postsynaptic NMJ (BTX, Fig. 5 B, E) and that of their coupling (overlay, Fig. 5 C, F). In line with the findings in soleus (but not in gastrocnemius) muscle, there was a significant diminution by 27.9% (p < 0.01) of postsynaptic NMJ in iNOS −/− compared to WT under conditions of NOX (Fig. 5 G), whereas CIH-effects compared to NOX were absent in WT or iNOS −/− mice. The presynaptic terminal, defined as the vACHT immunoreactive area, remained resistant against CIH- or genotype related effects (5H). The resulting percentage overlay area, a measure of NMJ integrity, was not significantly affected by CIH-intervention or iNOS −/− (Fig. 5 I). Mitochondrial ultrastructure in soleus muscle: The following ultrastructural mitochondrial abnormalities were quantified in soleus muscle of WT exposed to NOX (Fig. 6 A) or CIH (Fig. 6 B) as well as in iNOS −/ − mice in NOX (Fig. 6 C) or CIH (Fig. 6 D): Swollen matrix (Fig. 6 H), disruption of the outer mitochondrial membrane (Fig. 6 I), a complete loss of internal architecture (Fig. 6 J) and mitochondria with multi-lamellar bodies (Fig. 6 K). Effect of CIH in WT The percentage of damaged mitochondria, classified as < 50% filled with cristae, was significantly 1.8-fold higher in CIH vs. NOX (Fig. 6 E, left ), while an increase in percentage swollen mitochondria with CIH did not reach significances (Fig. 6 F, left ). The difference between CIH vs. NOX regarding the percentage mitochondria containing multi-lamellar bodies did not reach significance (12.8% vs. 6.6%; p > 0.05). Effect of iNOS −/− w/o CIH Somewhat reminiscent of CIH vs. NOX effects in WT, iNOS −/− compared to WT mice at NOX conditions revealed a significant 2.1-fold increase of damaged mitochondria (Fig. 6 E). Moreover, percentage of swollen mitochondria in iNOS −/− vs. WT mice was significantly increased under both conditions (NOX > 6-fold; CIH > 5-fold) (Fig. 6 F). Mitochondrial ultrastructure in gastrocnemius muscle: In gastrocnemius muscle, the abovementioned mitochondrial alterations were observed neither with CIH vs. NOX in WT nor with iNOS −/− of either condition (additional files 5–6). Correlations between NMJ fiber and mitochondrial morphology: In soleus muscle the NMJ area was significantly correlated with the percentage of damaged mitochondria (r = 0.584, p = 0.002) as well as with the ratio between type 1 and type 2a fibers (r = 0.397, p = 0.05) (Table 2 ). Table 2 Correlations of NMJ area and fragmented NMJ, percentage of damaged mitochondria, myofiber CSA and fiber type ratio (1/2a) in soleus muscles, n = 5–8 animals per group. Soleus muscle (n = 5–8 animals per group) NMJ area (µm²) Fragmented NMJ (%) Fiber type ratio; 1/2a Damaged mitochondria (%) r -0.710 0.584 0.394 p 0.000 0.002 0.051 NMJ area (µm²) r -0.642 -0.575 p 0.001 0.003 Fragmented NMJ (%) r 0.396 p 0.050 Transcripts of iNOS, SOCS3, SOD2 and pro-/antiapoptotic markers: Notably, mRNA expression of iNOS was undetectable in soleus (as well as in gastrocnemius) muscle of WT mice undergoing NOX or CIH intervention (Table 3 ). However, importantly, an iNOS expression was well detectable in the liver of WT mice, where it decreased significantly and massively by factor 0.12 (Table 3 ) after CIH-treatment. The absence of iNOS expression was proven in the liver and soleus (and gastrocnemius) muscle of iNOS −/− mice. Importantly, in WT mice, SOCS3 expression was found to be > 10-fold increased with CIH compared to NOX (Table 3 ). Similarly, iNOS −/− prompted a SOCS3 upregulation that was > 10-fold in NOX and > 4-fold in CIH as compared to WT at NOX (Table 3 ). Moreover, in WT mice, CIH compared to NOX led to an almost 50% decrease in mtSOD mRNA expression in soleus muscle. Similarly, iNOS −/− resulted in a 33% and a 45% decrease in SOD2 expression in soleus with NOX and CIH, respectively (Table 3 ). Furthermore, the screening for apoptotic markers (BAX, BCL2, caspase 3) showed neither CIH- nor iNOS −/− -related changes in soleus muscle (Table 3 ). Table 3 Relative gene expression in soleus muscle and liver of WT and iNOS −/− mice. iNOS SOCS3 SOD2 BAX BCL2 Caspase 3 Soleus muscle WT NOX 0.00 1.00 1.00 1.00 1.00 1.00 WT CIH 0.00 10.89 0.53 1.10 1.14 0.90 iNOS −/− NOX 0.00 10.45 0.75 1.23 1.01 1.09 iNOS −/− CIH 0.00 4.43 0.55 0.98 1.07 0.79 Liver WT NOX 1.00 WT CIH 0.12 Discussion Using long-term CIH exposure in mice as a model of OSA, the present study shows for the first time that CIH compared to NOX causes damage of potential functional relevance in ‘red’ (soleus) but not in ‘white’ (gastrocnemius) muscle. This comprises a reduction in area, in length and, by trend, in integrity of postsynaptic NMJ as well as in size (CSA) and fraction of type 2a fibers (at higher type-1 fiber fraction). Moreover, these changes were associated with considerable mitochondrial damage, which showed a significant correlation to (loss in) NMJ area (r=-0.71, p < 0.001) and were, again, limited to soleus muscle, while gastrocnemius revealed no significant mitochondrial damage. The present study furthermore included iNOS −/− mice into this analysis of CIH vs. NOX effects on skeletal muscle, in order to test the hypothesis that iNOS deficiency may at least in part protect against a pro-inflammatory/-oxidative effect through CIH, i.e. hypoxia-reoxygenation stress leading to ROS generation from various sources [ 36 ]. Contrary to expectation, our data demonstrate that, compared to WT, iNOS −/− by itself (i.e. under NOX conditions) also leads to highly significant postsynaptic NMJ area reduction and fragmentation in combination with mitochondrial damage and swelling, which surprisingly resemble and exceed those observed with CIH in WT mice. Notably, under the conditions of iNOS deficiency, CIH stress is able to further aggravate the damage at least in terms of a further reduction in postsynaptic NMJ area or length after normalization for fiber CSA or perimeter, respectively. The similarity between CIH (compared to NOX in WT) and iNOS −/− (compared to WT in NOX) was limited to NMJ and mitochondrial damage, while decreases in fiber CSA (including its correlation to NMJ) and centronucleation observed with CIH vs. NOX in WT were absent in iNOS −/− mice, i.e. they revealed no atrophy despite signs of denervation. As another striking similarity, we found a > 10-fold increase in SOCS3 expression with CIH vs. NOX in WT as well as with iNOS −/− vs. WT at NOX in (pooled samples of) soleus muscle. Available evidence qualifies SOCS3 as a candidate to mechanistically link mitochondrial damage to NMJ deterioration: SOCS3 upregulation has been demonstrated as an early event after skeletal muscle denervation by sciatic nerve transection [ 37 ]. This obviously occurs in response to local inflammatory signals, especially via IL6 which by itself, i.e. without denervation, causes upregulation of SOCS3 and of E3-ligases (atrogin-1, MURF-1) together with fiber atrophy all of which is abrogated by IL6 inhibition. At the same time, SOCS3 overexpression has been shown to cause mitochondrial damage like swelling or disruption in tibialis anterior muscle, which is reminiscent of what was presently observed in soleus muscle but not in gastrocnemius muscle. SOCS3 overexpression was, furthermore, associated with inhibited expression of mitochondrial genes, which included Smtck and Slc25a3 [ 38 ], but may also comprise mtSOD, which was presently found to be downregulated. As an inhibitor of leptin and insulin signaling, increased muscular SOCS3 expression has been suggested as a major contributor to mitochondrial dysfunction, impaired fatty acid oxidation, as associated with aging, metabolic syndrome and inflammation [ 39 – 41 ]. These severe metabolic effects in combination with previous evidence that SOCS3 overexpression dilates the sarcoplasmatic reticulum, dislocates and inhibits calcineurine (colocalized with SOCS3) and reduces skeletal muscle energy expenditure, oxygen uptake and activity [ 38 ] may contribute to muscle fiber atrophy as observed in case of CIH. While we found no evidence for increased apoptosis signals, a SOCS3 upregulation appears to be associated with impaired regenerative stem cell function in elderly humans [ 39 ] and may potentially play a role in the increased centronucleation presently observed with CIH. Moreover, the moderate decrease in mtSOD expression presently observed in soleus muscle with CIH and, to a lesser extent, with iNOS (NOX or CIH) might also play a role in fiber atrophy and mitochondrial deterioration: Sod1 −/− mice, used as a murine model of neuromuscular impairment in age-related muscle atrophy (sarcopenia), exhibit reduction in myofiber CSA of type IIa fibers [ 42 , 43 ]. Reduced CSA of type IIa fibers was, indeed, presently observed in association with the most marked mtSOD decrease (ca 50%), lower weight gain and alterations in mitochondrial ultrastructure and NMJ morphology. In addition, the observed shift in fiber metabolic phenotype, i.e. an increased type 1 at a decreased type 2 fiber fraction with both CIH (vs. WT) and iNOS −/− (vs. WT at NOX) might be attributed to decreased mtSOD expression, rather than to SOCS3 upregulation which decreases oxidative fiber characteristics [ 38 ]. Deficiency in mtSOD, representing impaired antioxidant defense, may also be involved in a remarkable number of age-related features which may originate from a loss of fast motoneurons followed by a reinnervation of slow motoneurons [ 44 ]. In the present study, fiber type ratio (type 1/type 2a) was significantly correlated with NMJ fragmentation and inversely correlated with NMJ size, pointing at a role of reinnervation in the fiber type shift. Thus, our observations with CIH may display some analogies to age-related neuromuscular deterioration involving SOCS3 and mtSOD. They are in line with previous studies in other rodent CIH models, revealing downregulation of mtSOD/SOD2 via downregulation of HIF-2α [ 45 ] and clinical observation of lower plasma CuZnSOD/SOD1 in OSA patients [ 46 ]. Our CIH-based OSA mouse model is, however, at variance with biopsy studies in OSA patients, which revealed no changes in tibialis anterior muscle fiber size compared to controls [ 11 ] or showed even enlarged diameters of type 2a fibers in quadriceps femoris at unaltered fiber type composition [ 47 ]. One should, however, bear in mind, that the CIH mouse model does not mimick certain OSA-inherent factors like sways in intrathoracic pressure and blood pCO 2 as well as ventilatory overshoots but at the same time involves more severe O 2 -desaturation without airway obstruction. Also the genetic background of mice may affect the degree of atrophy [ 48 ]. Even more important, our data provide first evidence for a strikingly differential effect of CIH-exposure between soleus and gastrocnemius i.e. (mixed) ’red’ and ‘white’ muscles, that has to be taken into account in translational studies. Indeed, in contrast to soleus muscle, gastrocnemius muscle revealed neither mitochondrial damage nor NMJ alterations (rather enlargement than shrinkage) with CIH. The differential exertion profiles between the postural soleus muscle (remaining recruited throughout during quiet standing) and the locomotor gastrocnemius muscle (providing fast forceful contractions) [ 49 ] may impact these muscle-specific findings in CIH and iNOS −/− mice and have likewise been implicated in massive muscle- (fiber-) specific differences in muscle aging or neurodegenerative disease [ 50 – 52 ]. However, our findings of compromised mitochondrial ultrastructure and gene expression in soleus muscle of CIH-mice may be in line with those in human palate muscle (a primary research focus within OSA pathophysiological), showing abnormal mitochondrial function and organization [ 53 ]. The observed close positive correlation of the fraction of damaged mitochondria to fragmented NMJ (or inverse correlation to NMJ area) reveals no clue for cause-effect relationship. As a first assumption, mitochondria-derived oxidative stress during CIH (hypoxia-reoxygenation stress) may compromise NMJ [ 54 ] acting in combination with other ROS sources like upregulated NOX2, as reported for the presently used CIH mouse model [ 55 ]. However, importantly, iNOS −/− at NOX largely mimicked the CIH effects, i.e. NMJ and mitochondrial damage together with SOCS3 up- and mtSOD downregulation and was despite the fact that iNOS mRNA expression in soleus or gastrocnemius muscles in WT was neither detectable with NOX nor with CIH exposure. Since, in contrast, WT mice revealed a hepatic iNOS expression, which was massively and significantly reduced (> 8-fold) with CIH compared to NOX, it is reasonable to assume that iNOS deficiency outside the skeletal muscle conveys both, the CIH and iNOS −/− effects. Thereby the NMJ damage, similarly observed with both these condition, strongly points towards an iNOS deficiency in peripheral nerves (i.e. in perikaryon of motoneurons or Schwann cells) as a cause of NMJ damage, though myeloid iNOS expression may also become muscle-protective [ 56 ]. Peripheral nerve injury may dramatically upregulate the low constitutive iNOS expression in Schwann cells, and iNOS deletion may result in smaller regenerating myelinated fibers and delayed reinnervation of muscle NMJ distal to the injury [ 57 ]. In fact, peripheral nerve dysfunction in patients suffering from OSA appears to be an early event [ 58 ], and denervation may precede muscular dysfunction, as suggested for human upper airway muscles [ 37 , 59 , 60 ] and supported by increased sarcolemmal N-CAM staining [ 15 ]. To date, no corresponding neuromuscular data exist for human locomotor muscle with OSA. However, they are needed to evaluate functional relevance of these alterations and to separate OSA-specific effects on NMJ, mitochondria, metabolism and related fiber dysfunction from the processes of aging and degenerative diseases [ 22 , 61 , 62 ]. Notably, the age of mice presently under test (four months) corresponded to early human adulthood (20–30 years) [ 63 ]. The conclusion that iNOS expression (outside skeletal muscle) may be neuro-protective and relevant for ‘red’ (aerobic) muscle function may be somewhat counterintuitive, as iNOS upregulation is resulting in a boost of NO, that is antimicrobial or antitumoral but also cytotoxic to normal tissue [ 64 ]. Furthermore it is causally implicated e.g. in insulin resistance and diabetes. Nonetheless, in humans a basal NO production rate (rendered mostly but not exclusively by nNOS and eNOS) is physiologically required (reviewed by [ 34 ]). There is evidence that NO may convey physiological oxidative signals [ 65 ] and a certain production by iNOS is required for neuroprotective antioxidative defense [ 66 ], e.g. through the ROS scavenging function of NO [ 64 , 66 ]. Data on skeletal muscle tissue are scarce, however, it was reported that iNOS deficiency leads to mitochondrial damage in myocardial dysfunction (adriamycin-based mouse model). Interestingly, this effect was abrogated by overexpression of mtSOD [ 67 , 68 ], which presently was found to be downregulated with both, CIH or iNOS deletion. As a limitation, this study includes no functional data regarding NMJ and skeletal muscle to challenge the relevance of morphological alterations. Moreover, our mouse model involved a limited CIH exposition of 5 days per week, which may allow adaptive or protective effects of 2 normoxic days per week. Nonetheless a previous study showed, that the pathophysiological changes of the clinical OSA, such as arterial hypertension, are accurately reflected by the here used CIH mouse model [ 55 ]. Conclusion In summary, this is the first study to demonstrate CIH as a model of moderate to severe OSA triggers NMJ and mitochondrial damage accompanied by fiber atrophy in slow-twitch muscle of WT mice, all of which may contribute to reduced exercise (aerobic) capacity in patients suffering from OSA. We furthermore demonstrate that iNOS deficiency, rather than yielding protection of skeletal muscle against CIH stress, leads to similar structural impairments of NMJ and mitochondria under normoxia and might contribute to the CIH effects in WT, putatively through compromised innervation. Abbreviations OSA - obstructive sleep apnea CIH - chronic intermittent hypoxia NMJ - neuromuscular junction NOX - normoxia CSA - cross-sectional area ATP - adenosine triphosphate TEM - transmission-electron-microscopy WT - wildtype iNOS - inducible-nitric-oxide-synthase SOCS3 - suppressor-of-cytokine-signaling-3 AHI - apnea–hypopnea index CPAP - continuous-positive-airway-pressure UCP - uncoupling protein ROS - reactive oxygen species RNS - reactive nitrogen species NO - nitric oxide BTX - α-bungarotoxin PFA - paraformaldehyde PBS - phosphate buffered saline HRP - horseradish peroxidase BSA - bovine serum albumin DAB - 3,3'-Diaminobenzidine vAChT - vesicular acetylcholine transporter Actb – actin beta GADPH - glyceraldehyde-3-phosphate dehydrogenase TBP - TATA-box binding protein SEM - standard error of the mean mtSOD – mitochondrial superdioxide dismutase BAX - BCL-2-associated X protein BCL2 - b-cell lymphoma 2 MURF-1 - Muscle RING-finger protein-1 sMtCK - sarcomeric mitochondrial creatine kinase Slc25a3 - Solute Carrier Family 25 Member 3 nNOS - neuronal-nitric-oxide-synthase eNOS - endothelial-nitric-oxide-synthase Declarations Ethics approval and consent to participate Animal experiments were approved by the regional board (RP Giessen, Hesse, Germany; Az: V 54-19 c 20 15 h 01 GI 20/10 Nr. 84/2011) in accordance with the German animal welfare law and the European legislation for the protection of animals used for scientific purposes (2010/63/EU). Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Materials used in this study are commercially available. Competing interests The authors declare that they have no competing financial interests. Funding This work was supported by grants from the von-Behring-Röntgen-Stiftung (Project 580071). Authors' contributions RK and WH conceived, managed and supervised the project, designed experiments and obtained funding. RS and NW contributed to the study design and edited the manuscript. LB and SM performed experiments and analyzed data with contributions from GB, MB and SK. LB and WH wrote the manuscript with input from all authors. The authors read and approved the final manuscript. Acknowledgements The authors gratefully acknowledge the expert laboratory assistance of Claudia Keppler, Michael Dreher, Steffi Zügel and Irmgard Dammshäuser. 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Values are given as mean+SEM; n=8-10 animals per group. additionalfile3.jpg The percentage of centronucleated fibers in gastrocnemius muscle is shown. Values are given as mean+SEM; n=8-10 animals per group. additionalfile4.jpg The post-synaptic NMJ area in gastrocnemius muscle is shown. Values are given as mean+SEM; n=8-10 animals per group. * p<0.05, significance between CIH and NOX. additionalfile5.jpg The percentage of damaged mitochondria in gastrocnemius muscle is shown. Values are given as mean+SEM; n=8-10 animals per group. additionalfile6.jpg The percentage of swollen mitochondria in gastrocnemius muscle is shown. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-102618","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":4480424,"identity":"0db8c311-42ab-4830-a630-f0a52fda154a","order_by":0,"name":"Laura Isabel Bannow","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYBACAwhlA8QJMC4PWIixAb+WNNK1HIZqYSBCizn72WMPPradj+ZnT2CTLihgiOafffaYdMUvBtl+HFose/LSDWe23c6d2fOATXqGAUPujHN5aZJn+xiMZ+KwxuBAjpk077bbuRtuAG3hMfif23CGx0yysYchccMBHFrOvwFpOZe7H6KFIXc+TMt+XFpugG05kLtBAqplA0hLww+gLbj8MuONmeTMf8m5M848bLYGadl4hi/ZsrFBwngGDlvM+XPMJD6cscvtb08+eJvnD0PuvDO8B282/LGR7cfhfSSAHBGMbRIE1aODPyTrGAWjYBSMguELAFv7W82WwXWcAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5770-5963","institution":"Philipps-Universität Marburg: Institute for Anatomy and Cell Biology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Laura","middleName":"Isabel","lastName":"Bannow","suffix":""},{"id":4480425,"identity":"b3c818fc-cdf0-452c-9deb-78e3f50de031","order_by":1,"name":"Gabriel A. Bonaterra","email":"","orcid":"","institution":"Department of Cell Biology, Institute for Anatomy and Cell Biologie, Philipps-University Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"A.","lastName":"Bonaterra","suffix":""},{"id":4480426,"identity":"2f718e5a-241b-4691-9e9f-495025a36e3e","order_by":2,"name":"Mirjam Bertoune","email":"","orcid":"","institution":"Department of Cell Biology, Institute for Anatomy and Cell Biology, Philipps-University Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mirjam","middleName":"","lastName":"Bertoune","suffix":""},{"id":4480427,"identity":"47cfd95c-dfb7-4fee-8098-7c3f589442e5","order_by":3,"name":"Sabrina Maus","email":"","orcid":"","institution":"Department of Cell Biology, Institute for Anatomy and Cell Biology, Philipps-University Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sabrina","middleName":"","lastName":"Maus","suffix":""},{"id":4480428,"identity":"6cb4a239-6598-4fe0-92fc-788c24c94e12","order_by":4,"name":"Richard Schulz","email":"","orcid":"","institution":"Department of Pulmonary Medicine, HELIOS Dr. Horst Schmidt Clinic, Wiesbaden","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Schulz","suffix":""},{"id":4480429,"identity":"a99ae45b-9e98-4a36-ad26-daab89016b17","order_by":5,"name":"Norbert Weissmann","email":"","orcid":"","institution":"Justus-Liebig University of Giessen, Excellence Cluster Cardiopulmonary Institute, Universities of Giessen and Marburg Lung Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Norbert","middleName":"","lastName":"Weissmann","suffix":""},{"id":4480430,"identity":"b567a0cb-4737-408a-8038-9efeae43a853","order_by":6,"name":"Simone Kraut","email":"","orcid":"","institution":"Justus-Liebig University of Giessen, Excellence Cluster Cardiopulmonary Institute, University of Giessen and Marburg Lung Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Simone","middleName":"","lastName":"Kraut","suffix":""},{"id":4480431,"identity":"9af270ec-5c46-4fad-9c71-3d7b7db90180","order_by":7,"name":"Ralf Kinscherf","email":"","orcid":"","institution":"Department of Cell Biology, Institute for Anatomy and Cell Biology, Philipps-University Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ralf","middleName":"","lastName":"Kinscherf","suffix":""},{"id":4480432,"identity":"a68cf9bc-01d0-4234-a146-f99a92a29c78","order_by":8,"name":"Wulf Hildebrandt","email":"","orcid":"","institution":"Department of Cell Biology, Institute for Anatomy and Cell Biology, Philipps-University Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wulf","middleName":"","lastName":"Hildebrandt","suffix":""}],"badges":[],"createdAt":"2020-11-03 21:42:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-102618/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-102618/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13395-022-00288-7","type":"published","date":"2022-02-12T13:45:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":3490058,"identity":"8196d6cd-682f-48a7-8f85-d6dc0f4c83bc","added_by":"auto","created_at":"2020-11-10 15:19:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":95423,"visible":true,"origin":"","legend":"Representative images (200fold magnification, scale bar = 50 µm) of soleus muscle cross sections stained for ATPase after preincubation at pH 4.55 of WT-NOX mouse (A), WT-CIH mouse (B), iNOS-/--NOX mouse (C) and iNOS-/--CIH mouse (D). Type 1 fibers are stained dark (white arrow), type 2a fibers are stained light-coloured (black star), and type 2x fibers are stained intermediate (black arrow head). (E) Overall myofiber CSA [µm²] and (F) CSA of type 2a myofibers in soleus muscle of WT and iNOS-/- mice. Fiber type distribution [%] in soleus muscle. Percentage of type 1, type 2a and type 2x fibers of WT mice after 6 weeks of CIH compared with NOX controls (G) and of iNOS-/- mice (H). Values are given as mean+SEM; n=8-14 animals per group. * p\u003c0.05, ** p\u003c0.01, *** p\u003c0.001, significance between CIH and NOX. ","description":"","filename":"OnlineFig.1.Png","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/cd3af651aed0f5855fbba37a.Png"},{"id":3490060,"identity":"5e6e7888-a9a9-4f70-99de-7f49026edb88","added_by":"auto","created_at":"2020-11-10 15:19:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":77249,"visible":true,"origin":"","legend":"Histogram of myofiber CSA in soleus muscle in WT mice. Values are given as mean+SEM; n=12-14 animals per group. * p\u003c0.05, significance between WT-CIH and WT-NOX.","description":"","filename":"Onlinefig2.Png","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/b9ac5eee2f170f745f27afb4.Png"},{"id":3490062,"identity":"a8cb4b87-f909-47b0-b4e3-73275660a9e4","added_by":"auto","created_at":"2020-11-10 15:19:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51264,"visible":true,"origin":"","legend":"The percentage of centronucleated fibers in soleus muscle is shown. Values are given as mean+SEM; n=8-14 animals per group. * p\u003c0.05, significance between CIH and NOX.","description":"","filename":"Onlinefig3.Png","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/52481957946e8a1f09033a4a.Png"},{"id":3490064,"identity":"3fd7d945-6912-40b4-ba9b-8d60b19a3cdf","added_by":"auto","created_at":"2020-11-10 15:19:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70275,"visible":true,"origin":"","legend":"Representative images (200fold magnification, scale bar=50µm) of soleus muscle cross sections stained for BTX of WT-NOX (A), WT-CIH (B) are shown. Postsynaptic NMJ area in soleus muscle (C). Postsynaptic NMJ area normalized to myofiber CSA (D) and NMJ length relative to myofiber perimeter (E) as well as percentage of fragmented NMJ in soleus muscle (F). Representative images (400fold magnification) of BTX-stained AChR distribution at muscular NMJ in soleus muscle (G-H). (G) Postsynaptic BTX-stained NMJ from WT-NOX mouse (white arrow). (H) Fragmented postsynaptic BTX-stained NMJ of WT-CIH mouse is (black arrow). Values are given as mean+SEM; n=8 animals per group. * p\u003c0.05, ** p\u003c0.01, *** p\u003c0.001, significance between CIH and NOX; # p\u003c0.05, ## p\u003c0.01, ### p\u003c0.001, significance between WT and iNOS-/-.","description":"","filename":"OnlineFig4.Png","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/159aaa5796832b16539627da.Png"},{"id":3490066,"identity":"1957aca2-402a-4ac8-8116-1304e5bd8fd4","added_by":"auto","created_at":"2020-11-10 15:19:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":17773,"visible":true,"origin":"","legend":"Confocal projection images of representative NMJ from the vastus muscle stained for postsynaptic AChR (BTX, A+D, red) and for nerve terminal vAChT (B+E, green). The merge is showing the colocalisation of BTX and vAChT (C+F). Example is given of an innervated muscular NMJ where the BTX-stained area is largely covered by the vAChT staining (A–C). In contrast, D–F are showing a NMJ where the AChR-stained areas are only partially covered by vAChT staining, representing denervation. Scale bar in panel F is 10µm. Mean postsynaptic (G) and presynaptic (H) NMJ areas and overlay areas of BTX and vAChT (I) in vastus muscle are shown. Values are given as mean+SEM; n=4-6 animals per group. ## p\u003c0.01, significance between WT and iNOS-/-.","description":"","filename":"Onlinefig5.Png","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/78936fad8b06682cda141cc5.Png"},{"id":3490068,"identity":"58360b25-6187-4968-8823-9147f1fd7b4f","added_by":"auto","created_at":"2020-11-10 15:19:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":335803,"visible":true,"origin":"","legend":"Representative TEM images of WT (A, B) and iNOS-/- (C, D) soleus muscle under CIH (B, D) vs. NOX (A, C) (scale bars=100nm). Percentage of damaged (E) and swollen mitochondria (F) are shown. Mitochondria presented various morphological abnormalities such as outer membrane rupture (I; black arrow), loss of cristae (J) or multi-lammelar bodies (K; black arrow head). G and H correspond to intact mitochondria completely (G) or partially (H) filled with cristae. Values are given as mean+SEM, n=7-9 animals per group. *** p\u003c0.001, significance between CIH and NOX, ### p\u003c0.001, significance between WT and iNOS-/-.","description":"","filename":"OnlineFig6.Png","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/7cfc44fdb11d868489002bb0.Png"},{"id":18161573,"identity":"53d5445c-031d-4012-bd94-25aceaa00d06","added_by":"auto","created_at":"2022-02-12 13:45:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1730467,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/c6fac7b1-dc1a-492d-a161-15626cc54d97.pdf"},{"id":3490057,"identity":"08510069-6c88-4a0c-a33d-67741e46bb5d","added_by":"auto","created_at":"2020-11-10 15:19:07","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":130987,"visible":true,"origin":"","legend":"The myofiber CSA in gastrocnemius muscle is shown. Values are given as mean+SEM; n=8-10 animals per group.","description":"","filename":"additionalfile2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/3bf422ce0dd53a1908325c2a.jpg"},{"id":3490059,"identity":"181c93d7-934a-4927-83a5-9946d663e463","added_by":"auto","created_at":"2020-11-10 15:19:08","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":135372,"visible":true,"origin":"","legend":"The percentage of centronucleated fibers in gastrocnemius muscle is shown. Values are given as mean+SEM; n=8-10 animals per group. ","description":"","filename":"additionalfile3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/612daa5f639fe0f1466b6ed6.jpg"},{"id":3490061,"identity":"fc527bc9-3835-4e52-a328-73f8ea1ee0f0","added_by":"auto","created_at":"2020-11-10 15:19:08","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":103332,"visible":true,"origin":"","legend":"The post-synaptic NMJ area in gastrocnemius muscle is shown. Values are given as mean+SEM; n=8-10 animals per group. * p\u003c0.05, significance between CIH and NOX.","description":"","filename":"additionalfile4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/51c012ebb146cab98a2af8aa.jpg"},{"id":3490063,"identity":"c43258a6-6e51-4863-ab42-4ffa77c325c2","added_by":"auto","created_at":"2020-11-10 15:19:09","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":126206,"visible":true,"origin":"","legend":"The percentage of damaged mitochondria in gastrocnemius muscle is shown. Values are given as mean+SEM; n=8-10 animals per group. ","description":"","filename":"additionalfile5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/bdc44d5471eb1805ee39ae96.jpg"},{"id":3490065,"identity":"f764811a-1f93-4251-beb1-3fdfea5a73fb","added_by":"auto","created_at":"2020-11-10 15:19:09","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":123160,"visible":true,"origin":"","legend":"The percentage of swollen mitochondria in gastrocnemius muscle is shown. Values are given as mean+SEM; n=8-10 animals per group. ","description":"","filename":"additionalfile6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/f7669a757d10d5bd199cb069.jpg"},{"id":3490067,"identity":"38f5a29d-58f4-446f-91c8-f3d275ccc7e2","added_by":"auto","created_at":"2020-11-10 15:19:09","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":29513,"visible":true,"origin":"","legend":"","description":"","filename":"tableadditional.docx","url":"https://assets-eu.researchsquare.com/files/rs-102618/v1/a0840f124433c5051b133634.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEffect of Chronic Intermittent Hypoxia (CIH) on Neuromuscular Junctions and Mitochondria in Slow- and Fast-Twitch Skeletal Muscle of Mice – Role of iNOS\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eObstructive sleep apnea (OSA), which describes a repetitive collapse of the upper airways during sleep, causes recurrent episodes of hypopnea or even apnea resulting in chronic intermittent hypoxia (CIH). Over the last two decades, the prevalence of sleep apnea has increased by a double-digit figure [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In a population-based study among the group of 40- to 85-year-old adults, 49.7% of men and 23.4% of women suffered from a moderate to severe OSA defined as an apnea\u0026ndash;hypopnea index (AHI)\u0026thinsp;\u0026ge;\u0026thinsp;15 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Due to the growing rate of obesity that is suspected as one of the strongest causal triggers, a further increase of sleep associated disorders is expected [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong various comorbidities, OSA patients often experience muscle fatigue and reduced physical performance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] resulting in limited daily activities and quality of life. In fact, recent meta-analyses showed that maximum oxygen uptake (aerobic capacity) under cycle ergometer test conditions is significantly reduced in patients with (severe) OSAS [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] compared to healthy controls and may be improved by continuous-positive-airway-pressure (CPAP) therapy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The limiting factors of O\u003csub\u003e2\u003c/sub\u003e-transport or muscle function responsible for an inverse relation between AHI and aerobic capacity have yet remained unclear, but may not include histomorphological muscle microvascularisation, which was found to be increases at least in the tibialis anterior muscle [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. While histomorphological data on locomotor muscle with OSA are surprisingly scarce, several studies in striated upper airway muscles have indicated various important alterations such as fiber type grouping [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], decreases in fiber cross-sectional area [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], muscle fiber atrophy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], centrally located nuclei [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] or abnormal mitochondrial distribution [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Thus, mechanisms underlying functional impairment of locomotor muscles have remained to be assessed. Notably, adequate muscle function is not only dependent on the integrity of muscle fibers themselves but also reliant on intact neuromuscular signal transmission via myelinated motor neuron, neuromuscular junction (NMJ) and postsynaptic sarcolemma [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. NMJ integrity and plasticity is considered critical for muscular function: While several studies have demonstrated a relationship between increases in pre- and postsynaptic areas and enhanced neuromuscular activity as well as increased fatigue resistance [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], age-related changes in NMJ, such as fragmentation or lowered NMJ area, are considered to contribute to sarcopenia [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moreover, investigations on mice lacking antioxidant enzymes (SOD\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) or overexpressing uncoupling proteins (UCP1) supported the hypothesis, that oxidative stress may trigger NMJ degenerations which occur in association with mitochondrial dysfunction [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Most relevant to OSA, a massive generation of reactive oxygen/nitrogen species (ROS/RNS) and related excessive oxidative and nitrosative stress has been attributed to repetitive nocturnal hypoxia-reoxygenation cycles in analogy to repeated ischemia and reperfusion injuries [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Indeed, evidence exists in OSA patients for increased production of superoxide, which was normalized under CPAP therapy [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], as well as for a decrease in anti-oxidative capacity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Moreover, studies in OSA patients as well as in CIH animal models detected an overexpression of inducible nitric oxide synthase (iNOS) via inflammatory triggers involving NF-κB activation, especially in neuronal or cardiovascular tissues, e.g. activated macrophages [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. iNOS may become a source of massive amounts of nitric oxide (NO, only limited by a lack of substrate or coenzymes) which as a highly reactive free radical forms peroxynitrite and other RNS compromising mitochondrial respiration, cell membrane integrity or insulin signaling [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. iNOS expression in skeletal muscle is observed in (type 2 fibers) of obese/diabetic adult, but rarely in healthy young subjects, unless exercising, while muscular iNOS expression in rodents was mainly detectable and studied in rats [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study used CIH as compared to normoxic control (NOX) as model of OSA in wildtype (WT) mice to investigate the long-term effect of OSA on skeletal muscle histomorphological measures of NMJ integrity, fiber size and composition as well as on transition electron microscopy (TEM) parameters of ultrastructural mitochondria integrity. Moreover, besides WT, we included iNOS-deficient (iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) mice into this analysis in order to evaluate the putatively risk-mediating role of iNOS within the pro-oxidative or -inflammatory stress arising from CIH-exposure. To the best of our knowledge, this is the first report on CIH-induced muscle-specific damage to NMJ, fiber histomorphology or mitochondrial ultrastructure in WT mice locomotor muscle, which unexpectedly is not inhibited by iNOS deficiency, but strikingly mimicked already at NOX.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCIH - mouse model of OSA:\u003c/h2\u003e \u003cp\u003eThe mouse model of OSA was based on the long-term exposure to CIH as described by Schulz et al. (Schulz et al. 2014). Male C57BL/6J-mice (WT, Charles River Deutschland GmbH, Sulzfeld, Germany) and iNOS-deficient mice (iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e; strain B6.129P2-Nos2\u003csup\u003etm1Lau\u003c/sup\u003e/J) aged 8\u0026ndash;10 weeks were exposed to a 6-week CIH profile (8\u0026nbsp;h/d 5 d/week) in a PC-controlled normobaric gas chamber which allowed alternating O\u003csub\u003e2\u003c/sub\u003e-concentration between 7% and 21% at cycles of 120\u0026nbsp;s corresponding to an AHI of 30\u0026nbsp;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, i.e. border between moderate to severe sleep apnea. NOX control conditions for age-matched WT and iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were rendered by the same chamber system flushed with room air (21% O\u003csub\u003e2\u003c/sub\u003e). CIH and control intervention were limited to daytime as mice are active at night. All animals were provided with standard diet and water ad libitum. All mice were weighed prior to intervention and immediately before euthanization.\u003c/p\u003e \u003cp\u003eAfter 6 weeks of CIH-/control intervention, mice were euthanized and the triceps surae (gastrocnemius, soleus and plantaris muscles) as well as the vastus lateralis muscle carefully removed, immediately shock frozen in liquid nitrogen-cooled isopentane and stored at -80\u0026nbsp;\u0026deg;C. Transversal cryosections of 7\u0026nbsp;\u0026micro;m (microtom Hyrax C60, Carl Zeiss AG, Oberkochen, -20\u0026nbsp;\u0026deg;C) were obtained for (immuno)histochemistry. Tibia length was carefully determined by a caliper.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMuscle fiber composition, size and centronucleation:\u003c/h2\u003e \u003cp\u003eMuscle fiber types (1, 2a, 2x) were identified via the acid-sensitive myofibrillar ATPase (Adenosine triphosphatase, Sigma-Aldrich Co. LLC, St. Louis, Missouri) staining at pH 4.55 as previously described (Friedmann-Bette et al. 2010) in randomly distributed two to three images taken at 200-fold magnification by the Zeiss Axio Imager.M2 microscope (Carl Zeiss AG; Oberkochen, Germany) after digitalization by the imaging system Axio-Cam HRc/AxioVision (Carl Zeiss GmbH). Type-specific fiber cross-sectional area (CSA) was determined by manually encircling each cross-section of at least 100 fibers using standard imaging software ImageJ (Scion Image, National Institutes of Health, Bethesda, USA). In addition, transverse cryosections were stained by hematoxylin and eosin to determine the percentage of centronucleated fibers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePostsynaptic NMJ morphology and integrity:\u003c/h2\u003e \u003cp\u003eNMJ analyses were based on α-bungarotoxin (BTX) staining in cryosections after fixation with 4% PFA/PBS (paraformaldehyde/phosphate buffered saline pH 7.4) for 10\u0026nbsp;min and blocking of endogenous peroxidase with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Thereafter, PBS-washed sections were incubated overnight with biotinylated BTX (1:500; Invitrogen Eugene, USA) in a humidified chamber, PBS-washed, blocked with 2% bovine serum albumin (BSA)/PBS, PBS-washed again, and incubated with horseradish peroxidase (HRP)-conjugated Streptavidin (Jackson ImmunoResearch Laboratories. Inc., West Grove, USA). 3,3'-Diaminobenzidine (DAB) was used as a chromogen substrate. Nuclei were counterstained with Mayer\u0026rsquo;s Hematoxylin (Carl Roth GmbH, Germany).\u003c/p\u003e \u003cp\u003eNMJ were identified in three consecutive, complete cross-sections per muscle from digital images (200-fold magnification) obtained by the Zeiss Axio Imager.M2 microscope (Carl Zeiss AG; Oberkochen, Germany) combined with Axio-Cam HRc/AxioVision (Carl Zeiss GmbH). The NMJ length and area were determined manually together with the corresponding myofiber CSA and perimeter using ImageJ software (Scion Image, National Institutes of Health). Single NMJ were considered as \u0026ldquo;fragmented\u0026rdquo; when the BTX-stained area was divided into several sections. Further calculations included NMJ area per myofiber CSA, NMJ length per myofiber perimeter and the percentage of fragmented NMJ (McLoon et al. 2016). On average, 21\u0026thinsp;\u0026plusmn;\u0026thinsp;2 (16\u0026thinsp;\u0026plusmn;\u0026thinsp;3) NMJ were analyzed per soleus or gastrocnemius muscle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePre- and postsynaptic NMJ co-staining:\u003c/h2\u003e \u003cp\u003eTo analyze the innervation status by double staining of NMJ pre- and postsynapse, every 6th of 60 serial 7\u0026nbsp;\u0026micro;m transverse sections of the vastus muscle (WT: n\u0026thinsp;=\u0026thinsp;4 NOX, n\u0026thinsp;=\u0026thinsp;6 CIH; iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: n\u0026thinsp;=\u0026thinsp;6 NOX, n\u0026thinsp;=\u0026thinsp;6 CIH) was stained for immunofluorescence with biotin-XX-conjugated BTX (Invitrogen Eugene, USA) and RvAChT (vesicular acetylcholine transporter) antibody (Lee Eiden, Laborchargen-Nr.: bl. 6/97). In detail, after fixation for 10\u0026nbsp;min in 4% PFA/PBS, and blockage by 1% BSA/PBS for 30\u0026nbsp;min, sections were incubated overnight at 4\u0026nbsp;\u0026deg;C with biotinylated BTX (1:500) or vAChT 80259 (1:1000) in a humidified chamber. The next day, washing was followed by two hours incubation with Alexa Fluor\u0026reg; 488 labeled donkey anti-rabbit IgG \u003cem\u003e(\u003c/em\u003e1:200, MoBiTec GmbH, G\u0026ouml;ttingen, Germany\u003cem\u003e)\u003c/em\u003e or Cy3-conjugated streptavidin (1:200, Dianova GmbH, Hamburg, Germany) respectively. Finally, slides were mounted using Immu-Mount\u0026trade;, (Fisher Scientific GmbH, Schwerte, Germany) and glass coverslips. Control sections were treated similarly but with either no primary antibody to exclude non-specific staining of secondary antibody. Confocal images were taken by the scanning laser microscope C2 (Nikon GmbH, D\u0026uuml;sseldorf, Germany) using the software NIS-Elements AR 4.30.01 (Laboratory Imaging). Each NMJ was scanned in a 630-fold magnification at 250\u0026nbsp;Hz with an image size of 1024\u0026thinsp;\u0026times;\u0026thinsp;1024 pixels. Subsequently, Fiji software (National Institute of Health, Bethesda, USA) was used for morphometrical analysis, determining the BTX and vAChT immunolabeled areas as well as their overlap area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy (TEM):\u003c/h2\u003e \u003cp\u003eMitochondrial ultrastructure of soleus and gastrocnemius muscle was evaluated using TEM as previously described [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Images were taken at 10.000-fold magnification and ten random pictures from both muscles of each mouse were analyzed using ImageJ software (Scion Image, National Institutes of Health, Bethesda, USA). Mean mitochondrial size was determined by manually encircling all identified mitochondria. Alterations on mitochondrial architecture were assessed by the categorization \u0026ldquo;normal\u0026rdquo; or \u0026ldquo;damaged\u0026rdquo; as follows: Mitochondria showing a loss of more than 50% of the cristae or a disruption of more than 50% of the outer membrane were assigned to \u0026ldquo;damaged\u0026rdquo; or otherwise to \u0026ldquo;normal\u0026rdquo;. The results were given as the percentage of the total number of mitochondria. Moreover, the percentage of swollen mitochondria and mitochondria containing multi-lamellar bodies was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT-PCR:\u003c/h2\u003e \u003cp\u003eThe extraction of RNA was performed using peqGOLD Isolation Systems TriFast\u0026trade; (PEQLAB Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer\u0026rsquo;s instructions. RNA quality (OD260nm / OD280nm\u0026thinsp;=\u0026thinsp;1.7 to 2.0) and concentration were determined using the NanoDrop 2000c spectrophotometer (Thermo Scientific, Schwerte, Germany). RNA integrity was confirmed by lab-on-a-chip technology, using an RNA 6000 NanoChip kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Total RNA (0.7\u0026nbsp;\u0026micro;g) was then treated with 1 unit DNAse (Thermo Scientific, St. Leon-Rot, Germany; 30\u0026nbsp;min, 37\u0026nbsp;\u0026deg;C). Thereafter, reverse transcription of RNA was carried out with 500\u0026nbsp;ng oligo (dT)\u003csub\u003e12\u0026minus;\u0026thinsp;18\u003c/sub\u003e primer, 20 units of the Affinity Script multiple temperature cDNA synthesis (Agilent) and 24 units of Ribo Lock\u0026trade; RNAse inhibitor (Fermentas; 1\u0026nbsp;h, 42\u0026nbsp;\u0026deg;C) and 4\u0026nbsp;mM dNTP-Mix. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed in duplicate using the QuantiTect/primerAssays from QIAGEN GmbH (Hilden, Germany). To confirm the primer specificity and the presence of a single amplicon, a melting curve (55\u0026ndash;95\u0026nbsp;\u0026deg;C) of the amplified product was performed. For amplification and data analysis the Mx3005P\u0026trade; QPCR System (Stratagene) was used. For each sample, the relative amount was calculated by linear regression analysis from their respective standard curves which was generated from a pool of cDNA. Among the transcripts Actb, Gapdh and Tbp, Tbp was identified by the NormFinder software as the most stable reference gene for RNA normalization of gene of interest (additional file 1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistics:\u003c/h2\u003e \u003cp\u003eData are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Differences between CIH and NOX or iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and WT were detected by ANOVA and Student\u0026rsquo;s t-test (post-hoc) or, in case of not normally distributed data, by the Kruskal-Wallis ANOVA and Dunn's post-hoc test. A p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant. For all statistical procedures SigmaPlot 14.0 software (Systat Software, Inc, Chicago, USA) was used.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBody weight:\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEffect of CIH in WT\u003c/strong\u003e \u003cp\u003eIn WT mice, 6 weeks of CIH compared to NOX significantly reduced the pre- to post-interventional gain in body weight, i.e., the weight gain between 2 and 3.5 months of age (starting from a 9.1% higher baseline) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEffect of iNOS w/o CIH\u003c/strong\u003e \u003cp\u003eUnder NOX conditions, iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed a significantly lower weight gain compared to WT (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), notably, starting from by 10.7% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) higher pre-interventional body weight than WT mice. When exposed to CIH, iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice even showed a weight loss and thus differed significantly from NOX conditions compared to NOX (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eWeight (g) and weight changes (%) of WT and iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice pre- to post-intervention. Values are given as mean\u0026thinsp;+\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;6\u0026ndash;14 animals per group. * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, significance between CIH and NOX; \u003csup\u003e##\u003c/sup\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003csup\u003e###\u003c/sup\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, significance between WT and iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT NOX\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT CIH\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eiNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e NOX\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eiNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e CIH\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePre-intervention weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003e###\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e26.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePost-intervention weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e25.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e25.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e24.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight change (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e-7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFiber morphology and fraction in soleus muscle:\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEffect of CIH in WT\u003c/strong\u003e \u003cp\u003eThe mean CSA covering all fiber types in soleus muscle did not significantly differ between CIH intervention and NOX in WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B, E \u003cem\u003eleft\u003c/em\u003e), though it correlated significantly and positively with the weight gain (r\u0026thinsp;=\u0026thinsp;0.41; p\u0026thinsp;=\u0026thinsp;0.047) in WT of both interventional groups. A fiber type-specific analysis showed, that CIH compared to NOX led to a significant decrease in fiber CSA exclusively of type 2a fibers by 13.5% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cem\u003eright\u003c/em\u003e). Thereby, CIH as compared to NOX resulted in an increase in type 1 fiber fraction (37% vs. 31%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in association with a decrease in type 2a fiber fraction (44% vs. 53%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) at unchanged type 2x fiber fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B, G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003eA parallel CSA-specific analyses of fiber distribution showed that CIH compared to NOX significantly increased the fraction of small fibers (CSA\u0026thinsp;\u0026lt;\u0026thinsp;800\u0026micro;m\u0026sup2;) in soleus muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, there was a significantly higher fraction of centronucleated fibers with CIH compared to NOX (1.5% vs. 0.2%; p\u0026thinsp;=\u0026thinsp;0.01) in WT soleus muscle, as counted in HE stained sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cem\u003eleft\u003c/em\u003e). A significant inverse correlation was found between the fraction of centronucleated fibers and the CSA of the fiber population in WT undergoing CIH or NOX (r=-0.417; p\u0026thinsp;=\u0026thinsp;0.043).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of iNOS w/o CIH\u003c/b\u003e: In both conditions, NOX and CIH, iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e compared to WT was without any significant effect on fiber CSA of the total fiber population or that of fiber type 1, 2a or 2x. In iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, CIH as compared to NOX caused no significant changes in CSA of fibers in total or of type (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), while CIH-related changes in fiber composition partly resembled those in WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, C, H): CIH compared to NOX in iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice led to a decrease in type 2a fiber fraction (45% vs. 51%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and an associated non-significant increase in type 1 fiber fraction (39% vs. 35%) at unchanged type 2x fiber fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D, H). Concerning the centronucleation, no significant difference was detected between the two genotypes or between CIH and NOX in iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFiber morphology and fraction in gastrocnemius muscle:\u003c/h2\u003e \u003cp\u003eIn contrast to soleus muscle, the gastrocnemius muscle mostly consisting of fiber type 2x revealed no significant effect of CIH vs. NOX and iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e vs. WT or their combination with regard to CSA (additional file 2) or percentage of central nuclei (additional file 3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eNMJ morphology/integrity in soleus muscle:\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEffect of CIH in WT mice\u003c/strong\u003e \u003cp\u003eIn soleus muscle of WT mice BTX-staining of NMJ showed, that CIH compared to NOX led to a significantly by 37% smaller postsynaptic NMJ area (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C, \u003cem\u003eleft\u003c/em\u003e). When calculating NMJ area relative to fiber CSA, (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, \u003cem\u003eleft\u003c/em\u003e), the CIH compared to NOX significantly reduced postsynaptic NMJ size. An alternative normalizing of NMJ length to fiber perimeter similarly decreased NMJ size with CIH compared to NOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cem\u003eleft\u003c/em\u003e). No sigificant CIH-related change occurred regarding the fraction of fragmented NMJ in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cem\u003eleft\u003c/em\u003e, G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEffect of iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e w/o CIH\u003c/strong\u003e \u003cp\u003eIn comparison to WT, iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice demonstrated a significantly by 55% diminished NMJ area under the condition of NOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), with this effect being significant also upon normalization of postsynaptic NMJ area for fiber CSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) or, alternatively, of postsynaptic NMJ length for fiber perimeter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). CIH compared to NOX intervention in iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice led to an additional decrease in NMJ area (in absolute terms) by trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cem\u003eright\u003c/em\u003e), however, this effect reached significance when normalizing postsynaptic NMJ area for fiber CSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, \u003cem\u003eright\u003c/em\u003e) or, alternatively, postsynaptic NMJ length for fiber perimeter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cem\u003eright\u003c/em\u003e). Notably, a strikingly higher percentage of NMJ fragmentation was observed selectively with iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e as compared to WT under both conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cem\u003eright\u003c/em\u003e, H).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNMJ morphology/integrity in gastrocnemius and vastus muscle:\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eGastrocnemius muscle\u003c/strong\u003e \u003cp\u003eIn contrast to soleus muscle, gastrocnemius muscle in WT mice showed a significantly increased NMJ size with CIH as compared to NOX (72%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, additonal file 4). However, this difference was abolished when normalizing postsynaptic NMR area for fiber CSA. Moreover, unlike the soleus muscle, the gastrocnemius muscle in iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice revealed no significant alterations in postsynaptic NMJ area, and this was also true for both above-mentioned normalization of NMJ area or length for fiber CSA or perimeter, respectively.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eVastus muscle\u003c/strong\u003e \u003cp\u003eFor further evaluation of functional NMJ integrity, double fluorescent staining was used in vastus muscle to quantify the area of the NMJ presynaptic nerve terminal (vACHT-antibodies, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, D), of postsynaptic NMJ (BTX, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, E) and that of their coupling (overlay, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, F). In line with the findings in soleus (but not in gastrocnemius) muscle, there was a significant diminution by 27.9% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) of postsynaptic NMJ in iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e compared to WT under conditions of NOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), whereas CIH-effects compared to NOX were absent in WT or iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. The presynaptic terminal, defined as the vACHT immunoreactive area, remained resistant against CIH- or genotype related effects (5H). The resulting percentage overlay area, a measure of NMJ integrity, was not significantly affected by CIH-intervention or iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial ultrastructure in soleus muscle:\u003c/h2\u003e \u003cp\u003eThe following ultrastructural mitochondrial abnormalities were quantified in soleus muscle of WT exposed to NOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) or CIH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) as well as in iNOS\u003csup\u003e\u003cb\u003e\u0026minus;/\u003c/b\u003e\u0026minus;\u003c/sup\u003e mice in NOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) or CIH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD): Swollen matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), disruption of the outer mitochondrial membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI), a complete loss of internal architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ) and mitochondria with multi-lamellar bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEffect of CIH in WT\u003c/strong\u003e \u003cp\u003eThe percentage of damaged mitochondria, classified as \u0026lt;\u0026thinsp;50% filled with cristae, was significantly 1.8-fold higher in CIH vs. NOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cem\u003eleft\u003c/em\u003e), while an increase in percentage swollen mitochondria with CIH did not reach significances (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, \u003cem\u003eleft\u003c/em\u003e). The difference between CIH vs. NOX regarding the percentage mitochondria containing multi-lamellar bodies did not reach significance (12.8% vs. 6.6%; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEffect of iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e w/o CIH\u003c/strong\u003e \u003cp\u003eSomewhat reminiscent of CIH vs. NOX effects in WT, iNOS\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e compared to WT mice at NOX conditions revealed a significant 2.1-fold increase of damaged mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Moreover, percentage of swollen mitochondria in iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e vs. WT mice was significantly increased under both conditions (NOX\u0026thinsp;\u0026gt;\u0026thinsp;6-fold; CIH\u0026thinsp;\u0026gt;\u0026thinsp;5-fold) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial ultrastructure in gastrocnemius muscle:\u003c/h2\u003e \u003cp\u003eIn gastrocnemius muscle, the abovementioned mitochondrial alterations were observed neither with CIH vs. NOX in WT nor with iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e of either condition (additional files 5\u0026ndash;6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCorrelations between NMJ fiber and mitochondrial morphology:\u003c/h2\u003e \u003cp\u003eIn soleus muscle the NMJ area was significantly correlated with the percentage of damaged mitochondria (r\u0026thinsp;=\u0026thinsp;0.584, p\u0026thinsp;=\u0026thinsp;0.002) as well as with the ratio between type 1 and type 2a fibers (r\u0026thinsp;=\u0026thinsp;0.397, p\u0026thinsp;=\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelations of NMJ area and fragmented NMJ, percentage of damaged mitochondria, myofiber CSA and fiber type ratio (1/2a) in soleus muscles, n\u0026thinsp;=\u0026thinsp;5\u0026ndash;8 animals per group.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eSoleus muscle (n\u0026thinsp;=\u0026thinsp;5\u0026ndash;8 animals per group)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNMJ area (\u0026micro;m\u0026sup2;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFragmented NMJ (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFiber type ratio; 1/2a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDamaged mitochondria (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.710\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.584\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.394\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.051\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNMJ area (\u0026micro;m\u0026sup2;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.642\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.575\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFragmented NMJ (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.396\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.050\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003eTranscripts of iNOS, SOCS3, SOD2 and pro-/antiapoptotic markers:\u003c/h2\u003e \u003cp\u003eNotably, mRNA expression of iNOS was undetectable in soleus (as well as in gastrocnemius) muscle of WT mice undergoing NOX or CIH intervention (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, importantly, an iNOS expression was well detectable in the liver of WT mice, where it decreased significantly and massively by factor 0.12 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) after CIH-treatment. The absence of iNOS expression was proven in the liver and soleus (and gastrocnemius) muscle of iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003eImportantly, in WT mice, SOCS3 expression was found to be \u0026gt;\u0026thinsp;10-fold increased with CIH compared to NOX (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e prompted a SOCS3 upregulation that was \u0026gt;\u0026thinsp;10-fold in NOX and \u0026gt;\u0026thinsp;4-fold in CIH as compared to WT at NOX (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, in WT mice, CIH compared to NOX led to an almost 50% decrease in mtSOD mRNA expression in soleus muscle. Similarly, iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e resulted in a 33% and a 45% decrease in SOD2 expression in soleus with NOX and CIH, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, the screening for apoptotic markers (BAX, BCL2, caspase 3) showed neither CIH- nor iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-related changes in soleus muscle (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelative gene expression in soleus muscle and liver of WT and iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eiNOS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSOCS3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSOD2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBAX\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBCL2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCaspase 3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSoleus muscle\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT NOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT CIH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e NOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e CIH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLiver\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT NOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT CIH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eUsing long-term CIH exposure in mice as a model of OSA, the present study shows for the first time that CIH compared to NOX causes damage of potential functional relevance in \u0026lsquo;red\u0026rsquo; (soleus) but not in \u0026lsquo;white\u0026rsquo; (gastrocnemius) muscle. This comprises a reduction in area, in length and, by trend, in integrity of postsynaptic NMJ as well as in size (CSA) and fraction of type 2a fibers (at higher type-1 fiber fraction). Moreover, these changes were associated with considerable mitochondrial damage, which showed a significant correlation to (loss in) NMJ area (r=-0.71, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and were, again, limited to soleus muscle, while gastrocnemius revealed no significant mitochondrial damage.\u003c/p\u003e \u003cp\u003eThe present study furthermore included iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice into this analysis of CIH vs. NOX effects on skeletal muscle, in order to test the hypothesis that iNOS deficiency may at least in part protect against a pro-inflammatory/-oxidative effect through CIH, i.e. hypoxia-reoxygenation stress leading to ROS generation from various sources [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Contrary to expectation, our data demonstrate that, compared to WT, iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e by itself (i.e. under NOX conditions) also leads to highly significant postsynaptic NMJ area reduction and fragmentation in combination with mitochondrial damage and swelling, which surprisingly resemble and exceed those observed with CIH in WT mice. Notably, under the conditions of iNOS deficiency, CIH stress is able to further aggravate the damage at least in terms of a further reduction in postsynaptic NMJ area or length after normalization for fiber CSA or perimeter, respectively. The similarity between CIH (compared to NOX in WT) and iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (compared to WT in NOX) was limited to NMJ and mitochondrial damage, while decreases in fiber CSA (including its correlation to NMJ) and centronucleation observed with CIH vs. NOX in WT were absent in iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, i.e. they revealed no atrophy despite signs of denervation.\u003c/p\u003e \u003cp\u003eAs another striking similarity, we found a\u0026thinsp;\u0026gt;\u0026thinsp;10-fold increase in SOCS3 expression with CIH vs. NOX in WT as well as with iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e vs. WT at NOX in (pooled samples of) soleus muscle. Available evidence qualifies SOCS3 as a candidate to mechanistically link mitochondrial damage to NMJ deterioration: SOCS3 upregulation has been demonstrated as an early event after skeletal muscle denervation by sciatic nerve transection [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This obviously occurs in response to local inflammatory signals, especially via IL6 which by itself, i.e. without denervation, causes upregulation of SOCS3 and of E3-ligases (atrogin-1, MURF-1) together with fiber atrophy all of which is abrogated by IL6 inhibition. At the same time, SOCS3 overexpression has been shown to cause mitochondrial damage like swelling or disruption in tibialis anterior muscle, which is reminiscent of what was presently observed in soleus muscle but not in gastrocnemius muscle. SOCS3 overexpression was, furthermore, associated with inhibited expression of mitochondrial genes, which included Smtck and Slc25a3 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], but may also comprise mtSOD, which was presently found to be downregulated. As an inhibitor of leptin and insulin signaling, increased muscular SOCS3 expression has been suggested as a major contributor to mitochondrial dysfunction, impaired fatty acid oxidation, as associated with aging, metabolic syndrome and inflammation [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. These severe metabolic effects in combination with previous evidence that SOCS3 overexpression dilates the sarcoplasmatic reticulum, dislocates and inhibits calcineurine (colocalized with SOCS3) and reduces skeletal muscle energy expenditure, oxygen uptake and activity [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] may contribute to muscle fiber atrophy as observed in case of CIH. While we found no evidence for increased apoptosis signals, a SOCS3 upregulation appears to be associated with impaired regenerative stem cell function in elderly humans [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and may potentially play a role in the increased centronucleation presently observed with CIH.\u003c/p\u003e \u003cp\u003eMoreover, the moderate decrease in mtSOD expression presently observed in soleus muscle with CIH and, to a lesser extent, with iNOS (NOX or CIH) might also play a role in fiber atrophy and mitochondrial deterioration: \u003cem\u003eSod1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, used as a murine model of neuromuscular impairment in age-related muscle atrophy (sarcopenia), exhibit reduction in myofiber CSA of type IIa fibers [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Reduced CSA of type IIa fibers was, indeed, presently observed in association with the most marked mtSOD decrease (ca 50%), lower weight gain and alterations in mitochondrial ultrastructure and NMJ morphology. In addition, the observed shift in fiber metabolic phenotype, i.e. an increased type 1\u0026nbsp;at a decreased type 2 fiber fraction with both CIH (vs. WT) and iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (vs. WT at NOX) might be attributed to decreased mtSOD expression, rather than to SOCS3 upregulation which decreases oxidative fiber characteristics [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Deficiency in mtSOD, representing impaired antioxidant defense, may also be involved in a remarkable number of age-related features which may originate from a loss of fast motoneurons followed by a reinnervation of slow motoneurons [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In the present study, fiber type ratio (type 1/type 2a) was significantly correlated with NMJ fragmentation and inversely correlated with NMJ size, pointing at a role of reinnervation in the fiber type shift.\u003c/p\u003e \u003cp\u003eThus, our observations with CIH may display some analogies to age-related neuromuscular deterioration involving SOCS3 and mtSOD. They are in line with previous studies in other rodent CIH models, revealing downregulation of mtSOD/SOD2 via downregulation of HIF-2α [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and clinical observation of lower plasma CuZnSOD/SOD1 in OSA patients [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur CIH-based OSA mouse model is, however, at variance with biopsy studies in OSA patients, which revealed no changes in tibialis anterior muscle fiber size compared to controls [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] or showed even enlarged diameters of type 2a fibers in quadriceps femoris at unaltered fiber type composition [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. One should, however, bear in mind, that the CIH mouse model does not mimick certain OSA-inherent factors like sways in intrathoracic pressure and blood pCO\u003csub\u003e2\u003c/sub\u003e as well as ventilatory overshoots but at the same time involves more severe O\u003csub\u003e2\u003c/sub\u003e-desaturation without airway obstruction. Also the genetic background of mice may affect the degree of atrophy [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEven more important, our data provide first evidence for a strikingly differential effect of CIH-exposure between soleus and gastrocnemius i.e. (mixed) \u0026rsquo;red\u0026rsquo; and \u0026lsquo;white\u0026rsquo; muscles, that has to be taken into account in translational studies. Indeed, in contrast to soleus muscle, gastrocnemius muscle revealed neither mitochondrial damage nor NMJ alterations (rather enlargement than shrinkage) with CIH. The differential exertion profiles between the postural soleus muscle (remaining recruited throughout during quiet standing) and the locomotor gastrocnemius muscle (providing fast forceful contractions) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] may impact these muscle-specific findings in CIH and iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and have likewise been implicated in massive muscle- (fiber-) specific differences in muscle aging or neurodegenerative disease [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, our findings of compromised mitochondrial ultrastructure and gene expression in soleus muscle of CIH-mice may be in line with those in human palate muscle (a primary research focus within OSA pathophysiological), showing abnormal mitochondrial function and organization [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The observed close positive correlation of the fraction of damaged mitochondria to fragmented NMJ (or inverse correlation to NMJ area) reveals no clue for cause-effect relationship. As a first assumption, mitochondria-derived oxidative stress during CIH (hypoxia-reoxygenation stress) may compromise NMJ [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] acting in combination with other ROS sources like upregulated NOX2, as reported for the presently used CIH mouse model [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. However, importantly, iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e at NOX largely mimicked the CIH effects, i.e. NMJ and mitochondrial damage together with SOCS3 up- and mtSOD downregulation and was despite the fact that iNOS mRNA expression in soleus or gastrocnemius muscles in WT was neither detectable with NOX nor with CIH exposure. Since, in contrast, WT mice revealed a hepatic iNOS expression, which was massively and significantly reduced (\u0026gt;\u0026thinsp;8-fold) with CIH compared to NOX, it is reasonable to assume that iNOS deficiency outside the skeletal muscle conveys both, the CIH and iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e effects. Thereby the NMJ damage, similarly observed with both these condition, strongly points towards an iNOS deficiency in peripheral nerves (i.e. in perikaryon of motoneurons or Schwann cells) as a cause of NMJ damage, though myeloid iNOS expression may also become muscle-protective [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Peripheral nerve injury may dramatically upregulate the low constitutive iNOS expression in Schwann cells, and iNOS deletion may result in smaller regenerating myelinated fibers and delayed reinnervation of muscle NMJ distal to the injury [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In fact, peripheral nerve dysfunction in patients suffering from OSA appears to be an early event [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], and denervation may precede muscular dysfunction, as suggested for human upper airway muscles [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] and supported by increased sarcolemmal N-CAM staining [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To date, no corresponding neuromuscular data exist for human locomotor muscle with OSA. However, they are needed to evaluate functional relevance of these alterations and to separate OSA-specific effects on NMJ, mitochondria, metabolism and related fiber dysfunction from the processes of aging and degenerative diseases [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Notably, the age of mice presently under test (four months) corresponded to early human adulthood (20\u0026ndash;30\u0026nbsp;years) [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe conclusion that iNOS expression (outside skeletal muscle) may be neuro-protective and relevant for \u0026lsquo;red\u0026rsquo; (aerobic) muscle function may be somewhat counterintuitive, as iNOS upregulation is resulting in a boost of NO, that is antimicrobial or antitumoral but also cytotoxic to normal tissue [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Furthermore it is causally implicated e.g. in insulin resistance and diabetes. Nonetheless, in humans a basal NO production rate (rendered mostly but not exclusively by nNOS and eNOS) is physiologically required (reviewed by [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]). There is evidence that NO may convey physiological oxidative signals [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] and a certain production by iNOS is required for neuroprotective antioxidative defense [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], e.g. through the ROS scavenging function of NO [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Data on skeletal muscle tissue are scarce, however, it was reported that iNOS deficiency leads to mitochondrial damage in myocardial dysfunction (adriamycin-based mouse model). Interestingly, this effect was abrogated by overexpression of mtSOD [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], which presently was found to be downregulated with both, CIH or iNOS deletion.\u003c/p\u003e \u003cp\u003eAs a limitation, this study includes no functional data regarding NMJ and skeletal muscle to challenge the relevance of morphological alterations. Moreover, our mouse model involved a limited CIH exposition of 5 days per week, which may allow adaptive or protective effects of 2 normoxic days per week. Nonetheless a previous study showed, that the pathophysiological changes of the clinical OSA, such as arterial hypertension, are accurately reflected by the here used CIH mouse model [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this is the first study to demonstrate CIH as a model of moderate to severe OSA triggers NMJ and mitochondrial damage accompanied by fiber atrophy in slow-twitch muscle of WT mice, all of which may contribute to reduced exercise (aerobic) capacity in patients suffering from OSA. We furthermore demonstrate that iNOS deficiency, rather than yielding protection of skeletal muscle against CIH stress, leads to similar structural impairments of NMJ and mitochondria under normoxia and might contribute to the CIH effects in WT, putatively through compromised innervation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ch3\u003eOSA - obstructive sleep apnea\u003c/h3\u003e\n\u003ch3\u003eCIH - chronic intermittent hypoxia\u003c/h3\u003e\n\u003ch3\u003eNMJ - neuromuscular junction\u003c/h3\u003e\n\u003ch3\u003eNOX - normoxia\u003c/h3\u003e\n\u003ch3\u003eCSA - cross-sectional area\u003c/h3\u003e\n\u003ch3\u003eATP - adenosine triphosphate\u003c/h3\u003e\n\u003ch3\u003eTEM - transmission-electron-microscopy\u003c/h3\u003e\n\u003ch3\u003eWT - wildtype\u003c/h3\u003e\n\u003ch3\u003eiNOS - inducible-nitric-oxide-synthase\u003c/h3\u003e\n\u003ch3\u003eSOCS3 - suppressor-of-cytokine-signaling-3\u003c/h3\u003e\n\u003ch3\u003eAHI - apnea\u0026ndash;hypopnea index\u003c/h3\u003e\n\u003ch3\u003eCPAP - continuous-positive-airway-pressure\u003c/h3\u003e\n\u003ch3\u003eUCP - uncoupling protein\u003c/h3\u003e\n\u003ch3\u003eROS - reactive oxygen species\u003c/h3\u003e\n\u003ch3\u003eRNS - reactive nitrogen species\u003c/h3\u003e\n\u003ch3\u003eNO - nitric oxide\u003c/h3\u003e\n\u003ch3\u003eBTX - \u0026alpha;-bungarotoxin\u003c/h3\u003e\n\u003ch3\u003ePFA - paraformaldehyde\u003c/h3\u003e\n\u003ch3\u003ePBS - phosphate buffered saline\u003c/h3\u003e\n\u003ch3\u003eHRP - horseradish peroxidase\u003c/h3\u003e\n\u003ch3\u003eBSA - bovine serum albumin\u003c/h3\u003e\n\u003ch3\u003eDAB - 3,3'-Diaminobenzidine\u003c/h3\u003e\n\u003ch3\u003evAChT - vesicular acetylcholine transporter\u003c/h3\u003e\n\u003ch3\u003eActb \u0026ndash; actin beta\u003c/h3\u003e\n\u003ch3\u003eGADPH - glyceraldehyde-3-phosphate dehydrogenase\u003c/h3\u003e\n\u003ch3\u003eTBP - TATA-box binding protein\u003c/h3\u003e\n\u003ch3\u003eSEM - standard error of the mean\u003c/h3\u003e\n\u003ch3\u003emtSOD \u0026ndash; mitochondrial superdioxide dismutase\u003c/h3\u003e\n\u003ch3\u003eBAX - BCL-2-associated X protein\u003c/h3\u003e\n\u003ch3\u003eBCL2 - \u003cem\u003eb-cell lymphoma 2\u003c/em\u003e\u003c/h3\u003e\n\u003ch3\u003eMURF-1 - Muscle RING-finger protein-1\u003c/h3\u003e\n\u003ch3\u003esMtCK - sarcomeric mitochondrial creatine kinase\u003c/h3\u003e\n\u003ch3\u003eSlc25a3 - Solute Carrier Family 25 Member 3\u003c/h3\u003e\n\u003ch3\u003enNOS - neuronal-nitric-oxide-synthase\u003c/h3\u003e\n\u003ch3\u003eeNOS - endothelial-nitric-oxide-synthase\u003c/h3\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were approved by the regional board (RP Giessen, Hesse, Germany; Az: V 54-19 c 20 15 h 01 GI 20/10 Nr. 84/2011) in accordance with the German animal welfare law and the European legislation for the protection of animals used for scientific purposes (2010/63/EU).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Materials used in this study are commercially available.\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the von-Behring-R\u0026ouml;ntgen-Stiftung (Project 580071).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRK and WH conceived, managed and supervised the project, designed experiments and obtained funding. RS and NW contributed to the study design and edited the manuscript. LB and SM performed experiments and analyzed data with contributions from GB, MB and SK. LB and WH wrote the manuscript with input from all authors. The authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the expert laboratory assistance of Claudia Keppler, Michael Dreher, Steffi Z\u0026uuml;gel and Irmgard Dammsh\u0026auml;user.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eT. Young, P. E. Peppard, and S. Taheri, \u0026ldquo;Excess weight and sleep-disordered breathing,\u0026rdquo; \u003cem\u003eJournal of applied physiology (Bethesda, Md. : 1985)\u003c/em\u003e, vol. 99, no. 4, pp.\u0026nbsp;1592\u0026ndash;1599, 2005.\u003c/li\u003e\n\u003cli\u003eT. Young, P. E. Peppard, and D. J. 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Ittarat et al., \u0026ldquo;Manganese superoxide dismutase and inducible nitric oxide synthase modify early oxidative events in acute adriamycin-induced mitochondrial toxicity,\u0026rdquo; \u003cem\u003eMolecular cancer therapeutics\u003c/em\u003e, vol. 4, no. 7, pp.\u0026nbsp;1056\u0026ndash;1064, 2005.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"skeletal-muscle","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"skem","sideBox":"Learn more about [Skeletal Muscle](http://skeletalmusclejournal.biomedcentral.com/)","snPcode":"13395","submissionUrl":"https://submission.nature.com/new-submission/13395/3","title":"Skeletal Muscle","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"denervation, muscle atrophy, mitochondria, fiber type, oxidative stress, neuromuscular junction, iNOS","lastPublishedDoi":"10.21203/rs.3.rs-102618/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-102618/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Obstructive sleep apnea (OSA) imposes vascular and metabolic risks through chronic intermittent hypoxia (CIH) and impairs skeletal muscle performance. As studies addressing limb muscles are rare, the reasons for the lower exercise capacity are unknown. We hypothesize that CIH-related morphological alterations in neuromuscular junctions (NMJ) and mitochondrial integrity might be the cause of functional disorders in skeletal muscles.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Mice were kept under 6-weeks-CIH (alternating 7% and 21% O\u003csub\u003e2\u003c/sub\u003e-fractions every 30s, 8h/d, 5d/w) compared to normoxia (NOX). Analyses included neuromuscular junctions (NMJ) postsynaptic morphology and integrity, fiber cross-sectional area (CSA) and composition (ATPase), mitochondrial ultrastructure (transmission-electron-microscopy) and relevant transcripts (qRT-PCR). Beside wildtype (WT) we included inducible-nitric-oxide-synthase knockout mice (iNOS\u003csup\u003e-/-\u003c/sup\u003e) to evaluate whether iNOS is protective or risk-mediating. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e In WT soleus muscle, CIH vs. NOX reduced NMJ size (-37.0%, p\u0026lt;0.001) and length (-25.0%, p\u0026lt;0.05) together with fiber CSA of type IIa fibers (-14%, p\u0026lt;0.05) and increased centronucleated fiber fraction (p\u0026lt;0.001). Moreover, CIH vs. NOX increased the fraction of damaged mitochondria (1.8-fold, p\u0026lt;0.001). Compared to WT, iNOS\u003csup\u003e-/-\u003c/sup\u003e similarly decreased NMJ area and length with NOX (-55%, p\u0026lt;0.001 and -33%, p\u0026lt;0.05, respectively) or with CIH (-37%, p\u0026lt;0.05 and -29%, p\u0026lt;0.05), however, prompted no fiber atrophy. Moreover, increased fractions of damaged (2.1-fold, p\u0026lt;0.001) or swollen (\u0026gt;6-fold, p\u0026lt;0.001) mitochondria were observed with iNOS\u003csup\u003e-/-\u003c/sup\u003e vs.\u003csup\u003e \u003c/sup\u003eWT under NOX and similarly under CIH. Both, CIH- and iNOS\u003csup\u003e-/- \u003c/sup\u003emassively upregulated suppressor-of-cytokine-signaling-3 (SOCS3) \u0026gt;10-fold. None of these morphological alterations with CIH- or iNOS\u003csup\u003e-/- \u003c/sup\u003ewere detected in gastrocnemius muscle. Notably, iNOS expression was undetectable in WT muscle, unlike the liver, where it was massively decreased with CIH. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e CIH leads to NMJ and mitochondrial damage associated with fiber atrophy/centronucleation selectively in slow-twitch muscle of WT. This effect is largely mimicked by iNOS\u003csup\u003e-/-\u003c/sup\u003e at NOX (except for atrophy). Both conditions involve massive SOCS3 upregulation likely through denervation. In the absence of muscular iNOS expression in WT, this damage may arise from extramuscular, e.g. motoneuronal iNOS deficiency (through CIH or knockout) awaiting functional evaluation.\u003c/p\u003e","manuscriptTitle":"Effect of Chronic Intermittent Hypoxia (CIH) on Neuromuscular Junctions and Mitochondria in Slow- and Fast-Twitch Skeletal Muscle of Mice – Role of iNOS","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-11-10 15:19:05","doi":"10.21203/rs.3.rs-102618/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-01-05T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-12-02T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2020-12-02T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-11-23T00:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-11-22T00:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-11-19T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-11-02T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-11-01T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-11-01T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-10-31T00:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"skeletal-muscle","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"skem","sideBox":"Learn more about [Skeletal Muscle](http://skeletalmusclejournal.biomedcentral.com/)","snPcode":"13395","submissionUrl":"https://submission.nature.com/new-submission/13395/3","title":"Skeletal Muscle","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b6d653ac-58c1-485f-a6f0-681a4d5d14a7","owner":[],"postedDate":"November 10th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1027427,"name":"Orthopedics"}],"tags":[],"updatedAt":"2022-02-12T13:45:24+00:00","versionOfRecord":{"articleIdentity":"rs-102618","link":"https://doi.org/10.1186/s13395-022-00288-7","journal":{"identity":"skeletal-muscle","isVorOnly":false,"title":"Skeletal Muscle"},"publishedOn":"2022-02-12 13:45:24","publishedOnDateReadable":"February 12th, 2022"},"versionCreatedAt":"2020-11-10 15:19:05","video":"","vorDoi":"10.1186/s13395-022-00288-7","vorDoiUrl":"https://doi.org/10.1186/s13395-022-00288-7","workflowStages":[]},"version":"v1","identity":"rs-102618","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-102618","identity":"rs-102618","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}