Abstract
BackgroundObstructive 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.MethodsMice were kept under 6 weeks of CIH (alternating 7% and 21% O2 fractions every 30 s, 8 h/day, 5 days/week) 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 (RT-qPCR). Besides wildtype (WT), we included inducible nitric oxide synthase knockout mice (iNOS−/−) to evaluate whether iNOS is protective or risk-mediating.ResultsIn 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, p < 0.001) or swollen (> 6-fold, p < 0.001) mitochondria were observed with iNOS−/− vs. WT under NOX and similarly under CIH. Both, CIH- and iNOS−/− massively upregulated suppressor-of-cytokine-signaling-3 (SOCS3) > 10-fold without changes in IL6 mRNA expression. Furthermore, inflammatory markers like CD68 (macrophages) and IL1β were significantly lower in CIH vs. NOX. None of these morphological alterations with CIH- or iNOS−/− were detected in the gastrocnemius muscle. Notably, iNOS expression was undetectable in WT muscle, unlike the liver, where it was massively decreased with CIH.ConclusionCIH 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 without Il6 upregulation but accompanied by a decrease of macrophage density especially next to denervated endplates. 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.
Highlights
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)
Studies in OSA patients as well as in CIH animal models detected an overexpression of inducible nitric oxide synthase via inflammatory triggers involving NF-κB activation, especially in neuronal or cardiovascular tissues, e.g., activated macrophages [30,31,32]. 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 Reactive nitrogen species (RNS) compromising mitochondrial respiration, cell membrane integrity or insulin signaling [33]. iNOS expression in skeletal muscle is observed in 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 similarity between CIH and iNOS−/− was limited to neuromuscular junctions (NMJ) and mitochondrial damage, while decreases in fiber cross-sectional area (CSA) and centronucleation observed with CIH vs. NOX in WT were absent in iNOS−/− mice, i.e., they revealed no atrophy despite signs of denervation
Summary
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). 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 microvascularization, which was found to be increased at least in the tibialis anterior muscle [11]. 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 [30,31,32]. Obstructive sleep apnea (OSA) imposes vascular and metabolic risks through chronic intermittent hypoxia (CIH) and impairs skeletal muscle performance. We hypothesize that CIH-related morphological alterations in neuromuscular junctions (NMJ) and mitochondrial integrity might be the cause of functional disorders in skeletal muscles
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