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

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% O2-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, 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. 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.