Gestational Intermittent Hypoxia Induces Mitochondrial Impairment in the Geniohyoid Muscle of Offspring Rats

Abstract

Introduction Gestational intermittent hypoxia (IH), a hallmark of obstructive sleep apnea during gestation, alters respiratory neural control and diaphragm muscle contractile function in the offspring. The geniohyoid (GH) muscle is innervated by the respiratory-related hypoglossal nerve and plays a role in tongue traction and suckling, motor behaviors that then give way to chewing. Here, we aimed to investigate the effects of gestational exposure to IH on the muscle development and metabolism of GH and masseter muscles in male offspring rats. Materials and methods Pregnant Sprague-Dawley rats were exposed to IH (3-min periods of 4-21% O₂) for eight hours/day during gestational days 7-20. The GH and masseter muscles from 35-day-old male offspring (n = 6 in each group) were analyzed. Results Gestational IH induction reduced type IIA fiber size in the GH muscle of the offspring but not in the masseter muscle. Western blot analysis showed that gestational IH-induced significant downregulation of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1-alpha (PGC1α) protein in the GH muscle but not in the masseter muscle. Moreover, optic atrophy 1 and mitofusin-2 proteins were decreased and mitochondrial fission 1 protein levels were increased in the GH muscle of the offspring exposed to gestational IH. Mitochondrial adenosine triphosphate (ATP) synthase subunit alpha and transcriptional factor A (TFAM) were decreased in the GH muscle post-gestational IH. Conclusion These findings suggest that gestational IH-induced impaired mitochondrial metabolism and alteration of oxidative myofibers of the GH muscle in the pre-adolescent offspring, but not the masseter muscle, owing to the susceptibility of GH muscular mitochondria to gestational IH.

Keywords: geniohyoid muscle; intermittent hypoxia; masseter muscles; obstructive sleep apnea; offspring rat; pregnancy.

Figure 1. Anatomical location and histological images of the GH and MAS muscles of gestational IH offspring.

(a) Ventral image of rat GH and MAS muscles. (b) The upper panel: hematoxylin & eosin stain; the middle panel: modified Gomori trichrome stain; the lower panel: NADH stain. Data represent male offsprings (n = 6) in each group. Scale bar: 100 μm. NADH: nicotinamide adenine dinucleotide reductase; MAS: masseter muscle; DG: digastric muscle; MH: mylohyoid muscle; GH: geniohyoid muscle; HB: hyoid bone; GG: genioglossus muscle; HG: hyoglossus muscle; IH: intermittent hypoxia; H&E: hematoxylin & eosin.

Figure 2. Distribution pattern of muscle fiber type in the GH muscle of gestational IH offspring rats.

(a) Fiber type-specific immunohistochemical staining for type I, type IIA, type IIB, and type IID fibers with skeletal muscle membrane protein, laminin (red). Each panel shows a cross-sectional image of the GH muscle. Green areas indicate immuno-positive muscle fibers. (b) The graph indicates the percentage of muscle fiber type distribution in GH muscle from each group. (c) Histogram of the fiber size distribution of each muscle fiber type. Solid line and dotted line show N/N and IH/N groups, respectively. Data represent male offspring (n = 6) in each group. Scale bar: 50 μm. *p < 0.05 vs. the N/N group. GH: geniohyoid, IH: intermittent hypoxia.

Figure 3. Distribution pattern of muscle fiber type in the MAS muscle of gestational IH offspring rats.

(a) Fiber type-specific immunohistochemical staining for type I, type IIA, type IIB, and type IID fibers with skeletal muscle membrane protein, laminin (red). Each panel shows a cross-sectional image of the MAS muscle. Green areas indicate immuno-positive muscle fibers. (b) The graph indicates the percentage of muscle fiber type distribution in MAS muscle from each group. (c) Histogram of the fiber size distribution of each muscle fiber type. Solid line and dotted line show N/N and IH/N groups, respectively. Data represent male offspring (n = 6) in each group. Scale bar: 50 μm. MAS: masseter muscle, IH: intermittent hypoxia.

Figure 4. Quantitative polymerase chain reaction analysis in the GH and MAS muscles of offspring rats.

Gene expression of muscle fiber type-related factors (Myh1, Myh2, Myh7, Atp2a1, and Atp2a2) and hypoxic-inducible factors (Hif1a and Epas1) in GH (a) and MAS (b) muscle from N/N and IH/N groups. The relative expression levels of each gene were normalized to the level of Lbr expression, and the relative expression levels were set to 1 for the N/N group. Data represent male offspring (n = 6) in each group. *p < 0.05 vs. the N/N group. GH: geniohyoid, IH: intermittent hypoxia, MAS: masseter muscle.

Figure 5. Protein levels of mitochondrial metabolic markers in the GH and MAS muscles of offspring rats.

Western blot images for PPAR-gamma coactivator 1 (PGC1α), mitochondrial transcription factor A (TFAM), NADH:ubiquinone oxidoreductase complex assembly factor 1 (NDUFAF1), ATP5A1, optic atrophy 1 (OPA1), mitofusin (MFN)1, MFN2, mitochondrial fission 1 (FIS1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in GH (a) and MAS (b) muscles. The molecular weight markers were represented in the left line. The expression levels of the long and short isoforms of OPA1 were quantified together. The expression levels of the long and short isoforms of OPA1 were quantified together. The relative expression levels of each protein were normalized to the level of GAPDH expression, and the relative expression levels were set to 1 for the N/N group. Data represent male offspring (n = 6) in each group. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the N/N group. GH: geniohyoid; MAS: masseter muscle.