Effects of obesity upon genioglossus structure and function in obstructive sleep apnoea

Lung function

Forced spirometry (Warren E. Collins, Braintree, MA, USA) was obtained in all participants according to the SEPAR recommendations 15. Reference values were those of a Mediterranean population 16.

Genioglossus biopsy

In 21 of the 23 patients with OSAS, the GG biopsy was obtained under local anaesthesia (1% lidocaine with epinephrine). In no patient was anaesthesia injected in or near the site of biopsy. Through a 3 cm wide horizontal incision, 2 cm below the inferior limit of the chin, the milohyoid muscle was exposed and separated vertically in the midline. Then, the GG muscle was identified (close to the inner aspect of the mandible) and a piece of 4 by 4 mm (weighty 100–300 mg) was resected and sent to the laboratory, covered by a saline-soaked gauze. In all the control subjects, and in the remaining two patients with OSAS, the GG biopsy was obtained under general anaesthesia (propofol, midazolam and fentanyl) following the same surgical technique. In all participants, the GG biopsy was excised without the use of electrocautery. Complications derived from the procedure were always minor (local pain or limited bruise).

In vitro functional studies

Muscle strips were mounted immediately after biopsy in an organ bath (Radnoti Glass Technology, California, USA) containing standard Krebs-Ringer solution gassed with 95% oxygen and 5% carbon dioxide, and heated at 37°C 17–19. One end of the strip was tied to an immobile hook and the other to a calibrated high-sensitivity isometric force transducer (Radnoti Glass Technology, California, USA). Once in the tissue bath, muscle fibre length was adjusted by the micropositioner until maximal isometric twitch force responses were obtained (optimal fibre length (Lo)). Muscle strips were always stimulated (Model S‐48, Grass Instruments Co, Mass, USA) with supramaximal pulses of 1.0 ms delivered through platinum electrodes (Radnoti Glass Technology, California, USA). All studies were performed at Lo, after an equilibration period 5. Muscle force production was recorded for later analysis (Atlantis/Pegasus, Lakeshore Tech, Inc. Chicago, USA) of the maximum twitch tension and resistance to fatigue 5, 9, 20, 21. The absolute maximal forces were normalised to muscle cross-sectional area, as previously described 19.

Fibre-type distribution

After the functional protocol, GG samples were frozen in isopentane cooled with liquid nitrogen, and stored at −70°C until analysis. Then, 6 µm thick sections of the biopsies were cut in a cryostat (−20°C) (Cryocut 1800, Leica Instruments, Bensheim, Germany). Type I (“slow twitch”) and type II (“fast twitch”) fibres (and their subtypes IIa and IIb) were identified by the standard adenosine triphosphatase (ATPase) (at 9.4 and 4.6 pH pre-incubation). Following standard methodology, at pH 9.4 ATPase stain type I fibres are clear and type II fibres are darker, and at pH 4.6 ATPase stain type IIa fibres are also clear and type IIb are grey 22, 23. Fibre-type percentage (over 300 fibres) was determined using a microscope at 100 magnifying by two independent observers (Carl Zeiss, Essen, Germany). Because the reproducibility of these measurements was good (intra-class correlation coefficient of 0.92) 24, average data was used for analysis.

Statistical analysis

The statistical significance of differences between groups was assessed by 2‐way analysis of variance, followed by post-hoc contrast (Scheffe test) when appropriate. The Kruskall-Wallis test was used to compare the fibre-types distribution between groups. A p‐value <0.05 was considered significant. The mean values are based on the ones of each individual.

Clinical data

Table 1⇑ shows the clinical characteristics of the subjects studied. By design, BMI was different between groups. Yet, age was similar and all participants showed normal spirometric results (table 1⇑). All patients with OSAS (both obese and nonobese) had severe disease, as indicated by the high apnoea-hypopnea index (table 1⇑). Patients treated with CPAP used it during 7±1 h per night (range, 5–8 h) (table 1⇑).

In vitro contractile properties of the genioglossus

Table 2⇓ presents the maximum twitch tension values determined in all participants. Differences between groups were not statistically significant, irrespective of the presence of obesity or treatment with CPAP. By contrast, it was observed that the in vitro fatigability characteristics of the GG were very different in the two groups of patients with OSAS studied (fig. 1⇓). In nonobese patients, at diagnosis, the current authors' previous observations were reproduced 5 showing increased GG fatigability that was not observed in nonobese patients treated with CPAP (p<0.001) (fig. 1a⇓). In contrast, in obese patients, neither before or after treatment with CPAP, GG fatigability was different from that recorded in obese controls (p=0.84) (fig. 1b⇓). Finally, GG endurance was similar in nonobese and obese controls (p=0.38).

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Fig. 1.—

Time course of decrement in force production of the genioglossus (mean±sd) in response to repetitive stimulation at 40 Hz in a) nonobese and b) obese subjects studied (▪: control; •: obstructive sleep apnoea syndrome (OSAS) at diagnosis; ▴: OSAS after continuous positive airway pressure). Force was normalised to that produced during the first stimulus train. *: indicates p<0.05 between untreated OSAS and controls; ^#: indicates p<0.05 between untreated and treated patients with OSAS. For further explanations, see text.

Genioglossus fibre-type distribution

At diagnosis patients with OSAS showed a higher percentage of type II fibres than controls independently of obesity (p<0.05). This was mainly due to a increase in subtype IIb percentage (table 2 and 3⇑⇓). Interestingly, these differences were not observed between controls and patients on long-term treatment (>1 yr) with CPAP independently of obesity (table 2⇑). For technical reasons there were available data in fibres II subtypes in three subjects of the CPAP obese group.

Interpretation of findings

The current study provides three main observations of interest, each of which need separate discussion. First, obesity did not influence the structure or function of the GG by itself (compare control subjects, with and without obesity, in table 2⇑ and fig. 1⇑). This contrasts with previous studies indicating that obesity can alter both the structure and function of skeletal muscle 7, 8. Given the extraordinary plasticity of the muscle tissue, which rapidly changes in response to different functional demands 25, this observation suggests that obesity (in the absence of OSAS) does not impose a higher work load upon the GG. This interpretation, however, has to be taken with caution due to the relatively small number of subjects studied.

Second, GG endurance was very low only in nonobese (as opposed to obese) patients with OSAS (fig. 1⇑). It is well know that anatomical abnormalities of the upper airway are frequent in nonobese OSAS 27. These can easily pose their GG at a mechanically disadvantageous position that favours the development of fatigue. In contrast, in obese patients with OSAS, the narrowing of the upper airway may be more related to the deposition of fat tissue around the neck 28 than to a mechanical impairment of their upper airway dilator muscles (fig. 1⇑). The differences in GG endurance between both groups of patients should also be related to the cellular mechanisms involved in the process of muscle contraction and force generation. However, the capacity to generate force momentarily (twitch tension) was similar in both groups of patients (table 2⇑). Thus, differences are likely to be related to those cellular processes involved in the maintenance (rather than the generation) of force, such as calcium release and uptake, and adenosine triphosphate and phosphocreatine synthesis and breakdown, among others.

Third, and finally, in keeping with previous studies 5, 9, 25, 26 the fibre-type distribution of GG was abnormal in patients with OSAS (table 2⇑). At diagnosis, patients showed a predominance of fast twitch fibres. This percentage was modified in patients on CPAP treatment. It was found that the abnormal fibre-type distribution of the GG was independent of the presence of obesity, indicating that, somehow, this abnormal distribution is a characteristic of the disease itself. The closure of the upper airway that occurs repeatedly during sleep in these patients, as well as the arterial oxygen desaturation episodes that normally follow them, are two of the most characteristic features of OSAS 1, 29, 30. Both can influence muscle structure through their respective mechanical 25, 31, 32 or chemical 33 effects. Given that both were similarly distributed in the two groups of patients with OSAS studied here (table 1⇑), the current authors postulate that one or both may underlie the changes observed in their GG structure (table 2⇑). In keeping with this interpretation is the fact that treatment with CPAP (which prevents the apnoea and the arterial oxygen desaturation episodes) changes the fibre-type distribution in both groups (table 2⇑). This hypothesis, however, does not exclude other potential mechanisms (systemic inflammation, genetic background of the individual) which will have to explored in future studies.

Methodological limitations

There are two potential limitations of the current study deserving comments. First, the small number of subjects included in the obese control and nonobese on CPAP groups (due to difficulties in recruiting this kind of specific subjects) recommends that the results should be interpreted with caution. Second, the biopsy was not performed in the same patient before and after CPAP therapy. This fact does not allow us to establish solid interpretations on the consequences of CPAP treatment, nor to perform correlations between structure and function changes. It also does not exclude that the results could be explained by individual variability. Ethical reasons do not allow such surgical procedures to be performed twice.

Clinical implications

Treatment with CPAP is highly effective in OSAS 34. However, because it is cumbersome and expensive, new therapeutic alternatives are needed 35. The present authors found that GG endurance was abnormal (and might be correctable by CPAP treatment) only in nonobese patients with OSAS (fig. 1⇑). This may therefore indicate the need of different therapeutic strategies for these “different” pathogenic forms of OSAS (obese versus nonobese). For instance, serotoninergic drugs enhance upper airway electromyogram (EMG) activity in nonobese healthy volunteers 36 and seem to reduce the apnoea/hypopnoea index in nonobese patients with OSAS 37. The eventual uncovering of the cellular mechanisms underlying the abnormal endurance capacity observed in the study in nonobese patients with OSAS may lead to more target-oriented pharmacological therapies. Alternatively, the fact that CPAP normalised GG endurance only in nonobese patients (fig. 1⇑), raises the intriguing possibility of intermittent (weekly? monthly?) CPAP therapy. The current authors do not know yet what is the minimum duration of CPAP treatment required to achieve this effect, or for how long does this improvement last when CPAP support is stopped. However, these questions will have to be addressed in future studies. In any case, if efficacious, intermittent CPAP therapy would have tremendous clinical impact because about 50% of patients with OSAS are nonobese 1.

Conclusions

The current study suggests that only in nonobese subjects, the resistance to fatigue of the geniglossus is reduced (and might be correctable by continuous positive airway pressure). It was also observed that the fibre-type distribution of the geniglossus is abnormal in patients with obstructive sleep apnoea syndrome (independently of obesity). Therefore, these results suggest that, from a mechanistic perspective, there are at least two “different” forms of obstructive sleep apnoea syndrome and open new questions of potential clinical interest.