Shah et al. Respiratory Research (2019) 20:31 https://doi.org/10.1186/s12931-019-0999-9 RESEARCH Open Access Desmin and dystrophin abnormalities in upper airway muscles of snorers and patients with sleep apnea Farhan Shah1, Karl A. Franklin2, Thorbjörn Holmlund3, Eva Levring Jäghagen4, Diana Berggren3, Sture Forsgren1 and Per Stål1* Abstract Background: The pathophysiology of obstruction and swallowing dysfunction in snores and sleep apnea patients remains unclear. Neuropathy and to some extent myopathy have been suggested as contributing causes. Recently we reported an absence and an abnormal isoform of two cytoskeletal proteins, desmin, and dystrophin, in upper airway muscles of healthy humans. These cytoskeletal proteins are considered vital for muscle function. We aimed to investigate for muscle cytoskeletal abnormalities in upper airways and its association with swallowing dysfunction and severity of sleep apnea. Methods: Cytoskeletal proteins desmin and dystrophin were morphologically evaluated in the uvula muscle of 22 patients undergoing soft palate surgery due to snoring and sleep apnea and in 10 healthy controls. The muscles were analysed with immunohistochemical methods, and swallowing function was assessed using videoradiography. Results: Desmin displayed a disorganized pattern in 21 ± 13% of the muscle fibres in patients, while these fibers were not present in controls. Muscle fibres lacking desmin were present in both patients and controls, but the proportion was higher in patients (25 ± 12% vs. 14 ± 7%, p = 0.009). The overall desmin abnormalities were significantly more frequent in patients than in controls (46 ± 18% vs. 14 ± 7%, p < 0.001). In patients, the C-terminus of the dystrophin molecule was absent in 19 ± 18% of the desmin-abnormal muscle fibres. Patients with swallowing dysfunction had 55 ± 10% desmin-abnormal muscle fibres vs. 22 ± 6% in patients without swallowing dysfunction, p =0.002. Conclusion: Cytoskeletal abnormalities in soft palate muscles most likely contribute to pharyngeal dysfunction in snorers and sleep apnea patients. Plausible causes for the presence of these abnormalities is traumatic snoring vibrations, tissue stretch or muscle overload. Keywords: Muscle, Upper airway dysfunction, Dysphagia, Pathophysiology, Cytoskeletal abnormalities, desmin, dystrophin Background of the upper airway muscles to maintain patency during Obstructive sleep apnea is a highly prevalent disorder asso- sleep and for the commonly occurring swallowing dysfunc- ciated with adverse health consequences [1]. The disorder tion in snorers and sleep apnea patients [3–14]. Although is characterized by repetitive narrowing and collapse of the evidence of upper airway neuropathy exists in snorer and upper airways, with snoring and subsequent hypoxia [2]. sleep apnea patients, acquired muscle injuries as a contrib- Inadequate dilating muscle forces are suggested as an im- uting cause for muscle weakness and pharyngeal dysfunc- portant factor in the pathophysiology of obstructive sleep tion has gained less attention [11]. apnea. Nerve injuries due to traumatic snoring vibration We have earlier reported that a small subpopulation of have been proposed as one of the causes for the inability fibres in soft palate muscles of healthy humans lacked or had a truncated form of two cytoskeletal proteins, des- min, and dystrophin [15]. This is specifically interesting * Correspondence: [email protected] 1Department of Integrative Medical Biology, Laboratory of Muscle Biology, since these two cytoskeletal filaments are considered to Umeå University, SE-901 87 Umeå, Sweden be ubiquitous and vital for muscle function [16]. Desmin Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shah et al. Respiratory Research (2019) 20:31 Page 2 of 11 is the major intermediate filament (IF) in mature mus- manually according to the American Academy of Sleep cles [16]. It is located at the periphery of the Z-disc and Medicine recommendations. The definition of an apnea links the entire contractile apparatus to the subsarco- was a ≥ 90% cessation of airflow lasting at least 10 s, lemmal cytoskeleton, the cell nuclei and to other organ- while a hypopnea was defined as 50% reduction in air- elles such as mitochondria [17–19]. The network of flow compared with baseline, in combination with an desmin filaments supports the structural and mechanical oxygen desaturation of ≥3% [24]. integrity of the muscle cell during contraction and con- tributes to force transmission and load bearing [16]. Swallowing examination Dystrophin is localized in the inner part of the sarco- Swallowing function was investigated in all patients and lemma where it binds other cytoskeletal protein fila- voluntary controls using a videoradiographic examin- ments in the muscle cell to the surrounding extracellular ation (C-arm, Philips BV 29, field width 23 cm) in an up- matrix (ECM) through the cell membrane [20]. The right position and with lateral and frontal projections. C-terminus domain of the dystrophin molecule is associ- The subjects first swallowed a chewed solid bolus of ated with the membrane-spanning dystrophin-associated crisp bread and barium sulphate (Mixobar Esophagus; protein complex (DAPC), whereas its N-terminus inter- Astra) and then a liquid barium sulphate contrast bolus acts with actin filaments. The DAPC has significant roles (Mixobar High Density; Astra). All standard boluses in stabilizing sarcolemma and transmitting force gener- were repeated twice in each projection. The examina- ated in the muscle sarcomere to ECM [21]. In genetic tions were evaluated at full speed and slow motion by myopathies and animal gene knockout experiments, the two investigators blinded for the clinical findings of the absence of these proteins leads to progressive muscle subjects. Swallowing function was graded as 1. normal weakness [22, 23]. function, 2. mild dysfunction in the presence of one of Presence of abnormalities in cytoskeletal proteins in the following deviant features; premature leakage, velar upper-airway muscles of snoring and sleep apnea pa- dysfunction, residual or laryngeal penetration, 3. moder- tients and its impact on pharyngeal function has not ate dysfunction with two or more deviant features in been investigated. Therefore, we aimed to investigate for grade 2 or dysfunction of the upper esophageal sphinc- desmin and dystrophin abnormalities in soft palate mus- ter, the epiglottis or the propagation wave and, 4. severe cles of snoring and sleep apnea patients and to evaluate dysfunction with aspiration below the vocal cords [25]. whether these abnormalities relate to deviations in swal- lowing function and severity of obstructive sleep apnea. Tissue samples and immunohistochemistry In patients, the entire base of the uvula was resected in Methods connection with soft-palate surgery. In 3 of the cases, Patients and controls parts of the palatopharyngeus muscle were also available. Twenty-two consecutive patients (1 female, 21 males) The samples from voluntary controls were acquired referred for upper-airway surgery because of snoring and from the corresponding site by using punch biopsy tech- sleep apnea were included. The exclusion criteria were nique, except in one case where complete surgical resec- smoking, previous palatal surgery, systemic disease, tion of the uvula was performed. Since a punch biopsy medications, and drug abuse. The mean age was 45 years represents only a part of the muscle cross-section, aut- (range 29–60), and the mean body mass index (BMI) opsies were acquired from the entire base of uvula from was 28 kg/m2 (range 21–34). Ten voluntary controls, all 5 subjects who died accidentally (2 males and 3 females), males, mean age 38 years (range 30–51) and mean BMI mean age 54 years (range 46–75), mean BMI 25 kg/m2 24 kg/m2 (range 22–30), were recruited through adver- (range 21–31). The autopsies were used only as a tisements. The exclusion criteria were similar as in pa- cross-reference to determine that the punch biopsies tients, but also included habitual snoring and sleep were representative for the entire muscle cross-section. apnea. For reference, a biopsy from an arm muscle, bi- The exclusion criteria were similar to those of voluntary ceps brachii, and a thigh muscle, vastus lateralis, were controls. No medical history of snoring and sleep apnea acquired from two healthy adult male subjects. were reported, and all subjects had a normal craniofacial and oro-pharyngeal anatomy. All samples were taken Sleep apnea recordings within 12–24 h post-mortem, a delay that not affects All patients and voluntary controls underwent ambula- muscle morphology, muscle proteome and fibre typing tory overnight sleep apnea recordings (Embletta, Embla [26, 27]. systems, Kanata, Canada) using nasal cannula pressure, The muscle samples were cut into small pieces and ori- thoracic and abdominal respiratory effort, finger oxim- ented