A novel mouse model of obstructive sleep apnea by bulking agent-induced tongue enlargement results in left ventricular contractile dysfunction

Simon Lebek; Philipp Hegner; Christian Schach; Kathrin Reuthner; Maria Tafelmeier; Lars Siegfried Maier; Michael Arzt; Stefan Wagner

doi:10.1371/journal.pone.0243844

. 2020 Dec 10;15(12):e0243844. doi: 10.1371/journal.pone.0243844

A novel mouse model of obstructive sleep apnea by bulking agent-induced tongue enlargement results in left ventricular contractile dysfunction

Simon Lebek ¹, Philipp Hegner ¹, Christian Schach ¹, Kathrin Reuthner ¹, Maria Tafelmeier ¹, Lars Siegfried Maier ¹, Michael Arzt ¹, Stefan Wagner ^1,^*

Editor: Michael Bader²

PMCID: PMC7728202 PMID: 33301470

Abstract

Aims

Obstructive sleep apnea (OSA) is a widespread disease with high global socio-economic impact. However, detailed pathomechanisms are still unclear, partly because current animal models of OSA do not simulate spontaneous airway obstruction. We tested whether polytetrafluoroethylene (PTFE) injection into the tongue induces spontaneous obstructive apneas.

Methods and results

PTFE (100 μl) was injected into the tongue of 31 male C57BL/6 mice and 28 mice were used as control. Spontaneous apneas and inspiratory flow limitations were recorded by whole-body plethysmography and mRNA expression of the hypoxia marker KDM6A was quantified by qPCR. Left ventricular function was assessed by echocardiography and ventricular CaMKII expression was measured by Western blotting. After PTFE injection, mice showed features of OSA such as significantly increased tongue diameters that were associated with significantly and sustained increased frequencies of inspiratory flow limitations and apneas. Decreased KDM6A mRNA levels indicated chronic hypoxemia. 8 weeks after surgery, PTFE-treated mice showed a significantly reduced left ventricular ejection fraction. Moreover, the severity of diastolic dysfunction (measured as E/e’) correlated significantly with the frequency of apneas. Accordingly, CaMKII expression was significantly increased in PTFE mice and correlated significantly with the frequency of apneas.

Conclusions

We describe here the first mouse model of spontaneous inspiratory flow limitations, obstructive apneas, and hypoxia by tongue enlargement due to PTFE injection. These mice develop systolic and diastolic dysfunction and increased CaMKII expression. This mouse model offers great opportunities to investigate the effects of obstructive apneas.

Introduction

Obstructive sleep apnea (OSA) is a very common disease with high global socio-economic impact, since almost one billion people are affected worldwide [1]. It is frequently associated with heart failure, cardiac hypertrophy or atrial and ventricular arrhythmias [2–4]. Patients with OSA have been shown to develop worse outcome after myocardial infarction [5]. While treatment with ventilation-therapy may reduce apnea events, not all patients can tolerate it [6] and this treatment may even be harmful for selected patients (e.g. for patients with predominant central apneas) [7]. Thus, the development of novel and more specific treatment concepts is warranted. We have shown that the activity of Ca/Calmodulin-dependent protein kinase II (CaMKII), a key player in heart failure development [8, 9], was increased in the hearts of patients with sleep-disordered breathing, leading to contractile dysfunction and arrythmias [4]. Proposed mechanisms of CaMKII activation in OSA include increased transmural pressure gradients with increased atrial wall stress, increased sympathetic and parasympathetic activation, and reactive oxygen species (ROS) [3, 4, 10, 11]. Unfortunately, patient studies are not always suited to discriminate between different pathophysiological factors. Moreover, the analysis of heterogenous patient populations with comorbidities makes it difficult to interpret the data.

To avoid these pitfalls and to investigate OSA-dependent effects in the absence of potential confounders, appropriate animal models are urgently warranted. However, current animal models of OSA and sleep-disordered breathing in general struggle with many limitations including lack of airway obstruction (intermittent hypoxia models), artificial sedation, lack of availability in small rodents (e.g. mice) for investigation of transgenic animals [12–17].

Interestingly, New Zealand Obese mice have notable thicker tongues with a consecutive increased occurrence of spontaneous apneas and hypopneas [14, 17]. However, these mice also suffer from comorbidities like arterial hypertension, hyperinsulinemia and hypercholesterolemia, which makes it difficult to ascribe observations to airway obstruction [15].

Here we test the hypothesis that bulking agent injection into the tongue of lean C57BL/6 mice would result in the development of features of OSA, such as intermittent inspiratory flow limitation (IFL), apneas, and hypoxia. We also tested if the severity of these breathing disorders may correlate with the severity of systolic and diastolic dysfunction and cardiac CaMKII expression in these lean mice devoid of comorbidities.

Materials and methods

All detailed method protocols and data are available upon reasonable request by the corresponding author. All investigations are conformed to directive 2010/63/EU of the European Parliament. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1985) and to local institutional guidelines. The protocol was approved by the Committee on the Ethics of the government of Unterfranken, Bavaria, Germany (Protocol Number: 55.2-2532-2-512). All animals were euthanized by cervical dislocation during the light period, i.e. regular sleep time of the animals (usually in the afternoon).

Polytetrafluoroethylene injection into the tongue

Fifty-nine male C57BL/6 mice at an age of 8–12 weeks (Charles River Laboratories) were included into the study. 31 mice were allocated to polytetrafluoroethylene (PTFE; 35 μm particle size; Sigma Aldrich) injection into the tongue and 28 to control (no treatment, S1 Fig). PTFE has been frequently used as an inert bulking agent for the treatment of primary vesicoureteral reflux in patients [18].

Our approach was based on the findings of Brennick et al., who had measured pharyngeal structures of New Zealand Obese mice (NZO) with spontaneous OSA using MRI [14]. Interestingly, they report that NZO mice (aged 23 weeks, mean body weight 35.7 g) showed a significantly increased mean tongue volume to about 137 μl (compared to 104 μl in control animals). This corresponds to a mean increase of 33 μl tongue volume. Since the tongue volume is the most important determinant of pharyngeal airway size for OSA [19], we aimed to increase the tongue volume of our mice to a similar extent by PTFE injection into the base of the tongue. We used younger mice (mean body weight 27.7 g, only about 70% of the body weight compared to Brennick et al. [14]) to enable the 8-week follow-up observation period. Thus, we anticipated that an increase of about 20–25 μl tongue volume would result in a similar airway obstruction. PTFE is a solid substance (density 2.1 g/ml). 50 mg of PTFE was diluted to 100 μl (50% w/v) with glycerol (Sigma Aldrich). 100 μl of this dilution contains 24 μl pure PTFE, which almost exactly matches the aimed increase in tongue volume. Larger injection volumes were investigated in some test mice, but periprocedural mortality exceeded. Since we were not interested in less upper airway obstruction, we have not studied lower injection volumes.

The allocation to the two treatment groups was done on a random basis. For each day of surgery, a similar number of mice was allocated to both groups. Mice were treated 1 h before the PTFE injection with buprenorphine (0.1 mg/kg bodyweight (BW) intraperitoneal) for optimal analgesia. Mice were anesthetized using intraperitoneal injections of medetomidine (0.5 mg/kg), midazolam (5 mg/kg) and fentanyl 0.05 mg/kg BW). After establishment of anesthesia, the mice were placed in a supine position on a heating plate and body temperature was controlled by a rectal probe. In addition, anesthesia was controlled by continuous monitoring of respiration and ECG. The mouse tongue was manually pulled as far as possible out of the oral cavity to allow for access to the base of the tongue. By using a 27-gauge cannula, about 100 μl PTFE dilution were then injected in the base of the tongue. In order to increase the tongue diameter as balanced as possible, the total volume of 100 μl was divided into multiple injections into depots at the dorsal and ventral side of the tongue (S1 Video). At the end of the procedure, anesthesia was antagonized using intraperitoneal injections of atipamezole (2.5 mg/kg), flumazenil (0.5 mg/kg) and buprenorphine (0.1 mg/kg BW). Untreated littermates were used as control mice.

From 31 mice treated with PTFE, 6 mice had to be killed within 72 h because of surgery-related complications (e.g. bleeding into the tongue, extensive tongue enlargement or infection). In order to respect animals’ wellbeing and to avoid animal suffering, we performed everyday visual inspection of every mouse. In particular, we analyzed their skin, food intake, movements and interaction with other mice. If a mouse showed an abnormal behavior, we immediately sacrificed the animal (6 mice had to be sacrificed (S1 Fig)). All the other mice (25/31) showed no evidence of stress or pain and could be monitored for the whole observation period of 8 weeks.

Sonographic measurement of tongue diameter

Tongue size was measured by ultrasound during the PTFE injection procedure. Mice were placed in supine position onto a heating plate. The tongue was gripped with a tiny crocodile clip. Ultrasound gel was placed onto the murine throat, mandible and mouth, but not on nostrils to keep mice breathing. Thereafter, a 30 MHz center frequency transducer (Vevo3100 system from VisualSonics, Toronto, Canada) was placed at median position of the murine throat to measure the dorso-ventral tongue diameter in sagittal plane. For some recordings, the ultrasound head was rotated clockwise by 90° to also measure the lateral tongue diameters in the transversal plane (S2A Fig). Recordings were acquired with 56 frames/s (gain 30 dB). For optimal magnification, acquisition was performed with 10.00 mm depth and 15.36 mm width. We used the presetting of VisualSonics; thus, no calibration was required. By carefully stirring the tongue via the crocodile clip and comparing tongue movements with the other pharyngeal structures under sonographic recording, tongue surface was easily discriminated from surrounding tissue and tongue diameter was assessed. Similar measurements were done before and after PTFE injection in a standardized manner. All measurements were done by the same investigator; therefore, Kappa statistics cannot be reported. We did not use any fluorescence techniques to identify the area of injection.

Monitoring of spontaneous breathing

Two weeks after the PTFE injection, spontaneous breathing was recorded by whole-body plethysmography (Buxco Electronics, Harvard Bioscience, Holliston, MA, USA) and analysis of the continuous box flow (FinePointe software, version 2.4.6.9414). To test whether breathing parameters remain stable for the whole observation period, whole-body plethysmography was repeated for some mice 8 weeks after PTFE injection. Mice were placed in the whole-body plethysmography chamber (9 cm diameter and 8 cm height), which was designed with one whole-body plethysmography port, one port for a drinking bottle and one port for an air exhauster (0.2 liter per minute; Buxco bias flow regulator). The system was calibrated according to the manufactures guidelines. Since mice are nocturnal animals, continuous recordings (sampling frequency 1 kHz) were done for 8 h during day-time, the interval with the highest frequency and duration of sleep periods complying with the murine sleep cycle [20]. This constitutes an established alternative method for sleep apnea monitoring if polygraphy with electroencephalography is not possible [20]. In addition, a few mice were subjected to whole-body plethysmography during night-time to compare frequencies of apneas and inspiratory flow limitations when mice are awake.

For a subgroup of mice, inspiratory flow limitations (IFLs) were analyzed using FinePointe software as following: for each breath, we calculated the ratio of inspiration time and tidal volume. A breath was considered to be flow limited if this ratio was increased at least 2.576 standard deviations compared to a running mean of the last 100 breaths, which corresponds to the 99% confidence interval. This corresponds to either an increase in inspiration time or a decrease in tidal volume or a combination of both. The cut-off was chosen after comprehensive manual investigation of box flow recordings. In addition, to avoid false positive detections, the flow was only considered to be flow limited if its peak inspiratory flow was at least 2.576 standard deviations lower than the mean peak inspiratory flow of the last 100 breaths. The absolute frequency (/h) and the proportional frequency (%) of IFLs were calculated as number of IFLs normalized to either total observation period or total number of breaths, respectively.

We further calculated the number of IFL aggregates that were defined by 3 or more consecutive IFLs and reported their absolute frequency (/h).

For apnea detection, an automatic detection algorithm (apnea analysis module of FinePointe) was used. We defined an apnea as a cessation of breathing for at least 1 s [21]. The frequency of apneas (/h) was calculated as number of apneas normalized to total observation period. Apnea frequency was considered to be abnormally increased, when being at least 2 standard deviations greater than the mean apnea frequency in control mice.

Isolation of RNA and transcription into cDNA

RNA was isolated from ventricular myocardium using the RNeasy Mini Kit (Qiagen, catalog number 74106) like described in the manufacture’s manual. RNA was measured using spectrophotometry (A = 260 nm, NanoDrop™ 2000c, Thermo Scientific™) and cDNA was obtained from 1 μg RNA using random primers (Promega, catalog number C1181), PCR nucleotide mix (Promega, catalog number C1145), RNasin® ribonuclease inhibitor (Promega, catalog number N2115), reverse transcriptase (Promega, catalog number M170B), and reverse transcriptase 5x reaction buffer (Promega, catalog number M531A). The ingredients were incubated for 1 h at 37° C according to the manufactures’ guidelines.

Quantification of KDM6A and HIF1α

KDM6A and HIF1α mRNA expression were measured in ventricular myocardium of PTFE and control mice by real-time qPCR with cDNA (see above) on ViiA 7 real-time PCR system (Applied Biosystems). As described by Jung et al., HIF1α mRNA expression increases under hypoxic conditions in cardiomyocytes [22]. In contrast to HIF1α protein analysis, mRNA expression is less vulnerable to fluctuation due to the critical timing and method of the euthanasia, which is why this substrate was selected for analysis. Experiments were performed with TaqMan™ Fast Advanced Master Mix (Applied Biosystems) and the following settings were used accordingly to the manufacture’s manual: initial uracil-N-glycosylase incubation at 50° C (2 min), polymerase activation at 95° C (2 min), followed by 40 cycles with 95° C (1 s) and 60° C (20 s). Pre-designed TaqMan® Gene Expression Assays (Applied Biosystems) were used for quantification of HIF1α (assay ID Mm00468869_m1), KDM6A (assay ID Mm00801998_m1) and β-actin (assay ID Mm01205647_g1).

All samples were measured as triplicate and the average threshold cycle (Ct) was used for the comparative Ct relative quantification analysis method [23]. Therefore, the mean Ct of each target was subtracted from the corresponding mean Ct of the housekeeper β-actin, obtaining the delta Ct (dCt) value. Calculating 2^-dCt x 100 revealed the relative expression of each target (in % of β-actin).

Transthoracic echocardiography

Transthoracic echocardiography was performed blinded using a Vevo3100 (VisualSonics, Toronto, Canada) system with a 30 MHz center frequency transducer. The animals were initially anesthetized with 2% isoflurane (Isoflurane Vaporizer; VisualSonics, Toronto, Canada), while temperature-, respiration-, and ECG-controlled anesthesia was maintained with 1.5% isoflurane. Two-dimensional cine loops with frame rates of >200 frames/s of a long axis view and a short axis view at mid-level of the papillary muscles as well as M-mode loops of the short axis view were recorded. Left ventricular ejection fraction (parasternal long axis view M-mode), as well as peak early (E) and late (A) diastolic filling velocities (PW doppler) were measured. Tissue doppler mode was used to measure peak early diastolic (e’) and late diastolic (a’) mitral annular velocities. The ratio E/e’ was calculated to estimate the severity of diastolic dysfunction. Measurements were obtained by an examiner blinded to the treatment of the animals.

Western blots

Tris buffer was used to homogenize the whole left and right murine ventricle. The buffer contained (mmol/L) 20 Tris-HCl, 200 NaCl, 20 NaF, 8.9 Nonidet P-40 (Sigma Aldrich), 18.3 phenylmethanesulfonylfluoride (Sigma Aldrich), complete protease inhibitor cocktail (Roche) and complete phosphatase inhibitor cocktail (Roche). Protein concentration was measured by BCA assay (Pierce Biotechnology). Proteins were denaturated at 95° C for 5 min (500 rpm) in 2% β-mercaptoethanol (Sigma Aldrich). They were then separated on 8% SDS-polyacrylamide gels, transferred to a nitrocellulose membrane (GE Healthcare) and incubated overnight with the primary antibody at 4° C: mouse monoclonal anti-CaMKII (1:1000, BD Biosciences, catalog number 611293) and anti-GAPDH (1:20000, Abcam, catalog number G8795). After that, the samples were incubated for 1 h at room temperature with the secondary antibody (HRP-conjugated sheep anti-mouse IgG: 1:3000 for anti-CaMKII, 1:30000 for anti-GAPDH, GE Healthcare, catalog number NA931VS). After incubation with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore) for 5 min at room temperature, protein bands were developed onto Super XR-N X-ray films (Fujifilm) and scanned by ChemiDoc™ MP Imaging System (Bio-Rad). Mean densiometric values were determined using ImageJ.

Data analysis and statistics

All measurements and experiments were performed and analyzed blinded to the treatment group (control or PTFE) and to frequency of apneas. Experimental data are presented as means ± standard error of the mean (SEM). All statistical analyses were based on the number of mice and normal distribution was assessed by Shapiro-Wilk normality test. Parametric or non-parametric tests were applied to test for significant differences, depending on whether a variable was normally distributed or not. Parametric and non-parametric tests used for the comparison of two groups were Student’s t and Mann-Whitney test, respectively. Ordinary one-way ANOVA with Holm-Sidak’s post-hoc correction and Kruskal-Wallis test with Dunn’s post-hoc correction were used for comparisons of more than two groups that were either normally or not normally distributed, respectively. One-way repeated measures ANOVA with Holm-Sidak’s post-hoc correction was used for the comparison of paired data that was normally distributed. If more than two groups and two different factors were compared in a repeated measures design, mixed-effects model analysis with Holm-Sidak’s post-hoc correction was used. Chi-square test was used for the comparison of categorial data. The tests above as well as linear regression analyses were used in GraphPad Prism 8 to test for significance, as appropriate. Two-sided P-values below 0.05 were considered as statistically significant.

Results

Increased tongue diameter after PTFE injection

Injection of polytetrafluoroethylene (PTFE) into the tongue of lean male C57BL/6 mice at an age of 8–12 weeks led to a sustained and significant increase of the sagittal tongue diameter from 2.75±0.16 mm to 3.67±0.20 mm (N = 31; P = 0.002; Fig 1A and 1B). Interestingly, we observed a similar increase in transversal tongue diameter leading to a homogenous increase of cross-sectional tongue area from (in mm²) 9.23±0.41 to 19.90±0.86 (N = 5; P<0.001; S2A Fig). To investigate whether the PTFE depots remain in the tongue for the whole follow up period, we measured the tongue diameter again in a subgroup of mice at 8 weeks after injection. Interestingly, the tongue diameter was still significantly thicker (3.58±0.18 mm; N = 10; P = 0.04).

Fig 1 — a) Original ultrasound image of a murine tongue at median position in sagittal plane before and after polytetrafluorethylene (PTFE) injection. The green line indicates the contour of the tongue. b) Mean dorso-ventral tongue diameter of 31 mice before and after PTFE injection. Interestingly, the mean dorso-ventral tongue diameter remained significantly increased at 8 weeks after the injection (N = 10). c) Due to a random allocation, there was no difference in body weight between control (N = 20) and PTFE mice (N = 28) at baseline. Importantly, there was a similar and significant increase in body weight in both control (N = 19) and PTFE mice (N = 24) at 8 weeks after the injection. *—P<0.05 vs. pre injection (b) or corresponding basal value (c), Kruskal-Wallis test with Dunn’s post-hoc correction (b) and mixed-effects model analysis with Holm-Sidak’s post-hoc correction (c).

An increased tongue diameter may disturb food intake. Therefore, we monitored the body weight (BW) of a subset of mice for 8 weeks. At 8 weeks follow up, we observed a similar significant increase in BW in control (from 27.86±0.54 g to 31.29±0.64 g; N = 19; P<0.001; Fig 1C) and PTFE injected mice (from 27.72±0.61 g to 30.85±0.59 g; N = 24; P<0.001). Importantly, mixed-effects model analysis accounting for timepoint and intervention group revealed no difference in BW between PTFE and control mice, neither at baseline nor at 8 weeks follow up (P(time)<0.001; P(intervention) = 0.82; P(interaction) = 0.89; Fig 1C).

PTFE injection increases the frequency of inspiratory flow limitations and apneas

Two weeks after the PTFE injection, mice were subjected to whole-body plethysmography for apnea analyses (Fig 2A). Interestingly, sleeping PTFE mice (during day-time) exhibited a significantly increased frequency of apneas of 11.32±1.45 per h (N = 25), compared to their littermates with 6.27±0.80 apneas per h (N = 28; P = 0.004; Fig 2B). Moreover, the proportion of mice showing an abnormally increased apnea frequency above the cut-off of 14.75 apneas/h (mean apnea frequency of control mice + 2 standard deviations) was significantly increased in PTFE-injected mice (S2B Fig). Interestingly, 8 out of 25 PTFE mice but only 2 out of 28 control mice showed an abnormally increased apnea frequency (P = 0.02; S2B Fig). In order to further validate our novel method and the mechanism of the increase in apnea frequency, we conducted a linear regression analysis with the tongue diameter after PTFE injection and the corresponding apnea frequency (Fig 2C). Intriguingly, there was a significant positive correlation between those parameters (R² = 0.22; N = 25; P = 0.02). In addition, we investigated inspiratory flow limitations (IFLs) as a milder form of upper airway obstruction (Fig 2D and S3 Fig). When mice were awake (during night-time, 10 p.m.– 7 a.m.), control and PTFE mice showed a similar breathing pattern with a negligible number of IFLs and no IFL aggregates indicating that PTFE injection into the tongue does not induce a fixed upper airway obstruction (S4 Fig). In conscious mice, mean IFL frequency (/h) was 1.98±0.52 vs. 2.49±0.51 (PTFE vs. control; N = 5 for both; P = 1.00; in S4C Fig). Also, mean apnea frequency (/h) was very low and similar in awake PTFE and control mice (1.95±1.01 vs. 2.38±1.19; N = 5 for both; P = 1.00; S4C Fig). In contrast, when analyzing sleeping mice (during day-time), PTFE injection resulted in a significant increase in IFL frequency (/h) from 57.74±4.58 to 72.28±3.59 (P = 0.02) and IFL aggregate frequency from 2.24±0.37 to 3.76±0.43 (N = 11 vs. 15; P = 0.02; S3A Fig and Fig 2E). Moreover, the apnea frequency correlated significantly positive with the IFL aggregate frequency in sleeping mice (R² = 0.24; N = 26; P = 0.01; Fig 2E), suggesting that these apneas may be due to intermittent airway obstruction. The latter is further supported by the clustered occurrence of IFLs and apneas that can be found only in the murine sleeping period (S4 Fig). In accordance, the percentage of flow limited breaths was significantly increased in sleeping mice from 0.43±0.028 to 0.51±0.02 (PTFE vs. control, P = 0.04), leading to a significant positive correlation with the frequency of apneas (R² = 0.20; P = 0.02; S3B Fig). Importantly, the intermittent airway obstruction in sleeping mice remained stable for the whole observation period. Compared to the 2-week timepoint, frequencies of apneas (P = 0.71), IFLs (P = 0.38), and IFL aggregates (P = 0.95) were similar at 8 weeks after PTFE injection (N = 6 for all; S2C Fig).

PTFE injection resulted in decreased KDM6A and increased HIF1α expression

Lysine (K)-specific demethylase 6A (KDM6A) and hypoxia-inducible factor 1α (HIF1α) have been shown to be valuable markers for hypoxemia [22, 24–27]. We measured cardiac KDM6A and HIF1α mRNA expression by qPCR. KDM6A has been shown to be negatively correlated with the extent of hypoxemia [27]. We observed a significant reduction in the expression of KDM6A (in % relative to β-actin) in PTFE mice from 2.44±0.46 (N = 5) to 1.28±0.19 (N = 8; P = 0.02; Fig 3A), leading to a significantly negative correlation with the frequency of apneas (R² = 0.61; N = 13; P = 0.002; Fig 3A). This indicates that the observed apneas may be responsible to induce hypoxemia. Consistent observations were made with the established hypoxemia marker HIF1α. Compared to control, there was a significant increase in HIF1α mRNA (in % relative to β-actin) in PTFE mice from 6.20±1.01 (N = 5) to 16.47±1.95 (N = 8; P = 0.002; S5A Fig). Moreover, we found that the frequency of apneas strongly and significantly correlated with HIF1α mRNA expression (R² = 0.68; N = 13; P<0.001; S5A Fig). Interestingly, the tongue diameter also corelated significantly negative with KDM6A (R² = 0.35.; N = 13; P = 0.03; Fig 3B) and significantly positive with the HIF1α expression (R² = 0.62; N = 13; P = 0.001; S5B Fig), supporting our novel methodological approach of tongue enlargement with PTFE.

Fig 3 — KDM6A mRNA expression was analyzed by qPCR (normalized to β-actin) from hearts. a) Scatter plots of KDM6A mRNA expression in control (N = 5) and PTFE-treated (N = 8) animals (left panel). There was a significant downregulation of KDM6A mRNA expression after PTFE treatment. The level of KDM6A mRNA expression correlated significantly negative with the frequency of apneas, suggesting hypoxemia (right panel). b) Interestingly, the tongue diameter correlated significantly negative with the KDM6A expression, indicating upper airway obstruction to potentially induce apnea-dependent hypoxemia. *—P<0.05, Student’s t-test and linear regression analysis, as appropriate.

Systolic and diastolic dysfunction in PTFE-treated mice

Since OSA may impair systolic and diastolic contractile function [2, 28], we assessed cardiac function by echocardiography in mice at 8 weeks follow up (Fig 4 and Table 1). PTFE injected mice showed a significantly decreased left ventricular ejection fraction of 49.02±2.07% (N = 12) compared to control mice with 56.10±2.49% (N = 12; P = 0.04; Fig 4B). In accordance, left ventricular end-diastolic volume was significantly increased in PTFE mice (Table 1). The ejection fraction also correlated significantly negative with the frequency of apneas (R² = 0.19; P = 0.04; Fig 4B).

Fig 4 — a) Representative M-mode echocardiographic recordings and (b) mean data for ejection fraction, which was significantly reduced in polytetrafluoroethylene (PTFE) mice (N = 12 vs. 12; left panel). Furthermore, there was a significant negative correlation between the frequency of apneas and the ejection fraction (right panel). c) Representative recordings of the mitral annular ring velocity with a tissue doppler. d) Interestingly, the frequency of apneas correlated significantly with the ratio E/e’ (N = 24), suggesting a more pronounced diastolic dysfunction in mice with more frequent apneas. *—P<0.05, Student’s t-test and linear regression analysis, as appropriate.

Table 1. Echocardiographic parameters.

	Control	PTFE	P Value
	(N = 12)	(N = 12)	P Value
Heart rate (/min), mean±SEM	490.75±15.02	489.42±12.29	0.95^T
Cardiac output (ml/min), mean±SEM	18.47±1.15	19.16±0.89	0.64^T
Stroke volume (μl), mean±SEM	37.47±2.11	38.52±1.93	0.93^MW
LV end-diastolic diameter (mm), mean±SEM	3.98±0.07	4.22±0.09	0.06^T
LV end-diastolic volume (μl), mean±SEM	68.32±3.22	81.42±3.97	0.02^T
Diastolic anterior wall thickness (mm), mean±SEM	0.87±0.08	0.81±0.05	0.50^T
Diastolic posterior wall thickness (mm), mean±SEM	0.68±0.07	0.65±0.03	0.78^T

Open in a new tab

LV–left ventricular, MW–Mann-Whitney test, T–Student’s t-test.

Furthermore, we analyzed the ratio of the early diastolic filling velocity (E) and the peak early diastolic mitral annular velocity (e’) to estimate the severity of diastolic dysfunction. Interestingly, E/e’ correlated significantly positive with the frequency of apneas (R² = 0.19; N = 24; P = 0.04; Fig 4D).

Increased heart and lung weight in PTFE-treated mice

Contractile dysfunction is frequently accompanied by structural changes of the heart and clinical signs of heart failure. Therefore, we measured heart (HWs) and lung weights (LWs) and normalized it to the BW (Fig 5) at 8 weeks follow up that can be used to estimate cardiac hypertrophy and pulmonary edema, respectively [29, 30]. Interestingly, HW/BW (Fig 5A) was not only significantly increased from 0.59±0.03% (N = 19) to 0.68±0.03% in PTFE injected mice (N = 24; P = 0.03), but also correlated significantly positive with the frequency of apneas (R² = 0.10; N = 39; P = 0.045). Importantly, this increase in heart weight appears to be rather due to eccentric hypertrophy with enlarged left ventricular end-diastolic volume and not due to hypertrophic wall thickening (Table 1).

In accordance with reduced left ventricular contractile function, PTFE-treated mice also showed a significant increase in LW/BW (Fig 5B) consistent with lung edema. Compared to control, LW/BW was significantly increased in PTFE-treated mice (0.64±0.02%; N = 24 vs. 0.56±0.02%; N = 19; P<0.001). In addition, LW/BW correlated significantly positive with the frequency of apneas (R² = 0.13; N = 39; P = 0.02).

Increased CaMKII expression in PTFE-treated mice

Increased CaMKII expression has been shown in multiple studies to be implemented in the pathogenesis of heart failure, hypertrophy, and arrhythmias [8, 9, 31], and has also been found in patients with sleep-disordered breathing [4]. Therefore, we analyzed CaMKII expression in ventricular homogenates of PTFE-treated (N = 6) and control mice (N = 9) at 8-week follow up by Western blotting (Fig 6). Interestingly, compared to control, PTFE-treated mice showed a significant increase in CaMKII expression normalized to GAPDH (5.01±0.61 vs. 2.51±0.66, P = 0.02; Fig 6B). Furthermore, the CaMKII/GAPDH expression correlated significantly positive with the frequency of apneas (R² = 0.28; P = 0.04; Fig 6C).

Fig 6 — a) Original Western blots investigating Ca/calmodulin-dependent protein kinase II (CaMKII) expression in ventricular homogenates. b) Mean densitometric values show a significantly increased CaMKII expression in polytetrafluoroethylene (PTFE) injected mice (N = 6 vs. 9). c) Interestingly, there was also a significant positive correlation between the frequency of apneas and CaMKII expression (N = 15). *—P<0.05, Student’s t-test (b) and linear regression analysis (c).

Discussion

In this study, we describe a novel mouse model of OSA by injecting polytetrafluoroethylene (PTFE) into the tongue of lean male C57BL/6 mice. These mice develop IFLs with an increased frequency of apneas. At 8 weeks follow up, PTFE-treated mice show increased ventricular expression of the novel hypoxia marker KDM6A, mild systolic and diastolic contractile dysfunction, signs of cardiac eccentric hypertrophy and pulmonary edema, that were accompanied by CaMKII overexpression. In the absence of co-morbidities, this mouse model may be suitable to investigate the mechanisms of spontaneous obstructive sleep apnea.

OSA is a widespread disease with increasing prevalence [1]. Since it is frequently associated with multiple disorders like heart failure or arrhythmias leading to increased morbidity and mortality, it takes on even greater socio-economic significance [2, 3]. To date, the treatment of OSA is mainly limited to continuous positive airway pressure (CPAP), but acceptance of CPAP in patients with low symptom burden is limited [6] and treatment with positive pressure ventilation (adaptive servo-ventilation) may be even harmful for specific patient populations (e.g. for patients with predominant central apneas) [7]. It is consequently essential to find novel therapeutic concepts for the treatment of OSA and OSA-related diseases. Therefore, it is necessary to get a better understanding about the mechanisms of OSA affecting the cardiovascular system. Although there are multiple studies analyzing the mechanisms OSA in humans, they always struggle with the problem of patient heterogenicity and several comorbidities as potential confounders, which makes it difficult to interpret mechanistic data. To avoid those pitfalls, research of OSA in animal models is important [16].

Current models of sleep apnea are limited

Despite the urgent need for animal models of sleep apnea, current animal models struggle with many limitations [12–17, 32].

A very common animal model is tracheotomy with intermittent tracheal occlusion, which has already been used in several animals like dogs [33], baboons [34], and rats [16, 20]. Linz et al. have reported tracheal occlusion with a negative pressure of -80 mbar in anesthetized pigs resulting in increased blood pressure and oxidative stress, activation of fibrotic pathways and subsequently an increased burden of atrial fibrillation [12]. However, while this model may be applied in otherwise healthy animals thereby excluding the confounding of comorbidities, it is currently not available in mice, excluding the opportunity to test novel hypotheses in genetically modified animals. Also, this model requires deep medical sedation and analgesia of the animals, which may interfere with the apnea-dependent sleep fragmentation with sudden awakening (arousal) and consecutive alteration of the autonomous nervous system [3, 16]. Additionally, Crossland et al. have reported a rodent model of OSA by chronic intermittent tracheal occlusion in rats during their sleep cycle (9 AM to 5 PM) [20]. However, all models of tracheal occlusion require substantial material and personal investment that precluded application in large animal cohorts for extended periods of follow up. Another rat model of OSA mimicking airway obstruction was first described by Farré et al. [35]. Rats were placed awake in a setup with two chambers split by a latex neck collar and airway obstruction was induced by interruption of bias flow in the head chamber [35, 36]. While the authors observed remarkable decreases in oxygen saturation, this model is hardly comparable to clinical OSA since rats were placed awake in the chamber [35, 36]. Although this model requires few animal manipulation and the setup is easy to handle, it is very time intensive since animals have to be placed in the obstruction chambers every day for at least three weeks [35, 36].

Chronic intermittent hypoxemia (CIH) is another very frequently used method for investigation of sleep-disordered breathing in animals [16, 37]. Although this method gave valuable insights into the pathophysiology of sleep-disordered breathing, the optimal gas composition is still under discussion [16]. Moreover, it completely lacks important aspects of the pathophysiology like airway obstruction. OSA, is mainly characterized by obstruction of the upper airway with consecutive inefficient breathing effort, intrathoracic pressure swings, which are followed by intermittent hypoxemia and β-adrenergic stress during sudden awakening [3]. This may be one explanation, why some clinical findings of patients with sleep-disordered breathing have not been recapitulated by CIH in animals [3, 16, 37].

For instance, sustained sleep-related arterial hypertension has been shown to occur in dogs only in combination with upper airway obstruction (induced by tracheal occlusion), but not following repetitive arousals or hypoxemia alone [3, 16, 38].

Moreover, short-term CIH was found to even protect the heart by inducing ischemic preconditioning and increasing cardiac contractility [39]. But even after long term CIH exposure, the basal cardiac phenotype may depend on the specific CIH protocol applied and may require additional interventions like ischemia/reperfusion to induce detectable damage [40].

Beside these limitations, CIH also requires substantial material and personal investment (cost-intensive ventilation chamber, huge amount of gas to be exchanged) rendering long term studies for larger animal cohorts very difficult, which limits high throughput animal research [16].

PTFE tongue injection induces obstructive apneas in mice

Considering the difficulties of current animal models, novel mouse models are clearly warranted. Interestingly, obesity, as one of the most common risk factors for OSA, is accompanied by a narrowed upper airway in humans and several animals [13, 14, 16, 17]. Looking at the structures in the upper airway, Schwab et al. have found that an increased tongue volume is the most important risk structure for OSA and is even independent from sex, age, race, craniofacial size, and parapharyngeal fat [19]. In accordance, New Zealand Obese mice have also an increased tongue volume with consecutive apneas and hypopneas [14, 16, 17]. However, these mice also suffer from OSA-independent diseases like arterial hypertension, hyperinsulinemia and hypercholesterolemia, which makes it difficult to ascribe observations to airway obstruction [15].

Thus, it was a reasonable concept to induce intermittent airway obstruction by increasing the cross-sectional area of the tongue. PTFE is an inert substance that can be used for treating primary vesicoureteral reflux by narrowing the ureterovesical junction [18]. We show here that a single PTFE injection procedure not only resulted in increased tongue diameters that remain increased across the whole follow up period (Fig 1B) without signs of inflammation or disturbance of food intake, but also that PTFE-treated mice develop spontaneous IFLs and abnormally increased apneas. According to the sustained increase in tongue diameter, we could demonstrate that the increased frequencies of IFLs and apneas remained stable for the whole 8-week observation period (S2C Fig). Importantly, PTFE injection into the tongue does not lead to a fixed upper airway obstruction (S4 Fig). In sleeping mice, only about 0.50% of all breaths in PTFE mice were flow limited (S3B Fig). In accordance with the intermittent nature of obstructive breathing abnormalities, the PTFE-induced IFLs and apneas occurred in clusters and only in sleeping mice (S4A Fig). In contrast, awake PTFE mice exhibit a regular breathing pattern without airway obstruction (S4B Fig). Moreover, we observed no IFL aggregates and similar frequencies of IFLs and apneas after PTFE treatment during awake periods (S4C Fig). Therefore, hypoxia is likely not present in awake mice.

Similar observations were made by Philip et al., who injected liquid collagen into the uvula, tongue, and pharyngeal walls of monkeys leading to more frequent hypopneas during sleep [41]. Since monkeys are not a very common animal model, and are more difficult in housing, this model was not established in OSA research [41]. Additionally, polyacrylamide and sodium hyaluronate injection into the palate of rabbits and rats, respectively, has been shown to induce obstructive sleep apnea by upper airway obstruction [42, 43]. Unfortunately, the availability of appropriate knock-out or transgene models is very limited in both rabbits and rats. In this context, future studies investigating sodium hyaluronate injection into the palate of mice may lead to another promising animal model of OSA.

Therefore, we report here a unique mouse model of OSA that avoids many pitfalls of former models. Our model develops spontaneous apneas and does not require anesthesia or additional interventions. This could be important, when investigating OSA-specific features like sudden awakening with consecutive β-adrenergic stress. Further, the mouse models offer great opportunities by investigating specific knock out or transgene mice.

Importantly, our model is not only very effective in inducing obstructive apneas, but also very efficient since a single intervention resulted in a sustained airway obstruction with IFLs and apneas across the whole observation period of 8 weeks. Thus, large cohorts of animals can be investigated with relatively low material and personal requirements.

Consequences of PTFE-injection on contractile function

Since OSA is frequently associated to systolic and diastolic dysfunction [2, 28], we performed echocardiography to complete the basal characterization of our new model (Fig 4). PTFE-treated mice show mild systolic and diastolic contractile dysfunction. In addition, these mice show signs of eccentric left ventricular hypertrophy (increased HW/BW ratio and increased LV end-diastolic volume) and congestive heart failure (increased LW/BW ratios). Moreover, CaMKII expression was significantly increased, which is a hallmark for hypertrophy, contractile dysfunction and arrhythmias [8, 9, 31].

Since obstructive sleep apnea may result in the development of arterial hypertension with increased cardiac afterload, this may partly explain the cardiac phenotype of the present model. In fact, rodent OSA models showed OSA-dependent development of arterial hypertension [16, 32, 44].

However, all our mice subjected to PTFE injection had been healthy at baseline. Thus, all potential pathophysiologic changes that may have developed during the 8 weeks observation period, such as increased blood pressure, impaired myocardial contractility or impaired sleep with chronic sympathetic stress, are secondary to the PTFE-induced intermittent airway obstruction during sleep. Consequently, all cardiovascular effects can be (directly or indirectly) attributed to OSA. This differentiates our model from obese and diabetic mouse models of sleep apnea, for instance, where OSA-independent comorbidities confound the experiments. Future studies using our model may address the relative contribution of the different pathophysiological alterations secondary to OSA individually.

The cardiovascular dysfunction developed by mice in our model is rather modest. A reduction of ejection fraction from 56.10±2.49% in control to 49.02±2.07% in PTFE mice may be detectable, but its pathophysiological relevance may be low. There are mouse models of systolic heart failure like transverse aortic constriction that would result in a much larger degree of systolic dysfunction [45]. Moreover, if compared to patients, the magnitude of ejection fraction observed in our PTFE mice would still be in the normal to subnormal range. On the other hand, we have observed many clinical features that can be found in patients with heart failure with preserved ejection fraction or patients with hypertension and hypertensive heart disease [46, 47]. Importantly, arterial hypertension and heart failure with preserved ejection fraction are very common in patients with OSA and not only found in those patients at the far end of extremely severe intermittent airway obstruction [48–50].

The frequency of apnea events and the increase with PTFE-injection was rather modest in our model. In contrast, many other animal models exceed the severity of human OSA, partly because the consequences are to be detected within a few weeks [12, 16, 32, 33]. We have extended our observation period to a long duration of 8 weeks, other mouse models usually perform OSA protocols (e.g. CIH, tracheal occlusion) for about 3–5 weeks [12, 16, 20, 32, 35, 36]. We did this to model the human situation more closely, where mild intermittent airway obstruction may result in the development of pathophysiological sequelae only after years. Despite this mild increase in intermittent airway obstruction, we show here that the frequency of apneas correlated significantly with the severity of contractile dysfunction and other features of the heart failure (heart and lung weight, Figs 4 and 5), suggesting a causal relationship. On the other hand, we cannot exclude that other factors following OSA that are not directly related to intermittent airway obstruction may also potentially contribute to the phenotype of these mice.

Limitations

Although we conclusively report that PTFE-injected mice show features of OSA, we have not simultaneously monitored tidal volume and breathing effort. Therefore, we cannot exclude that some of the detected apneas may be a consequence of dysregulation by the central nervous system. However, we demonstrated a significant correlation between IFLs and apneas, suggesting that the majority of apneas observed in this model is obstructive in nature. In addition, the cut-off of 1 s for detection of apneas was arbitrarily chosen. We have not continuously monitored arterial oxygen concentration to show that 1 s apneas would result in arterial desaturation. Intriguingly, KDM6A has recently been reported to be a novel and high sensitive hypoxic marker that is decreased upon hypoxia [27]. Importantly, we have observed that mRNA expression of KDM6A was significantly decreased in ventricular myocardium of PTFE-treated mice, indicating that hypoxia may be present. Since there was also a significant negative correlation between the tongue diameter and KDM6A expression, upper airway obstruction via tongue enlargement seems to potentially induce apnea-dependent hypoxemia. Our findings were confirmed by mRNA expression analysis of hypoxia-inducible factor 1α (HIF1α) that was upregulated in the ventricular myocardium of PTFE-treated mice, further suggesting that arterial hypoxia may be present [22, 24–26]. HIF1α has been shown to be a valuable marker for hypoxemia [22, 25, 26]. It enhances the transcription of either adaptive or deleterious genes, depending on the intensity and duration of hypoxemia [25, 26]. In addition to the regulation of HIF1α by protein stabilization [51], several in vivo studies showed increased levels of HIF1α mRNA in human, rats, and mice that were exposed to hypoxia [22, 24, 26]. Moreover, HIF1α mRNA expression is less vulnerable to fluctuation due to the critical timing and method of the euthanasia compared to HIF1α protein [22, 24–26, 51]. Interestingly, HIF1α mRNA expression has been shown to be already increased after 30 min of hypoxia (at 7% O₂) in vivo in mice [24]. Moreover, the total increase of HIF1α mRNA expression to about 2.5-fold was also comparable to a model of CIH [26].

We have not directly monitored sleep cycles by electroencephalography, which would have required a substantial additional methodological effort. On the other hand, performing sleep apnea monitoring without electroencephalography during the usual rodent sleep cycle (day-time, e.g. 9 AM to 5 PM) has been shown to be feasible [20]. Additionally, we have shown here that IFLs do not occur with a uniform distribution across the monitoring interval. Instead, they form clusters when mice were supposed to sleep, while no IFL aggregates and only a very low frequency of IFLs was observed at night-time when mice were awake. Nevertheless, further investigations are required to directly correlate IFLs and apneas with sleep.

Conclusions

In conclusion, we describe here the first mouse model showing spontaneous IFLs, obstructive apneas and hypoxia by tongue enlargement due to PTFE injection in the absence of co-morbidities. These mice develop systolic and diastolic dysfunction and increased CaMKII expression. This model circumvents many problems of current animal models of sleep apnea. It is feasible and readily available to many researchers, which renders it an ideal tool to investigate mechanisms of OSA especially in the context of genetically modified mice, which may help identify novel treatment strategies that are urgently needed.

Supporting information

S1 Fig. Study flowchart.

Study flowchart showing the allocation of 59 mice in total. 31 mice were subjected to tongue enlargement by PTFE and 28 littermates were used as control animals. 16 mice (5 control vs. 11 PTFE mice) were used for some proof of principle experiments that are part of the Supporting information. Since they are not part of the main manuscript, they are not shown in this study flowchart.

(TIF)

Click here for additional data file.^{(208.2KB, tif)}

S2 Fig. Increased cross-sectional tongue area and sustained OSA at 8 weeks after PTFE injection.

a) Original ultrasound image of a murine tongue in transversal plane before and after (PTFE) injection (left panel). Interestingly, we observed a strong increase in the lateral tongue diameter. The mean data for 5 animals is shown in right panel. PTFE injection resulted in a significant increase in both lateral (transversal plane) and dorso-ventral (sagittal plane) tongue diameters. Cross-sectional tongue area was calculated by lateral diameter*dorso-ventral diameter*0.25*π estimating an elliptical shape of the tongue. b) After PTFE injection, the proportion of mice showing an abnormally increased apnea frequency (cut-off 14.75 apneas/h) was significantly increased. c) Importantly, frequencies of apneas, IFLs, and IFL aggregates remained stable for the whole 8-week observation period (N = 6). *—P<0.05 vs. pre injection (a) or control (b), one-way repeated measures ANOVA with Holm-Sidak’s post-hoc correction (a+c) and Chi-square test (b).

(TIF)

Click here for additional data file.^{(1MB, tif)}

S3 Fig. Increased IFL frequency in mice after PTFE injection.

a) PTFE-injected mice showed a significant increase in the absolute frequency of inspiratory flow limitations (IFLs/h). b) In accordance, we observed a significantly increased percentage of flow limited breaths in PTFE mice that also correlated significantly positive with the frequency of apneas. *—P<0.05, Student’s t-test and linear regression analysis, as appropriate.

(TIF)

Click here for additional data file.^{(326.9KB, tif)}

S4 Fig. Breathing characteristics in conscious mice are unaltered by PTFE injection.

a) Average breath frequency, and the number of IFL and apneas were calculated for 30 min intervals during a 22 h observation period (from 10 p.m. to 8 p.m. the other day) in a PTFE-treated mouse. Time of activity of conscious mice can be easily discriminated from sleep time by monitoring average breathing frequency. An increased number of IFLs was typically accompanied by a concomitant increase in the number of apneas only during sleep time. b) Original box flow recordings of a control (upper panel) and a PTFE-treated mouse (lower panel) measured by whole-body plethysmography in conscious mice. Both mice showed a similar breathing pattern, indicating that tongue enlargement due to PTFE injection does not induce upper airway obstruction in conscious mice. c) Mean data for IFL and apnea frequency during activity time. In conscious mice (activity time at night), a negligible number of apneas and IFL could be detected with no difference between PTFE-treated and control animals. Kruskal-Wallis test with Dunn’s post-hoc correction.

(TIF)

Click here for additional data file.^{(817.5KB, tif)}

S5 Fig. HIF1α mRNA expression is upregulated in PTFE mice.

HIF1α mRNA expression was analyzed by qPCR (normalized to β-actin) from hearts. a) Scatter plots of HIF1α mRNA expression in control (N = 5) and PTFE-treated (N = 8) animals (left panel). There was a significant upregulation of HIF1α mRNA expression after PTFE treatment. The level of HIF1α mRNA expression correlated significantly with the frequency of apneas (right panel). b) Interestingly, the tongue diameter correlated significantly positive with the HIF1α expression, indicating hypoxemia due to PTFE-dependent tongue enlargement. *—P<0.05, Mann-Whitney test and linear regression analysis, as appropriate.

(TIF)

Click here for additional data file.^{(361.8KB, tif)}

S1 Raw images. Gels.

(PDF)

Click here for additional data file.^{(4.3MB, pdf)}

S1 Video. Tongue injection procedure.

(MOV)

Click here for additional data file.^{(34.5MB, mov)}

Acknowledgments

We greatly appreciate the expert technical assistance of Gabriela Pietrzyk, Thomas Sowa, and Felicia Radtke.

Non-standard abbreviations and acronyms

BW: body weight
CaMKII: Ca/calmodulin-dependent protein kinase II
CIH: chronic intermittent hypoxemia
HIF1α: hypoxia-inducible factor 1α
HW: heart weight
IFL: inspiratory flow limitation
KDM6A: lysine (K)-specific demethylase 6A
LW: lung weight
OSA: obstructive sleep apnea
PTFE: polytetrafluoroethylene

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

SL was funded by the Max Weber scholarship. SW was funded by DFG grants WA 2539/4-1, 5-1, 7-1, and 8-1. LSM was funded by DFG grants MA 1982/5-1 and 7-1. SW and LSM were also funded by the DFG SFB 1350 grant (Project Number 387509280, TPA6), and were supported by the ReForM C program of the faculty. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Benjafield AV, Ayas NT, Eastwood PR, Heinzer R, Ip MSM, Morrell MJ, et al. Estimation of the global prevalence and burden of obstructive sleep apnoea. A literature-based analysis. Lancet Respir Med. 2019; 7: 687–698. 10.1016/S2213-2600(19)30198-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Gottlieb DJ, Yenokyan G, Newman AB, O'Connor GT, Punjabi NM, Quan SF, et al. Prospective study of obstructive sleep apnea and incident coronary heart disease and heart failure. The sleep heart health study. Circulation. 2010; 122: 352–360. 10.1161/CIRCULATIONAHA.109.901801 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Rossi VA, Stradling JR, Kohler M. Effects of obstructive sleep apnoea on heart rhythm. Eur Respir J. 2013; 41: 1439–1451. 10.1183/09031936.00128412 [DOI] [PubMed] [Google Scholar]
4.Lebek S, Pichler K, Reuthner K, Trum M, Tafelmeier M, Mustroph J, et al. Enhanced CaMKII-Dependent Late INa Induces Atrial Proarrhythmic Activity in Patients With Sleep-Disordered Breathing. Circ Res. 2020; 126: 603–615. 10.1161/CIRCRESAHA.119.315755 [DOI] [PubMed] [Google Scholar]
5.Buchner S, Eglseer M, Debl K, Hetzenecker A, Luchner A, Husser O, et al. Sleep disordered breathing and enlargement of the right heart after myocardial infarction. Eur Respir J. 2015; 45: 680–690. 10.1183/09031936.00057014 [DOI] [PubMed] [Google Scholar]
6.McEvoy RD, Antic NA, Heeley E, Luo Y, Ou Q, Zhang X, et al. CPAP for prevention of cardiovascular events in obstructive sleep apnea. N Engl J Med. 2016; 375: 919–931. 10.1056/NEJMoa1606599 [DOI] [PubMed] [Google Scholar]
7.Cowie MR, Woehrle H, Wegscheider K, Angermann C, d'Ortho M-P, Erdmann E, et al. Adaptive servo-ventilation for central sleep apnea in systolic heart failure. N Engl J Med. 2015; 373: 1095–1105. 10.1056/NEJMoa1506459 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Sossalla S, Fluschnik N, Schotola H, Ort KR, Neef S, Schulte T, et al. Inhibition of elevated Ca2+/calmodulin-dependent protein kinase II improves contractility in human failing myocardium. Circ Res. 2010; 107: 1150–1161. 10.1161/CIRCRESAHA.110.220418 [DOI] [PubMed] [Google Scholar]
9.Fischer TH, Herting J, Tirilomis T, Renner A, Neef S, Toischer K, et al. Ca2+/calmodulin-dependent protein kinase II and protein kinase A differentially regulate sarcoplasmic reticulum Ca2+ leak in human cardiac pathology. Circulation. 2013; 128: 970–981. 10.1161/CIRCULATIONAHA.113.001746 [DOI] [PubMed] [Google Scholar]
10.Wagner S, Ruff HM, Weber SL, Bellmann S, Sowa T, Schulte T, et al. Reactive oxygen species-activated Ca/calmodulin kinase IIdelta is required for late I(Na) augmentation leading to cellular Na and Ca overload. Circ Res. 2011; 108: 555–565. 10.1161/CIRCRESAHA.110.221911 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Dybkova N, Wagner S, Backs J, Hund TJ, Mohler PJ, Sowa T, et al. Tubulin polymerization disrupts cardiac beta-adrenergic regulation of late INa. Cardiovasc Res. 2014; 103: 168–177. 10.1093/cvr/cvu120 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Linz D, Hohl M, Nickel A, Mahfoud F, Wagner M, Ewen S, et al. Effect of renal denervation on neurohumoral activation triggering atrial fibrillation in obstructive sleep apnea. Hypertension. 2013; 62: 767–774. 10.1161/HYPERTENSIONAHA.113.01728 [DOI] [PubMed] [Google Scholar]
13.Fleury Curado T, Pho H, Berger S, Caballero-Eraso C, Shin M-K, Sennes LU, et al. Sleep-disordered breathing in C57BL/6J mice with diet-induced obesity. Sleep. 2018; 41: 1–9. 10.1093/sleep/zsy089 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Brennick MJ, Pack AI, Ko K, Kim E, Pickup S, Maislin G, et al. Altered upper airway and soft tissue structures in the New Zealand Obese mouse. Am J Respir Crit Care Med. 2009; 179: 158–169. 10.1164/rccm.200809-1435OC [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Ortlepp JR, Kluge R, Giesen K, Plum L, Radke P, Hanrath P, et al. A metabolic syndrome of hypertension, hyperinsulinaemia and hypercholesterolaemia in the New Zealand obese mouse. Eur J Clin Invest. 2000; 30: 195–202. 10.1046/j.1365-2362.2000.00611.x [DOI] [PubMed] [Google Scholar]
16.Chopra S, Polotsky VY, Jun JC. Sleep Apnea Research in Animals. Past, Present, and Future. Am J Respir Cell Mol Biol. 2016; 54: 299–305. 10.1165/rcmb.2015-0218TR [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Baum DM, Morales Rodriguez B, Attali V, Cauhapé M, Arnulf I, Cardot P, et al. New Zealand Obese Mice as a Translational Model of Obesity-related Obstructive Sleep Apnea Syndrome. Am J Respir Crit Care Med. 2018; 198: 1336–1339. 10.1164/rccm.201801-0162LE [DOI] [PubMed] [Google Scholar]
18.Diamond DA, Mattoo TK. Endoscopic treatment of primary vesicoureteral reflux. N Engl J Med. 2012; 366: 1218–1226. 10.1056/NEJMct1108922 [DOI] [PubMed] [Google Scholar]
19.Schwab RJ, Pasirstein M, Pierson R, Mackley A, Hachadoorian R, Arens R, et al. Identification of upper airway anatomic risk factors for obstructive sleep apnea with volumetric magnetic resonance imaging. Am J Respir Crit Care Med. 2003; 168: 522–530. 10.1164/rccm.200208-866OC [DOI] [PubMed] [Google Scholar]
20.Crossland RF, Durgan DJ, Lloyd EE, Phillips SC, Reddy AK, Marrelli SP, et al. A new rodent model for obstructive sleep apnea. Effects on ATP-mediated dilations in cerebral arteries. Am J Physiol Regul Integr Comp Physiol. 2013; 305: R334–342. 10.1152/ajpregu.00244.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Simeone KA, Hallgren J, Bockman CS, Aggarwal A, Kansal V, Netzel L, et al. Respiratory dysfunction progresses with age in Kcna1-null mice, a model of sudden unexpected death in epilepsy. Epilepsia. 2018; 59: 345–357. 10.1111/epi.13971 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Jung F, Palmer LA, Zhou N, Johns RA. Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes. Circ Res. 2000; 86: 319–325. 10.1161/01.res.86.3.319 [DOI] [PubMed] [Google Scholar]
23.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25: 402–408. 10.1006/meth.2001.1262 [DOI] [PubMed] [Google Scholar]
24.Wiener CM, Booth G, Semenza GL. In vivo expression of mRNAs encoding hypoxia-inducible factor 1. Biochem Biophys Res Commun. 1996; 225: 485–488. 10.1006/bbrc.1996.1199 [DOI] [PubMed] [Google Scholar]
25.Belaidi E, Joyeux-Faure M, Ribuot C, Launois SH, Levy P, Godin-Ribuot D. Major role for hypoxia inducible factor-1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea. J Am Coll Cardiol. 2009; 53: 1309–1317. 10.1016/j.jacc.2008.12.050 [DOI] [PubMed] [Google Scholar]
26.Yuan G, Khan SA, Luo W, Nanduri J, Semenza GL, Prabhakar NR. Hypoxia-inducible factor 1 mediates increased expression of NADPH oxidase-2 in response to intermittent hypoxia. J Cell Physiol. 2011; 226: 2925–2933. 10.1002/jcp.22640 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Chakraborty AA, Laukka T, Myllykoski M, Ringel AE, Booker MA, Tolstorukov MY, et al. Histone demethylase KDM6A directly senses oxygen to control chromatin and cell fate. Science. 2019; 363: 1217–1222. 10.1126/science.aaw1026 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Chami HA, Resnick HE, Quan SF, Gottlieb DJ. Association of incident cardiovascular disease with progression of sleep-disordered breathing. Circulation. 2011; 123: 1280–1286. 10.1161/CIRCULATIONAHA.110.974022 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Parker JC, Townsley MI. Evaluation of lung injury in rats and mice. Am J Physiol Lung Cell Mol Physiol. 2004; 286: L231–246. 10.1152/ajplung.00049.2003 [DOI] [PubMed] [Google Scholar]
30.Hamano G, Yamamoto K, Takami Y, Takeshita H, Shimosato T, Moritani T, et al. Effects of Low-Dose Sacubitril/Valsartan on Different Stages of Cardiac Hypertrophy in Salt-Loaded Hypertensive Rats. J Cardiovasc Pharmacol. 2019; 73: 282–289. 10.1097/FJC.0000000000000662 [DOI] [PubMed] [Google Scholar]
31.Neef S, Dybkova N, Sossalla S, Ort KR, Fluschnik N, Neumann K, et al. CaMKII-dependent diastolic SR Ca2+ leak and elevated diastolic Ca2+ levels in right atrial myocardium of patients with atrial fibrillation. Circ Res. 2010; 106: 1134–1144. 10.1161/CIRCRESAHA.109.203836 [DOI] [PubMed] [Google Scholar]
32.Dematteis M, Godin-Ribuot D, Arnaud C, Ribuot C, Stanke-Labesque F, Pépin J-L, et al. Cardiovascular consequences of sleep-disordered breathing. Contribution of animal models to understanding the human disease. ILAR J. 2009; 50: 262–281. 10.1093/ilar.50.3.262 [DOI] [PubMed] [Google Scholar]
33.Brooks D, Horner RL, Kozar LF, Render-Teixeira CL, Phillipson EA. Obstructive sleep apnea as a cause of systemic hypertension. Evidence from a canine model. J Clin Invest. 1997; 99: 106–109. 10.1172/JCI119120 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.White SG, Fletcher EC, Miller CC. Acute systemic blood pressure elevation in obstructive and nonobstructive breath hold in primates. J Appl Physiol. 1995; 79: 324–330. 10.1152/jappl.1995.79.1.324 [DOI] [PubMed] [Google Scholar]
35.Farré R, Nácher M, Serrano-Mollar A, Gáldiz JB, Alvarez FJ, Navajas D, et al. Rat model of chronic recurrent airway obstructions to study the sleep apnea syndrome. Sleep. 2007; 30: 930–933. 10.1093/sleep/30.7.930 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Rubies C, Dantas A-P, Batlle M, Torres M, Farre R, Sangüesa G, et al. Aortic remodelling induced by obstructive apneas is normalized with mesenchymal stem cells infusion. Sci Rep. 2019; 9: 11443 10.1038/s41598-019-47813-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Farré R, Montserrat JM, Gozal D, Almendros I, Navajas D. Intermittent Hypoxia Severity in Animal Models of Sleep Apnea. Front Physiol. 2018; 9: 1556 10.3389/fphys.2018.01556 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.O'Donnell CP, Ayuse T, King ED, Schwartz AR, Smith PL, Robotham JL. Airway obstruction during sleep increases blood pressure without arousal. J Appl Physiol. 1996; 80: 773–781. 10.1152/jappl.1996.80.3.773 [DOI] [PubMed] [Google Scholar]
39.Naghshin J, McGaffin KR, Witham WG, Mathier MA, Romano LC, Smith SH, et al. Chronic intermittent hypoxia increases left ventricular contractility in C57BL/6J mice. J Appl Physiol. 2009; 107: 787–793. 10.1152/japplphysiol.91256.2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Joyeux-Faure M, Stanke-Labesque F, Lefebvre B, Béguin P, Godin-Ribuot D, Ribuot C, et al. Chronic intermittent hypoxia increases infarction in the isolated rat heart. J Appl Physiol. 2005; 98: 1691–1696. 10.1152/japplphysiol.01146.2004 [DOI] [PubMed] [Google Scholar]
41.Philip P, Gross CE, Taillard J, Bioulac B, Guilleminault C. An animal model of a spontaneously reversible obstructive sleep apnea syndrome in the monkey. Neurobiol Dis. 2005; 20: 428–431. 10.1016/j.nbd.2005.03.024 [DOI] [PubMed] [Google Scholar]
42.Lu H-y, Dong F, Liu C-y, Wang J, Liu Y, Xiao W. An animal model of obstructive sleep apnoea-hypopnea syndrome corrected by mandibular advancement device. Eur J Orthod. 2015; 37: 284–289. 10.1093/ejo/cju041 [DOI] [PubMed] [Google Scholar]
43.Liu Y, Gao L, Lv W, Lin L, Wang Y, He H, et al. Histological, Ultrastructural, and Physiological Evaluation of a Rat Model of Obstructive Sleep Apnea Syndrome. Med Sci Monit. 2019; 25: 1806–1813. 10.12659/MSM.913056 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Tietjens JR, Claman D, Kezirian EJ, Marco T de, Mirzayan A, Sadroonri B, et al. Obstructive Sleep Apnea in Cardiovascular Disease. A Review of the Literature and Proposed Multidisciplinary Clinical Management Strategy. J Am Heart Assoc. 2019; 8: e010440 10.1161/JAHA.118.010440 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Toischer K, Rokita AG, Unsöld B, Zhu W, Kararigas G, Sossalla S, et al. Differential cardiac remodeling in preload versus afterload. Circulation. 2010; 122: 993–1003. 10.1161/CIRCULATIONAHA.110.943431 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Ponikowski P, Voors AA, Anker SD, Bueno H, Cleland JGF, Coats AJS, et al. 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur Heart J. 2016; 37: 2129–2200. 10.1093/eurheartj/ehw128 [DOI] [PubMed] [Google Scholar]
47.Williams B, Mancia G, Spiering W, Agabiti Rosei E, Azizi M, Burnier M, et al. 2018 ESC/ESH Guidelines for the management of arterial hypertension. Eur Heart J. 2018; 39: 3021–3104. 10.1093/eurheartj/ehy339 [DOI] [PubMed] [Google Scholar]
48.Korcarz CE, Peppard PE, Young TB, Chapman CB, Hla KM, Barnet JH, et al. Effects of Obstructive Sleep Apnea and Obesity on Cardiac Remodeling. The Wisconsin Sleep Cohort Study. Sleep. 2016; 39: 1187–1195. 10.5665/sleep.5828 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Arias MA, García-Río F, Alonso-Fernández A, Mediano O, Martínez I, Villamor J. Obstructive sleep apnea syndrome affects left ventricular diastolic function. Effects of nasal continuous positive airway pressure in men. Circulation. 2005; 112: 375–383. 10.1161/CIRCULATIONAHA.104.501841 [DOI] [PubMed] [Google Scholar]
50.Shim CY, Kim D, Park S, Lee CJ, Cho H-J, Ha J-W, et al. Effects of continuous positive airway pressure therapy on left ventricular diastolic function. A randomised, sham-controlled clinical trial. Eur Respir J. 2018; 51: 1701774 10.1183/13993003.01774-2017 [DOI] [PubMed] [Google Scholar]
51.Schofield CJ, Ratcliffe PJ. Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol. 2004; 5: 343–354. 10.1038/nrm1366 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0243844.r001

Decision Letter 0

Michael Bader

14 Oct 2020

PONE-D-20-28615

Obstructive sleep apnea by bulking agent-induced tongue enlargement results in left ventricular contractile dysfunction

PLOS ONE

Dear Dr. Wagner,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Nov 28 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Michael Bader

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please modify the title to ensure that it is meeting PLOS’ guidelines (https://journals.plos.org/plosone/s/submission-guidelines#loc-title). In particular, the title should be "specific, descriptive, concise, and comprehensible to readers outside the field" and in this case we feel that the animal model used should be included. Please amend both the title on the online submission form (via Edit Submission) and the title in the manuscript so that they are identical.

3. Please clarify whether the method of euthanasia used was cervical dislocation.

4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

5. Thank you for stating the following in the Competing Interests section:

'I have read the journal's policy and the authors of this manuscript have the following competing interests: MA received grant support from ResMed, the ResMed Foundation, and Philips Respironics as well as lecture and consulting fees from ResMed, Philips Respironics, Boehringer-Ingelheim, NRI, Novartis and Bresotec. There are no other competing interests to declare.'

a. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these.

Please note that we cannot proceed with consideration of your article until this information has been declared.

b. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The main aim of the present manuscript by Lebek and collaborators is the description of a new model of obstructive sleep apnea (OSA) in rodents by injecting polytetrafluoroethylene in the base of their tongue, thebe increasing its size. By mean of plethysmography, the authors found that apneas are doubled in OSA mice. The number of apneas correlate with systolic and diastolic function, and hypoxia molecular markers. Tongue size remained stable for 8 weeks.

The manuscript is well written and, in general, easy to follow.

Other OSA animal models have been published and are discussed. However, some others needing few manipulation or setup are not mentioned (eg, Rubies et al. Sci Rep. 2019;9:11443). Amongst similar models, the authors acknowledge injection of a variety of inert or biological substances in the tongue or larynges of large (monkey) and small (rabbit, rat) animals. The main advance of the present model is its use in mice and the possibility to use transgenic animals. Whether the model developed in rat (hyaluronate injection) could be used in mice has not been studied.

I have several major comments.

One of my most important concerns is whether apneas/flow limitations occur during sleep only. The authors’ state so on the basis of a subjective assessment of flow patterns during light and dark periods, but objective data is warranted. Flow and apnea recordings during awake periods (night time, darkness) in both groups, and formal comparisons, are warranted.

Some of the authors claims are not sufficiently supported and need more supporting data. The authors state that the model was very effective. According to figure 2b, there was a large overlap in the number of apneas in control and OSA mice. In which percentage of OSA animals the number of apneas was higher than normal (ie, “normal” could be defined as the mean+2SD apneas in the control group)?

The authors also claim that a single injection results in sustained airway obstruction at 8 weeks. Nevertheless, the authors show that tongue size remains stable from baseline to 8 weeks. One may argue that, while tongue size remains stable, mice keep growing and, thereby, the relative obstruction (and thereby, apnea effectiveness) is lower at the 8-week timepoint. The authors do only demonstrate a significant increase in apnea frequency a the 2-week timepoint, but not at the 8-week timepoint.

In addition to those animals that had to be sacrificed and data on normal weight gain, was any evidence of stress or pain evident?

The number of induced apneas is rather modest: on average, less than doubles the number of apneas. In contrast, other animal models and OSA in human increase the number of apneas by several-fold. However, the authors show a remarkable cardiovascular affectation, including systolic dysfunction. Could the authors discuss?

Could other factors play a role? The authors claim that the cardiovascular effects of the present OSA model may not be caused by comorbidities. However, OSA promotes an increase in bloop pressure, and resistant hypertension (Tietjens et al. J Am Heart Assoc 2019;8: e010440), but blood pressure is not tested.

Statistical analyses are, in general, appropriate. Was normality assessed? Were paired t-test or repeated measures ANOVA performed with those with >1 measurement per animal?

Considering that this paper is mainly describing a new method, it may be informative providing a recording of tongue injections.

Were mice sacrificed during the light or dark period?

How was tongue echography performed?

Were ventricular samples obtained from the left or right ventricle? Could the authors show the full WB lane?

Both in the introduction and the discussion, the authors claim that CPAP may be harmful to OSA patients on the basis of SERVE-HF trial. However, the authors statement might be misleading and should be corrected. The Cowie et al. trial did include patients with predominant central apneas, in contrast to an OSA population.

Reviewer #2: I do have some comments related to some of sections of the manuscript:

*Title: The title of the paper does reflect what was done but does not seem to follow the overall rationale of the manuscript. I would suggest editing the title to match what was done with greater accuracy. Make clear to the reader that this was done in mice, with the aim to present a novel animal model of OSA.

*Methods

1- How was the 100 uL amount determined? Were other amounts tested previously? If yes, please include this in the paper and how the investigators reached a final decision to use 100 uL.

2- The plethysmography was done during the daytime. I realize that mice are nocturnal animals, and I would stress in the methods sections that the recordings were done during the sleep cycle. On a related note, what happens to levels of hypoxia etc when they are awake?

3- Based on figure 1 a , it seems very arbitrary how tongue volume was measured. My questions to the authors are : How were the images standardized? Was a specific magnification used? Were all these measurements done by the same investigator? Was there a calibration? Any Kappa statistics to be reported? Are there any fluorescence techniques to show the areas of injection? This should be added to the paper? Finally, the imaging seems to be in 2D, while you are referring to tongue volume. If a 3D measurement was done, more detail is needed about how the different planes of space were oriented, etc.

The whole paper is based on the increase tongue volume, it would be beneficial to have more details about how the tongue volume was assessed.

*Discussion/Conclusions

Based on the authors findings on the increased heart and lung weight, significant increases in CaMKII and KDM6A, it is quite clear to me that these mice developed cardiovascular morbidity from the intervention. That being the case, I disagree that this is a purely a model of OSA in mice. It can be argued that this is on the far end of extremely severe OSA, which may be encountered in heart failure patients. I would suggest acknowledging these findings and adapting the text to reflect that. Still, these data suggest a novel approach for a future valid OSA model, however it poses the question whether this animal model would be able to represent the burden of solely due to OSA.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Dec 10;15(12):e0243844. doi: 10.1371/journal.pone.0243844.r002

Author response to Decision Letter 0

27 Nov 2020

Response to the Editors

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by a You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Response: We have resubmitted all required documents.

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

Response: We have revised the manuscript according to PLOS ONE's style requirements.

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: We have revised the title page according to the journal guidelines.

Response: We appreciate this important comment and have revised the title accordingly. It now reads “A novel mouse model of obstructive sleep apnea by bulking agent-induced tongue enlargement results in left ventricular contractile dysfunction”. Moreover, we have also revised the short title that now reads “A novel mouse model of sleep apnea with contractile dysfunction”.

3. Please clarify whether the method of euthanasia used was cervical dislocation.

Response: Indeed, we used cervical dislocation for euthanasia. We therefore state on page 6 in lines 85-87:

“All animals were euthanized by cervical dislocation during the light period, i.e. regular sleep time of the animals (usually in the afternoon).”

Response: We now provide the full unedited gels for Fig 6 in the Supporting Information, labeled “S1 Gels. Raw images.”, according to the journal guidelines. There are no other blot/gel image data related to this manuscript. In addition, all figures adhere to the journal guidelines.

Response: We now provide the full unedited gels underlying Fig 6 in the Supporting Information, labeled “S1 Gels. Raw images.”. There are no other blot/gel image data related to this manuscript.

5. Thank you for stating the following in the Competing Interests section:

Please note that we cannot proceed with consideration of your article until this information has been declared.

Response: We confirm that our competing interests do not alter our adherence to all PLOS ONE policies on sharing data and materials. Accordingly, we have added to the Competing Interests on page 29 in lines 629-630:

“This does not alter our adherence to PLOS ONE policies on sharing data and materials.”

b. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

Response: We have included our updated Competing Interests statement in our cover letter.

Response: We have included in the revised manuscript captions for our Supporting Information files at the end of our manuscript on pages 41-42 and have updated all in-text citations according to the journal guidelines.

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

5. Review Comments to the Author

Response to the Reviewers

The manuscript is well written and, in general, easy to follow.

Response: We thank the reviewer for these important comments. Indeed, several animal models of OSA have been published. We have discussed these different models and approaches without being exhaustive. We agree with the reviewer that the model of Rubies et al. is interesting and needs to be included in our discussion. We have therefore added the following sentence on page 21 lines 435-438 to the discussion section of the revised manuscript.

“Another rat model of OSA mimicking airway obstruction was first described by Farré et al. [35]. Rats were placed awake in a setup with two chambers split by a latex neck collar and airway obstruction was induced by interruption of bias flow in the head chamber [35,36].”

Moreover, we now also discuss that the previously published rat model of upper airway obstruction by hyaluronate injection may also be applicable to mice. You can read on page 24 in lines 500-505:

“Additionally, polyacrylamide and sodium hyaluronate injection into the palate of rabbits and rats, respectively, has been shown to induce obstructive sleep apnea by upper airway obstruction [42,43]. Unfortunately, the availability of appropriate knock-out or transgene models is very limited in both rabbits and rats. In this context, future studies investigating sodium hyaluronate injection into the palate of mice may lead to another promising animal model of OSA.”

I have several major comments.

Response: We appreciate this important comment. Indeed, it is essential to delineate whether inspiratory flow limitations (IFLs) and apneas occur during sleep only. To account for this, we have performed novel experiments for the revised version of the manuscript by measuring breathing patterns (whole-body plethysmography) during night-time, darkness, when mice are awake. To also investigate the shift from awake breathing to sleep-related breathing we have, furthermore, extended the recording period to 22 hours (from 10 p.m. to 8. p.m. the other day). As obvious from the original recording (novel figure a) in S4 Fig) IFLs and apneas do not occur during night-time (10 p.m. – 7 a.m. the other day) in awake PFTE-treated mice. In accordance, no airway obstruction was observed during this period (b) in S4 Fig) In sharp contrast, as evident from the original recording (novel figure a) in S4 Fig), the same PTFE-treated mouse showed a substantial increase in IFL/apnea frequency during the light period (7 a.m. – 8 p.m.), when mice are sleeping. Interestingly, the shift from awake to sleep time may be even inferred from the strong decrease in breathing frequency occurring around 7 a.m. and remaining low during daytime. The lack of IFL and apneas occurrence during night-time/awake time was also evident from mean data analyzed from 5 mice (novel figure c) in S4 Fig). During night-time, we observed a very low frequency of IFLs and apneas in PTFE-treated mice that was comparable to control mice (P=1.00 for both, c) in S4 Fig). Importantly, no IFL aggregates were observed in awake mice.

We have added the novel data to the results section of the revised manuscript on page 15 in lines 310-316:

“When mice were awake (during night-time, 10 p.m. – 7 a.m.), control and PTFE mice showed a similar breathing pattern with a negligible number of IFLs and no IFL aggregates indicating that PTFE injection into the tongue does not induce a fixed upper airway obstruction (S4 Fig). In conscious mice, mean IFL frequency (/h) was 1.98±0.52 vs. 2.49±0.51 (PTFE vs. control; N=5 for both; P=1.00; c) in S4 Fig). Also, mean apnea frequency (/h) was very low and similar in awake PTFE and control mice (1.95±1.01 vs. 2.38±1.19; N=5 for both; P=1.00; c) in S4 Fig).”

Moreover, we discuss these findings in the revised version of the manuscript on page 23-24 in lines 487-495:

“Importantly, PTFE injection into the tongue does not lead to a fixed upper airway obstruction (S4 Fig). In sleeping mice, only about 0.50% of all breaths in PTFE mice were flow limited (b) in S3 Fig). In accordance with the intermittent nature of obstructive breathing abnormalities, the PTFE-induced IFLs and apneas occurred in clusters and only in sleeping mice (a) in S4 Fig). In contrast, awake PTFE mice exhibit a regular breathing pattern without airway obstruction (b) in S4 Fig). Moreover, we observed no IFL aggregates and similar frequencies of IFLs and apneas after PTFE treatment during awake periods (c) in S4 Fig). Therefore, hypoxia is likely not present in awake mice.”

Although we can now demonstrate that the PTFE-induced apneas and IFLs occur mainly during the murine sleep time, we cannot correlate the apneas and IFLs with definite sleep, e.g. using EEG. We have accounted for this aspect in the limitations section on page 28 in lines 595-603:

“We have not directly monitored sleep cycles by electroencephalography, which would have required a substantial additional methodological effort. On the other hand, performing sleep apnea monitoring without electroencephalography during the usual rodent sleep cycle (day-time, e.g. 9 AM to 5 PM) has been shown to be feasible [20]. Additionally, we have shown here that IFLs do not occur with an uniform distribution across the monitoring interval. Instead, they form clusters when mice were supposed to sleep, while no IFL aggregates and only a very low frequency of IFLs was observed at night-time when mice were awake. Nevertheless, further investigations are required to directly correlate IFLs and apneas with sleep.”

Response: We thank the reviewer for this important comment. Indeed, there was a substantial variation in the in the number of apneas in control and PTFE mice, which is not unusual for this type of measurement. As suggested by the reviewer, we have performed a novel analysis for the revised version of the manuscript: by using the mean±2SD of the control group, a cut-off of 14.75 apneas/h was used to discriminate between normal or abnormal increased apnea frequency. Interestingly, 8 out of 25 PTFE mice (32%) but only 2 out of 28 control mice showed an abnormal increased apnea frequency (P=0.02, Chi-square test). We believe that this substantial and significant increase in the number of abnormally breathing mice after PTFE treatment is convincing. We have added the novel analysis to the result section (page 15, lines 299-304) and b) in novel S2 Fig:

“Moreover, the proportion of mice showing an abnormally increased apnea frequency above the cut-off of 14.75 apneas/h (mean apnea frequency of control mice + 2 standard deviations) was significantly increased in PTFE-injected mice (b) in S2 Fig). Interestingly, 8 out of 25 PTFE mice but only 2 out of 28 control mice showed an abnormally increased apnea frequency (P=0.02; b) in S2 Fig).”

Response: We thank the reviewer for this important comment. Indeed, it is essential to ensure that IFL and apnea frequency remains stable for the whole 8-week follow-up period. We can now show in the revised version of the manuscript novel data comparing frequencies of IFLs, IFL aggregates, and apneas at the 2-week with the 8-week timepoint (novel figure c) in S2 Fig). Importantly, all parameters (IFLs, IFL aggregates, apneas) remain stable for the whole observation period and no significant difference was observed. We have added this finding to the results section on page 16 in lines 327-331:

“Importantly, the intermittent airway obstruction in sleeping mice remained stable for the whole observation period. Compared to the 2-week timepoint, frequencies of apneas (P=0.71), IFLs (P=0.38), and IFL aggregates (P=0.95) were similar at 8 weeks after PTFE injection (N=6 for all; c) in S2 Fig).”

Moreover, we have discussed this observation on page 23 in lines 485-487:

“According to the sustained increase in tongue diameter, we could demonstrate that the increased frequencies of IFLs and apneas remained stable for the whole 8-week observation period (c) in S2 Fig).”

In addition, to those animals that had to be sacrificed and data on normal weight gain, was any evidence of stress or pain evident?

Response: We thank the reviewer for this important comment. In order to respect animals’ wellbeing and to avoid animal suffering, we performed everyday visual inspection of every mouse in the study. In particular, we analyzed their skin, food intake, movements and interaction with other mice. If a mouse showed an abnormal behavior, we immediately sacrificed the animal (only 6 mice had to be sacrificed (S1 Fig)). All the other mice (25/31) showed no evidence of stress or pain and could be monitored for the whole observation period. To account for this, we added to the method section on page 8 in lines 127-134:

“From 31 mice treated with PTFE, 6 mice had to be killed within 72 h because of surgery-related complications (e.g. bleeding into the tongue, extensive tongue enlargement or infection). In order to respect animals’ wellbeing and to avoid animal suffering, we performed everyday visual inspection of every mouse. In particular, we analyzed their skin, food intake, movements and interaction with other mice. If a mouse showed an abnormal behavior, we immediately sacrificed the animal (6 mice had to be sacrificed (S1 Fig)). All the other mice (25/31) showed no evidence of stress or pain and could be monitored for the whole observation period of 8 weeks.”

Response: This is an important comment. We have discussed the severity of intermittent airway obstruction in comparison to other animal models and its impact on pathophysiology on page 26, lines 552-565 of the discussion section of the revised manuscript:

“The frequency of apnea events and the increase with PTFE-injection was rather modest in our model. In contrast, many other animal models exceed the severity of human OSA, partly because the consequences are to be detected within a few weeks [12,16,33,32]. We have extended our observation period to a long duration of 8 weeks, other mouse models usually perform OSA protocols (e.g. CIH, tracheal occlusion) for about 3-5 weeks [12,16,35,36,20,32]. We did this to model the human situation more closely, where mild intermittent airway obstruction may result in the development of pathophysiological sequelae only after years. Despite this mild increase in intermittent airway obstruction, we show here that the frequency of apneas correlated significantly with the severity of contractile dysfunction and other features of the heart failure (heart and lung weight, Figs 4 and 5), suggesting a causal relationship. On the other hand, we cannot exclude that other factors following OSA that are not directly related to intermittent airway obstruction may also potentially contribute to the phenotype of these mice.”

Response: We thank the reviewer for this important comment. We agree with the reviewer that obstructive sleep apnea may result in the development of arterial hypertension with increased cardiac afterload. In fact, this may partly explain the cardiac phenotype of the present model. However, all animals subjected to PTFE injection had been healthy C57BL/6 mice at baseline. Thus, all potential pathophysiologic changes that may have developed during the 8 weeks observation period, let it be increased blood pressure, impaired myocardial contractility or impaired sleep with chronic sympathetic stress, are secondary to the PTFE-induced intermittent airway obstruction during sleep. Consequently, all cardiovascular effects can be (directly or indirectly) attributed to OSA. This differentiates our model from obese and diabetic mouse models of sleep apnea, for instance, where OSA-independent comorbidities confound the experiments. Future studies using our model may address the relative contribution of the different pathophysiological alterations secondary to OSA individually.

To account for this, we have added this aspect to the revised version of the manuscript on page 25 in lines 526-539:

“Since obstructive sleep apnea may result in the development of arterial hypertension with increased cardiac afterload, this may partly explain the cardiac phenotype of the present model. In fact, rodent OSA models showed OSA-dependent development of arterial hypertension [16,32,44].

Statistical analyses are, in general, appropriate. Was normality assessed? Were paired t-test or repeated measures ANOVA performed with those with >1 measurement per animal?

Response: We thank the reviewer for this important comment. In the revised version of the manuscript, we have now tested all data for normal distribution using Shapiro-Wilk normality test. Consequently, we used a parametric or a non-parametric test, depending on whether a variable was normally distributed or not, respectively. Moreover, one may argue that in some figures longitudinal observations are presented and paired t-tests or repeated measures ANOVA may be considered. All these data sets are discussed here:

1) in b) in Fig 1, data for 8 weeks was only available for a subgroup of mice, which hampers the use of a repeated measures ANOVA.

2) in c) in Fig 1, a two factor repeated measures study design was present. As recommended by the reviewer we have now performed a mixed-effects model analysis with Holm-Sidak’s post-hoc correction.

3) In novel S2 Fig, longitudinal data is compared, wherefore we used one-way repeated measures ANOVA with Holm-Sidak’s post-hoc correction.

We have therefore revised the methods section on page 13 in lines 255-272 now describing this new statistical analysis:

“All measurements and experiments were performed and analyzed blinded to the treatment group (control or PTFE) and to frequency of apneas. Experimental data are presented as means ± standard error of the mean (SEM). All statistical analyses were based on the number of mice and normal distribution was assessed by Shapiro-Wilk normality test. Parametric or non-parametric tests were applied to test for significant differences, depending on whether a variable was normally distributed or not. Parametric and non-parametric tests used for the comparison of two groups were Student’s t and Mann-Whitney test, respectively. Ordinary one-way ANOVA with Holm-Sidak’s post-hoc correction and Kruskal-Wallis test with Dunn’s post-hoc correction were used for comparisons of more than two groups that were either normally or not normally distributed, respectively. One-way repeated measures ANOVA with Holm-Sidak’s post-hoc correction was used for the comparison of paired data that was normally distributed. If more than two groups and two different factors were compared in a repeated measures design, mixed-effects model analysis with Holm-Sidak’s post-hoc correction was used. Chi-square test was used for the comparison of categorial data. The tests above as well as linear regression analyses were used in GraphPad Prism 8 to test for significance, as appropriate. Two-sided P-values below 0.05 were considered as statistically significant.”

Considering that this paper is mainly describing a new method, it may be informative providing a recording of tongue injections.

Response: We appreciate this important comment. We have performed a video recording of the surgical procedure of PTFE tongue injection. It can be found in the revised version of the manuscript as Supporting Information named “S1 Video”. Tongue injection procedure”.

Were mice sacrificed during the light or dark period?

Response: This is an important comment. All mice were sacrificed during the light period, i.e. regular sleep time of the animals. We have added this information on page 6 in lines 85-87:

“All animals were euthanized by cervical dislocation during the light period, i.e. regular sleep time of the animals (usually in the afternoon).”

How was tongue echography performed?

Response: We thank the reviewer for this comment and have now added the novel paragraph “Sonographic measurement of tongue diameter” to the revised method section on pages 8-9 in lines 136-153:

“Tongue size was measured by ultrasound during the PTFE injection procedure. Mice were placed in supine position onto a heating plate. The tongue was gripped with a tiny crocodile clip. Ultrasound gel was placed onto the murine throat, mandible and mouth, but not on nostrils to keep mice breathing. Thereafter, a 30 MHz center frequency transducer (Vevo3100 system from VisualSonics, Toronto, Canada) was placed at median position of the murine throat to measure the dorso-ventral tongue diameter in sagittal plane. For some recordings, the ultrasound head was rotated clockwise by 90° to also measure the lateral tongue diameters in the transversal plane (a) in S2 Fig). Recordings were acquired with 56 frames/s (gain 30 dB). For optimal magnification, acquisition was performed with 10.00 mm depth and 15.36 mm width. We used the presetting of VisualSonics; thus, no calibration was required. By carefully stirring the tongue via the crocodile clip and comparing tongue movements with the other pharyngeal structures under sonographic recording, tongue surface was easily discriminated from surrounding tissue and tongue diameter was assessed. Similar measurements were done before and after PTFE injection in a standardized manner. All measurements were done by the same investigator; therefore, Kappa statistics cannot be reported. We did not use any fluorescence techniques to identify the area of injection.”

Were ventricular samples obtained from the left or right ventricle? Could the authors show the full WB lane?

Response: We thank the reviewer for this comment. In order to save enough material for protein analyses, we used the whole left and right ventricle for homogenization. We have added this information to the method section (page 12, line 237). In addition, we now show the full Western blot gels of CaMKII and GAPDH as Supporting Information “S1 Gels. Raw images”.

Response: We thank the reviewer for this comment and apologize this misunderstanding. We have specified the statements in the introduction and in the discussion accordingly.

It now reads on page 4 in lines 48-50 in the introduction:

“While treatment with ventilation-therapy may reduce apnea events, not all patients can tolerate it [6] and this treatment may even be harmful for selected patients (e.g. for patients with predominant central apneas) [7].”

We have also specified the statement in the discussion on page 20 in lines 405-409:

“To date, the treatment of OSA is mainly limited to continuous positive airway pressure (CPAP), but acceptance of CPAP in patients with low symptom burden is limited [6] and treatment with positive pressure ventilation (adaptive servo-ventilation) may be even harmful for specific patient populations (e.g. for patients with predominant central apneas) [7].”

Reviewer #2: I do have some comments related to some of sections of the manuscript:

*Methods

1- How was the 100 uL amount determined? Were other amounts tested previously? If yes, please include this in the paper and how the investigators reached a final decision to use 100 uL.

Response: This is an important comment. Our approach was based on the findings of Brennick et al., who had measured pharyngeal structures of New Zealand Obese mice (NZO) with spontaneous OSA using MRI (Brennick MJ, Pack AI, Ko K, Kim E, Pickup S, Maislin G, et al. Altered upper airway and soft tissue structures in the New Zealand Obese mouse. Am J Respir Crit Care Med. 2009; 179: 158–169). Interestingly, they report that NZO mice (aged 23 weeks, mean body weight 35.7 g) showed a significantly increased mean tongue volume to about 137 µl (compared to 104 µl in control animals). This corresponds to a mean increase of 33 µl tongue volume. Since the tongue volume is the most important determinant of pharyngeal airway size for OSA (Schwab RJ, Pasirstein M, Pierson R, Mackley A, Hachadoorian R, Arens R, et al. Identification of upper airway anatomic risk factors for obstructive sleep apnea with volumetric magnetic resonance imaging. Am J Respir Crit Care Med. 2003; 168: 522–530), we aimed to increase the tongue volume of our mice to a similar extent by PTFE injection into the base of the tongue. We used younger mice (mean body weight 27.7 g, only about 70% of the body weight compared to Brennick et al.) to enable the 8-week follow-up observation period. Thus, we anticipated that an increase of about 20-25 µl tongue volume would result in a similar airway obstruction. PTFE is a solid substance (density 2.1 g/ml). 50 mg of PTFE was diluted to 100 µl (50% w/v) with glycerol (Sigma Aldrich). 100 µl of this dilution contains 24 µl pure PTFE, which almost exactly matches the aimed increase in tongue volume. Larger PTFE injection volumes were investigated in some test mice, but periprocedural mortality exceeded. Since we were not interested in less upper airway obstruction, we have not studied lower injection volumes.

We have added this information to the method section of the revised version of the manuscript on pages 6-7 in lines 94-110:

“Our approach was based on the findings of Brennick et al., who had measured pharyngeal structures of New Zealand Obese mice (NZO) with spontaneous OSA using MRI [14]. Interestingly, they report that NZO mice (aged 23 weeks, mean body weight 35.7 g) showed a significantly increased mean tongue volume to about 137 µl (compared to 104 µl in control animals). This corresponds to a mean increase of 33 µl tongue volume. Since the tongue volume is the most important determinant of pharyngeal airway size for OSA [19], we aimed to increase the tongue volume of our mice to a similar extent by PTFE injection into the base of the tongue. We used younger mice (mean body weight 27.7 g, only about 70% of the body weight compared to Brennick et al. [14]) to enable the 8-week follow-up observation period. Thus, we anticipated that an increase of about 20-25 µl tongue volume would result in a similar airway obstruction. PTFE is a solid substance (density 2.1 g/ml). 50 mg of PTFE was diluted to 100 µl (50% w/v) with glycerol (Sigma Aldrich). 100 µl of this dilution contains 24 µl pure PTFE, which almost exactly matches the aimed increase in tongue volume. Larger injection volumes were investigated in some test mice, but periprocedural mortality exceeded. Since we were not interested in less upper airway obstruction, we have not studied lower injection volumes.”

Response: We thank the reviewer for this helpful comment. We now explain in the method section on page 9 in lines 164-167 that recordings were conducted during the murine sleep cycle:

“Since mice are nocturnal animals, continuous recordings (sampling frequency 1 kHz) were done for 8 h during day-time, the interval with the highest frequency and duration of sleep periods complying with the murine sleep cycle [20].”

Moreover, we have added novel data to the revised version of the manuscript investigating breathing characteristics at night-time (activity time), when mice are awake (S4 Fig). Intriguingly, awake mice showed no IFL aggregates and only very low frequencies of IFLs and apneas with no differences between the control and PTFE-treated animals. Consistently, the level of intermittent hypoxia should be negligible in PTFE-treated awake mice and comparable to control mice.

We have added this aspect to the revised discussion section on pages 23-24 in lines 487-495:

3- Based on figure 1 a, it seems very arbitrary how tongue volume was measured. My questions to the authors are: How were the images standardized? Was a specific magnification used? Were all these measurements done by the same investigator? Was there a calibration? Any Kappa statistics to be reported? Are there any fluorescence techniques to show the areas of injection? This should be added to the paper? Finally, the imaging seems to be in 2D, while you are referring to tongue volume. If a 3D measurement was done, more detail is needed about how the different planes of space were oriented, etc.

The whole paper is based on the increase tongue volume, it would be beneficial to have more details about how the tongue volume was assessed.

Response: We appreciate this important comment and agree with the reviewer that measurement of the tongue size should be described more in detail. Therefore, we have added a novel paragraph “Sonographic measurement of tongue diameter” to the methods section of the revised manuscript.

On pages 8-9, lines 136-153 the text reads as following:

We agree with the reviewer that it would have been better to measure tongue volume instead of diameters only. However, a precise measurement of tongue volume by 3D ultrasound would require the measurement of tongue length in addition to dorso-ventral and lateral diameter. Since the tip of the tongue was gripped with a tiny crocodile clip, the ultrasound head would not be able to reach it. Therefore, precise measurement of tongue length is not possible. Nevertheless, for the revised version of the manuscript we have now performed novel experiments to measure dorso-ventral and lateral tongue diameter in the same animal by rotating the ultrasound head 90 degrees to measure the transversal plane. In panel a) in S2 Fig we now report dorso-ventral and lateral tongue diameters and calculated cross-sectional area following PTFE injection (novel panel a) in S2 Fig). Intriguingly, both tongue diameters increased in parallel and to a similar extent resulting in a significant increase of cross-sectional tongue area (a) in S2 Fig).

We have added these novel findings to the results section on page 14 in lines 278-280:

“Interestingly, we observed a similar increase in transversal tongue diameter leading to a homogenous increase of cross-sectional tongue area from (in mm²) 9.23±0.41 to 19.90±0.86 (N=5; P<0.001; a) in S2 Fig).”

Moreover, to avoid misunderstanding, we have revised the entire manuscript and clarified our terminology: we now use either tongue diameter, cross-sectional area or volume, as appropriate.

*Discussion/Conclusions

Response: We appreciate this important comment. However, we disagree on the statement that our model would be at the far end of extremely severe OSA.

On page 26, lines 540-551 of the discussion section of the revised manuscript we explain this matter. The text reads as following:

“The cardiovascular dysfunction developed by mice in our model is rather modest. A reduction of ejection fraction from 56.10±2.49% in control to 49.02±2.07% in PTFE mice may be detectable, but its pathophysiological relevance may be low. There are mouse models of systolic heart failure like transverse aortic constriction that would result in a much larger degree of systolic dysfunction [45]. Moreover, if compared to patients, the magnitude of ejection fraction observed in our PTFE mice would still be in the normal to subnormal range. On the other hand, we have observed many clinical features that can be found in patients with heart failure with preserved ejection fraction or patients with hypertension and hypertensive heart disease [46,47]. Importantly, arterial hypertension and heart failure with preserved ejection fraction are very common in patients with OSA and not only found in those patients at the far end of extremely severe intermittent airway obstruction [48–50].“

In addition, we state on page 25, lines 526-539:

With respect to the severity of intermittent airway obstruction we also state on page 26, lines 552-565:

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Response: We agree publishing the complete peer review history.

Response: We have used PACE digital diagnostic tool to ensure that all figures meet PLOS requirements.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(54.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0243844.r003

Decision Letter 1

Michael Bader

30 Nov 2020

A novel mouse model of obstructive sleep apnea by bulking agent-induced tongue enlargement results in left ventricular contractile dysfunction

PONE-D-20-28615R1

Dear Dr. Wagner,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Michael Bader

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have provided a comprehensive and excellent reply to all my comments. They are to be commended for their effort. I have no further comments.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. doi: 10.1371/journal.pone.0243844.r004

Acceptance letter

Michael Bader

2 Dec 2020

PONE-D-20-28615R1

A novel mouse model of obstructive sleep apnea by bulking agent-induced tongue enlargement results in left ventricular contractile dysfunction

Dear Dr. Wagner:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Michael Bader

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Study flowchart.

(TIF)

Click here for additional data file.^{(208.2KB, tif)}

S2 Fig. Increased cross-sectional tongue area and sustained OSA at 8 weeks after PTFE injection.

(TIF)

Click here for additional data file.^{(1MB, tif)}

S3 Fig. Increased IFL frequency in mice after PTFE injection.

(TIF)

Click here for additional data file.^{(326.9KB, tif)}

S4 Fig. Breathing characteristics in conscious mice are unaltered by PTFE injection.

(TIF)

Click here for additional data file.^{(817.5KB, tif)}

S5 Fig. HIF1α mRNA expression is upregulated in PTFE mice.

(TIF)

Click here for additional data file.^{(361.8KB, tif)}

S1 Raw images. Gels.

(PDF)

Click here for additional data file.^{(4.3MB, pdf)}

S1 Video. Tongue injection procedure.

(MOV)

Click here for additional data file.^{(34.5MB, mov)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(54.2KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0243844.ref001] 1.Benjafield AV, Ayas NT, Eastwood PR, Heinzer R, Ip MSM, Morrell MJ, et al. Estimation of the global prevalence and burden of obstructive sleep apnoea. A literature-based analysis. Lancet Respir Med. 2019; 7: 687–698. 10.1016/S2213-2600(19)30198-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref002] 2.Gottlieb DJ, Yenokyan G, Newman AB, O'Connor GT, Punjabi NM, Quan SF, et al. Prospective study of obstructive sleep apnea and incident coronary heart disease and heart failure. The sleep heart health study. Circulation. 2010; 122: 352–360. 10.1161/CIRCULATIONAHA.109.901801 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref003] 3.Rossi VA, Stradling JR, Kohler M. Effects of obstructive sleep apnoea on heart rhythm. Eur Respir J. 2013; 41: 1439–1451. 10.1183/09031936.00128412 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref004] 4.Lebek S, Pichler K, Reuthner K, Trum M, Tafelmeier M, Mustroph J, et al. Enhanced CaMKII-Dependent Late INa Induces Atrial Proarrhythmic Activity in Patients With Sleep-Disordered Breathing. Circ Res. 2020; 126: 603–615. 10.1161/CIRCRESAHA.119.315755 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref005] 5.Buchner S, Eglseer M, Debl K, Hetzenecker A, Luchner A, Husser O, et al. Sleep disordered breathing and enlargement of the right heart after myocardial infarction. Eur Respir J. 2015; 45: 680–690. 10.1183/09031936.00057014 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref006] 6.McEvoy RD, Antic NA, Heeley E, Luo Y, Ou Q, Zhang X, et al. CPAP for prevention of cardiovascular events in obstructive sleep apnea. N Engl J Med. 2016; 375: 919–931. 10.1056/NEJMoa1606599 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref007] 7.Cowie MR, Woehrle H, Wegscheider K, Angermann C, d'Ortho M-P, Erdmann E, et al. Adaptive servo-ventilation for central sleep apnea in systolic heart failure. N Engl J Med. 2015; 373: 1095–1105. 10.1056/NEJMoa1506459 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref008] 8.Sossalla S, Fluschnik N, Schotola H, Ort KR, Neef S, Schulte T, et al. Inhibition of elevated Ca2+/calmodulin-dependent protein kinase II improves contractility in human failing myocardium. Circ Res. 2010; 107: 1150–1161. 10.1161/CIRCRESAHA.110.220418 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref009] 9.Fischer TH, Herting J, Tirilomis T, Renner A, Neef S, Toischer K, et al. Ca2+/calmodulin-dependent protein kinase II and protein kinase A differentially regulate sarcoplasmic reticulum Ca2+ leak in human cardiac pathology. Circulation. 2013; 128: 970–981. 10.1161/CIRCULATIONAHA.113.001746 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref010] 10.Wagner S, Ruff HM, Weber SL, Bellmann S, Sowa T, Schulte T, et al. Reactive oxygen species-activated Ca/calmodulin kinase IIdelta is required for late I(Na) augmentation leading to cellular Na and Ca overload. Circ Res. 2011; 108: 555–565. 10.1161/CIRCRESAHA.110.221911 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref011] 11.Dybkova N, Wagner S, Backs J, Hund TJ, Mohler PJ, Sowa T, et al. Tubulin polymerization disrupts cardiac beta-adrenergic regulation of late INa. Cardiovasc Res. 2014; 103: 168–177. 10.1093/cvr/cvu120 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref012] 12.Linz D, Hohl M, Nickel A, Mahfoud F, Wagner M, Ewen S, et al. Effect of renal denervation on neurohumoral activation triggering atrial fibrillation in obstructive sleep apnea. Hypertension. 2013; 62: 767–774. 10.1161/HYPERTENSIONAHA.113.01728 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref013] 13.Fleury Curado T, Pho H, Berger S, Caballero-Eraso C, Shin M-K, Sennes LU, et al. Sleep-disordered breathing in C57BL/6J mice with diet-induced obesity. Sleep. 2018; 41: 1–9. 10.1093/sleep/zsy089 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref014] 14.Brennick MJ, Pack AI, Ko K, Kim E, Pickup S, Maislin G, et al. Altered upper airway and soft tissue structures in the New Zealand Obese mouse. Am J Respir Crit Care Med. 2009; 179: 158–169. 10.1164/rccm.200809-1435OC [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref015] 15.Ortlepp JR, Kluge R, Giesen K, Plum L, Radke P, Hanrath P, et al. A metabolic syndrome of hypertension, hyperinsulinaemia and hypercholesterolaemia in the New Zealand obese mouse. Eur J Clin Invest. 2000; 30: 195–202. 10.1046/j.1365-2362.2000.00611.x [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref016] 16.Chopra S, Polotsky VY, Jun JC. Sleep Apnea Research in Animals. Past, Present, and Future. Am J Respir Cell Mol Biol. 2016; 54: 299–305. 10.1165/rcmb.2015-0218TR [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref017] 17.Baum DM, Morales Rodriguez B, Attali V, Cauhapé M, Arnulf I, Cardot P, et al. New Zealand Obese Mice as a Translational Model of Obesity-related Obstructive Sleep Apnea Syndrome. Am J Respir Crit Care Med. 2018; 198: 1336–1339. 10.1164/rccm.201801-0162LE [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref018] 18.Diamond DA, Mattoo TK. Endoscopic treatment of primary vesicoureteral reflux. N Engl J Med. 2012; 366: 1218–1226. 10.1056/NEJMct1108922 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref019] 19.Schwab RJ, Pasirstein M, Pierson R, Mackley A, Hachadoorian R, Arens R, et al. Identification of upper airway anatomic risk factors for obstructive sleep apnea with volumetric magnetic resonance imaging. Am J Respir Crit Care Med. 2003; 168: 522–530. 10.1164/rccm.200208-866OC [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref020] 20.Crossland RF, Durgan DJ, Lloyd EE, Phillips SC, Reddy AK, Marrelli SP, et al. A new rodent model for obstructive sleep apnea. Effects on ATP-mediated dilations in cerebral arteries. Am J Physiol Regul Integr Comp Physiol. 2013; 305: R334–342. 10.1152/ajpregu.00244.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref021] 21.Simeone KA, Hallgren J, Bockman CS, Aggarwal A, Kansal V, Netzel L, et al. Respiratory dysfunction progresses with age in Kcna1-null mice, a model of sudden unexpected death in epilepsy. Epilepsia. 2018; 59: 345–357. 10.1111/epi.13971 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref022] 22.Jung F, Palmer LA, Zhou N, Johns RA. Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes. Circ Res. 2000; 86: 319–325. 10.1161/01.res.86.3.319 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref023] 23.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25: 402–408. 10.1006/meth.2001.1262 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref024] 24.Wiener CM, Booth G, Semenza GL. In vivo expression of mRNAs encoding hypoxia-inducible factor 1. Biochem Biophys Res Commun. 1996; 225: 485–488. 10.1006/bbrc.1996.1199 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref025] 25.Belaidi E, Joyeux-Faure M, Ribuot C, Launois SH, Levy P, Godin-Ribuot D. Major role for hypoxia inducible factor-1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea. J Am Coll Cardiol. 2009; 53: 1309–1317. 10.1016/j.jacc.2008.12.050 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref026] 26.Yuan G, Khan SA, Luo W, Nanduri J, Semenza GL, Prabhakar NR. Hypoxia-inducible factor 1 mediates increased expression of NADPH oxidase-2 in response to intermittent hypoxia. J Cell Physiol. 2011; 226: 2925–2933. 10.1002/jcp.22640 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref027] 27.Chakraborty AA, Laukka T, Myllykoski M, Ringel AE, Booker MA, Tolstorukov MY, et al. Histone demethylase KDM6A directly senses oxygen to control chromatin and cell fate. Science. 2019; 363: 1217–1222. 10.1126/science.aaw1026 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref028] 28.Chami HA, Resnick HE, Quan SF, Gottlieb DJ. Association of incident cardiovascular disease with progression of sleep-disordered breathing. Circulation. 2011; 123: 1280–1286. 10.1161/CIRCULATIONAHA.110.974022 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref029] 29.Parker JC, Townsley MI. Evaluation of lung injury in rats and mice. Am J Physiol Lung Cell Mol Physiol. 2004; 286: L231–246. 10.1152/ajplung.00049.2003 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref030] 30.Hamano G, Yamamoto K, Takami Y, Takeshita H, Shimosato T, Moritani T, et al. Effects of Low-Dose Sacubitril/Valsartan on Different Stages of Cardiac Hypertrophy in Salt-Loaded Hypertensive Rats. J Cardiovasc Pharmacol. 2019; 73: 282–289. 10.1097/FJC.0000000000000662 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref031] 31.Neef S, Dybkova N, Sossalla S, Ort KR, Fluschnik N, Neumann K, et al. CaMKII-dependent diastolic SR Ca2+ leak and elevated diastolic Ca2+ levels in right atrial myocardium of patients with atrial fibrillation. Circ Res. 2010; 106: 1134–1144. 10.1161/CIRCRESAHA.109.203836 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref032] 32.Dematteis M, Godin-Ribuot D, Arnaud C, Ribuot C, Stanke-Labesque F, Pépin J-L, et al. Cardiovascular consequences of sleep-disordered breathing. Contribution of animal models to understanding the human disease. ILAR J. 2009; 50: 262–281. 10.1093/ilar.50.3.262 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref033] 33.Brooks D, Horner RL, Kozar LF, Render-Teixeira CL, Phillipson EA. Obstructive sleep apnea as a cause of systemic hypertension. Evidence from a canine model. J Clin Invest. 1997; 99: 106–109. 10.1172/JCI119120 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref034] 34.White SG, Fletcher EC, Miller CC. Acute systemic blood pressure elevation in obstructive and nonobstructive breath hold in primates. J Appl Physiol. 1995; 79: 324–330. 10.1152/jappl.1995.79.1.324 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref035] 35.Farré R, Nácher M, Serrano-Mollar A, Gáldiz JB, Alvarez FJ, Navajas D, et al. Rat model of chronic recurrent airway obstructions to study the sleep apnea syndrome. Sleep. 2007; 30: 930–933. 10.1093/sleep/30.7.930 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref036] 36.Rubies C, Dantas A-P, Batlle M, Torres M, Farre R, Sangüesa G, et al. Aortic remodelling induced by obstructive apneas is normalized with mesenchymal stem cells infusion. Sci Rep. 2019; 9: 11443 10.1038/s41598-019-47813-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref037] 37.Farré R, Montserrat JM, Gozal D, Almendros I, Navajas D. Intermittent Hypoxia Severity in Animal Models of Sleep Apnea. Front Physiol. 2018; 9: 1556 10.3389/fphys.2018.01556 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref038] 38.O'Donnell CP, Ayuse T, King ED, Schwartz AR, Smith PL, Robotham JL. Airway obstruction during sleep increases blood pressure without arousal. J Appl Physiol. 1996; 80: 773–781. 10.1152/jappl.1996.80.3.773 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref039] 39.Naghshin J, McGaffin KR, Witham WG, Mathier MA, Romano LC, Smith SH, et al. Chronic intermittent hypoxia increases left ventricular contractility in C57BL/6J mice. J Appl Physiol. 2009; 107: 787–793. 10.1152/japplphysiol.91256.2008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref040] 40.Joyeux-Faure M, Stanke-Labesque F, Lefebvre B, Béguin P, Godin-Ribuot D, Ribuot C, et al. Chronic intermittent hypoxia increases infarction in the isolated rat heart. J Appl Physiol. 2005; 98: 1691–1696. 10.1152/japplphysiol.01146.2004 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref041] 41.Philip P, Gross CE, Taillard J, Bioulac B, Guilleminault C. An animal model of a spontaneously reversible obstructive sleep apnea syndrome in the monkey. Neurobiol Dis. 2005; 20: 428–431. 10.1016/j.nbd.2005.03.024 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref042] 42.Lu H-y, Dong F, Liu C-y, Wang J, Liu Y, Xiao W. An animal model of obstructive sleep apnoea-hypopnea syndrome corrected by mandibular advancement device. Eur J Orthod. 2015; 37: 284–289. 10.1093/ejo/cju041 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref043] 43.Liu Y, Gao L, Lv W, Lin L, Wang Y, He H, et al. Histological, Ultrastructural, and Physiological Evaluation of a Rat Model of Obstructive Sleep Apnea Syndrome. Med Sci Monit. 2019; 25: 1806–1813. 10.12659/MSM.913056 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref044] 44.Tietjens JR, Claman D, Kezirian EJ, Marco T de, Mirzayan A, Sadroonri B, et al. Obstructive Sleep Apnea in Cardiovascular Disease. A Review of the Literature and Proposed Multidisciplinary Clinical Management Strategy. J Am Heart Assoc. 2019; 8: e010440 10.1161/JAHA.118.010440 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref045] 45.Toischer K, Rokita AG, Unsöld B, Zhu W, Kararigas G, Sossalla S, et al. Differential cardiac remodeling in preload versus afterload. Circulation. 2010; 122: 993–1003. 10.1161/CIRCULATIONAHA.110.943431 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref046] 46.Ponikowski P, Voors AA, Anker SD, Bueno H, Cleland JGF, Coats AJS, et al. 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur Heart J. 2016; 37: 2129–2200. 10.1093/eurheartj/ehw128 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref047] 47.Williams B, Mancia G, Spiering W, Agabiti Rosei E, Azizi M, Burnier M, et al. 2018 ESC/ESH Guidelines for the management of arterial hypertension. Eur Heart J. 2018; 39: 3021–3104. 10.1093/eurheartj/ehy339 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref048] 48.Korcarz CE, Peppard PE, Young TB, Chapman CB, Hla KM, Barnet JH, et al. Effects of Obstructive Sleep Apnea and Obesity on Cardiac Remodeling. The Wisconsin Sleep Cohort Study. Sleep. 2016; 39: 1187–1195. 10.5665/sleep.5828 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243844.ref049] 49.Arias MA, García-Río F, Alonso-Fernández A, Mediano O, Martínez I, Villamor J. Obstructive sleep apnea syndrome affects left ventricular diastolic function. Effects of nasal continuous positive airway pressure in men. Circulation. 2005; 112: 375–383. 10.1161/CIRCULATIONAHA.104.501841 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref050] 50.Shim CY, Kim D, Park S, Lee CJ, Cho H-J, Ha J-W, et al. Effects of continuous positive airway pressure therapy on left ventricular diastolic function. A randomised, sham-controlled clinical trial. Eur Respir J. 2018; 51: 1701774 10.1183/13993003.01774-2017 [DOI] [PubMed] [Google Scholar]

[pone.0243844.ref051] 51.Schofield CJ, Ratcliffe PJ. Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol. 2004; 5: 343–354. 10.1038/nrm1366 [DOI] [PubMed] [Google Scholar]

PERMALINK

A novel mouse model of obstructive sleep apnea by bulking agent-induced tongue enlargement results in left ventricular contractile dysfunction

Simon Lebek

Philipp Hegner

Christian Schach

Kathrin Reuthner

Maria Tafelmeier

Lars Siegfried Maier

Michael Arzt

Stefan Wagner

Roles

Abstract

Aims

Methods and results

Conclusions

Introduction

Materials and methods

Polytetrafluoroethylene injection into the tongue

Sonographic measurement of tongue diameter

Monitoring of spontaneous breathing

Isolation of RNA and transcription into cDNA

Quantification of KDM6A and HIF1α

Transthoracic echocardiography

Western blots

Data analysis and statistics

Results

Increased tongue diameter after PTFE injection

Fig 1. Increased tongue diameter after PTFE injection.

PTFE injection increases the frequency of inspiratory flow limitations and apneas

Fig 2. PTFE injection into the tongue induces apneas and inspiratory flow limitations.

PTFE injection resulted in decreased KDM6A and increased HIF1α expression

Fig 3. KDM6A mRNA expression is decreased in PTFE mice.

Systolic and diastolic dysfunction in PTFE-treated mice

Fig 4. Systolic and diastolic dysfunction in PTFE mice.

Table 1. Echocardiographic parameters.

Increased heart and lung weight in PTFE-treated mice

Fig 5. Heart and lung weight in control and PTFE injected mice.

Increased CaMKII expression in PTFE-treated mice

Fig 6. Increased CaMKII expression in PTFE mice.

Discussion

Current models of sleep apnea are limited

PTFE tongue injection induces obstructive apneas in mice

Consequences of PTFE-injection on contractile function

Limitations

Conclusions

Supporting information

Acknowledgments

Non-standard abbreviations and acronyms

Data Availability

Funding Statement

References

Decision Letter 0

Michael Bader

Roles

Author response to Decision Letter 0

Decision Letter 1

Michael Bader

Roles

Acceptance letter

Michael Bader

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases