# Chronic intermittent hypoxia triggers cardiac fibrosis: Role of epididymal white adipose tissue senescent remodeling?

Suzain Naushad, Jonathan Gaucher, Zaineb Mezdari, Maximin Détrait, Elise Belaidi, Yanyan Zhang, Guillaume Vial, Sophie Bouyon, Gabor Czibik, Maria Pini, et al.

► To cite this version:

Suzain Naushad, Jonathan Gaucher, Zaineb Mezdari, Maximin Détrait, Elise Belaidi, et al. Chronic intermittent hypoxia triggers cardiac fibrosis: Role of epididymal white adipose tissue senescent remodeling?. Acta Physiologica, 2024, 240 (11), pp.e14231. 10.1111/apha.14231. hal-04759160

HAL Id: hal-04759160

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Submitted on 28 Jan 2026

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# Chronic intermittent hypoxia triggers cardiac fibrosis: Role of epididymal white adipose tissue senescent remodeling?

Suzain Naushad<fnref n="1" />

Jonathan Gaucher<fnref n="2" />

Zaineb Mezdari<fnref n="1" />

Maximin Détrait<fnref n="2" />

Elise Belaidi<fnref n="2" />

Yanyan Zhang<fnref n="1" />

Guillaume Vial<fnref n="2" />

Sophie Bouyon<fnref n="2" />

Gabor Czibik<fnref n="1" />

Maria Pini<fnref n="1" />

Sahar Aldekwer<fnref n="2" />

Hao Liang<fnref n="1" />

Véronique Pelloux<fnref n="4" />,<fnref n="5" />

Judith Aron-Wisnewsky<fnref n="4" />,<fnref n="5" />

Renaud Tamisier<fnref n="2" />

Jean-Louis Pépin<fnref n="2" />

Geneviève Derumeaux<fnref n="1" />,<fnref n="3" />

Daigo Sawaki<fnref n="1" />

Claire Arnaud<fnref n="2" />

<figure><img src="image_2.png" /></figure>

## Correspondence

Claire Arnaud, Université Grenoble Alpes, INSERM U1300, Laboratoire HP2, Faculté de Médecine de Grenoble, Place du Commandant Naï, 38700 La Tronche, Grenoble, France.

Email: claire.arnaud@univ-grenoble-alpes.fr

## Funding information

RHU CARMMA, Grant/Award Number: ANR-15-RHUS-0003; Fondation Université Grenoble Alpes, Grant/Award Number: ANR-19-P3IA-0003; ANR-IDEX-UGA Initiative de Recherche Stratégique, Grant/Award Number: ANR-IDEX-UGA-IRS; Fondation pour la Recherche Médicale, Grant/Award Number:

## Abstract

**Aim:** Obstructive sleep apnea (OSA) is a growing health problem affecting nearly 1 billion people worldwide. The landmark feature of OSA is chronic intermittent hypoxia (CIH), accounting for multiple organ damage, including heart disease. CIH profoundly alters both visceral white adipose tissue (WAT) and heart structure and function, but little is known regarding inter-organ interaction in the context of CIH. We recently showed that visceral WAT senescence drives myocardial alterations in aged mice without CIH. Here, we aimed at investigating whether CIH induces a premature visceral WAT senescent phenotype, triggering subsequent cardiac remodeling.

**Methods:** In a first experiment, 10-week-old C57bl6J male mice (n=10/group) were exposed to 14 days of CIH (8 h daily, 5%–21% cyclic inspired oxygen fraction, 60 s per cycle). In a second series, mice were submitted to either epididymal WAT surgical lipectomy or sham surgery before CIH exposure. Finally, we used p53 deficient mice or Wild-type (WT) littermates, also exposed to the same CIH protocol. Epididymal WAT was assessed for fibrosis, DNA damages, oxidative stress, markers of senescence (p16, p21, and p53), and inflammation by RT-qPCR and histology, and myocardium was assessed for fibrosis and cardiomyocyte hypertrophy.

**Results:** CIH-induced epididymal WAT remodeling characterized by increased fibrosis, oxidative stress, DNA damage response, inflammation, and increased expression of senescent markers. CIH-induced epididymal WAT remodeling was associated with subtle and early myocardial interstitial fibrosis. Both epididymal WAT surgical lipectomy and p53 deletion prevented CIH-induced myocardial fibrosis.

<footnote n="1">¹Université Paris Est Créteil, INSERM U955, Créteil, France</footnote>

<footnote n="2">²Université Grenoble Alpes, INSERM U1300, CHU Grenoble Alpes, HP2, Grenoble, France</footnote>

<footnote n="3">³Department of Physiology, AP-HP, Henri Mondor Hospital, FHU-SENEC, Créteil, France</footnote>

<footnote n="4">⁴Nutrition and Obesities, Systemic Approaches, NutriOmics, Laboratory, Sorbonne University, Paris, France</footnote>

<footnote n="5">⁵Nutrition Department, CRNH Ile de France, AP-HP, Pitie-Salpêtrière Hospital, Paris, France</footnote>
EQU202003010186; Agence Nationale de la Recherche, Grant/Award Number: 'ANR-19-CE14-0037-01'; 'ANR-20-CE14-0029-01; Fond de dotation Agir pour les Maladies Chroniques; France Life Imaging, Grant/Award Number: ANR-11-INBS-0006

**Conclusion:** Short-term exposure to CIH induces epididymal WAT senescent remodeling and cardiac interstitial fibrosis, the latter being prevented by lipectomy. This finding strongly suggests visceral WAT senescence as a new target to mitigate OSA-related cardiac disorders.

KEYWORDS

cardiac remodeling, cellular senescence, chronic intermittent hypoxia, obstructive sleep apnea, visceral white adipose tissue

# 1 | INTRODUCTION

Obstructive sleep apnea (OSA) is one of the most frequent chronic disorders affecting nearly one billion people worldwide.¹ OSA is characterized by the repetitive occurrence of partial or complete upper airway obstruction during sleep, leading to chronic intermittent hypoxia (CIH), which represents the major detrimental factor inducing OSA-related cardiometabolic complications and mortality.²⁻⁵ In lean rodents, CIH contributes to cardiac remodeling, mainly characterized by myocardial hypertrophy, interstitial fibrosis, and secondary contractile dysfunction.⁶ At the molecular level, CIH is the key trigger of oxidative stress, sympathetic over-activity, inflammation, and activation of the hypoxia-inducible transcription factor 1 (HIF1).⁵ This has been consistently demonstrated in rodents⁷⁻⁹ and healthy humans¹⁰ exposed to CIH. In addition, CIH affects several metabolic organs, including the adipose tissue, which may per se mediate noxious effects of CIH to remote organs, such as the vessels and the heart.¹¹ Indeed, we previously demonstrated in rodents that CIH profoundly alters the visceral white adipose tissue (WAT), induces shrinkage of adipocytes, increases β-adrenergic-induced lipolysis and inflammation together with abnormal WAT insulin signaling leading to systemic insulin resistance.¹²⁻¹⁴ Surgical removal of epididymal WAT (lipectomy) mitigates CIH-induced atherosclerosis.¹²

The adipose tissue is at the nexus of pathways involved in longevity, genesis of age-related chronic diseases, metabolic dysfunction, and low-grade inflammation.¹⁵ Now, a strong association between visceral adiposity and cardiac alterations is established.¹⁵,¹⁶ In example, we recently highlighted the role of visceral WAT senescence in the process of aging-induced cardiac fibrosis.¹⁷ Interestingly, recent studies and meta-analysis point out that OSA accelerates biological aging through induction of cellular senescence.¹⁸⁻²¹ Cellular senescence was discovered initially as a phenotype of terminal growth arrest, and later recognized as a key biological process underlying beneficial (e.g., tumor suppression and clearance of senescent cells) and deleterious outcomes (e.g., driving inflammation,

degeneration late in life).²² Metabolically active senescent cells enter an irreversible cell-cycle arrest mediated predominantly by the p16Ink4a/pRB pathway in response to telomere shortening (replicative senescence) and/or the p53/p21 pathway in response to stress stimuli such as oxidative stress (premature senescence). Senescent cells display a complex phenotype including altered morphology and changes in gene expression together with the upregulation of senescence-associated beta-galactosidase (SA-β-gal). All these alterations influence profoundly the secretory properties of many cell types, including in the adipose tissue, and induce a senescence-associated secretory phenotype or SASP.²³ The SASP-secreted proteins promote neighboring cell damage²⁴ and trigger adverse effects on remote tissues.²⁵

In the present study, we hypothesized that CIH induces WAT senescence and remodeling, which contributes to the induction of cardiac alterations.

# 2 | RESULTS

## 2.1 | CIH induces early structural remodeling of epididymal WAT

We first demonstrated that 2 weeks of CIH induced an early remodeling of epididymal WAT, characterized by fibrosis (Figure 1A) together with oxidative stress (Figure 1B), DNA damage response (Figure 1C), and immune cell infiltration (i.e., CD45, CD68, and CD3 positive cells, Figure 1D-F, respectively). Interestingly, WAT remodeling occurred out of any modification of mice body weight (Figure S2A), adipose fat pad weights (Figure S2B), nor adiposity index (Figure S2C). However, mice exposed to CIH exhibited increased fasting blood glucose (Figure S2D) and systemic insulin resistance (Figure S2E,F). In addition, CIH significantly induced HIF-1 target gene expression in epididymal WAT, such as vascular endothelial growth factor (VEGF), pyruvate dehydrogenase kinase 1 (Pdk1), and glucose transporter protein type 1 (Scl2a1) (Figure S2G). Of note, this short CIH
exposure did not increase WAT beta3-adrenergic receptor expression, nor lipolysis that might be indicative of WAT sympathetic activation (data not shown).

Using high-throughput transcriptomic analysis, we uncovered that CIH altered the expression of 5129 genes in the epididymal WAT (3574 upregulated and 1555 downregulated). Analysis of molecular signatures based on differentially expressed genes revealed that top upregulated genes belonged to DNA repair, protein secretion, and p53 pathway, whereas downregulated genes were related to adipogenesis, oxidative phosphorylation, and protein secretion (Figure 1G). Furthermore, transcription factor (TFs) binding site analysis (TFBS) showed a deregulation of TP53 target genes upon CIH exposure (Figure 1H, Figure S3). Altogether, these transcriptomic findings suggest cellular senescence in the epididymal WAT of mice exposed to CIH.

## 2.2 | CIH early induces cellular senescence in epididymal WAT

To further confirm hallmarks of cellular senescence,²³ we investigated the impact of 2 weeks of CIH on the protein expression of the senescence markers p16, p21, and p53 in the whole mice epididymal WAT (Figure 2A-C) and in the various cell types of WAT (Figure 2D-I). While we found an increase in the senescence markers p16, p21, and p53 in the whole epididymal WAT, we identified an increase in p21 positive staining in adipocyte progenitors (PDGFRα) (Figure 2D), and a higher frequency of p53 positive cells in monocytes/macrophages (Figure 2H) and T lymphocytes (Figure 2I). In line with these results in rodents, we observed a similar pattern of senescent markers expression in subcutaneous WAT obtained from healthy volunteers exposed to 14-night CIH and lean OSA patients (Figure 2J).

## 2.3 | Short-term CIH-induced epididymal WAT senescence contributes to cardiac fibrosis

In parallel to epididymal WAT remodeling and senescence, we highlighted an early cardiac remodeling, characterized by increased interstitial fibrosis, evidenced by Sirius red staining (collagen) (Figure 3A) and vimentin staining (fibroblast) (Figure 3B). Importantly, this subtle cardiac remodeling was not associated with alterations in both systolic and diastolic cardiac function assessed by echocardiography at that early time point (Table 1). In addition, we did not observe any change in heart weight (Figure S4A), nor increase in expression of genes

involved in cardiac hypertrophy (Figure S4B) between normoxic and hypoxic animals, while the cardiomyocyte cross-sectional area was increased under CIH exposure (Figure S4C).

As we recently highlighted the role of visceral WAT senescence in age-related cardiac fibrosis,¹⁷ we investigated whether CIH-induced epididymal WAT senescence could also contribute to cardiac fibrosis. As a proof-of-concept, we performed a surgical removal of epididymal WAT (Figure S1C,D). Epididymal WAT removal significantly reduced CIH-induced myocardial interstitial fibrosis, evidenced by Sirius red (Figure 3C) and vimentin (Figure 3D) staining, but not cardiomyocyte hypertrophy (Figure S5A-C). Of note, blunted cardiac fibrosis by visceral lipectomy was not mediated by metabolic improvements, such as body weight (Figure S5D) and fat pad weight (Figure S5E) reduction, or increased insulin sensitivity (Figure S5F-H). Since the epididymal WAT transcriptomic analysis highlighted an upregulation of p53-linked pathways by CIH (Figure 1G,H), we then subjected p53-deficient mice to CIH (Figure S1E,F). Interestingly, p53 deletion prevented adipose tissue senescence as assessed by the reduction of the senescence marker p16 in the whole epididymal WAT (data not shown) and reduced CIH-induced collagen accumulation compared to N (Figure 3E). Collectively, these results strongly support our hypothesis that short-term exposure to CIH early induces epididymal WAT senescence, which in turn promotes cardiac fibrosis.

## 2.4 | Upregulation of senescence-related gene expression is not associated with an increase in circulating pro-fibrotic or pro-inflammatory markers

Whereas we found an increased expression of several senescence-associated secretory phenotype (SASP) genes in visceral WAT of mice exposed to CIH, such as the transforming growth factor (TGF)-β (Tgfβ), plasminogen activator inhibitor (PAI)-1 (Serpine 1) and interleukin (IL)-1β (IL1β) (Figure 4A), plasma levels of MCP1, IL-6, TNF-α, IL-1β, IL-10, VEGF, and osteopontin were under the detection levels (data not shown). In line with the lack of CIH-induced WAT weight modification, we did not observe any change in transcript or circulating levels of leptin (Figure 4B,C, respectively). Of note, CIH significantly reduced plasma adiponectin levels and blunted adiponectin mRNA expression in visceral WAT (Figure 4D,E). Finally, ex-vivo epididymal WAT secretome analysis showed that p53-deficiency resulted in preserved adiponectin secretion but no significant change in leptin levels in response to CIH exposure (Figure 4F,G).
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FIGURE 1 CIH induces early structural remodeling of epididymal WAT. Epididymal WAT fibrosis was evaluated on paraffin-embedded sections through analysis of Sirius red staining (A) and oxidative damage was evaluated through 4-Hydroxy-2-Nonenal (4-HNE) staining (B). Quantification is expressed as percentage of tissue area. (C) DNA damage was analyzed in epididymal WAT from N and CIH mice through γH2AX positive cells quantification. Results are expressed as percentage of DAPI. Inflammation was evaluated through staining of leukocytes (number of CD45 positive cells per section) (D), monocytes/macrophages (number of CD68 positive cells per section) (E), and T lymphocytes (number of CD3 positive cells per section) (F). N=5 to 6 mice per group, n=3 to 6 images per mice. Data are expressed as individual values from each section, together with mean ± SEM and were analyzed using unpaired t-tests or Mann-Whitney test (GraphPad Prism Software). Differences between groups were considered statistically significant at *p* < 0.05. (G) Molecular signatures analysis showing the top five hallmark gene sets category enriched in upregulated (left panel – in red) and downregulated genes upon CIH (right panel – in green). The number of dysregulated genes in our transcriptome over the total number of genes for each molecular signature category is indicated on the graph. (*H) TF analysis showing the top five enriched TFs associated with TF binding sites (TFBS) detected at the promoter of genes that are present in TRANSFAC and JASPAR databases. The number of dysregulated genes in our transcriptome over the total number of TFBS associated with each TF target gene is indicated on the graph.

## 2.5 | Short-term CIH induces cardiac fibroblast activation through epididymal WAT secretion

Although, except for adiponectin, we did not identify a specific molecule or protein secreted by the epididymal WAT and responsible for CIH-induced early cardiac remodeling, our ex-vivo experiments (Figure 5A) confirmed the contributing role of CIH epididymal WAT secretome and plasma on cardiac fibroblast activation. Indeed, primary cardiac fibroblast incubated with plasmas from CIH mouse for 24 h exhibited fibroblast differentiation, as evidenced by a significant increase in αSMA staining, and a higher proliferation rate, as demonstrated by an increased number of EdU-positive cells compared to fibroblasts stimulated with plasma from normoxia animals (Figure 5B,C). Interestingly, while we did not observe any changes in fibroblast differentiation, primary cardiac fibroblasts incubated with epididymal WAT secretome from CIH mice also exhibited an increased proliferation rate (Figure 5D,E). Conversely, fibroblasts incubated with plasma derived from mice subjected to WAT lipectomy and CIH exhibited a decrease in αSMA staining and in the number of EdU-positive cells compared to plasma from CIH mice (Figure 5F,G). These findings underscore the contribution of epididymal WAT on cardiac fibroblast activation and support the contributing role of epididymal WAT in the development of interstitial cardiac fibrosis.

## 3 | DISCUSSION

This proof-of-concept study adds important new information on the established impact of CIH on the adipose tissue<sup>11,26</sup> and heart.<sup>5,6</sup> First, we found that cardiac fibrosis and adipose tissue remodeling occur early after the initiation of a short-term exposure to CIH. Second, we demonstrated that cellular senescence in WAT is a primary event occurring before systemic inflammation. Third, we investigated the role of WAT senescence by

genetic deletion of p53, a marker of cellular senescence, which prevented CIH-induced cardiac fibrosis. Fourth, we added evidence to the contribution of adipose tissue senescent remodeling on cardiac fibrosis, showing that epididymal WAT lipectomy prevents CIH-induced myocardial interstitial fibrosis. Finally, we uncovered that CIH modified plasma composition through epididymal WAT secretion, leading to cardiac fibroblast activation. Collectively, our data open new avenues for therapeutic interventions targeting adipose tissue cellular senescence to mitigate cardiac remodeling during OSA. This should be first tested in murine models and further validated in patients.

### 3.1 | Early impact of CIH on visceral WAT

Several lines of evidence identified OSA as an independent risk factor for metabolic disturbances including diabetes and fatty liver diseases.<sup>5,11,26</sup> CIH-induced adipose tissue dysfunction represents a key player in the organ crosstalk leading to cardiometabolic complications.<sup>11,26</sup> Indeed, previous preclinical studies in rodent models reported that visceral WAT is highly sensitive to long-term CIH exposure (at least 6 weeks), with structural remodeling, presence of shrunken adipocytes,<sup>12,27,28</sup> browning<sup>12</sup> and inflammation.<sup>12,13</sup> WAT is a sensitive tissue, known to be altered by several stressors, such as obesity,<sup>29</sup> aging,<sup>17</sup> and heart failure.<sup>30</sup> Here, our work demonstrates that short-term CIH is also a trigger of WAT remodeling and senescence.

Our study extends our current knowledge on CIH-induced WAT remodeling by highlighting that CIH-dependent alterations occur as early as 2-week post-exposure. First, we evidenced WAT gene dysregulation (more than 5000 transcripts). Interestingly, top upregulated genes belong to DNA repair, p53 pathway, and protein secretion. Thus, our transcriptomic analysis is indicative of highly expressed genes involved in
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senescence process, beyond those involved in mitochondrial dysfunction, disruption of metabolic processes, induction of apoptosis, and oxidative stress, as previously

reported.31 Second, this altered transcriptomic landscape matches with the increased protein expression of senescence markers (p16, p21, and p53) in various cell types,
FIGURE 2 CIH induces premature senescence in epididymal WAT. Epididymal WAT immunofluorescence for p16 (A), p21 (B), and p53 (C) (senescent markers in red; counterstaining with DAPI in white or blue). Quantification of individual senescent markers is expressed as number of positive cells per section, N=4 to 5 mice per group, n=3 to 6 sections per mice. Representative immunofluorescence images and quantification of epididymal WAT co-staining of p21 (green, D-F) or p53 (green, G-I) with marker of adipocyte precursor platelet-derived growth factor receptor alpha, PDGFRα (red, D and G), CD68 monocytes/macrophages (red, E and H) or CD3 T lymphocytes (red, F and I). Sections were counterstained with DAPI (blue). Double-positive cells are expressed as individual values from each section together with mean ± SEM, N=4 to 5 mice per group, n=3 to 6 sections per mice. Results were analyzed using unpaired t-test or Mann-Whitney test (GraphPad Prism Software). Differences between groups were considered statistically significant at *p* < 0.05. Representative images showing p21 senescent markers in the human adipose tissue from healthy volunteers exposed to 14 nights Air or CIH and OSA patients (J).

including adipocyte progenitors, monocytes, and CD3-T
lymphocytes, together with increased WAT fibrosis, DNA
damage response, oxidative stress, and local inflammation
(recruitment of inflammatory cells). Our in vivo find-
ings in mice are aligned with prior in vitro study demon-
strating that CIH induces a senescent-like phenotype in
primary human preadipocytes, with elevated SA-β-gal
activity together with nuclear co-localization of γH2AX
and p16.²¹ Finally, our translational data in humans show
that healthy volunteers exposed to 14 nights of CIH and
lean OSA patients exhibit high levels of senescent mark-
ers within subcutaneous WAT, which has recently been
shown to exhibit more cellular senescence than visceral
depots in the context of obesity in patients.³² Altogether,
our preclinical and translational data allow us to identify
cellular senescence as a key orchestrator of the WAT re-
sponse to CIH exposure.

**3.2 | Short-term CIH triggers early cardiac fibrosis and tackles the role of epididymal WAT senescence**

Interestingly, we observed a modest but significant
increase in myocardial interstitial fibrosis after only
2 weeks of CIH (but no evidence of diastolic or systolic
dysfunction). Thus, our model exposing young animals
out of any other additional myocardial stress captures
the inception of subtle and early cardiac fibrosis,
before overt cardiac dysfunction occurring after longer
exposure duration.⁶ Our data raise the question of the
causality between WAT senescence and cardiac fibrosis.
Indeed, we might discuss the role of other triggers such
as increased sympathetic tone, energy metabolism
dysregulation, or the role of other organs such as the
liver. However, as a proof-of-concept, we demonstrated
that cardiac fibrosis was prevented by epididymal WAT
lipectomy in vivo in mice. This was strengthened by our
ex-vivo experiments demonstrating that CIH-induced
cardiac fibroblast activation was prevented in cells
stimulated with plasma from lipectomized animals.
Furthermore, p53 genetic deletion also prevented CIH-
induced interstitial myocardial fibrosis, supporting

a link between CIH-related WAT senescence and
cardiac remodeling. These data obtained in young
mice subjected to CIH broaden the pathophysiological
relevance of a crosstalk between senescent WAT and
heart, previously reported in natural aging.¹⁷ In addition,
our study shed new mechanistic light in results obtained
in a recent meta-analysis in rodents, reporting that long-
term CIH exposure (at least 6 weeks) induces cardiac
remodeling and contractile dysfunction.⁶ Of note, these
data in rodent models are consistent with studies in
OSA patients showing an association between sleep-
disordered breathing and myocardial fibrosis,³³,³⁴ with
or without reduced ejection fraction.³⁵⁻³⁷

**3.3 | Evidence of a crosstalk between
visceral WAT and heart?**

In light of our findings indicating cardiac fibroblast activation by epididymal WAT secretome and plasma from CIH mice but not lipectomized mice, it is reasonable to consider a crosstalk between visceral WAT and the heart in the context of CIH. Despite the fact that our study revealed a modest CIH-induced increase in gene expression of pro-inflammatory/pro-fibrotic factors (TNFα, IL1β, TGFβ, and serpin), these transcript data were not associated with significant changes in circulating SASP factors at 14 days after the initiation of CIH. This does not preclude an unidentified SASP factor or an earlier release of pro-fibrotic circulating factors by WAT. Indeed, such an observation is in line with reports showing that the SASP composition varies dramatically depending on the length of time after senescence induction,²³ and also depends on the origin of the pro-senescence stimulus and the cell type.³⁸ For instance, in the context of high-fat diet (HFD)-induced adipose tissue senescence, a very early but transient increase in TGF-β gene expression was found within the adipose tissue,²⁹ concomitant with increased circulating levels.³⁹ As in the context of CIH, this temporal pattern of HFD-related SASP showed a pro-fibrotic secretome occurring before any inflammatory signaling.²⁹ Such a transition from an early TGF-β-dependent secretome to a pro-inflammatory secretome
<figure><img src="image_42.png" /><figcaption>FIGURE 3 Short-term CIH-induced epididymal WAT remodeling contributes to cardiac fibrosis. Sirius red and vimentin (red) staining of myocardial paraffin-embedded sections, from mice exposed to 14 days normoxia (N) or CIH (A and B); from mice submitted to epididymal WAT removal (Lipec) or Sham surgery (Sham), followed by 14 days of CIH (C and D) and from P53 KO or Wild-type mice, exposed to N or CIH (E). Quantifications are based on N=4 to 6 mice per group, n=3 to 6 sections per mice. Results are expressed as individual values from each section together with mean ± SEM and were analyzed using unpaired t-tests or Mann-Whitney test (GraphPad Prism Software). Differences between groups were considered statistically significant at *p* < 0.05.</figcaption></figure>

has been reported in vitro to be governed by fluctuation of Notch1 activity.⁴⁰,⁴¹

In parallel, CIH did not change the expression and cir-
culating levels of leptin, but decreased both gene expres-
sion and secretion of adiponectin by visceral WAT. Notably,

our murine findings are in line with low levels of circulat-
ing adiponectin in OSA patients⁴² and in healthy subjects
exposed to 14 nights of CIH¹⁰. Given this, it is tempting to
propose that adiponectin may act as a WAT-derived guard-
ian of cardiac integrity, with CIH undermining its levels,
<table><caption>TABLE 1 Echographic parameters of mice exposed to 2 weeks N or CIH.</caption><thead><tr><th></th><th>N</th><th>CIH</th></tr></thead><tbody><tr><td>Heart rate, bpm</td><td>420 ± 14</td><td>393 ± 9</td></tr><tr><td>E/A</td><td>1.507 ± 0.064</td><td>1.545 ± 0.067</td></tr><tr><td>IVSd, mm</td><td>0.744 ± 0.020</td><td>0.735 ± 0.021</td></tr><tr><td>IVSs, mm</td><td>1.015 ± 0.020</td><td>1.007 ± 0.021</td></tr><tr><td>LVDd, mm</td><td>4.025 ± 0.094</td><td>3.872 ± 0.127</td></tr><tr><td>LVDs, mm</td><td>3.047 ± 0.094</td><td>2.871 ± 0.123</td></tr><tr><td>LVPWd, mm</td><td>0.767 ± 0.033</td><td>0.729 ± 0.020</td></tr><tr><td>LVPWs, mm</td><td>1.011 ± 0.046</td><td>0.925 ± 0.026</td></tr><tr><td>EF, %</td><td>48.897 ± 1.472</td><td>51.437 ± 2.135</td></tr><tr><td>FS, %</td><td>24.392 ± 0.869</td><td>25.933 ± 1.274</td></tr><tr><td>LV mass corrected, mg</td><td>115.112 ± 4.750</td><td>103.227 ± 4.888</td></tr></tbody></table>

Note: Echocardiographic parameters of mice exposed to 2 weeks N or CIH (n=10/group). E/A, ratio of the early (E) to late (A) filling velocities, IVS, interventricular septum, LVD, left ventricular diameter, LVPW, left ventricular posterior wall, EF, ejection fraction, FS, fractional shortening, LV mass, left ventricular mass, d, parameters obtained in diastole, s, parameters obtained in systole.

hence permitting initiation and development of cardiac fibrosis.⁴³ In support to that idea, previous studies demonstrated that adiponectin exerts protective effect against cardiac fibrosis in other experimental models than CIH⁴⁸ (i.e., transverse aortic constriction,⁴⁴ diabetes,⁴⁵ angiotensin II⁴⁶).

## 3.4 | Clinical perspectives

Our data support the development of adjunctive geroprotective strategies in OSA.⁴⁷ This might be of major interest considering the poor adherence to OSA first-line continuous positive airway pressure (CPAP) therapy.⁴⁸ In addition, extending the findings of the role of WAT senescence in inducing metabolic disturbances in obese patients,³² geroprotective strategies might also have an impact on OSA-induced systemic insulin resistance. Furthermore, as in other chronic diseases, it is now recommended to use aggressive risk reduction management strategies in OSA patients, including CPAP plus weight loss and physical activity rehabilitation programs.⁴⁹⁻⁵¹ Our data suggest that geroprotective strategies merit to be included in these combination treatment packages.

# 4 | MATERIALS AND METHODS

All the materials submitted conform to good publishing practice in physiology.⁵²

## 4.1 | Ethical approval

All experiments in rodents were performed in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (decree 2013-118 and orders of February 1, 2013) and have received approval from the ethical committee of the Universite Grenoble Alpes and the "Ministère de l'enseignement supérieur et de la recherche" (APAFIS#20468-2019060611431556v1).

## 4.2 | Animals

Ten-week-old C57bl6J male mice (Charles River, France) were randomly exposed to CIH or normoxic condition (N) for 14 days (n=10 mice per group) to assess subsequent visceral (epididymal) WAT senescence and cardiac remodeling (Figure S1A,B).

In a second set of experiments, 10-week-old C57bl6J male mice (Charles River, France) were first randomized into two groups, submitted to a surgical removal of epididymal WAT (Lipec) or sham surgery (Sham). After 4 weeks of recovery, Sham and Lipec mice (n=10 mice per group) were then exposed to CIH for 14 days to investigate the contributing role of visceral WAT on CIH-induced cardiac remodeling (Figure S1C,D).

Finally, in a third set of experiments, 13-week-old p53 knockout mice (p53KO, n=19) and their Wild-type (WT) littermates (p53WT, n=18) were also randomized to CIH or N exposure to investigate the contribution of senescence on visceral WAT remodeling and subsequent cardiac fibrosis by CIH (Figure S1E,F).

Animals were housed under standard conditions in conventional cages with ad libitum access to water and chow diet, at constant temperature (22°C), with a 12-h light–dark cycle. Mice were weighted before exposure to N or CIH and weekly thereafter.

## 4.3 | Intermittent hypoxia

Animals were exposed to CIH or N as previously described.⁵³ Briefly, mice were submitted to either 60s IH cycles (inspired oxygen fraction oscillating from 21% to 5% every 30s) or air–air cycles (21% O₂), for 8h during sleeping time (Figure S1B).

## 4.4 | Insulin tolerance test

The week before mice euthanasia, an intraperitoneal (i.p.) insulin tolerance test (ipITT) was performed to assess
<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /><figcaption>FIGURE 4 Upregulation of senescence-related gene expression was not associated with an increase in circulating pro-fibrotic or pro-inflammatory markers. (A) Relative mRNA expression of genes involved in fibrosis and inflammation (*Tgfb*, *Serpine1*, *Tnfa*, *Il1b*) in visceral WAT of mice exposed to normoxia (N) or CIH. The mRNA expression was normalized to Actin mRNA levels and expressed as fold change compared to normoxic mice using $2^{\Delta\Delta C_t}$ method (where $C_t$ is threshold cycle), *N* = 6 mice per group. Relative mRNA expression of leptin (*Lep*) (B) and adiponectin (*AdipoQ*) (E) in epididymal WAT from mice exposed to 14 days N and CIH, *N* = 6 mice per group. Plasma levels of leptin (C) and adiponectin (D) from C57bl6 mice exposed to 14 days N and CIH, *N* = 10 mice per group. Adiponectin (F) and leptin (G) levels in epididymal WAT supernatants from p53 knockout mice (p53KO) and Wild-type littermate (p53WT) exposed to 14 days N and CIH. Data are expressed as individual values together with mean ± SEM and were analyzed using unpaired t-tests, Mann-Whitney test, or two-way ANOVA (GraphPad Prism Software). Differences between groups were considered statistically significant at *p* < 0.05.</figcaption></figure>

whole-body insulin sensitivity. Briefly, mice were fasted for 6 h, and insulin was administrated (0.5 U/kg total body weight, i.p.). Blood samples were drawn from the tail vein

at different time points (0–15 to 30–45–60 min) to determine the blood glucose concentrations using a glucometer (One Touch VERIO).
<figure><img src="image_50.png" /><figcaption>FIGURE 5 Short-term CIH induces cardiac fibroblast activation via epididymal WAT secretion. (A) Primary cardiac fibroblasts obtained from neonatal mice were incubated 24 h with plasma or epididymal WAT secretome from normoxic (N) or CIH animals. (B) αSMA positive area and (C) percentage of EdU-positive cells of primary cardiac fibroblast incubated with plasma from N and CIH exposed mice. (D) αSMA positive area and (E) percentage of EdU-positive cells of primary cardiac fibroblast incubated with epididymal WAT supernatants from N and CIH exposed mice. (F) αSMA positive area and (G) percentage of EdU-positive cells of primary cardiac fibroblast incubated with plasma from mice submitted to epididymal WAT removal (Lipec) or Sham surgery (Sham), followed by 14 days of CIH. \*p < 0.05, \daggerp < 0.01.</figcaption></figure>

## 4.5 | Echocardiography

Cardiac echography was performed by a trained technician after 2 weeks exposure to CIH or N, using a Vevo 2100 imaging system (22–55 MHz MS550D transducer, Visual Sonics). Mice were anesthetized with 1% isoflurane supplemented with O₂. The body temperature,

respiratory rate, and electrocardiogram (ECG) were monitored, and the heart rate was maintained at least at 400 beats/min during the measurements of cardiac parameters. All parameters were measured by a two-dimensional parasternal short-axis imaging plane of M-mode traces close to the papillary muscle level. Diastolic function was estimated by the calculation of E/A ratio
by Doppler measurements of the mitral valve inflow in apical 4-chamber view.

## 4.6 | Epididymal WAT lipectomy

Mice were anesthetized with intraperitoneal administration of ketamine-xylazine mixture (100 mg·kg⁻¹/10 mg·kg⁻¹ body weight), and a 1-cm single abdominal midline incision was made. Bilateral epididymal fat pads were lifted from the peritoneal cavity onto a sterilized and humidified surgical drape, dissected with an electronic scalpel, and removed without damaging the testicular blood supply, as previously described.¹⁷ The Sham operation was performed in the same manner without fat pads removal. The abdominal peritoneum was closed with prolene sutures and the skin was closed with silk sutures (Angiotech, USA). The mice were given 2.5 mg/kg buprenorphine (Sigma-Aldrich, France) intraperitoneally and let recover from surgery. After one month of recovery, mice were exposed to CIH for 14 days.

## 4.7 | Blood and tissue sampling

At the end of CIH exposure, mice were anesthetized with intraperitoneal ketamine-xylazine mixture administration (100 mg·kg⁻¹/10 mg·kg⁻¹). Blood was collected by inferior vena cava puncture, centrifuged and plasma was stored at -80°C. Epididymal, inguinal, and peri-renal WATs and heart were rapidly weighted and conditioned for further experiments. Adiposity index was calculated as the sum of adipose tissue weights (epididymal + inguinal + perirenal) reported to body weight. Bilateral epididymal fat pads were either snap frozen in liquid nitrogen, then stored at -80°C for mRNA study, or fixed in 4% formaldehyde solution (Sigma-Aldrich, France), embedded in paraffin, then cut into 5 µm sections for immunohistochemistry, or incubated in Dulbecco's modified Eagle's medium (DMEM) for secretome investigations. Similarly, hearts were either snap frozen in liquid nitrogen, then stored at -80°C for mRNA/protein studies, or fixed in 4% formaldehyde solution (Sigma-Aldrich, France), embedded in paraffin, then cut into 5 µm sections for immunohistochemistry.

## 4.8 | RNA extraction

RNA was extracted from snap-frozen tissue using the mini RNeasy kit (QIAGEN, France) for the adipose tissue or RNeasy fibrous tissue mini kit (QIAGEN, France) for the heart. The RNA concentration was measured with NanoDrop ND-2000kit (QIAGEN, France). RNA was

then used to quantify gene expression by Microarray or RT-qPCR.

## 4.9 | Microarray experiment, gene expression analysis, and data availability

Microarray experiment, gene expression, and functional analysis have been performed as previously described.⁵⁴⁻⁵⁶ Briefly, the EnrichR analysis tools (https://doi.org/10.1002/cpz1.90, https://doi.org/10.1093/nar/gkw377) were used to identify transcription factor binding site (TFBS) at the promoter of significant differentially expressed genes using TRANSFAC and JASPAR databases. Results were ranked by p value computed from the Fischer exact test which is a proportion test that assumes a binomial distribution and independence for probability of any gene belonging to any set. The data are publicly available in the National Center for Biotechnology Information Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE104128.

## 4.10 | RT-qPCR

Total RNA (0.5–1.0 µg) was reversely transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit and a thermal cycler SimpliAmp 7 (Applied Biosystems, CA, USA) according to the manufacturer's instructions. Quantitative real-time PCR was performed and analyzed using TaqMan® assays with Universal PCR Master Mix (Applied Biosystems, France). β-Actin was used as internal standard and expressed as fold change compared to the levels of normoxic mice. TaqMan oligos used are listed in Table S1.

## 4.11 | Histology

Epididymal adipose tissue and heart were fixed in 4% formaldehyde and embedded in paraffin. Heart tissue sections were stained with Sirius red staining for interstitial fibrosis measurement, as previously described.¹⁷,⁵³ Four to 6 mice per group and 3 to 6 images per sections were analyzed. Images were acquired using a Zeiss Axioplan M2 Imaging microscope, and fibrosis was measured as red fibrotic area divided by total surface area. In order to determine cardiomyocytes cross-sectional area, tissue sections were stained with wheat-germ agglutinin (WGA, Alexa Fluor 488 conjugated). Staining was performed following standard procedures, and images were acquired using a Zeiss Axioplan M2 Imaging microscope.¹⁷,⁵³ Cross-sectional area (CSA) analysis (4 to
6 mice per group, up to 300 cardiomyocytes per mice) was performed by a blinded experimenter using Image J and CSA were expressed in µm². Sections of epididymal adipose tissue were stained with Sirius red staining for fibrosis analysis. 4-Hydroxynonenal (4-HNE) immunostaining was performed to determine oxidative stress and immunofluorescent stainings were performed to visualize adipocytes precursors (PDGFRα), inflammatory cells (CD45, leukocytes; CD68, monocytes/macrophages; CD3; T lymphocytes), and senescence (p16; p21; p53), as previously described.¹⁷,²⁹ Antibodies used are listed in Table S2.

## 4.12 | WAT explants

Epididymal adipose tissue (0.1 g) was minced and incubated for 1 h at 37°C prior to transfer into a new plate with fresh medium (DMEM, 4.5 g glucose, glutamine, containing 1% free fatty acid bovine serum albumin and 1% antibiotic and antimycotic solution). The supernatant was collected 24 h after incubation and stored at -80°C until analysis of WAT secretome.

## 4.13 | Plasma and WAT secretome analysis

Plasma leptin and adiponectin were analyzed by ELISA (R&D Systems, USA). Plasma and WAT conditioned media were quantified for a select panel of pro-inflammatory and pro-fibrotic factors (MCP-1, IL-6, TNFα, IL-1β, IL-10, and VEGF) using a 7-plex Luminex bead-based multiplex assay, according to the manufacturer's instructions (Bio-Techne, UK).

## 4.14 | Cell Culture and Treatments

Primary neonatal mouse cardiac fibroblast culture was performed with the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical, Lakewood, NJ) according to the manufacturer's instructions as described previously.¹⁷ Briefly, neonatal C57/bl6JRj mice (Janvier) were euthanized by decapitation, and their ventricles were separated and minced in an ice-cold balanced salt solution. Minced hearts were incubated in a balanced salt solution containing trypsin for 24 h at 4°C, and then further digested with 300 U/mL of collagenase type 2 for 30 to 45 minutes at 37°C with agitation. The cell suspension was filtered with 70 µm mesh and passed onto dishes with Medium 199 (Gibco and Life Technologies, France), supplemented with 10% fetal calf serum. Thirty minutes later,

floating cells were removed, and the attached cells were considered as cardiac fibroblasts. The purified cardiac fibroblasts were then passed onto 12-well culture plates at a density of 2.10⁵ cells/well or culture slide (Lab-Tek™ II Chamber Slide 8 well, ThermoFisher, France). After serum starvation for 24 h, the cells were stimulated with supernatant of epididymal WAT explants or plasma (50% or 25% volume of total medium respectively) for 24 h.

## 4.15 | Immunofluorescent staining and EdU detection

For immunofluorescent staining, cells cultured on chamber slides were fixed in 1% paraformaldehyde (PFA) followed with permeabilization in 1% Triton X-100. After blocking with 30% goat serum, first antibodies mouse monoclonal anti-alpha-smooth muscle (Sigma A2547) and rabbit anti-vimentin (Abcam, ab92547) were applied with antibody diluent (Dako) for 1 h at room temperature in humidified chamber. Corresponding secondary antibodies goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 555 (Invitrogen) were used to visualize the signal. For ethynyl deoxyuridine (EdU) labeling, EdU was added at the concentration of 10 µM and incubated for 2 h. The incorporated EdU was detected using the Click-iT EdU Alexa Fluor 647 imaging kit (Invitrogen) according to the manufacturer's protocol. Slides were mounted with fluorescent mounting media with DAPI (4',6-diamidino-2-phenylindole; Abcam). Fluorescent pictures were taken with a Zeiss Axio Imager M2 fluorescent microscope and processed with equipped Zen imaging software.

## 4.16 | Human adipose tissue sampling

Subcutaneous adipose tissue biopsies were collected from healthy volunteers exposed to 14 nights of CIH or ambient air as previously described,¹⁰ and from lean OSA patients (ADISAS, NCT01196845). All subjects provided written informed consent approved by the ethical committee at the Grenoble University Hospital Center (Grenoble, France). The samples were fixed in 4% formaldehyde solution (Sigma-Aldrich, France), embedded in paraffin, and then cut into 5 µm sections for immunohistochemistry, as described above.

## 4.17 | Statistical analyses

Statistical analyses were performed using GraphPad Prism Software (San Diego, California, USA). For each
comparison group, normality and equal variance were
tested using Shapiro-Wilk and Bartlett tests, respec-
tively. Depending on the distribution of data, an un-
paired 2-tailed t-test or a Mann-Whitney test was used
to compare 2 groups. The effect of p53 deletion and im-
pact of CIH were analyzed by two-way ANOVA, with a
subsequent Sidak's post-hoc test. Data are expressed as
mean ± SEM. A p value <0.05 was considered statisti-
cally significant.

5 | CONCLUSION

Our present results represent a step forward in our under-
standing of the detrimental effect of CIH-induced WAT
remodeling on cardiac fibrosis. Specifically, we provide
the proof-of-concept for an early WAT senescence, inde-
pendent of circulating inflammation in both rodents ex-
posed to a short duration of CIH and in human adipose
tissue biopsies. Furthermore, we uncovered a clinical rel-
evant noxious crosstalk between senescent adipose tissue
and heart contributing to cardiac fibrosis. Taken together,
these results highlight the interest to early identify and
target WAT senescence in OSA patients in order to pre-
vent CIH-induced early cardiac remodeling.

**5.1 | Study Limitations**

While 14 days CIH increases gene expression of several
pro-inflammatory and pro-fibrotic factors in the WAT
(TNFα, IL1β, TGFβ, and serpin), it does not alter plasmatic
concentrations of these “prototypical” SASP factors. It is
important to note that our targeted multiplex approach
in the plasma has not allowed the identification of other
SASP factors that could have been secreted by the adipose
tissue in response to CIH¹⁵. We cannot exclude the role
of other factors in this CIH-induced systemic dysregula-
tion, such as liver or kidney dysfunction. Therefore, our
present study cannot make a firm conclusion on specific
circulating factors that could explain the adipose-heart
crosstalk under CIH, and further studies are needed to
clarify the nature of factors secreted by the adipose tissue
and involved in the initiation of cardiac fibrosis.

AUTHOR CONTRIBUTIONS

**Suzain Naushad:** Conceptualization; investigation; formal analysis; data curation; visualization; writing – review and editing; writing – original draft. **Jonathan Gaucher:** Conceptualization; investigation; formal analysis; data curation; visualization; software; writing – original draft; writing – review and editing. **Zaineb Mezdari:** Investigation; formal analysis. **Maximin Détrait:** Conceptualization;

formal analysis; data curation; visualization; writing – review and editing. **Elise Belaidi:** Conceptualization; writing – review and editing. **Yanyan Zhang:** Investigation; methodology; writing – review and editing. **Guillaume Vial:** Investigation; writing – review and editing. **Sophie Bouyon:** Investigation; formal analysis. **Gabor Czibik:** Conceptualization; investigation; writing – review and editing; formal analysis. **Maria Pini:** Conceptualization; investigation; formal analysis; writing – review and editing. **Sahar Aldekwer:** Investigation. **Hao Liang:** Investigation. **Véronique Pelloux:** Investigation. **Judith Aron-Wisnewsky:** Investigation; writing – review and editing. **Renaud Tamisier:** Resources; writing – review and editing. **Jean-Louis Pépin:** Resources; supervision; writing – review and editing. **Geneviève Derumeaux:** Conceptualization; funding acquisition; project administration; supervision; validation; writing – original draft; writing – review and editing. **Daigo Sawaki:** Conceptualization; investigation; formal analysis; methodology; project administration; visualization; validation; supervision; writing – review and editing; writing – original draft. **Claire Arnaud:** Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; supervision; validation; visualization; writing – review and editing; writing – original draft.

ACKNOWLEDGMENTS

We thank C. Gandolphe and X. Decrouy (IMRB Imagery
platform), M. Surenaud (IMRB U955 Luminex platform,
UPEC) and J. Seguin (Opticalimaging platform LIOPA,
Paris Descartes University) for technical contribution.

FUNDING INFORMATION

This work was supported by the Agence Nationale de la
Recherche grant (ANR-20-CE14-0029-01), the Fond de
dotation Agir pour les Maladies Chroniques, and partly
by France Life Imaging grant (ANR-11-INBS-0006). JG
was the recipient of a postdoctoral fellowship from the
ANR-IDEX-UGA Initiative de Recherche Stratégique
(ANR-IDEX-UGA-IRS, Project HypoClock). JG and JLP
are partially funded by the Agence Nationale pour la
Recherche (ANR-19-CE14-0037-01). SN was partially
funded by the “e-health and integrated care and trajecto-
ries medicine and MIAI artificial intelligence” Chairs of ex-
cellence from the Grenoble Alpes University Foundation
(ANR-19- P3IA-0003). GD was partially funded by an
RHU CARMMA grant (ANR-15-RHUS-0003) and a
grant from the Fondation pour la Recherche Médicale
(EQU202003010186).

CONFLICT OF INTEREST STATEMENT

The authors have no conflict of interest.
DATA AVAILABILITY STATEMENT

The data underlying this article are available in the article and in its online supplementary material.

ORCID

Claire Arnaud https://orcid.org/0000-0003-0964-9423

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Naushad S, Gaucher J, Mezdari Z, et al. Chronic intermittent hypoxia triggers cardiac fibrosis: Role of epididymal white adipose tissue senescent remodeling? *Acta Physiol.* 2024;240:e14231. doi:[10.1111/apha.14231](https://doi.org/10.1111/apha.14231)