Sleep fragmentation exacerbates myocardial ischemia-reperfusion injury via hypothalamic paraventricular nucleus-resident OX1R-mediated sympathetic hyperactivity in adult mice

Abstract

Background/Objective

Sleep fragmentation (SF) is a prevalent sleep disorder with an increased risk of cardiovascular diseases. Although epidemiological studies have shown a strong link between SF and adverse cardiac outcomes, specific central neural mechanisms through which SF exacerbates myocardial ischemia-reperfusion injury (MI/RI) are unclear. This study investigated the role of orexin receptor 1 (OX1R) in the hypothalamic paraventricular nucleus (PVN) in SF-induced aggravation of MI/RI and the underlying mechanism using a mouse model.

Materials and methods

C57BL/6 mice were subjected to chronic SF for 16 weeks before MI/RI modeling. Cardiac function was assessed by echocardiography. Sympathetic activity was evaluated based on the heart rate variability analysis. Molecular changes were evaluated by western blotting, qRT-PCR, and immunohistochemistry. The in vivo functional role of OX1R signaling was determined by administering OX1R-specific antagonist SB-334867 via stereotaxic injection into the PVN in the experimental groups of mice.

Results

SF mice exhibited significantly worse cardiac dysfunction and larger infarct areas following MI/RI compared to the controls. This was accompanied by enhanced sympathetic nerve activity and elevated catecholamine levels. Specifically, SF upregulated OX1R expression in the PVN and increased the levels of neuronal activation markers such as c-Fos. Pharmacological blockade of OX1R in the PVN significantly ameliorated SF-induced cardiac dysfunction, reduced the infarct size, and suppressed sympathetic hyperactivity post-MI/RI.

Conclusions

SF may aggravate MI/RI through PVN OX1R-associated sympathetic hyperactivation. Pharmacological inhibition of OX1R improved cardiac outcomes, supporting the involvement of the PVN OX1R-sympathetic pathway in sleep disruption related cardiac injury.

KEY MESSAGES

1. SF exacerbates MI/RI by upregulating OX1R in the hypothalamic paraventricular nucleus, leading to sympathetic hyperactivity and worsened cardiac outcomes.

2. Selective blockade of OX1R in the hypothalamic paraventricular nucleus effectively attenuates SF–induced sympathetic overactivation and myocardial injury.

3. Our findings implicate the PVN OX1R-sympathetic axis in the pathogenesis of SF-induced cardiac injury, providing a basis for future mechanistic research.

Introduction

Sleep is essential for maintaining physiological and psychological health and plays a critical role in homeostasis. Sleep fragmentation (SF) is characterized by frequent and brief awakenings during sleep, which disrupts sleep continuity, and leads to multisystem dysfunction [1,2]. Epidemiological studies have shown that SF is associated with an increased risk of hypertension, coronary artery disease, and heart failure [3–5]. These findings suggest that SF is a significant contributor to cardiovascular pathophysiology.

Myocardial ischemia-reperfusion injury (MI/RI) is a major clinical challenge because of its complex pathogenesis and poor prognosis. MI/RI is caused by tissue damage during restoration of blood flow after ischemia [6,7]. The key pathological features of MI/RI include cardiomyocyte apoptosis and necrosis [8], inflammatory infiltration [7], microvascular dysfunction [9], and oxidative stress [10], all of which contribute to impaired cardiac function and increased risk of heart failure [11]. Hyperactivation of the sympathetic nervous system is a key pathological mechanism that contributes to these processes [11–13] and is associated with increased myocardial oxygen demand, vasoconstriction, arrhythmogenesis, and inflammation [6,11].

The CNS plays a key role in regulating the autonomic nervous system and the paraventricular nucleus (PVN) of the hypothalamus is a key integrative center of autonomic control [14,15]. The PVN coordinates responses to stress by integrating signals from various brain regions and the periphery, including inflammation and metabolic stress [16]. PVN exerts its effects through activation of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system, leading to increased secretion of catecholamines and vascular reactivity [17]. Hyperactivity of the PVN is implicated in cardiovascular diseases such as hypertension and heart failure [15,18,19]. Furthermore, activation of the ventrolateral part of the ventromedial hypothalamus (VMHvl) enhances cardiac sympathetic activity through the PVN-superior cervical ganglion axis, leading to worsening of post-infarction outcomes [15]. Chronic SF increases sympathetic activity through disrupted sleep architecture, intermittent hypoxia, and activation of the HPA axis, leading to elevated catecholamine levels [20–22]. Given the PVN’s central role in autonomic regulation, SF may aggravate MI/RI by disturbing hypothalamic signaling, particularly within the PVN. However, the neural mechanisms involved in this process remain to be clarified.

PVN activity is regulated by multiple neurotransmitters and neuropeptides, including glutamate, γ-aminobutyric acid (GABA), and norepinephrine, orexin, vasopressin, and thyrotropin-releasing hormone [23–26]. Among these, the orexin signaling system, especially orexin receptor 1 (OX1R), has emerged as a critical regulator of PVN excitability and autonomic output [27–30]. OX1R is expressed in the PVN neurons and is involved in the regulation of arousal, energy homeostasis, and autonomic function [31]. SF activates orexinergic neurons in the lateral hypothalamus (LH), which in turn stimulates OX1R in the PVN [32]. This suggests that elevated OX1R signaling within the PVN during SF may aggravate MI/RI by enhancing sympathetic neuronal activity and promoting cardiovascular dysfunction.

Therefore, in this study, we aimed to determine the role of PVN OX1R signaling in SF-aggravated MI/RI and its underlying mechanisms. Towards this, we established an SF model in the adult C57BL/6 mice to determine changes in OX1R expression levels, sympathetic neuronal biomarkers (tyrosine hydroxylase), CNS activation (c-Fos), cardiac function, and heart rate variability. Furthermore, we evaluated whether pharmacological modulation of PVN OX1R signaling would ameliorate MI/RI in the SF model mice.

Materials and methods

Animal ethics statement

All animal experiments were conducted in accordance with the ARRIVE guidelines 2.0 and approved by the Animal Ethics Committee of Xiangya Hospital, Central South University (Changsha, China; license code: 2021111240). All animal procedures were performed in accordance with the Guidelines for the Euthanasia of Animals, 2020 Edition, published by the American Veterinary Medical Association. At the end of the experiments, mice were euthanized by inhalation of sevoflurane until complete unconsciousness, followed by cervical dislocation to ensure death. Six-to-eight-week old male C57BL/6 mice weighing 20–25 g were obtained from the SJA Laboratory (Hunan, China). Mice were housed in standard plastic cages under controlled conditions (temperature: 22-26 °C; humidity: 50-60%; 12-hour light/dark cycle) and provided unrestricted access to food and water. The mice were also allowed a 1-week acclimation period to adjust in the laboratory environment prior to the experiments.

Animal grouping

To investigate whether chronic SF aggravates MI/RI, adult male C57BL/6 mice (6–8 weeks old) were randomly assigned into the following four groups (n = 9 per group): (1) Sham group mice, which underwent thoracotomy without coronary artery ligation or reperfusion; (2) SF+Sham group mice, which were subjected to SF for 16 consecutive weeks using a motorized sleep deprivation apparatus, followed by thoracotomy without coronary artery ligation or reperfusion; (3) MI/RI group mice, which underwent 30 min of myocardial ischemia followed by reperfusion but did not undergo prior SF exposure; and (4) SF+MI/RI group mice, which were pre-exposed to 16 weeks of SF prior to MI/RI modeling. This grouping was designed to evaluate whether SF alone affected cardiac structure and function, and exacerbated cardiac injury following MI/RI.

To further assess the impact of SF on cardiac autonomic regulation, particularly sympathetic neuronal activity, additional mice from the following four groups (n = 8–10 per group) were implanted with subcutaneous bioelectrical leads in the lower left chest and the right pectoral muscle: (1) Sham group, (2) SF+Sham group, (3) MI/RI group, and (4) SF+MI/RI group. Telemetric recordings of heart rate and heart rate variability (HRV) parameters were obtained under resting conditions and after MI/RI to evaluate the sympathetic tone and the contribution of SF to sympathetic hyperactivity.

To investigate the role of OX1R signaling in the PVN of the hypothalamus in SF-induced sympathetic activation and cardiac injury, we included the following three groups of mice (n = 6 per group): (1) MI/RI group mice, which underwent myocardial ischemia for 30 min followed by reperfusion without SF exposure or PVN intervention; (2) SF+MI/RI+Vehicle group mice, which were exposed to 16 weeks of SF, followed by bilateral stereotaxic implantation of microinjection cannulas targeting the PVN, and were administered vehicle solution one week prior to MI/RI modeling; and (3) SF+MI/RI+SB-334867 group mice, which received microinjection of the selective OX1R antagonist SB-334867 into the PVN following the same protocol. This design aimed to determine whether pharmacological blockade of OX1R in the PVN attenuated SF-induced cardiac dysfunction and sympathetic overactivation.

Randomization was conducted by an independent biostatistician using the SPSS 25.0 software (IBM, USA). Group assignments were concealed in sequentially numbered opaque envelopes until the end of the study. All surgical procedures, data collection, and analyses were performed by investigators blinded to group allocation.

Sleep fragmentation (SF) model

SF was performed in mice according to our previously published protocol [33]. Briefly, mice were housed in a sleep deprivation chamber (Model ZL-013; Anhui Yaokun Biotechnology). During the light phase (ZT0-12) when the mice are usually inactive and asleep, a motorized bar was swept through the cage at 2-minute intervals to interrupt sustained sleep. During dark phase (ZT12-24) when the mice are usually active, the bar remained stationary allowing undisturbed sleep. The control mice were housed in identical chambers without bar movement.

Myocardial ischemia-reperfusion injury (MI/RI) model

MI/RI was induced in mice according to a previously established protocol [33]. Briefly, mice were anesthetized and subsequently underwent endotracheal intubation and mechanical ventilation. A left lateral mini-thoracotomy was performed by making a small incision in the chest wall between the ribs at the fourth or fifth intercostal space to expose the heart. The left anterior descending (LAD) coronary artery was ligated with a 10-0 prolene suture (Ningbo Medical, Lingqiao) that was placed approximately 2–3 mm from its origin. Successful occlusion in the coronary artery was confirmed by ST-segment elevation on an electrocardiogram and a color change in the myocardium distal to the ligation site. The sutures were released after 30 min of ischemia to initiate reperfusion and was visually confirmed. After surgery, mice were kept on a warming pad to prevent hypothermia and promote faster recovery. Then, they were returned to their cages and provided with ad libitum access to moistened food and water throughout the reperfusion phase. Sham-operated control mice underwent the same surgical procedures, except ligation of the LAD.

Echocardiography assessments

Transthoracic echocardiography was performed to assess cardiac function in mice after MI/RI. Mice were lightly anesthetized and placed on the examination table in a supine position. Their heart rates were maintained between 400 and 500 beats per minute. No specific temperature regulatory methods were used. After depilation of the chest area, ultrasound gel was applied to ensure adequate acoustic coupling. Echocardiography was performed using a Mindray M9CV system (Shenzhen Mindray Bio-Medical Electronics) equipped with an L16-4Hs (4–16 MHz) transducer. M-mode images were acquired at the level of the papillary muscles and heart functional parameters such as left ventricular ejection fraction (LVEF), LV fractional shortening (LVFS), LV internal diameter at systole (LVIDs) and diastole (LVIDd). LVEF and LVFS were calculated using the standard formulas according to previously reported protocols [33].

Estimation of cardiac autonomic function

Telemetry monitoring was used to assess cardiac autonomic function in mice. After anesthesia, mice were implanted with a sterilized telemetry probe (MT 10B, AD Instruments) beneath their dorsal skin. The electrodes of the probes were attached to the sternocleidomastoid muscles and extended from the suprasternal notch to a point just behind the xiphoid process. After wound closure, the animals were placed on a telemetry recording platform and permitted unrestricted movement during data collection. Continuous electrocardiographic (ECG) signals were recorded for 2–10 min using the LabChart software (version 8.1.24, AD Instruments, Australia) and HRV analysis was performed with the built-in modules of the software. Time-domain analysis included estimation of the standard deviation of RR intervals (SDNN). Frequency-domain analysis included quantifying the low-frequency (LF, 0.20–0.75 Hz) and high-frequency (HF, 0.75–2.5 Hz) components.

Bilateral PVN microinjection

SF mice underwent stereotaxic cannula implantation surgery as previously described [34–38]. Mice were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and positioned in a stereotaxic apparatus. After skin incision and craniotomy above the PVN, microinjection cannulas (Shanghai Yuyan Scientific Instrument Co., YAN-3100) were slowly inserted in the bilateral PVN coordinates (AP = −0.5 mm; ML = ± 0.2 mm; DV = −4.2 mm from dura mater) and secured with dental cement (Shanghai Yuyan Scientific Instrument Co., YAN-2947). The cannula placements were verified using methylene blue labeling and examination of coronal brain sections under bright-field microscopy (Figure S1), to confirm accurate targeting and assess potential drug diffusion within the PVN region.

After 16 weeks of SF intervention, the mice received daily microinjections of SB-334867 or vehicle for one week prior to MI/RI modeling. The injection system consisted of a 0.5 μL Hamilton microsyringe (Shanghai Yuyan Scientific Instrument Co., Model 7000.5 KH SYR) connected to the cannula via a PE-20 polyethylene tubing. SB-334867 was slowly administered at 30 nL/min into the bilateral PVN (70 nL per site), whereas the control mice received equivalent volumes of the vehicle volumes. After completing the injections, the cannulas remained in position for 10 min before gradual withdrawal. All the procedures were conducted under sterile conditions according to standard stereotaxic surgery protocols.

TTC staining

Myocardial infarct size was evaluated following MI/RI. After excision, hearts were thoroughly rinsed with phosphate-buffered saline (PBS) to remove residual blood. Then, they were stored at −20 °C for 15 min to stiffen the tissues that would aid with sectioning. Then, the hearts were sliced horizontally into five equal sections. The slices were incubated in 1% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma, T8877) at 37 °C for 20 ± 5 min, followed by fixation in 4% paraformaldehyde for 24 h. The viable myocardium stained red, but the infarcted tissue remained unstained or pale. Infarct size was quantified using ImageJ software (version 1.53t) and expressed as the percentage of infarcted area relative to total left ventricular area [33]. The coefficient of variation (CV) was additionally reported to describe relative within-group dispersion of infarct size.

RT-qPCR

Total RNA was extracted from the PVN using the E.N.Z.A. Total RNA Kit (Transgene, ER501-01-V2) according to the manufacturer’s instructions. Reverse transcription was performed using the First Strand cDNA Reverse Transcription SuperMix (Transgene, AU341-02-V2). Quantitative real-time PCR was conducted in an ABI QuantStudio 7 system (Applied Biosystems) using the SYBR Green qPCR SuperMix (GeneCopoeia, QP001). GAPDH was used as the internal control. Specific qPCR primers for the target genes of interest and GAPDH are listed in Supplementary Table S1. Threshold cycle (Ct) values were used to estimate the relative gene expression with the 2-^ΔΔCt method after normalization with GAPDH mRNA expression levels.

Immunofluorescence staining

Mice were anesthetized using sevoflurane inhalation. Then, transcardial perfusion was performed with 0.01 M PBS to clear blood from the tissues. Subsequently, perfusion was performed with 4% paraformaldehyde solution for fixation. The brain tissues were harvested and immersed in 4% paraformaldehyde solution for an additional 24 h at 4 °C. Then, they were sequentially dehydrated in 0.01 M PBS containing 15% and 30% sucrose at 4 °C. The brain tissues were then embedded in optimal cutting temperature compound and sectioned into 30 μm thick coronal slices using a cryostat (Leica CM1950, Wetzlar, Germany). For the immunofluorescence assay, PVN sections were first washed in 0.1 M PBS for 10 min, blocked with 5% goat serum at room temperature for 60 min, and then incubated overnight at 4 °C with primary antibodies such as rabbit monoclonal anti-c-Fos (Abcam, 1:1000) and rabbit polyclonal anti-OX1R-488 (Alomone labs, 1:250). After washing thrice in 0.1 M PBS for 10 min each, the sections were incubated with Alexa Fluor 488 or 594-labeled secondary antibodies for 1 h in the dark. Then, the sections were mounted on slides and images were acquired using a fluorescence microscope to visualize fluorescent signals.

Immunohistochemical analysis

Paraffin-embedded murine cardiac tissue sections were incubated at 60 °C for 6 h, followed by dewaxing in xylene and sequential rehydration with graded ethanol solutions (twice with absolute ethanol and once each with 95%, 85%, and 75% ethanol). Antigen retrieval was performed by immersing slides in preheated retrieval buffer and boiling in the water bath for 20 min. After cooling, endogenous peroxidase activity was blocked with a 3% hydrogen peroxide solution. The sections were then incubated overnight at 4 °C with a rabbit monoclonal anti-TH antibody (Abcam, ab137869). Diaminobenzidine was used for color development. Protein localization and staining intensity were visualized using a light microscope.

Western blotting

Total protein was extracted from the bilateral PVN tissues using the RIPA lysis buffer (NCM, WB2100) supplemented with PMSF (Byotime, ST507) and phosphatase inhibitors (NCM, P002). Protein concentrations were determined using a BCA protein assay kit (NCM, WB6501). Equal amounts of protein (30 μg per lane) were separated by SDS-PAGE (4-20%, Beyotime) and transferred onto PVDF membranes (Millipore). The membranes were then blocked with 5% skim milk in PBST and incubated overnight at 4 °C with primary antibodies against tyrosine hydroxylase (TH, Abcam, 1:1000), c-Fos (Abcam, 1:2000), or OX1R (Alomone labs, 1:500). GAPDH (Beyotime, 1:1000) was used as the loading control. Subsequently, the blots were incubated HRP-conjugated AffiniPure goat anti-rabbit IgG (H + L) (1:10000, Jackson, 111-035-144). The protein bands were developed with the ECL reagent (MCE, HY-K1005). Protein bands were acquired using the GE IMAGEQUANTMT 800 CCD imager and quantified using the ImageJ software (version 1.53t). The target protein signals were normalized to GAPDH.

ELISA to estimate plasma and myocardial norepinephrine and epinephrine levels

Blood samples from mice were collected from the orbital vein and transferred into the EDTA-treated tubes (1.5 mg/ml) to isolate plasma samples. Furthermore, heart tissue lysates were prepared from 100 mg LV myocardial samples. Plasma concentrations of norepinephrine (NE) and epinephrine (EPI) were measured using enzyme-linked immunosorbent assay (ELISA) kits from Wuhan Huamei Biotech Co. LTD.,(CUSABIO) (CSB-E07870m and CSB-E08679) according to the manufacturer’s protocols. NE and EPI levels in the tissue homogenates were assessed using ELISA kits from the Shanghai Jianglai Biotechnology (JL13969-48T and JL11194-48T). The absorbance was measured at 450 nm using a microplate reader (MQX 200, BioTek Instruments).

Statistical analysis

Statistical analysis was performed using the GraphPad Prism 9.0 (GraphPad Software Inc., USA) and SPSS 25.0 (IBM, USA) software. Statistical data are presented as mean ± standard error of the mean (SEM) and based on at least three independent experiments. The comparisons between two groups were assessed using a two-tailed unpaired Student’s t-test. Differences among three or more groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test for pairwise group comparisons. p < 0.05 was considered statistically significant.

Results

Chronic SF exacerbates MI/RI and excessive infiltration of inflammatory cells in the myocardium

To determine whether sleep disturbance exacerbates MI/RI, C57BL/6 mice were subjected to chronic SF for 16 weeks prior to MI/RI modeling (Figure 1A). Then, we assessed whether SF independently affected cardiac function. Cardiac functional parameters, including LVEF, LVFS, LVIDd, and LVIDs, were comparable between the Sham and SF+Sham groups and did not show statistically significant differences (Figure 1B–F). Furthermore, TTC staining demonstrated absence of myocardial necrosis in the SF+Sham group compared to the Sham group (Figure 1G–H). This confirmed that chronic SF did not directly alter cardiac structure or function. However, the SF+MI/RI group mice exhibited decreased LVEF (one-way ANOVA, F_{(3, 32)} =68.87, P_adj < 0.0001) and LVFS (one-way ANOVA, F_{(3, 32)} = 41.64, P_adj = 0.0303) as well as increased LVIDs (one-way ANOVA, F_{(3, 32)} = 45.37, P_adj = 0.0385) compared to the MI/RI group (Figure 1B–F). Moreover, TTC staining results demonstrated that the infarct areas were significantly larger in the SF+MI/RI group compared to the MI/RI group (one-way ANOVA, F_{(3, 12)} = 278.3, P_adj < 0.0001) (Figure 1G–H). H&E staining data demonstrated extensive loss of cardiomyocytes and higher infiltration of inflammatory cells in the SF+MI/RI group compared to MI/RI group (Figure 1I). Collectively, these findings suggested that although chronic SF alone did not directly alter the myocardial structure and function, it significantly exacerbated both functional deterioration and structural damage following MI/RI. This indicated that sleep disturbance was a significant risk factor for adverse post-MI outcomes.

Figure 1.

Chronic SF exacerbates MI/RI-induced cardiac dysfunction and myocardial injury in mice.

(A) Schematic representation of the experimental protocol. C57BL/6 mice underwent chronic SF for 16 weeks, followed by MI/RI modeling. Echocardiographic assessments and tissue collection were performed at the designated time points. (B) Representative M-mode echocardiographic tracings show the status of left ventricular function in the Sham, SF, MI/RI, and SF+MI/RI groups of mice. (C-F) Echocardiography assessment of cardiac functional parameters, (C) LVEF, (D) LVFS, (E) LVIDd, and (F) LVIDs in the Sham, SF, MI/RI, and SF+MI/RI groups of mice. Data are presented as mean ± SEM; n = 8–10 mice per group. (G) Representative images of TTC-stained cardiac sections show infarct areas (pale regions) and viable myocardium (red regions) in Sham, SF, MI/RI, and SF+MI/RI groups of mice. Coefficients of variation (CVs) for infarct size were 50.6% (Sham), 16.1% (SF+Sham), 13.9% (MI/RI), and 6.3% (SF+MI/RI). Sham group was due to its near-zero infarct size rather than true variability. Scale bar (left panel) = 2000 μm. (H) Quantitative analysis shows the infarct size expressed as percentage of total left ventricular area in the Sham, SF, MI/RI, and SF+MI/RI groups of mice. Data are presented as mean ± SEM; n = 6–8 mice per group. (I) Representative H&E-stained left ventricular sections demonstrate the myocardial structural changes and infiltration status of the inflammatory cells. The boxed regions represent areas visualized at higher magnification. Scale bar (upper panel) = 2000 μm; scale bar (lower panel) = 100 μm. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. * P_adj < 0.05, ** P_adj < 0.01, *** P_adj < 0.001, **** P_adj < 0.0001.

SF induces cardiac sympathetic hyperactivity

Sympathetic hyperactivity is a characteristic feature of several sleep disorders and frequently compromises cardiovascular function [39]. To evaluate cardiac autonomic regulation, bioelectric leads were subcutaneously implanted in the lower left chest and right pectoral muscle (Figure 2A). Our analysis demonstrated that SF significantly elevated HR compared with the Sham group (one-way ANOVA, F_{(3, 36)} = 71.28, P_adj < 0.0001), and this effect was more pronounced following MI/RI (one-way ANOVA, F_{(3, 36)} = 71.28, P_adj = 0.001) (Figure 2B and C). Heart rate variability parameters in both the frequency and time domains were used to assess cardiac autonomic function by estimating the ratio of LF/HF and SDNN. The LF/HF ratio was significantly higher in the SF+Sham group compared to the Sham group (one-way ANOVA, F_{(3, 36)} = 27.99, P_adj < 0.0001) and further augmented by MI/RI (one-way ANOVA, F_{(3, 36)} = 27.99, P_adj = 0.0007) (Figure 2D and E). Conversely, SDNN was significantly reduced in the SF+Sham group compared to the Sham group (one-way ANOVA, F_{(3, 26)} = 19.37, P_adj = 0.0243) (Figure 2F and G).

Figure 2.

SF induces cardiac sympathetic hyperactivity and autonomic dysfunction.

(A) Schematic representation shows the placement of bioelectric leads that were subcutaneously implanted in the lower left chest and the right pectoral muscle for continuous electrocardiographic monitoring. (B) Representative ECG tracings show the heart rate patterns in the Sham, SF+Sham, MI/RI, and SF+MI/RI groups of mice. (C) Quantitative analysis of resting HR in the Sham, SF+Sham, MI/RI, and SF+MI/RI groups of mice. HR is expressed in beats per minute (bpm). Data are presented as mean ± SEM; n = 8–10 mice per group. (D) Representative power spectral density plots show the frequency domain measures of HRV for the low frequency (LF) and high frequency (HF) components in the Sham, SF+Sham, MI/RI, and SF+MI/RI groups of mice. (E) Quantitative analysis of the LF/HF ratio in Sham, SF+Sham, MI/RI, and SF+MI/RI groups. Data are presented as mean ± SEM; n = 8–10 mice per group. (F) Representative Poincaré plots demonstrate the time domain measures of HRV in the Sham, SF+Sham, MI/RI, and SF+MI/RI groups of mice. (G) Quantitative analysis of standard deviation of normal-to-normal intervals (SDNN) in the Sham, SF+Sham, MI/RI, and SF+MI/RI groups of mice. SDNN is expressed in milliseconds and is a measure of overall HRV. Data are presented as mean ± SEM; n = 8–10 mice per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. * P_adj < 0.05, ** P_adj < 0.01, *** P_adj < 0.001, **** P_adj < 0.0001.

SF enhances sympathetic nerve activity in the myocardium and PVN

Since sympathetic nervous system plays a pivotal role in MI/RI pathogenesis, we further elucidated the mechanisms underlying SF-mediated alterations in the cardiac sympathetic function. Immunohistochemical analysis demonstrated that the density of TH-positive fibers in the myocardium was significantly higher in the SF+Sham group compared to the Sham group (one-way ANOVA, F_{(3, 12)} = 52.77, P_adj < 0.0001) and further elevated in the SF+MI/RI group after MI/RI modeling (one-way ANOVA, F_{(3, 12)} = 52.77, P_adj < 0.0001) (Figure 3A and B). Western blotting results confirmed that the expression levels of the myocardial TH protein were significantly elevated in the SF+Sham group compared to the Sham group (one-way ANOVA, F_{(3, 8)} = 26.14, P_adj = 0.0189) and further augmented in the SF+MI/RI group after MI/RI modeling (one-way ANOVA, F_{(3, 8)} = 26.14, P_adj = 0.0067) (Figure 3C and D). These protein-level changes were accompanied by corresponding changes in the neurotransmitter levels of the sympathetic nervous system. ELISA results showed that the myocardial and plasma norepinephrine levels were significantly higher in the SF group mice than in the Control group mice (Unpaired t test, t = 4.90, p = 0.0004) and the plasma epinephrine levels were significantly elevated in the SF group compared with the Control group (Unpaired t test, t = 4.67, p = 0.0005) (Figure 3E and F). These findings collectively demonstrate that SF serves as a critical driver of enhanced sympathetic activity and promotes both local cardiac sympathetic innervation and systemic catecholamine release. To investigate the central mechanisms underlying this SF-induced sympathetic activation, we analyzed neuronal activity in the PVN, which is a crucial regulator of both cardiovascular function as well as sleep-wake control [40,41]. Immunostaining data demonstrated that the number of c-Fos-positive neurons were significantly higher in the PVN region of the SF mice compared to the PVN region of the Control group mice (Unpaired t test, t = 7.87, p < 0.0001) (Figure 3G and H). This demonstrated significant alterations in the CNS activation status.

Figure 3.

SF enhances sympathetic nerve activity in the myocardium and PVN.

(A) Representative immunohistochemical staining demonstrates TH-positive sympathetic nerve fibers (brown) in the myocardial tissues in the Sham, SF+Sham, MI/RI, and SF+MI/RI groups of mice. The boxed regions are displayed at higher magnification. Scale bar (upper panel) = 2000 μm; scale bar (lower panel) = 100 μm. (B) Quantitative analysis of TH-positive fiber density in the myocardial tissues of the Sham, SF+Sham, MI/RI, and SF+MI/RI groups of mice. Data are presented as mean ± SEM; n = 3 mice per group. (C) Representative western blot shows TH protein expression in the myocardial tissue lysates from the Sham, SF+Sham, MI/RI, and SF+MI/RI groups of mice. GAPDH is used as the loading control. (D) Quantitative assessment of the relative expression levels of TH protein normalized to GAPDH in the myocardial tissues in the Sham, SF+Sham, MI/RI, and SF+MI/RI groups of mice. Data are presented as mean ± SEM; n = 3 mice per group. (E) ELISA results show the levels of myocardial norepinephrine (NE) and epinephrine (EPI) in the Control and SF groups of mice. Data are presented as mean ± SEM, n = 7 mice per group. (F) ELISA results show the plasma levels of NE and EPI in in the Control and SF groups of mice. Data are presented as mean ± SEM; n = 7 mice per group. (G) Representative immunofluorescence staining shows the c-Fos-positive neurons (red) in the PVN region of the Control and SF groups of mice. The nuclei were counterstained with DAPI (blue). Scale bar = 200 μm. (H) Quantitative analysis of the number of c-Fos-positive neurons in the PVN of the Control and SF groups of mice. Data are presented as mean ± SEM; n = 6 mice per group. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc multiple-comparisons test (B, D),with significance reported as adjusted P-values (P_adj): * P_adj < 0.05, ** P_adj < 0.01, *** P_adj < 0.001, **** P_adj < 0.0001. Unpaired two-tailed Student’s t-test was used for pairwise comparisons (E, F, H), with significance reported as P-values: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

SF upregulates OX1R expression in the PVN

Various receptors within the PVN play significant roles in neuronal activation and signal transduction [29,39–42]. Our previous data showed that SF activates neurons in the PVN. Therefore, we used RT-qPCR analysis to systematically screen key receptors involved in neural signaling within the PVN, including mineralocorticoid receptor (MR), N-methyl-D-aspartate receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), orexin receptor 2 (OX2R), and OX1R. RT-qPCR results demonstrated that relative to the Control group, SF group mice exhibited significantly higher OX1R mRNA levels in the PVN region (Unpaired t test, t = 8.38, p < 0.0001), but the mRNA expression levels of other receptors (MR, NMDAR, AMPAR, and OX2R) remained unchanged (Figure 4A). This suggested that OX1R was a potential key mediator in SF-induced PVN activation. To further validate this finding, we performed immunofluorescence staining of OX1R. Compared to the Control group, PVN region in the SF group mice exhibited significantly larger areas of OX1R-specific immunoreactivity (Unpaired t test, t = 4.26, p = 0.0131) (Figure 4B and C). Western blot analysis also demonstrated that OX1R protein expression levels were significantly elevated in the PVN region of the SF mice relative to the Control group (Unpaired t test, t = 4.18, p = 0.0139) (Figure 4D and E).

Figure 4.

SF upregulates OX1R expression in the PVN.

(A) RT-qPCR analysis results show the relative mRNA expression levels of neural signaling receptors in the PVN region, including mineralocorticoid receptor (MR), N-methyl-D-aspartate receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), orexin receptor 2 (OX2R), and OX1R in the Control and SF groups of mice. The expression levels were normalized to GAPDH. The data are expressed as mean ± SEM; n = 6 mice per group. (B) Representative immunofluorescence staining images show OX1R expression (green) in the PVN region of the Control and SF groups of mice. The nuclei were stained with DAPI in blue. The boxed regions are shown at higher magnification. Scale bar = 100 μm. (C) Quantification of OX1R-positive signal area in the PVN region of the Control and SF groups of mice. The data are expressed as mean ± SEM; n = 3 mice per group. (D) Representative western blot shows expression levels of the OX1R protein in the PVN tissue lysates from the Control and SF groups of mice. GAPDH was used as loading control. (E) Quantification of relative OX1R protein levels normalized to GAPDH in the PVN tissue lysates from the Control and SF groups of mice. The data are expressed as mean ± SEM; n = 3 mice per group. Statistical analysis was performed using unpaired two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001.

Blockade of OX1R in PVN attenuates SF-aggravated MI/RI

To further validate the functional role of OX1R in SF-induced aggravation of MI/RI, OX1R signaling was inhibited using a specific antagonist SB-334867, which is extensively used to investigate the role of OX1R in various pathophysiological processes. In our experimental design (Figure 5A), stereotaxic techniques were used to implant microinjection cannulas in the PVN region of mice, followed by administration of SB-334867 or vehicle for one week prior to MI/RI modeling. Compared to the MI/RI group, the SF+MI/RI+Vehicle group demonstrated pronounced cardiac dysfunction characterized by significantly reduced LVEF (one-way ANOVA, F_{(2, 15)} = 64.53, P_adj = 0.0027) and LVFS (one-way ANOVA, F_{(2, 15)} =32.99, P_adj = 0.036) (Figure 5B and E), higher loss of myocardial cells and infiltration of inflammatory cells based on H&E staining (Figure 5F), significantly larger myocardial infarct area based on TTC staining (one-way ANOVA, F_{(2, 9)} = 53.81, P_adj = 0.0001) (Figure 5G and H). In addition, HRV analysis revealed elevated resting HR, an increased LF/HF ratio, and reduced SDNN (Figure 5I–M). Conversely, compared to the SF+MI/RI+Vehicle group, the SF+MI/RI+SB-334867 group exhibited significantly improved cardiac function, which was manifested by significantly higher LVEF (one-way ANOVA, F_{(2, 15)} = 64.53, P_adj < 0.0001) and LVFS (one-way ANOVA, F_{(2, 15)} = 32.99, P_adj  < 0.0001), and significantly decreased LVIDd (one-way ANOVA, F_{(2, 15)} = 21.26, P_adj  < 0.0001) (Figure 5B–E), significant reduction in the loss of myocardial cells and infiltration of inflammatory cells (Figure 5F) (one-way ANOVA, F_{(2, 9)} = 53.81, P_adj < 0.0001) (Figure 5G and H), and suppressed sympathetic nerve activity based on lower HR (one-way ANOVA, F_{(2, 12)} =14.36, P_adj  = 0.0006) and LF/HF ratio (one-way ANOVA, F_{(2, 12)} = 37.55, P_adj  < 0.0001), and elevated SDNN (one-way ANOVA, F_{(2, 12)} = 12.31, P_adj  = 0.0012) (Figure 5I–M). These findings collectively established that OX1R signaling was a pivotal pathway in SF-induced aggravation of MI/RI, and targeted blockade of OX1R in the PVN region effectively ameliorates SF-induced sympathetic hyperactivity and mitigates exacerbated cardiac injury.

Figure 5.

Blockade of OX1R in PVN attenuates SF-induced aggravation of MI/RI.

(A) Schematic diagram of the experimental design. Microinjection cannulas were stereotaxically implanted in the PVN region, followed by pretreatment with OX1R antagonist SB-334867 or vehicle for one week prior to MI/RI modeling. (B) Representative M-mode echocardiographic images of the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. (C-E) Echocardiography assessments of cardiac function parameters such as (C) LVEF, (D) LVFS, and (E) LVIDd in the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. The data are expressed as mean ± SEM; n =6 mice per group. (F) Representative H&E-stained images of left ventricular sections showing myocardial structure and infiltration of inflammatory cells in the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. Scale bar (upper panel) = 2000 μm; scale bar (lower panel) = 100 μm. (G) Representative images of TTC-stained heart sections show the infarct areas (pale) and viable myocardium (red) in MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. Coefficients of variation (CVs) for infarct size were 22.1% (MI/RI), 11.0% (SF+MI/RI+Vehicle), and 7.2% (SF+MI/RI+SB-334867). (H) Quantification of infarct size as a percentage of left ventricular area in the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. The data are expressed as mean ± SEM; n = 4 mice per group. (I) Representative ECG traces for all the groups of mice. (J) Quantification of resting HR in beats per minute (bpm) in the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. The data are expressed as mean ± SEM; n = 5 mice per group. (K) Representative power spectral density plots show the frequency domain parameters of HRV for all the groups of mice. (L) Quantification of LF/HF ratio in the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. The data are expressed as mean ± SEM; n = 5 mice per group. (M) Quantification of SDNN in milliseconds in the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. The data are expressed as mean ± SEM; n = 5 mice per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. * P_adj < 0.05, ** P_adj < 0.01, *** P_adj < 0.001, **** P_adj < 0.0001.

SB-334867 protects against aggravated MI/RI by inhibiting PVN neuronal activation and sympathetic excitation

Next, we investigated the mechanistic role of OX1R by using the OX1R-specific antagonist SB-334867 and systematically assessing the expression levels of neuronal activation marker c-Fos, sympathetic neuronal activity marker TH, and OX1R in the PVN region across various experimental groups. Western blot analysis demonstrated that relative to the MI/RI group, the SF+MI/RI+Vehicle group mice exhibited significantly higher c-Fos expression in the PVN (one-way ANOVA, F_{(2, 6)} = 23.07, P_adj = 0.0279), increased TH expression in the myocardial tissue (one-way ANOVA, F_{(2, 6)} = 8.64, P_adj = 0.0187), and upregulated OX1R expression in the PVN (one-way ANOVA, F_{(2, 6)} = 7.51, P_adj  = 0.0338) (Figure 6A–D). These findings were further corroborated by immunofluorescence staining, which showed significantly higher number of c-Fos-positive neurons in the PVN of the SF+MI/RI+Vehicle group mice (one-way ANOVA, F_{(2, 9)} = 160.8, P_adj  < 0.0001) (Figure 6E and F). Immunohistochemistry experiments demonstrated enhanced TH-positive sympathetic fiber density in the myocardial tissue(one-way ANOVA, F_{(2, 6)} = 306.3, P_adj < 0.0001) (Figures 6G and H). This demonstrated that SF exacerbated MI/RI-induced PVN activation in the CNS and peripheral sympathetic neuronal activity in the heart. To test whether SB-334867 could reverse these effects, Western blot analysis showed that SB-334867 pretreatment significantly attenuated SF-induced c-Fos overexpression in the PVN (one-way ANOVA, F_{(2, 6)} = 23.07, P_adj = 0.0012), which was further confirmed by immunofluorescence staining (one-way ANOVA, F_{(2, 9)} = 160.8, P_adj < 0.0001). Similarly, Western blot analysis demonstrated a marked reduction in TH expression in myocardial tissue in the SF+MI/RI+SB-334867 group compared to the SF+MI/RI+Vehicle group (one-way ANOVA, F_{(2, 6)} = 8.64, P_adj = 0.0428), and this finding was corroborated by immunohistochemistry (one-way ANOVA, F_{(2, 6)} = 306.3,P_adj < 0.0001). Compared to the SF+MI/RI+Vehicle group, SB-334867 pretreatment effectively suppressed SF-induced hyperactivation of both the central nervous system (PVN) and the peripheral sympathetic nervous system (heart). Notably, Western blot analysis showed that the OX1R expression levels in the PVN were comparable between the SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups. This indicated that SB-334867 exerted its cardioprotective effects through functional antagonism of OX1R signaling rather than receptor downregulation, thereby resulting in the inhibition of PVN activation and sympathetic excitation and subsequent amelioration of SF-induced exacerbation of MI/RI-induced cardiac injury.

Figure 6.

SB-334867 protects against SF-induced aggravation of MI/RI by inhibiting PVN neuronal activation and sympathetic excitation.

(A) Representative western blots show c-Fos expression levels in the PVN tissue lysates, TH expression levels in the myocardial tissue lysates, and OX1R expression in the PVN tissue lysates from the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. GAPDH was used as loading control. (B-D) Quantification of relative protein expression levels of (B) c-Fos in PVN, (C) TH in myocardium, and (D) OX1R in PVN, normalized to GAPDH. The data are expressed as mean ± SEM, n = 3 mice per group. (E) Representative immunofluorescence staining images show the c-Fos-positive neurons (red) in the PVN region of the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. The nuclei were stained with DAPI in blue. Scale bar = 200μm. (F) Quantification of c-Fos-positive neurons in the PVN region of the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. The data are expressed as mean ± SEM; n = 4 mice per group. (G) Representative immunohistochemical staining images show the TH-positive sympathetic nerve fibers (brown) in the myocardial tissues from the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. Scale bar (upper panel) = 2000 μm; scale bar (lower panel) = 100 μm. (H) Quantification of TH-positive fiber density in the myocardial tissue of the MI/RI, SF+MI/RI+Vehicle and SF+MI/RI+SB-334867 groups of mice. The data are expressed as mean ± SEM. n = 3 mice per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. * P_adj < 0.05, ** P_adj < 0.01, *** P_adj < 0.001, **** P_adj < 0.0001.

Discussion

Our study demonstrates that SF exacerbates MI/RI through hyperexcitation of the sympathetic nervous system via upregulation of PVN-resident OX1R. The cardiovascular system is sensitive to changes in sleep quality. Several studies have suggested that SF exacerbates myocardial damage through enhanced inflammation and oxidative stress [42,43]. Hypothalamus, a central regulator of the autonomic nervous system, plays a crucial role in SF-induced cardiovascular pathology by regulating sympathetic and parasympathetic neuronal activity, which influences cardiac function. Previous investigations have demonstrated that OX1R plays a key role in enhancing survival and suppressing apoptosis of the neuronal cells by activating the AKT and ERK signaling pathways [44,45]. This protective mechanism may also extend to cardiomyocytes under ischemic conditions. Furthermore, disruption of orexin signaling is associated with sleep disorders such as REM sleep behavior disorder, where damage to orexin neurons impairs their protective functions [46,47]. Our study focused on SF-induced upregulation of hypothalamic OX1R in the context of MI/RI and the results provide important insights into the mechanism by which sleep disorders disrupt the protective functions of the orexin system and exacerbate cardiac injury through autonomic dysregulation. Our data suggests that targeting the upregulated hypothalamic OX1R may represent a new therapeutic approach to mitigate the adverse cardiovascular effects of SF.

The 16-week SF protocol used in this study was designed to model chronic sleep disruption and its cumulative physiological effects. Previous studies have indicated that the autonomic response to sleep loss. Shorter periods of SF or sleep deprivation may transiently suppress sympathetic activity [48,49], whereas prolonged exposure leads to sustained sympathetic overactivation and cardiovascular dysfunction [33,50]. Both 12-week and 16-week-protocols have been validated in the literature as chronic SF models. In this study, we chose the 16-week protocol to represent long-term exposure, consistent with our previously published work that prolonged SF increases sympathetic activity [33]. Our data showed that prolonged SF increased the LF/HF ratio, suggestive of altered autonomic regulation. However, this metric should be interpreted cautiously, as its physiological relevance in rodents is debated. In mice, LF power reflects both sympathetic and parasympathetic input, and LF/HF does not reliably indicate “sympathovagal balance” as in humans. In our study, the elevated LF/HF ratio, along with increased c-Fos expression in the PVN and higher norepinephrine and TH levels in cardiac tissue, indicate enhanced sympathetic activity. These results suggest that SF promotes PVN-mediated sympathetic hyperactivation, which may contribute to increased cardiac vulnerability. Poor sleep habits aggravate cardiovascular injury, which is associated with the dysregulation of the neuroendocrine system [51,52]. The underlying mechanism involves SF-induced disruption of autonomic nervous system homeostasis. Our data showed that SF-induced upregulation of OX1R in the PVN aggravated cardiac injury by inducing sympathetic hyperactivity. Thus, our results provide new insights into mechanisms by which SF exacerbates cardiac damage.

The autonomic neural network regulates cardiovascular function [53] and the sleep states reciprocally influence autonomic activity, including heart rate, blood pressure, and myocardial metabolism [21,54]. In this study, we demonstrate that upregulation of OX1R in the hypothalamic PVN promotes sympathetic excitation, thereby exacerbating MI/RI, although effects on other physiological processes were not assessed in this study. Further investigations are necessary to determine whether OX1R upregulation in other brain regions may influence cardiovascular function or involve autonomic regulatory mechanisms during specific physiological states.

Consistent with the classical neuroendocrine regulatory mechanisms [28,55], upregulated OX1R exerts both local and distant effects by acting on the PVN neurons and the peripheral sympathetic nervous system to promote sympathetic excitation and aggravate myocardial damage. OX1R belongs to the orexin receptor family and is widely expressed throughout the central nervous system [56,57]. Ligand binding to OX1R triggers activation of multiple signaling cascades, which regulate critical processes such as wakefulness, blood pressure, and metabolism [58]. Previous studies have demonstrated that OX1R activation in specific brain regions enhances sympathetic nerve activity [28]. Based on these findings, OX1R upregulation may participate in SF-induced cardiac injury through sympathetic overactivation, although this inference remains to be validated using genetic approaches.

OX1R is expressed in various brain regions and peripheral organs, including hypothalamus, medulla, heart, and blood vessels [59–61]. In this study, we demonstrate that upregulated OX1R in the PVN significantly influences sympathetic nerve activity and cardiac function. Furthermore, our data showed that blockade of PVN-specific OX1R blockade significantly alleviated SF-induced aggravation of MI/RI, including significant improvement in cardiac function and reduced structural damage to the myocardium. This demonstrated that the OX1R-sympathetic axis in the PVN plays a pivotal role in SF-induced aggravation of MI/RI.

From a clinical perspective, our results suggest an association between sleep disruption, hypothalamic OX1R activation, and adverse cardiac outcomes, rather than an immediately applicable therapeutic target. Because SB-334867 is not clinically approved and shows limited CNS penetration, further studies using brain-permeant OX1R modulators and genetic models are needed to clarify its translational relevance. Overall, these results highlight the importance of adequate sleep in maintaining cardiovascular health.

This study has several limitations that warrant consideration. First, although our data demonstrated that SF-induced upregulation of OX1R was a crucial mediator in exacerbating MI/RI-induced myocardial damage through sympathetic hyperactivity, clinical validation of altered OX1R expression and sympathetic activity in patients with sleep disorders remains unexplored because of ethical constraints. Therefore, there is an urgent need to develop non-invasive methodologies to establish clinical relevance of our findings. Second, only adult male C57BL/6J mice were used to minimize variability related to hormonal fluctuations and the estrous cycle. Nevertheless, the lack of female mice limits generalizability. Given the known cardioprotective effects of estrogen, future studies including both sexes are warranted to explore sex-specific differences in SF exacerbated MI/RI [62–64]. Third, although we partially established the causal relationship between SF-induced OX1R upregulation and sympathetic nervous system-mediated cardiac injury, we did not conduct experiments using a PVN-specific OX1R knockout to validate this relationship. Moreover, upstream regulatory mechanisms governing the brain-heart interactions in the context of MI/RI and SF requires further investigations. Fourth, despite our evidence suggesting that OX1R upregulation in the PVN directly contributes to SF-induced aggravation of MI/RI, we did not comprehensively investigate the long-term cardiac remodeling effects after modulation of OX1R activity. Furthermore, establishment of robust in vivo therapeutic intervention models represent a critical gap that will guide our future research endeavors. Fifth, this study did not compare different durations of SF, so it remains unclear whether autonomic and cardiac responses change with longer or shorter exposure. Future studies should include multiple time points to explore how the effects of SF vary over time.

In summary, SF may enhance PVN OX1R signaling and sympathetic hyperactivation, which could exacerbate myocardial injury following MI/RI. Pharmacological inhibition of OX1R alleviated these effects, supporting the involvement of the PVN OX1R-sympathetic pathway in sleep disruption related cardiac injury.

Funding Statement

National Natural Science Foundation of China (Grant Nos. 82170291, 81800058, 82400356, 82400351 and 82200324) and the Hunan Provincial Natural Science Foundation of China (Grant No. 2025JJ60764, 2023JJ40926).

Ethics approval and consent to participate

All animal experiments were conducted in accordance with the ARRIVE guidelines 2.0 and were approved by the Animal Ethics Committee of the Xiangya Hospital, Central South University (Changsha, China; license code: 2021111240). The manuscript does not contain clinical studies or patient data.

Consent for publication

Yes.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Availability of data and materials

The data presented in this study are available on request from the corresponding authors.