Chronic intermittent hypoxia aggravated diabetic cardiomyopathy through LKB1/AMPK/Nrf2 signaling pathway

Bingbing Liu; Jianchao Si; Kerong Qi; Dongli Li; Tingting Li; Yi Tang; Ensheng Ji; Shengchang Yang

doi:10.1371/journal.pone.0296792

. 2024 Mar 7;19(3):e0296792. doi: 10.1371/journal.pone.0296792

Chronic intermittent hypoxia aggravated diabetic cardiomyopathy through LKB1/AMPK/Nrf2 signaling pathway

Bingbing Liu ^1,^#, Jianchao Si ^1,^#, Kerong Qi ^1,^#, Dongli Li ¹, Tingting Li ¹, Yi Tang ¹, Ensheng Ji ^1,^2,^*, Shengchang Yang ^1,^2,^*

Editor: Michael Bader³

PMCID: PMC10919874 PMID: 38452099

Abstract

Chronic intermittent hypoxia (CIH) may play an important role in the development of diabetic cardiomyopathy (DCM). However, the exact mechanism of CIH-induced myocardial injury in DCM remains unclear. In vivo, the db/db mice exposed to CIH were established, and in vitro, the H9C2 cells were exposed to high glucose (HG) combined with intermittent hypoxia (IH). The body weight (BW), fasting blood glucose (FBG) and food intake were measured every two weeks. The glycolipid metabolism was assessed with the oral glucose tolerance test (OGTT) and insulin resistance (IR). Cardiac function was detected by echocardiography. Cardiac pathology was detected by HE staining, Masson staining, and transmission electron microscopy. The level of reactive oxygen species (ROS) in myocardial tissue was detected by dihydroethidium (DHE). The apoptosis was detected by TUNEL staining. The cell viability, ROS, and the mitochondrial membrane potential were detected by the cell counting kit-8 (CCK-8) assay and related kits. Western blotting was used to analyze the liver kinase B1/AMP-activated protein kinase/ nuclear factor-erythroid 2-related factor 2 (LKB1/AMPK/Nrf2) signaling pathway. CIH exposure accelerated glycolipid metabolism disorders and cardiac injury, and increased the level of cardiac oxidative stress and the number of positive apoptotic cells in db/db mice. IH and HG decreased the cell viability and the level of mitochondrial membrane potential, and increased ROS expression in H9C2 cells. These findings indicate that CIH exposure promotes glycolipid metabolism disorders and myocardial apoptosis, aggravating myocardial injury via the LKB1/AMPK/Nrf2 pathway in vitro and in vivo.

Introduction

Diabetic cardiomyopathy (DCM) is a representative complication of type 2 diabetes mellitus (T2DM), which is mainly manifested as systolic and diastolic dysfunction along with cardiac fibrosis, glycolipid metabolism disorders, and elevated oxidative stress [1, 2]. Recent studies have shown that sleep-disordered breathing, especially obstructive sleep apnea (OSA), is closely related to glycolipid metabolism disorders and T2DM [3]. OSA is independently related to metabolic syndrome and insulin resistance (IR), which is strongly associated with the risk of adverse cardiovascular events [4]. OSA can lead to left ventricular diastolic and systolic dysfunction [5], which is associated with DCM [6]. Therefore, we explored the role of OSA in the pathogenesis of DCM, aiming to provide potential prevention and treatment approaches for DCM patients with OSA.

Chronic intermittent hypoxia (CIH) is a typical pathophysiological feature of OSA, which is associated with T2DM and cardiovascular disease [7, 8]. Badran et al. [9] found that CIH could aggravate the level of oxidative stress in intermittent hypoxia diabetic mice, further impairing endothelial dysfunction of db/db mice. Through the model of chronic intermittent hypoxia combined with the diabetes mellitus, Peng and Hu [10] found that CIH aggravated glycolipid metabolism disorders and the level of oxidative stress in the liver and kidney of diabetic rats, further aggravating the damage of the liver and kidney. At present, it is unclear whether CIH could be a factor in accelerating the progression of DCM.

Oxidative stress plays a crucial role in the pathogenesis of DCM [11]. The nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway is the most important endogenous antioxidant stress pathway, which has a positive effect on resisting oxidative damage and the inflammatory response in DCM [12]. The AMP-activated protein kinase (AMPK)/Nrf2 pathway is related to antioxidant stress damage to the heart and brain. The downstream product of the AMPK/Nrf2 pathway, heme oxygenase-1 (HO-1), is a classic antioxidant enzyme and has a good inhibitory effect on oxidative stress and inflammation [13, 14]. Liver kinase B1 (LKB1), as the main upstream protein kinase of AMPK, promotes the phosphorylation and activation of AMPK, which is associated with the growth and metabolism of cells. The LKB1/AMPK signal transduction pathway plays an important role in glycolipid metabolism [15]. Related studies have shown that CIH inhibits the expression of the LKB1/AMPK pathway [16, 17]. In addition, CIH can induce ROS production and disrupt the oxidative/antioxidant balance of cells [18], and excessive ROS can inhibit AMPK inactivation by inhibiting the phosphorylation of LKB1 [19]. Therefore, we speculated that the LKB1/AMPK/Nrf2 signaling pathway may play a key role in the progression of CIH exacerbating DCM.

Materials and methods

Animals and treatment

Male diabetic db/db mice and their non-diabetic db/m mice (4-5weeks old) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. (License number: SCXK(Su)2016-0010). Mice were housed in a standard environment which was maintained on a 12 h light/dark cycle at constant room temperature (22±2°C) with free access to food and water. All procedures involving animal and experimental protocols were approved by the Animal Care and Use Committee of Medical Ethics of Hebei University of Chinese Medicine (on Mar. 21, 2023; Permit No. DWLL202203123).

The db/db mice (n = 16) were randomly divided into the db/db group and CIH group. The db/m mice were used as the control group (n = 8). The treatment of CIH was started as previously described [9]. Briefly, db/db mice were placed in special cages with a controlled gas delivery system that regulated the flow of air, nitrogen, and oxygen into the cages. The concentration of oxygen in the chamber for the CIH group was changed from 21% to 5%, 8 h/day for 8 weeks. The db/m and db/db group mice were kept under air-conditioning. After exposure to CIH for 8 weeks, all mice were euthanized by cervical dislocation after intraperitoneal injection of 30 mg/kg of sodium pentobarbital. Then, the cardiac tissue was isolated.

The measurement of biochemical parameters

The levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in mice were detected by an automatic biochemical analyzer (Changchun Huili Biotech Co., Ltd., Changchun, China). The myocardial tissue was collected to prepare myocardial homogenate, and the myocardial superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels in each group of mice were detected by using related kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The levels of creatine kinase myocardial band (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin Ⅰ (cTnI) were detected by automatic blood biochemical detector.

Insulin resistance (IR) and Oral glucose tolerance test (OGTT)

The insulin level was detected by using a mouse insulin ELISA (abcam, UK). The following formula was used to calculate IR according to the following formula: Homeostatic Model Assessment for IR (HOMA-IR) = fasting insulin (μIU/L) × fasting glucose (mmol/L)/22.5. For the OGTT test, all mice were fasted for 15 h and given 2 g/kg of 20% glucose solution after FBG measurement. Blood samples were taken from tail veins at 15, 30, 60, and 120 min after gavage to measure the blood glucose level. The glucose-time curve was plotted and the area under the curve (AUC 0–120 min) was calculated.

Echocardiography

The mice were anesthetized by inhalation with isoflurane (2%) and fixed in the supine position after full anesthesia. B-mode is the basic imaging mode of ultrasonic imaging, in which images of the anatomical structure of animals are used to locate the long and short axes of mice. M-mode was used to measure the left ventricular end diastolic diameter (LVDd), left ventricular end systolic diameter (LVDs), left ventricular fractional shortening (LVFS), and left ventricular ejection fraction (LVEF). Color Doppler-mode was used to measure the velocity ratio of the E peak to the A peak in the cardiac mitral valve (E/A).

Histological examination

The heart tissue was fixed in 4% paraformaldehyde for 24 h for soaking, fixation, and dehydration, and then the wax-soaked tissue was embedded in an embedding machine. The sections were sliced on a paraffin slicer at a slice thickness of 4μm. The sections were stained in a dye solution and the morphological changes of the heart tissue were observed under the microscope.

Tissue blocks of about 1 mm³ in size were removed from the apex of the heart and fixed in 2.5% glutaraldehyde (pH 7.4). Ultrathin sections were made and the ultrastructure was observed by electron microscopy.

The heart was fixed on ice in 4% paraformaldehyde for 2 h, and then transferred to a 30% sucrose solution for dehydration at 4˚C overnight, embedded with OCT, sliced with a frozen microtome with a thickness at 5μm, and fixed in cold acetone for 10min.

Measurement of GLUT4 and Nrf2 expression

The frozen sections of the myocardium of mice were a sealed with 5% BSA blocking buffer for 1h at room temperature, GLUT4 (1:100, 66846-1-Ig, Proteintech, China) and Nrf2 (1:100, A0674, BOSTER, China) antibody was diluted and dropped onto the sections, and incubated at room temperature for 2 h. PBS was washed, and the diluted fluorescently labeled secondary antibody was added for 1h at 37˚C for protection from light, and the nuclear was stained with diluted DAPI. The film was sealed and placed under confocal microscope to observe and photograph.

TUNEL staining

The heart tissue embedded in paraffin was sectioned, dewaxed in xylene solution, hydrated with ethanol from a high to low concentration, washed three times with distilled water with phosphoric acid buffer, and incubated with TUNEL reagent at 37˚C for 1h, observed under a fluorescence microscope.

Dihydroethidium staining

The myocardial tissues of mice in each group were taken for frozen sections, and incubated in 10μmol/L DHE solution for 90 min under dark conditions at 37˚C, washed with PBS, and photographed under fluorescence microscope after sealing. The fluorescence absorbance value was calculated as the level of intracellular ROS.

Cell culture and treatment

The H9C2 cells were purchased from Procell Life Science & Technology Co., Ltd (Cat. No.: CL-0089; Wuhan, China). The Metformin was purchased from Shanghai yuanye Bio-Technology Co., Ltd (Cat. No.: B25331; Shanghai, China). The A-769662 was purchased from MedChemExpress LLC (Cat. No.: 844499-71-4; Shanghai, China). The H9C2 cells were divided into three groups: control (CON) group, high glucose (HG) group, and IH+HG group. The H9C2 cells were seeded in 24-well plates. The H9C2 cells were used to induce DCM in vitro under the HG condition. We cultured the H9C2 cells with 30mM of HG for 48 h. The IH+HG group was cultured in the hypoxic chamber of IH cells, and the oxygen concentration in the hypoxic chamber was cycled between 21%-1%, 10 min/cycle, and cultured for 48h.

Cell viability

The H9C2 cells were prepared into a cell suspension and inoculated into 96-well plates at density of 5×10⁴ cells/well. The Cell Counting Kit-8 (C0037, Beijing, China) was used to detect cell viability, CCK-8 solution (10μL) was added to each well, and the cells were incubated at 37°C for 1 h. The absorbance value of each well was measured at 450 nm with a microplate reader, and the cell activity was calculated.

Measurement of ROS production

The H9C2 cells were seeded in 24-well culture plates at a density of 5×10⁴ cells in each well and incubated for 24 hours before treatment. Assay for ROS Assay Kit (S0033S, Beyotime, China), dilute DCFH-DA with serum-free DMEM medium at the ratio of 1:1000, add diluted with DCFH-DA 200 μL to each well, the cells were incubated in 5%CO₂ and 37°C for 20 min and washed three times with serum-free cell culture medium. Finally, the cells were observed and photographed under an inverted fluorescence microscope and analyzed with Image J software.

Measurement of mitochondrial membrane potential

The H9C2 cells were seeded in 24-well culture plates at a density of 5×10⁴ cells in each well and incubated for 24 h before treatment. Mitochondrial Membrane Potential and Apoptosis Detection Kit with Mito-tracker Red CMXRos and Annexin V-FITC (C1071M, Beyotime, China) was used to detect the mitochondrial membrane potential. The medium and JC-1 working solution were added in equal proportions, fully mixed, and incubated at 37°C for 30 min. After incubation, the liquid was removed and the cells were washed three times. Finally, the cell membrane potential was observed and photographed under an inverted fluorescence microscope.

Western blotting

The protein concentration was quantified with the BCA Protein Assay Kit. Then 4× protein gel electrophoresis loading buffer was added to the protein samples, which were denatured at 100°C for 5 min. The SDS-PAGE gel was prepared, samples were added for electrophoresis, and electricity was transferred to PVDF membrane. Seal with 5% skim milk powder sealing solution for 2 h, adding primary antibody and incubating overnight at 4°C: Tubulin (1:10000, GTX628802) and Caspase-3(1:1000, #14220) from CST; p-AMPK (1:2000, AP0116) and HO-1(1:1000, A1346) from ABclonal; PI3K (1:1000, AF6241) from Affinity; LKB1 (1:1000, 10746-1-AP), p-LKB1 (1:5000, 80127-1-RR), AMPK (1:500, 10929-2-AP), p-AKT (1:5000, 66444-1-Ig), AKT (1:5000, 60203-2-Ig), GLUT4 (1:3000, 66846-1-Ig) from Proteintech; Bax (1:1000, GB12690) from Seville; Bcl-2 (1:1000, BA0412) and Nrf2 (1:1000, A0674) from BOSTER; Lamin-B1(1:1000, SI17-06) from Huabio. After washing, the membrane was incubated for 1 h with second antibody (1:8000, Servicebio, China) and the proteins were detected with ECL luminescent solution. The gray value was analyzed by Image J software.

Statistical analysis

Data are expressed as means ± S.E.M. The results were analyzed by SPSS 21.0 software. One-way ANOVA was used for intergroup mean comparison. The p < 0.05 was considered to be statistically significant.

Results

Effects of CIH exposure on the metabolic characteristics of db/db mice

As shown in Fig 1A and 1B, compared to the db/m group, the levels of BW, FBG and food intake in the db/db group and the CIH group were increased (p < 0.01). Compared to the db/db group, the BW of the CIH group was decreased (p < 0.05). There was no statistical significance in FBG between the db/db group and the CIH group, but the fluctuation of FBG in the CIH group was increased. Compared to the db/m group, the food intake of db/db group and CIH group was significantly increased (p < 0.01, Fig 1C). Compared to the db/db group, the food intake of the CIH group was decreased (p < 0.05). The changes in lipid metabolism in the db/db mice are shown in Fig 1D–1F. Compared to the db/m group, the levels of TC, TG, and LDL-C in the db/db group and CIH group were increased (p < 0.05). Compared to the db/db group, the levels of TC, TG, and LDL-C in the CIH group were increased (p < 0.05). As shown in Fig 1G and 1H, after 0–30 min of intragastric administration of glucose solution, the level of blood glucose in each group was increased; and after lavage for 30–120 min, the level of blood glucose was gradually decreased. Compared to the db/m group, the level of blood glucose in the db/db group and CIH group was significantly increased (p < 0.01). Compared to the db/db group, the level of blood glucose in the CIH group was increased after 15min of intragastric glucose solution (p < 0.05). Compared to the db/m group, the AUC value of the OGTT in the db/db group and CIH group was significantly increased (p < 0.01). Compared to the db/db group, the AUC value of the OGTT in the CIH group was increased (p < 0.05).

Effects of CIH exposure on the insulin-signaling pathway of db/db mice

As shown in Fig 2A, compared to the db/m group, the level of HOMA-IR in the db/db group and the CIH group was significantly increased (p < 0.01). Compared to the db/db group, the level of HOMA-IR was increased in the CIH group (p < 0.05). Compared to the db/m group, the expression of PI3K and P-AKT was decreased in the db/db group and CIH group (p < 0.05, Fig 2B–2D). Compared to the db/db group, the expression of PI3K and P-AKT in the CIH group was decreased (p < 0.05). As shown in Fig 2E–2H, the expression of GLUT4 in the CIH group was determined by immunofluorescence and Western blotting. Compared to the db/m group, the expression of GLUT4 in the db/db group and the CIH group was decreased (p < 0.05). Compared to the db/db group, the expression of GLUT4 in the CIH group was decreased (p < 0.05).

Effects of CIH exposure on cardiac pathophysiology of db/db mice

As shown in Fig 3A, compared to the db/m group, the results showed that myocardial disorders in the db/db group and the CIH group. Compared to the db/db group, CIH exposure aggravated the myocardial disorders. Masson staining was used to evaluate the myocardial fibers of db/db mice. Compared to the db/m group, the area of myocardial tissue fibrosis was increased in the db/db group and the CIH group (p < 0.01, Fig 3C). Compared to the db/db group, the area of myocardial tissue fibrosis was increased in CIH group (p < 0.05). Compared to the db/m group, the expression of collagen Ⅰ and collagen Ⅲ in the db/db group and the CIH group increased (p < 0.05, p < 0.01, Fig 3B–3E), and that of the CIH group showed a further increase compared to the db/db group (p < 0.05).

Effects of CIH exposure on cardiac function of db/db mice

The effect of CIH exposure on the cardiac function of db/db mice by echocardiography (Fig 4A and 4B). The levels of LVEF and LVFS are important indicators of left ventricular systolic function [20], which is the most commonly used method to evaluate ventricular function. Compared to the db/m group, the LVEF and LVFS of the db/db group and the CIH group were significantly decreased (p < 0.01, Fig 4C and 4D). Compared to the db/db group, the LVEF and LVFS of the CIH group were decreased (p < 0.05). Compared to the db/m group, the LVDd and LVDs of the db/db group and the CIH group were significantly increased (p < 0.01, Fig 4E and 4F). Compared to the db/db group, the LVDd and LVDs of the CIH group were increased (p < 0.05). The E/A ratio is an important indicator of left ventricular diastolic function. Compared to the db/m group, the ratio of E/A in the db/db group and the CIH group was significantly decreased (p < 0.01, Fig 4G). Compared to the db/db group, the ratio of E/A in the CIH group was decreased (p < 0.05). DCM causes an increase in the serum levels of cTnI, LDH, and CK-MB [21]. Consistent with this, compared to the db/m group, the levels of LDH, CK-MB and cTnI in the db/db group and CIH group were increased (p < 0.05, Fig 4H–4J). Compared to the db/db group, the levels of LDH, CK-MB and cTnI of the CIH group were increased (p < 0.05).

Effects of CIH exposure on cardiac apoptosis of db/db mice

Compared to the db/m group, the number of positive apoptotic cells in the db/db group and CIH group was significantly increased (p < 0.01, Fig 5A and 5B). Compared to the db/db group, the number of positive apoptotic cells in the CIH group was increased (p < 0.05). The protein expression of Bax, Bcl-2, and Caspase-3 was detected by western blotting (Fig 5C). Compared to the db/m group, the Bax/Bcl-2 ratio was increased in the db/db group and CIH group (p < 0.01, Fig 5D). Compared to the db/db group, the Bax/Bcl-2 ratio in the CIH group was increased (p < 0.05). Caspase-3 is a major executive protein of apoptosis and is involved in the apoptosis of diabetic cardiomyocytes [22]. Compared to the db/m group, the cleaved-caspase-3/pro-caspase-3 ratio was significantly increased in the db/db group and the CIH group (p < 0.05, Fig 5E). Compared to the db/db group, the cleaved-caspase-3/pro-caspase-3 ratio in the CIH group was increased (p < 0.05).

Effects of CIH exposure on cardiac oxidative stress of db/db mice

As shown in Fig 6A and 6C, compared to the db/m group, the levels of SOD and GSH-Px were decreased in the db/db group and the CIH group (p < 0.05). Compared to the db/db group, the levels of SOD and GSH-Px in the CIH group were decreased (p < 0.05). Compared to the db/m group, the level of MDA was increased in the db/db group and the CIH group (p < 0.05, Fig 6B). Compared to the db/db group, the level of MDA of the CIH group was increased (p < 0.05). Moreover, ROS expression in cardiac tissues was detected by DHE (Fig 6D and 6E). Compared to the db/m group, the expression of ROS in the db/db group and CIH group was significantly increased (p < 0.01). Compared to the db/db group, the expression of ROS in the CIH group was increased (p < 0.05). As shown in Fig 6F, the mitochondrial structure of the db/m group was intact and clear, whereas the mitochondrial structure of the db/db group and CIH group was deformed. Compared to the db/db group, mitochondrial swelling and vacuolar degeneration were observed in the CIH group.

Effects of CIH exposure on the LKB1/AMPK/Nrf2 signaling pathway of db/db mice

The AMPK/Nrf2/HO-1 pathway has been implicated in oxidative stress damage in the heart and LKB1 is an upstream kinase of AMPK. Studies have confirmed that oxidative stress reduces LKB1 activity, which in turn reduces AMPK activity [23]. Compared to the db/m group, the p-LKB1/LKB1 and p-AMPK/AMPK ratio in the db/db group and CIH group was decreased (p < 0.05, Fig 7A–7C). Compared to the db/db group, the ratio of p-LKB1/LKB1 and p-AMPK/AMPK in the CIH group was decreased (p < 0.05). Compared to the db/m group, the expression of Nrf2 and HO-1 in the db/db group and the CIH group was decreased (p < 0.05, Fig 7D and 7E). Compared to the db/db group, the protein expression of Nrf2 and HO-1 in the CIH group was decreased (p < 0.05). Furthermore, the expression of nuclear-Nrf2 was detected by immunofluorescence and western blot (Fig 7F–7I). Compared to the db/m group, the expression of nuclear-Nrf2 in the db/db group and the CIH group was decreased (p < 0.01). Compared to the db/db group, the expression of nuclear-Nrf2 in the CIH group was decreased (p < 0.05).

Fig 7 — (A-E) Representative blot images and quantitative analysis of LKB1, AMPK, Nrf2, HO-1. (F-G) Representative blot images and quantitative analysis of Nuclear-Nrf2. (H-I) Representative images of Nuclear-Nrf2 and the quantitative analysis for Nuclear-Nrf2 (scale bar, 100 μm). n = 3. Data are presented as means ± S.E.M. *p < 0.05 and **p < 0.01 vs. db/m group; ^Φp < 0.05 and ^ΦΦp < 0.01 vs. db/db group.

Effects of IH and HG on the cell viability, ROS production, and mitochondrial membrane potential of H9C2 cells

As shown in Fig 8A and 8B, cells were used treated with different concentrations of HG for 24 and 48 h; we selected a final concentration of 30mM HG for 48h. The IH+HG group was treated with IH for 48h, compared to the CON group, and the cell viability of the HG group and the IH+HG group was decreased (p < 0.05, Fig 8C). Compared to the HG group, the cell viability of the IH+HG group was decreased (p < 0.05). As shown in Fig 8D and 8E, compared to the CON group, the cell membrane potential of the HG group and the IH+HG group was significantly decreased (p < 0.01). Compared to the HG group, the cell membrane potential of the IH+HG group was decreased (p < 0.05). As shown in Fig 8F and 8G, compared to the CON group, the expression of ROS in the HG group and the IH+HG group was significantly increased (p < 0.01). Compared to the HG group, the expression of ROS in the IH+HG group was increased (p < 0.05). As shown in Fig 8H–8K, compared to the CON group, the expression of p-LKB1/LKB1 and p-AMPK/AMPK and Nrf2 in the HG group and IH+HG group was decreased (p < 0.05, p < 0.01). Compared to the HG group, the expression of p-LKB1/LKB1 and p-AMPK/AMPK and Nrf2 in IH+HG group was decreased (p < 0.05).

Effects of IH and HG on the LKB1/AMPK/Nrf2 signaling pathway of H9C2 cells

As shown in Fig 9A and 9B, compared to the CON group, the cell membrane potential of the IH+HG group was decreased (p < 0.01). Compared to the IH+HG group, the cell membrane potential of the Metformin group and the A-769662 group was increased (p < 0.01). As shown in Fig 9C and 9D, compared to the CON group, the expression of ROS in the IH+HG group was significantly increased (p < 0.01). Compared to the IH+HG group, the expression of ROS in the Metformin group and the A-769662 group was decreased (p < 0.05). As shown in Fig 9E–9G, compared to the CON group, the expression of p-AMPK/AMPK and Nrf2 in the IH+HG group was decreased (p < 0.05). Compared to the IH+HG group, the expression of p-AMPK/AMPK and Nrf2 in the Metformin group and the A-769662 group was increased (p < 0.05).

Discussion

This study mainly discussed the mechanism by which CIH aggravates IR and glycolipid metabolism disorders through oxidative stress, inducing myocardial cell apoptosis, leading to cardiac dysfunction, and further resulting in the development and deterioration of DCM. It provides a new method for the pathogenesis of OSA with DCM.

The db/db mice are congenital obese type 2 DCM mice with leptin deficiency caused by gene mutation [24], characterized by hyperinsulinemia, hyperlipidemia and myocardial hypertrophy [25], which has been widely used to study the metabolic pathogenesis and activated inflammatory mechanisms of type 2 DCM [26]. As a common respiratory disease, OSA often coexists with T2DM, and can promote the disease progression of T2DM and its complications [27]. CIH is the core pathological basis of OSA, associated with IR and glycolipid metabolism disorders in OSA patients, which is an important risk factor for cardiovascular diseases. Clinical studies suggest that OSA can increase the level of blood glucose, decreasing insulin sensitivity and aggravating IR in T2DM [28, 29]. In our experiment, after 8 weeks of CIH exposure, db/db mice had an increase in the fluctuation of blood glucose and impaired glucose tolerance, which is a potential risk factor for accelerating the occurrence and development of DCM.

Myocardial disorders and myocardial fibrosis are the characteristic pathological changes of DCM myocardial remodeling. Recently, it was shown that the hypoxia-induced inflammatory response aggravates myocardial cell damage [30]. Under the stimulation of inflammatory factors, abnormal deposition of extracellular matrix and increased content of collagen fibers jointly promote the occurrence and development of myocardial fibrosis, and ultimately participate in the destruction of cardiac structure and function [31]. Our results showed that collagen fibers were increased in CIH group, suggesting that CIH exposure may aggravate the progression of myocardial fibrosis in DCM. To assess the association between CIH and cardiac dysfunction of DCM, echocardiography was used to measure cardiac function. Goes CM found that repeated episodes of hypoxia can lead to left ventricular dysfunction [32]. Our results showed that CIH exposure could cause left ventricular systolic and diastolic dysfunction in db/db mice. The level of myocardial enzyme is used to measure the damage degree of myocardial cells indirectly. We found that the serum levels of CK-MB, LDH and cTnI were significantly reduced after CIH exposure, suggesting that CIH further aggravated myocardial injury in DCM.

Oxidative stress injury of myocardial tissue is a key factor in the occurrence and development of DCM [33]. Oxidative stress injury is the main pathological mechanism leading to the occurrence and progression of DCM [34]. CIH is associated with increased ROS and oxidative stress [35]. Oxidative stress is up-regulated in OSA patients and may adversely affect cardiovascular disease [36]. ROS in the human heart is derived from mitochondrial production and NADPH [37]. ROS can cause molecular damage, leading to mitochondrial dysfunction and reducing the oxidative capacity of fatty acids, which results in lipid accumulation, fibrosis, diastolic dysfunction, and even heart failure, all of which are related to the occurrence of DCM [38, 39]. The mitochondrial structure of db/db mice is swollen and irregular, suggesting CIH exposure aggravating the mitochondrial structural damage, increasing the production of ROS and promoting the progression of DCM. Previous studies have shown that under IH conditions, H9C2 cardiomyocytes are also damaged, ROS is markedly increased, oxidative stress in cardiomyocytes is increased, mitochondria are seriously fragmented, the membrane potential is decreased, and the mitochondrial function is impaired. Together, these data show that CIH has serious damaging effects on db/db mice and H9C2 cardiomyocytes [40–42].

Glucose metabolism disorders are correlated with oxidative stress, which reduces antioxidant levels and further aggravates oxidative stress. Oxidative stress is related to the activation of PI3K/AKT/GLUT4 signaling pathway. Under the condition of IR, the utilization of glucose by the heart is significantly reduced, which makes the heart more dependent on fatty acid oxidation for energy metabolism. GLUT4 is mainly expressed in skeletal muscle, myocardium and fat cells. The stimulation of GLUT4 transportation will be an important step in improving cardiac glucose metabolism. CIH could increase the level of oxidative stress and block the activation of PI3K/AKT/GLUT4 signaling pathway, further aggravating glucose metabolism disorders and oxidative stress damage of myocardial cells, and ultimately affecting the cardiac function of DCM [43]. Studies have shown that CIH exposure could reduce the mRNA and protein levels of GLUT4 in skeletal muscle, which may occur simultaneously in liver, adipose tissue and even all tissues of the body [44]. After CIH exposure, our results showed that the expression of GLUT4 was significantly reduced, aggravating glucose metabolism disorders, oxidative stress, and suggesting an increase in the cardiac dysfunction of DCM.

Myocardial apoptosis is an important mechanism of myocardial dysfunction in DCM [45]. Myocardial apoptosis is the main cause of myocardial cell loss, myocardial remodeling and dysfunction in diabetic animal models and patients [22]. Caspase-3 is an important cysteine protease that participates in apoptosis directly after activation. We found that TUNEL positive cells, the ratio of cleaved-caspase-3/pro-caspase-3 and Bax/Bcl-2 were significantly increased after exposure to CIH. Consistent with previous studies [46–48], in this study, IH-induced H9C2 cardiomyocyte injury was closely related to decreasing the expression of anti-apoptotic protein and increasing the expression of pro-apoptotic protein, suggesting that CIH exposure can increase myocardial apoptosis in DCM.

Nrf2 is one of the therapeutic targets for DCM, which plays an important role in enhancing myocardial antioxidant, anti-inflammatory, anti-fibrosis and anti-apoptosis abilities. Several studies have shown that Nrf2 can be activated by AMPK. The activation of Nrf2 can activate downstream HO-1, which quickly and effectively removed excessive ROS, reducing the myocardial injury of DCM. AMPK is a central protein that coordinates metabolism and energy and regulates a variety of lipid metabolization-related enzymes and transcription factors. The AMPK/Nrf2 signaling pathway plays an important role in reducing oxidative stress response and inhibiting mitochondria mediated apoptosis [49]. LKB1, as the upstream kinases of AMPK, has been proven to activate AMPK. The expression of p-LKB1 is positively correlated with AMPK activity. We found that CIH exposure reduced the expression of p-LKB1, p-AMPK and Nrf2 in vitro and in vivo, suggesting that CIH exposure may aggravate oxidative stress and promote glycolipid metabolism disorders by inhibiting LKB1/AMPK/Nrf2 signaling pathway, and further aggravate the cardiac function impairment of DCM. Therefore, the inhibition of oxidative stress and apoptosis as therapeutic targets is crucial for the prevention and treatment of DCM.

Conclusion

In conclusion, CIH could aggravate oxidative stress, promoting glycolipid metabolism disorders and leading to myocardial apoptosis by inhibiting cardiac LKB1/AMPK/Nrf2 signaling pathway in vitro and in vivo, further aggravating cardiac function injury of DCM (Fig 10).

Supporting information

S1 Graphical abstract

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S1 Raw images

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pone.0296792.s002.pdf^{(1.4MB, pdf)}

S1 Data

(XLSX)

pone.0296792.s003.xlsx^{(15.7KB, xlsx)}

Data Availability

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

Funding Statement

This work was supported by the National Natural Science Foundation of China (82274617), the Hebei Natural Science Foundation (H2022423352, H2022423370), the Science and Technology Project of Hebei Education Department (QN2023159) and the Medical Science Research Projects of Health Commission of Hebei Province (20231573). Author Contributions: Conceptualization: Shengchang Yang, Ensheng Ji. Data curation: Bingbing Liu, Jianchao Si, Shengchang Yang, Ensheng Ji. Formal analysis: Dongli Li, Tingting Li, Yi Tang, Shengchang Yang, Ensheng Ji. Funding acquisition: Shengchang Yang, Ensheng Ji. Investigation: Dongli Li, Tingting Li, Yi Tang. Methodology: Bingbing Liu. Project administration: Shengchang Yang, Ensheng Ji. Software: Kerong Qi. Visualization: Jianchao Si. Writing – original draft: Bingbing Liu, Jianchao Si, Kerong Qi. Writing – review & editing: Shengchang Yang, Ensheng Ji.

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PLoS One. doi: 10.1371/journal.pone.0296792.r001

Decision Letter 0

Michael Bader

20 Oct 2023

PONE-D-23-28549Chronic intermittent hypoxia aggravated diabetic cardiomyopathy through LKB1/AMPK/Nrf2 signaling pathwayPLOS ONE

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Reviewer #1: Although chronic intermittent hypoxia (CIH) exposure promotes glycolipid metabolism disorders and myocardial apoptosis, aggravating the myocardial injury via liver kinase B1 (LKB1)/5' adenosine monophosphate-activated protein kinas (AMPK)/nuclear erythroid 2-related factor 2 (Nrf2) pathway in vitro and in vivo is interesting, numbers of points need clarifying and certain statements require further justification. These are given below.

<point>

1. The authors described, “All procedures involving animal and experimental protocols were approved by the Hebei University of Chinese Medicine Animal Care and Use Committee.” (lines 99–102) without showing approval date and number(s).

2. In Fig. 1, what age and gender of mice were used should be clarified.

3. In Fig. 2, what age and gender of mice were used should be clarified.

4. In Fig. 3, what age and gender of mice were used should be clarified.

5. In Fig. 4, what age and gender of mice were used should be clarified.

6. In Fig. 5, what age and gender of mice were used should be clarified.

7. In Fig. 6, what age and gender of mice were used should be clarified. In addition, scale bar(s) should be added in Fig. 6F.

8. In Fig. 7, what age and gender of mice were used should be clarified.

9. In Fig. 8, what age and gender of mice were used should be clarified.

10. In Suppl. Fig. 2, the arrow did not indicate any band/signal in Tubulin “55KD”.

11. In Suppl. Fig. 7, “dh/m”, “db/db”, and “CIH” did not match the band position. The 110 KD band in “Nuclear-Nrf2” did not fit in the figure.

12. In Suppl. Fig. 8, the 54 KD band(s) in “p-LKB1” were not clear.

13. “Dihydroethidium” (line 35) should be changed to “dihydroethidium”.

14. “Ismail Laher [9]” (line 66) should be changed to “Baran et al. [9]” or “Laher and his colleagues [9]”.

15. “Hu k [10]” (line 69) should be changed to “Peng and Hu [10]”.

16. “37°C” (lines 153, 160, and 165) should be changed to “37 ˚C”.

17. Concerning intermittent hypoxia in cardiomyocytes, several paperers (Hu, Z. et al. Sleep Breath doi: 10.1007/s11325-023-02863-8. Online ahead of print; Chen, Q. et al. Sleep Breath 27, 129-136, 2023; Dong, N. & Liu, W.Y. Sleep Breath 27, 399-410; Takasawa, S. et al. Int. J. Mol. Sci. 23, 8782, 2022; Takasawa, S. et al. Int. J. Mol. Sci. 23, 12414, 2022; Moulin, S. et al. Antioxidants 11, 1462, 2022) have recently been reported. The authors should cite the papers and make some discussions.

18. There are some reports concerning human cardiomyocytes in intermittent hypoxia such as Regev, D. et al. Int. J. Mol. Sci. 23, 10272, 2022, Huang, J. et al. Sleep Breath 27, 1005-1011, 2023; Moulin, S. et al. Antioxidants 11, 1462, 2022; Chen, Q. et al. Sleep Breath 27, 129-136, 2023; Dong, N. & Liu, W.Y. Sleep Breath 27, 399-410). The authors should cite the papers and make some discussions.</point>

Reviewer #2: Liu et al. reported on the effects of chronic intermittent hypoxia (CIH) on diabetic cardiomyopathy. Using in vivo and in vitro models they concluded that CIH aggravated diabetic cardiomyopathy through LKB1/AMPK/Nrf2 signaling pathway resulting in cardiomyocyte apoptosis and increased myocardial fibrosis. Overall, it is a relevant topic, as sleep perturbations and diabetes are highly prevalent clinical disorders which may negatively influence each other and understanding of pathomechanisms underlying these interactions is of great importance. However, this paper needs to be improved, as it lacks some important information, especially in the method section and the presentation of the results is sometimes unclear.

Below please find more detailed comments:

- Introduction: Page 11: “…and excessive ROS can inhibit AMPK inactivation by inhibiting the phosphorylation of LKB1”. Here, I think you meant ROS can inhibit AMPK activation, or?

- Methods, page 11: only male animals were used. Was there any reason not to include females? Including only males is a limitation of this study. How old were the mice at the beginning of the experiment?

- Methods, page 11: “24 db/db mice were randomly divided into three groups (n=8): db/m group, db/db group, and CIH group”. Db/db mice cannot serve as a source of db/m mice. Please rewrite this sentence.

- Methods, page 11: “The concentration of oxygen in the chamber for the CIH group was changed from 21% to 5%, 8 h/day for 8 weeks”. -> How long was one cycle of normoxia-hypoxia-normoxia? Did you measure the extent of hypoxemia in blood of mice under intermittent hypoxia?

- Methods, page 12: How did the authors measure glucose levels?

- Methods, page 13: the authors describe paraffin embedment of heart specimens for subsequent histological stainings, but in the next method section, frozen sections are mentioned. The method of frozen section preparation is missing.

- Methods, page 12: echocardiographic measurements are not precisely described: what view was used? What mode was used? E.g. M-mode for chamber diameters, B-mode for Doppler measurements.

- Methods, page 12: Did you measure thickness of the left ventricular walls and the potential impact of diabetes and IH on this parameter?

- Methods: The authors present results for CK-MB, troponin and LDH serum levels, the corresponding section in the Methods how these parameters were measured is missing.

- Methods, page 15: “we cultured the H9C2 cells with different concentrations of HG for 48 h.” -> what were the concentrations of HG?

- Methods, page 17: Statistical analysis: did the authors check for normal distribution? What method was used for the comparisons between two groups?

- Results, page 23: the results showed that metformin improved membrane potential and decreased oxidative stress in cells which was associated with increased p-AMPK/AMPK and Nrf2 levels. Did this have any impact on the cell viability/apoptosis?

- Graphical abstract: according to the findings, not only cardiomyocyte apoptosis, but also aggravated fibrosis may lead to cardiac dysfunction; fibrosis is not illustrated here.

- Throughout the whole manuscript there are sentences which can hardly be understood (below only a couple of examples of them). Please improve this as it might be very confusing for the reader.

Results, page 17: “Compared to the db/db group, the BW of the CIH group was decreased (p<0.05), but there was no statistical significance in FBG and the fluctuation of blood glucose in the CIH group was increased.” -> This sentence is unclear.

Results, page 19: “As shown in Fig.3A, the results showed that myocardial disorders in the db/db group and the CIH group compared to the db/m group.” -> This sentence is unclear.

Figure 8, legend: “The cell viability of H9C2 cells was treated with IH and HG for 24 h and 48h.” -> This sentence is unclear.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2024 Mar 7;19(3):e0296792. doi: 10.1371/journal.pone.0296792.r002

Author response to Decision Letter 0

30 Nov 2023

Response

Dear Professor:

Thank you very much for your comments and professional advice. These opinions help to improve the academic rigor of our article. Based on your suggestion and request, we have made explanations. Thank you very much for providing a new perspective for our research. We appreciate your valuable comments and hope to receive your approval, which we wish to be considered for publication as an original article in “PLOS ONE”.

Journal Requirements:

Question 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Answer: Thanks for your kind suggestions. According to the style requirements of PLOS ONE, we have made revisions to the manuscript. The detailed modifications are as follows:

1. The level 1 heading for all major sections (Abstract, Introduction, Materials and methods, Results, Discussion, Conclusions, References) have been revised to 18pt font and bold type marked in red.

2. The level 2 headings for sub-sections of major sections have been revised to 16pt font and bold type marked in red.

3. This manuscript has been revised to double-space paragraph format.

4. The figure files have been revised to “Fig1 .tif”, “Fig2 .tif”, “Fig3.tif”, “Fig4 .tif”, “Fig5 .tif”, “Fig6 .tif”, “Fig7 .tif”, “Fig8 .tif”, “Fig9 .tif”, “Fig10 .tif”.

5. In aspect of “Figure Citations”, we made a detailed revision in the “Results” of the manuscript and marked in red.

6. In aspect of “References”, we made a detailed revision in the “References” of the manuscript and marked in red.

7. We added the part of “Supporting information” in the manuscript and marked in red.

Question 2: Thank you for stating the following financial disclosure:

Answer: Thanks for your kind suggestions. Thank you for reminding us that we have added the roles of funders Shengchang Yang and Ensheng Ji marked in red. Funders Shengchang Yang and Ensheng Ji were involved in the data curation, formal analysis and project administration in the study. Thank you very much for your valuable advice.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Answer: Thanks for your kind suggestions. Thank you for reminding us that we have added the role of funder statement in cover letter marked in red. Thank you very much for your valuable advice.

Question 3: PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ？？

Answer: Thanks for your kind suggestions. We set up an ORCID iD, thank you.

ORCID iD for the corresponding authors:

Shengchang Yang yscdekaoyan@163.com 0000-0002-2002-4945

Ensheng Ji jesphy@126.com 0000-0002-5858-7567

Question 4: Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well.

Answer: Thanks for your kind suggestions. Thank you for reminding us that we have added the full name of ethics committee and ethical license number in “Methods” marked in red. All procedures involving animal and experimental protocols were approved by the Animal Care and Use Committee of Medical Ethics of Hebei University of Chinese Medicine, and obtained informed written. Thank you very much for your valuable advice.

Question 5: PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

Answer: Thanks for your kind suggestions. We have added the original uncropped and unadjusted images in a PDF and upload it for a supplement file.

In cover letter, we have noted that the blot/gel image data are in Supporting Information marked in red.

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

Answer: Thanks for your kind suggestions. The original contributions presented in the study are all included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Reviewer #1:

Question 1: The authors described, “All procedures involving animal and experimental protocols were approved by the Hebei University of Chinese Medicine Animal Care and Use Committee.” (lines 99–102) without showing approval date and number(s).

Answer: Thanks for your kind suggestions. Thank you for reminding us that we have added the approval date and number in “Methods” marked in red. Thank you very much for your valuable advice.

Question 2: In Fig. 1, what age and gender of mice were used should be clarified.

Answer: Thanks for your kind suggestions. We have added age and gender of mice in Fig. 1 according to your suggestion. Thank you very much for your valuable advice

Question 3: In Fig. 2, what age and gender of mice were used should be clarified.

Answer: Thanks for your kind suggestions. We have added age and gender of mice in Fig. 2 according to your suggestion. Thank you very much for your valuable advice

Question 4: In Fig. 3, what age and gender of mice were used should be clarified.

Answer: Thanks for your kind suggestions. We have added age and gender of mice in Fig. 3 according to your suggestion. Thank you very much for your valuable advice

Question 5: In Fig. 4, what age and gender of mice were used should be clarified.

Answer: Thanks for your kind suggestions. We have added age and gender of mice in Fig.4 according to your suggestion. Thank you very much for your valuable advice

Question 6: In Fig. 5, what age and gender of mice were used should be clarified.

Answer: Thanks for your kind suggestions. We have added age and gender of mice in Fig. 5 according to your suggestion. Thank you very much for your valuable advice

Question 7: In Fig. 6, what age and gender of mice were used should be clarified. In addition, scale bar(s) should be added in Fig. 6F.

Answer: Thanks for your kind suggestions. We have added age and gender of mice in Fig. 6 according to your suggestion. Thank you very much for your valuable advice. In addition, according to your suggestions, we have added scale bar(s) in New-Fig6.tif.

Question 8: In Fig. 7, what age and gender of mice were used should be clarified.

Answer: Thanks for your kind suggestions. We have added age and gender of mice in Fig. 7 according to your suggestion. Thank you very much for your valuable advice

Question 9: In Fig. 8, what age and gender of mice were used should be clarified.

Answer: Thanks for your kind suggestions. The result shown in Fig 8 was carried out on H9C2 cell experiments. Thank you very much for your valuable advice.

Question 10: In Suppl. Fig. 2, the arrow did not indicate any band/signal in Tubulin “55KD”.

Answer: Thanks for your kind suggestions. According to your suggestions, we relabeled Tubulin “55KD” with arrows in Suppl. Fig. 2. Thank you very much for your valuable advice.

Question 11: In Suppl. Fig. 7, “dh/m”, “db/db”, and “CIH” did not match the band position. The 110 KD band in “Nuclear-Nrf2” did not fit in the figure.

Answer: Thanks for your kind suggestions. According to your suggestions, we have matched the “dh/m”, “db/db”, and “CIH” with the band position in Suppl. Fig.7.

We have conducted WB results and replaced the representative image of “Nuclear-Nrf2” in Suppl. Fig. 7, as shown in New Fig. 7. Thank you very much for your valuable advice.

Question 12: In Suppl. Fig. 8, the 54 KD band(s) in “p-LKB1” were not clear.

Answer: Thanks for your kind suggestions. We have conducted WB results and replaced the representative image of “p-LKB1” in Suppl. Fig. 8, as shown in New Fig. 8. Thank you very much for your valuable advice.

Question 13: “Dihydroethidium” (line 35) should be changed to “dihydroethidium”.

Answer: Thank you for your review and guidance. As recommended by reviewer, " Dihydroethidium " has been changed into " dihydroethidium" in line 35 marked in red.

Question 14: “Ismail Laher [9]” (line 66) should be changed to “Baran et al. [9]” or “Laher and his colleagues [9]”.

Answer: Thanks for your kind suggestion. As recommended by reviewer, " Ismail Laher [9]" has been changed into " Baran et al. [9]" in line 66 marked in red.

Question 15: “Hu k [10]” (line 69) should be changed to “Peng and Hu [10]”.

Answer: Thank you for your review and guidance. As recommended by reviewer, " Hu k [10]" has been changed into " Peng and Hu [10]" in line 69 marked in red.

Question 16: “37°C” (lines 153, 160, and 165) should be changed to “37 ˚C”.

Answer: Thanks for your kind suggestion. As recommended by reviewer, "37°C" has been changed into " 37 ˚C" marked in red.

Question 17: Concerning intermittent hypoxia in cardiomyocytes, several paperers (Hu, Z. et al. Sleep Breath doi: 10.1007/s11325-023-02863-8. Online ahead of print; Chen, Q. et al. Sleep Breath 27, 129-136, 2023; Dong, N. & Liu, W.Y. Sleep Breath 27, 399-410; Takasawa, S. et al. Int. J. Mol. Sci. 23, 8782, 2022; Takasawa, S. et al. Int. J. Mol. Sci. 23, 12414, 2022; Moulin, S. et al. Antioxidants 11, 1462, 2022) have recently been reported. The authors should cite the papers and make some discussions.

Question 18: There are some reports concerning human cardiomyocytes in intermittent hypoxia such as Regev, D. et al. Int. J. Mol. Sci. 23, 10272, 2022, Huang, J. et al. Sleep Breath 27, 1005-1011, 2023; Moulin, S. et al. Antioxidants 11, 1462, 2022; Chen, Q. et al. Sleep Breath 27, 129-136, 2023; Dong, N. & Liu, W.Y. Sleep Breath 27, 399-410). The authors should cite the papers and make some discussions.

Answer: Thanks for your kind suggestions. We have cited the papers and make some discussions marked in red in the manuscript according to your suggestions. The detailed modifications are as follows:

1.In the second paragraph of “Discussion”, we have cited the paper such as “Takasawa, S. et al. Int. J. Mol. Sci. 23, 8782, 2022”

2.In the third paragraph of “Discussion”, we have cited the paper such as “Regev, D. et al. Int. J. Mol. Sci. 23, 10272, 2022” and make some discussions marked in red.

3. In the fourth paragraph of “Discussion”, we have cited three papers such as Takasawa, S. et al. Int. J. Mol. Sci. 23, 12414, 2022; Hu, Z. et al. Sleep Breath; Dong, N. & Liu, W.Y. Sleep Breath and make some discussions marked in red.

4. In the sixth paragraph of “Discussion”, we have cited three papers such as Chen, Q. et al. Sleep Breath; Moulin, S. et al. Antioxidants 11, 1462, 2022; Huang, J. et al. Sleep Breath 27, 1005-1011, 2023 and make some discussions marked in red.

Reviewer #2:

Question 1: Introduction: Page 11: “…and excessive ROS can inhibit AMPK inactivation by inhibiting the phosphorylation of LKB1”. Here, I think you meant ROS can inhibit AMPK activation, or?

Answer: Thank you very much for your valuable advice. We agree with your suggestion, and excessive ROS can inhibit AMPK activation. And we have consulted the relevant references [1-5]. The references are as follows:

[1] Zhang J, Zhu Y, Lai C, Du H, Tang K. Expression of sirtuin type 3 in locus ceruleus is associated with long-term intermittent hypoxia-induced neurocognitive impairment in mice. Neuroreport 2020;31(3):220-5.

[2] Wu Y, Duan X, Gao Z, Yang N, Xue F. AICAR attenuates postoperative abdominal adhesion formation by inhibiting oxidative stress and promoting mesothelial cell repair. PLoS One. 2022 Sep 1;17(9):e0272928.

[3] Jiang P, Ren L, Zhi L, Yu Z, Lv F, Xu F, et al. Negative regulation of ampk signaling by high glucose via e3 ubiquitin ligase mg53. Mol Cell 2021;81(3):629-37.

[4] Li Q, Tuo X, Li B, Deng Z, Qiu Y, Xie H. Semaglutide attenuates excessive exercise-induced myocardial injury through inhibiting oxidative stress and inflammation in rats. Life Sci. 2020 Jun 1;250:117531.

[5] Chen X, Li X, Zhang W, He J, Xu B, Lei B, et al. Activation of AMPK inhibits inflammatory response during hypoxia and reoxygenation through modulating JNK-mediated NF-κB pathway. Metabolism. 2018 Jun;83:256-270.

Question 2: Methods, page 11: only male animals were used. Was there any reason not to include females? Including only males is a limitation of this study. How old were the mice at the beginning of the experiment?

Answer: Thanks for your kind suggestions. We have used male mice in the study. Before using male mice, we consulted many related literatures and found that male mice were used in a large number of studies, and female mice were easily influenced by hormones, which complicated many factors [1-3]. In addition, combined with the clinical studies, we found that male OSA patients are at a higher risk of developing DM than female patients [4-5]. Finally, we chose male mice for study. In future studies, we will appropriately add female mice to observe the corresponding indicators. Thank you very much for your valuable advice.

These mice were 4 to 5 weeks old at the beginning of the experiment in the “Animals and treatment”of “Materials and methods” marked in red. Thank you very much for your valuable advice. The references are as follows:

[1] Badran M, Abuyassin B, Golbidi S, Ayas N, Laher I. Uncoupling of Vascular Nitric Oxide Synthase Caused by Intermittent Hypoxia. Oxid Med Cell Longev. 2016;2016:2354870.

[2] Takahashi N, Yoshida H, Kimura H, Kamiyama K, Kurose T, Sugimoto H, Imura T, Yokoi S, Mikami D, Kasuno K, Kurosawa H, Hirayama Y, Naiki H, Hara M, Iwano M. Chronic hypoxia exacerbates diabetic glomerulosclerosis through mesangiolysis and podocyte injury in db/db mice. Nephrol Dial Transplant. 2020 Oct 1;35(10):1678-1688.

[3] Takahashi N, Yoshida H, Kimura H, Kamiyama K, Kurose T, Sugimoto H, Imura T, Yokoi S, Mikami D, Kasuno K, Kurosawa H, Hirayama Y, Naiki H, Hara M, Iwano M. Chronic hypoxia exacerbates diabetic glomerulosclerosis through mesangiolysis and podocyte injury in db/db mice. Nephrol Dial Transplant. 2020 Oct 1;35(10):1678-1688.

[4] Tao Y, Li X, Yang G, Wang L, Lian J, Chang Z. Gender Differences in the Association Between Obstructive Sleep Apnea and Diabetes. Diabetes Metab Syndr Obes. 2021 Nov 23;14:4589-4597.

[5] Chung F, Liao P, Yang Y, Andrawes M, Kang W, Mokhlesi B, Shapiro CM. Postoperative sleep-disordered breathing in patients without preoperative sleep apnea. Anesth Analg. 2015 Jun;120(6):1214-24.

Question 3: Methods, page 11: “24 db/db mice were randomly divided into three groups (n=8): db/m group, db/db group, and CIH group”. Db/db mice cannot serve as a source of db/m mice. Please rewrite this sentence.

Answer: Thank you very much for your valuable advice. We have rewritten this sentence according to your suggestion in the second paragraph of “Animals and treatment”of “Materials and methods” marked in red. The detailed modifications are as follows: The db/db mice (n=16) were randomly divided into the db/db group and CIH group. The db/m mice were used as the control group (n=8).

Question 4: Methods, page 11: “The concentration of oxygen in the chamber for the CIH group was changed from 21% to 5%, 8 h/day for 8 weeks”. -> How long was one cycle of normoxia-hypoxia-normoxia? Did you measure the extent of hypoxemia in blood of mice under intermittent hypoxia?

Answer: Thank you very much for your valuable advice. One cycle of normoxia-hypoxia-normoxia was 5 minutes. In the first 3.5 minutes, 100% nitrogen was injected into the device to reduce the oxygen concentration to 5%, and in the next 1.5 minutes, oxygen was injected to gradually restore the oxygen concentration to 21%.

Thanks for your kind suggestions, we did not measure the extent of hypoxemia in the blood of mice with intermittent hypoxia. However, the oxygen concentration in the hypoxic chamber during modeling is guaranteed at 21% to 5%, and the model is stable, and the laboratory has also published relevant model articles [1-3]. We will be focused on the extent of hypoxemia in our future study. Thank you very much for your valuable advice. The references are as follows:

[1] Li D, Si J, Guo Y, Liu B, Chen X, Qi K, et.al. Danggui-Buxue decoction alleviated vascular senescence in mice exposed to chronic intermittent hypoxia through activating the Nrf2/HO-1 pathway. Pharm Biol. 2023 Dec;61(1):1041-1053.

[2] Bingbing L, Jieru LI, Jianchao SI, Qi C, Shengchang Y, Ensheng JI. Ginsenoside Rb1 alleviates chronic intermittent hypoxia-induced diabetic cardiomyopathy in db/db mice by regulating the adenosine monophosphate-activated protein kinase/Nrf2/heme oxygenase-1 signaling pathway. J Tradit Chin Med. 2023 Oct;43(5):906-914.

[3] Song JX, Zhao YS, Zhen YQ, Yang XY, Chen Q, An JR, et.al. Banxia-Houpu decoction diminishes iron toxicity damage in heart induced by chronic intermittent hypoxia. Pharm Biol. 2022 Dec;60(1):609-620.

Question 5: Methods, page 12: How did the authors measure glucose levels?

Answer: Thank you very much for your valuable advice. Blood glucose levels are measured every two weeks. The mice were fasted for 15 hours at 18:00 the night before each blood sugar measurement, and the blood glucose was measured at 9:00 the next morning.

Question 6: Methods, page 13: the authors describe paraffin embedment of heart specimens for subsequent histological stainings, but in the next method section, frozen sections are mentioned. The method of frozen section preparation is missing.

Answer: Thank you very much for your valuable advice. We have added the method of frozen section in the third paragraph of “Histological examination” of “Materials and methods” marked in red according to your suggestion. The detailed modifications are as follows: The heart was fixed on ice in 4% paraformaldehyde for 2 h, and then transferred to a 30% sucrose solution for dehydration at 4˚C overnight, embedded with OCT, sliced with a frozen microtome with a thickness at 5μm, and fixed in cold acetone for 10min.

Question 7: Methods, page 12: echocardiographic measurements are not precisely described: what view was used? What mode was used? E.g. M-mode for chamber diameters, B-mode for Doppler measurements.

Answer: Thank you very much for your valuable advice. We have precisely described the echocardiographic measurements in “Echocardiography” of “Materials and methods” marked in red according to your suggestion. The detailed modifications are as follows: B-mode is the basic imaging mode of ultrasonic imaging, in which images of the anatomical structure of animals are used to locate the long and short axes of mice. M-mode was used to measure the left ventricular end diastolic diameter (LVDd), left ventricular end systolic diameter (LVDs), left ventricular fractional shortening (LVFS), and left ventricular ejection fraction (LVEF). Color Doppler-mode was used to measure the velocity ratio of the E peak to the A peak in the cardiac mitral valve (E/A).

Question 8: Methods, page 12: Did you measure thickness of the left ventricular walls and the potential impact of diabetes and IH on this parameter?

Answer: Thank you very much for your valuable advice. We have measured the thickness of the left ventricular end systolic/diastolic anterior wall thickness in the experiment. Compared to the db/m group, the LVAWs and LVAWd of the db/db group and the CIH group were increased (p < 0.01). Compared to the db/db group, the LVAWs and LVAWd of the CIH group were increased (p < 0.05). The results suggested that CIH increased thickness of the left ventricular walls in db/db mice

Question 9: Methods: The authors present results for CK-MB, troponin and LDH serum levels, the corresponding section in the Methods how these parameters were measured is missing.

Answer: Thank you very much for your valuable advice. We have added the method of the serum levels of CK-MB, LDH and cTnI in “The measurement of biochemical parameters” of “Materials and methods” marked in red according to your suggestion. The detailed modifications are as follows: The levels of creatine kinase myocardial band (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin Ⅰ (cTnI) were detected by automatic blood biochemical detector.

Question 10: Methods, page 15: “we cultured the H9C2 cells with different concentrations of HG for 48 h.” -> what were the concentrations of HG?

Answer: Thank you very much for your valuable advice. We have added the concentrations of HG in “Cell culture and treatment” of “Materials and methods” marked in red according to your suggestion. The detailed modifications are as follows: We cultured the H9C2 cells with 30mM of HG for 48 h.

Question 11: Methods, page 17: Statistical analysis: did the authors check for normal distribution? What method was used for the comparisons between two groups?

Answer: Thanks for your kind suggestions. The normal distribution has been checked. One-way ANOVA was used for data comparison between groups, and LSD was used for statistical analysis when the variance of pairwise comparison was homogeneous. Dunnett's T3 was used for statistical analysis of variance heterogeneity. The P<0.05 was statistically significant. Thank you very much for your valuable advice.

Question 12: Results, page 23: the results showed that metformin improved membrane potential and decreased oxidative stress in cells which was associated with increased p-AMPK/AMPK and Nrf2 levels. Did this have any impact on the cell viability/apoptosis?

Answer: Thanks for your kind suggestion. Metformin can improve the cell viability and reduce apoptosis. We have provided data on the effect of metformin on cell viability and apoptosis. And we found that metformin improved the cell viability and alleviated apoptosis. Thank you very much for your valuable advice.

Question 13: Graphical abstract: according to the findings, not only cardiomyocyte apoptosis, but also aggravated fibrosis may lead to cardiac dysfunction; fibrosis is not illustrated here.

Answer: Thanks for your kind suggestion. We have revised the Graphical abstract and fibrosis has been illustrated. The detailed modifications are as follows:

Question 14: Throughout the whole manuscript there are sentences which can hardly be understood (below only a couple of examples of them). Please improve this as it might be very confusing for the reader.

Answer: Thank you for your review and guidance. Our manuscript has been send to native English speaking editors for improving writing style and checking the whole manuscript as showed below.

Answer: Thank you for your review and guidance. We have rewritten this sentence marked in red according to your suggestion. Our manuscript has been send to native English speaking editors for improving writing style and checking the whole manuscript. The detailed modifications are as follows: Compared to the db/db group, the BW of the CIH group was decreased (p < 0.05). There was no statistical significance in FBG between the db/db group and the CIH group, but the fluctuation of FBG in the CIH group was increased.

Results, page 19: “As shown in Fig.3A, the results showed that myocardial disorders in the db/db group and the CIH group compared to the db/m group.” -> This sentence is unclear.

Answer: Thank you for your review and guidance. We have rewritten this sentence marked in red according to your suggestion. Our manuscript has been send to native English speaking editors for improving writing style and checking the whole manuscript. The detailed modifications are as follows: As shown in Fig.3A, compared to the db/m group, the results showed that myocardial disorders in the db/db group and the CIH group.

Figure 8, legend: “The cell viability of H9C2 cells which was treated with IH and HG for 24 h and 48h.” -> This sentence is unclear.

Answer: Thank you for your review and guidance. We have rewritten this sentence marked in red according to your suggestion. Our manuscript has been send to native English speaking editors for improving writing style and checking the whole manuscript. The detailed modifications are as follows: The H9C2 cells were treated with IH and HG for 24 and 48 h, and cell viability of H9C2 cells was measured.

Attachment

Submitted filename: 11.29 respone to reviewers.docx

pone.0296792.s004.docx^{(740.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0296792.r003

Decision Letter 1

Michael Bader

19 Dec 2023

Chronic intermittent hypoxia aggravated diabetic cardiomyopathy through LKB1/AMPK/Nrf2 signaling pathway

PONE-D-23-28549R1

Dear Dr. Yang,

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

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

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

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

Kind regards,

Michael Bader

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: All the points that I pointed out were suitably revised in PONE-D-23-28549R1. It is a very nice paper. Congratulations!

Reviewer #2: Dear Authors,

thank you very much for all answers and corresponding changes in the revised manuscript.

**********

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Reviewer #1: No

Reviewer #2: No

**********

PLoS One. doi: 10.1371/journal.pone.0296792.r004

Acceptance letter

Michael Bader

28 Feb 2024

PONE-D-23-28549R1

PLOS ONE

Dear Dr. Yang,

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

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

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PERMALINK

Chronic intermittent hypoxia aggravated diabetic cardiomyopathy through LKB1/AMPK/Nrf2 signaling pathway

Bingbing Liu

Jianchao Si

Kerong Qi

Dongli Li

Tingting Li

Yi Tang

Ensheng Ji

Shengchang Yang

Roles

Abstract

Introduction

Materials and methods

Animals and treatment

The measurement of biochemical parameters

Insulin resistance (IR) and Oral glucose tolerance test (OGTT)

Echocardiography

Histological examination

Measurement of GLUT4 and Nrf2 expression

TUNEL staining

Dihydroethidium staining

Cell culture and treatment

Cell viability

Measurement of ROS production

Measurement of mitochondrial membrane potential

Western blotting

Statistical analysis

Results

Effects of CIH exposure on the metabolic characteristics of db/db mice

Fig 1. Effects of CIH exposure on the metabolic characteristics of male db/db mice at 12 to 13 weeks.

Effects of CIH exposure on the insulin-signaling pathway of db/db mice

Fig 2. Effects of CIH exposure on the insulin-signaling pathway of male db/db mice at 12 to 13 weeks.

Effects of CIH exposure on cardiac pathophysiology of db/db mice

Fig 3. Effects of CIH exposure on cardiac histopathology of male db/db mice at 12 to 13 weeks.

Effects of CIH exposure on cardiac function of db/db mice

Fig 4. Effects of CIH exposure on cardiac function of male db/db mice at 12 to 13 weeks.

Effects of CIH exposure on cardiac apoptosis of db/db mice

Fig 5. Effects of CIH exposure on cardiac apoptosis of male db/db mice at 12 to 13 weeks.

Effects of CIH exposure on cardiac oxidative stress of db/db mice

Fig 6. Effects of CIH exposure on cardiac oxidative stress of male db/db mice at 12 to 13 weeks.

Effects of CIH exposure on the LKB1/AMPK/Nrf2 signaling pathway of db/db mice

Fig 7. Effects of CIH exposure on the LKB1/AMPK/Nrf2 signaling pathway of male db/db mice at 12 to 13 weeks.

Effects of IH and HG on the cell viability, ROS production, and mitochondrial membrane potential of H9C2 cells

Fig 8. Effects of IH and HG on the cell viability, ROS production, and mitochondrial membrane potential of H9C2 cells.

Effects of IH and HG on the LKB1/AMPK/Nrf2 signaling pathway of H9C2 cells

Fig 9. Effects of IH and HG on the LKB1/AMPK/Nrf2 signaling pathway of H9C2 cells.

Discussion

Conclusion

Fig 10. The possible mechanism diagram of DCM is aggravated by CIH.

Supporting information

Data Availability

Funding Statement

References

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Author response to Decision Letter 0

Decision Letter 1

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Acceptance letter

Michael Bader

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Associated Data

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Data Availability Statement

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