Introduction

Obstructive sleep apnea syndrome (OSAS), which is characterized by chronic, repetitive short cycles of oxygen desaturation followed by rapid reoxygenation (chronic intermittent hypoxia, CIH), has become a public health concern because it leads to poor sleep quality and clinical complications. Among the complications associated with OSAS, cardiovascular issues are the most severe [1]. Patients with OSAS who do not accept effective treatment have a higher rate of cardiovascular mortality [2]. Myocardial apoptosis, which can lead to cardiovascular dysfunction, can also be induced by chronic intermittent hypoxia [3, 4]. Therefore, inhibition of myocardial apoptosis could improve cardiovascular complications caused by OSAS.

NO is an important signaling molecule in the human body that participates in several physiological functions including sleep regulation [5]. NO and NO synthases (the neuronal, endothelial, and inducible isoforms of nitric oxide synthase, known as nNOS, eNOS, and iNOS, respectively) are known to be important regulators of the cardiovascular system [6]. After ischemia or hypoxia, normal metabolism of NO and NOS activity is disrupted, leading to a loss of redox equilibrium, which is associated with pathological damage in the cardiovascular system [7]. These results, together with the fact that chronic hypoxia is the most characteristic pathophysiological change associated with OSAS, suggest that the NO metabolic pathways might be involved in CIH-induced cardiovascular damage, and that restoration of normal NO metabolism may be a potential treatment for cardiovascular complications induced by CIH.

Telmisartan, an angiotensin II type 1 receptor blocker (ARB), is used to treat high blood pressure. Recently, pleiotropic effects of telmisartan were reported in several preclinical and clinical studies, which showed beneficial effects of telmisartan for conditions other than hypertension [8, 9]. Compared to other ARB antihypertensive drugs, telmisartan has some unique biological activities, including regulation of NO metabolites [10] and attenuation of ischemic myocardial injury [11]. However, the therapeutic potential of telmisartan in OSAS remains unknown. Here, we report the results of our investigation into the protective effect of telmisartan in a rodent model of intermittent hypoxia-induced myocardial apoptosis.

Methods

Animal model and experimental design

Forty male Sprague–Dawley (SD) rats (220–250 g) were purchased from the experimental animal center of Wuhan University (Wuhan, China). Animals were kept in a departmental animal facility on a 12:12-h light–dark cycle under standard laboratory conditions (temperature 25 ± 2 °C, humidity 60 ± 5 %). Rats were provided with standard rodent chow and allowed free access to water. The experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Tongji Medical College at Huazhong University of Science and Technology. Rats were randomly divided into four groups (n = 10 each): normoxia + vehicle, normoxia + telmisartan, CIH + vehicle, and CIH + telmisartan. Rats were administered either telmisartan (10 mg/kg dissolved in double-distilled water) or vehicle by oral gavage prior to exposure to intermittent hypoxia on every day of the 8-week experimental period.

Intermittent hypoxia exposure

CIH was performed using custom-built chambers (OxyCycler A84, BioSpherix, Redfield, NY, USA) connected to a supply of O2 and N2 gas. The CIH protocol was as follows: O2 level was reduced from 21 to 8 % over a period of 120 s, held at 8 % for 120 s, returned to 21 % over a period of 50 s, and held at 21 % for 300 s. The rats were exposed to intermittent hypoxia for 8 h/day (during the day) on 7 days/week over 8 weeks. For the normoxic group, rats were placed in similar chambers under normoxic conditions. Within 24 h after the last exposure, rats were killed with pentobarbital sodium (40 mg/kg administered by intraperitoneal injection). The left ventricular free wall was excised from each rat, perfused with cold PBS, and preserved in liquid nitrogen or 10 % formalin for in vitro analyses.

Western blotting

Protein abundance of nNOS, eNOS, iNOS, and 3-nitrotyrosine (3-NT) were determined by Western blotting, which was performed according to routine procedures. Briefly, after tissue homogenization, proteins were extracted from the left ventricular free wall sample using RIPA lysis buffer (Beyotime, Jiangsu, China) containing a protease inhibitor cocktail to prevent protein degradation. Protein concentration was determined using a Bradford protein assay kit (Bio-Rad, Hercules, CA). Proteins (30 μg/band) were separated via 10 % SDS-PAGE and transferred to 0.45-μm nitrocellulose membranes (Bio-Rad). Membranes were blocked in 5 % non-fat dry milk in TBST (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05 % Tween-20) for 1 h at room temperature. Membranes were incubated with rat monoclonal antibodies (Abgent, San Diego, CA, USA) against nNOS(1:500), eNOS(1:500), iNOS(1:500), and anti-3-NT(1:500) overnight at 4 °C, after which the membranes were incubated with a secondary antibody conjugated to horseradish peroxidase at room temperature for 2 h. Reactive proteins were analyzed with an ECL Western blotting detection system. All experiments were performed three or more times.

Measurement of NO production

NO content in myocardial tissue was detected using a commercially available NO assay kit (Keming Bioengineer Company, Suzhou, China) according to the manufacturer’s instructions. Because NO is very active and NO monomers have a very short existence within tissues, indirect methods were adopted to determine NO content in cardiac muscle tissue. Within tissue, NO is easily oxidized to NO2−, which reacts under acidic conditions with diazonium salts to form diazo compounds, which further couple with naphthyl-based ethylene diamine to form specific products with characteristic absorption peaks at 550 nm, through which NO content can be determined. Briefly, fresh tissue of the rat left ventricular wall (0.05 g) was homogenized with the extraction solution (0.5 mL) by centrifugation (12,000 rpm for 15 min at 4 °C), and 100 μL of the supernatant was collected and mixed with the specific reaction reagent. The resulting mixture was allowed to rest for 15 min at room temperature, after which its absorbance at 550 nm was measured using a microplate reader.

Lipid peroxidation assay

Intermittent hypoxia often leads to increased lipid peroxidation that causes tissue damage. Malondialdehyde (MDA) is commonly used to measure the effect of lipid peroxidation. In this study, oxidative stress was measured using a commercially available kit to detect the level of MDA production in myocardial tissue according to the manufacturer’s instructions (Keming Bioengineer Company, Suzhou, China).

Detection of plasma levels of C-reactive protein and interleukin 6

Changes in plasma levels of inflammatory cytokines were detected. We used ELISA kits (Neobioscience Technology Company, Beijing, China) to measure C-reactive protein (CRP) and interleukin 6 (IL-6) in the plasma according to the manufacturer’s instructions.

Terminal deoxynucleotidyl transferase dUTP nick end labeling staining

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for apoptotic cells in the left ventricle was used to evaluate myocardial injury. As described previously [12], a commercially available TUNEL staining kit (Boster Biological Technology, Ltd., China) was used to detect apoptosis according to the manufacturer’s instructions. At least three apoptotic cells were selected from each section of each group (three hearts per group) for photographing in the visual field (×400 magnification). The numbers of apoptotic cells and total cardiomyocytes were determined. The results are expressed as the percentage of apoptotic cells among the total cell population.

Statistical analysis

All data were expressed as mean ± SD. Data were statistically analyzed using one-way analysis of variance (ANOVA) for group comparisons. Student–Newman–Keuls post hoc tests were used when appropriate. A value of P < 0.05 was considered statistically significant.

Results

Treatment with telmisartan regulated NOS expression and limited excessive NO production

Immunoblot analysis confirmed that eNOS expression in the left ventricle was significantly decreased after CIH (P < 0.05; Fig. 1a), while iNOS expression was significantly increased (P < 0.05; Fig. 1b). There was no significant difference in nNOS abundance between the control and hypoxia groups (P > 0.05; Fig. 1c). Compared to the control group, NO synthesis was significantly elevated in the left ventricle after exposure to CIH (P < 0.05; Fig. 1d). Treatment with telmisartan abolished CIH-induced changes in eNOS and iNOS expression and inhibited excessive NO synthesis in the left ventricle after exposure to CIH, but had no effect on the expression of nNOS.

Fig. 1
figure 1

Chronic intermittent hypoxia (CIH) disrupted NOS/NO. After male SD rats were exposed to CIH for 8 weeks, the expression of eNOS was inhibited (a), while iNOS was activated (b) and NO was overproduced (d), but there was no significant difference in nNOS expression between the control group and the hypoxia group (c). Pre-treatment with telmisartan effectively inhibited CIH-induced iNOS activation and excessive production of NO, and preserved eNOS levels, but had no effect on nNOS. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; (I) normoxic, (II) normoxic + telmisartan, (III) hypoxia, and (IV) hypoxia + telmisartan. Results represent mean ± SD with means compared using one-way ANOVA. A value of P < 0.05 is considered statistically significant. ★P < 0.05 vs normoxia; ★★P < 0.05 vs hypoxia; &P > 0.05 vs normoxia; &&P > 0.05 vs hypoxia

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Telmisartan inhibited CIH-induced oxidation/nitration stress in the left ventricle

Oxidation/nitration stress is considered to play a key role in the pathophysiological process of CIH-induced tissue injury. We measured MDA production in the left ventricle after exposure to CIH as an indicator of oxidation stress. MDA levels were significantly elevated in the left ventricle of rats exposed to CIH as compared to the control group (P < 0.05; Fig. 2a). Furthermore, we found a significant increase in 3-NT protein expression in the left ventricle after CIH exposure (P < 0.05; Fig. 2b). The effects of CIH on MDA levels and 3-NT protein expression were suppressed by the treatment with telmisartan.

Fig. 2
figure 2

MDA levels and 3-NT protein expression are increased in the left ventricular (LV) myocardium by chronic intermittent hypoxia (CIH). Intermittent hypoxia significantly increased expression of MDA (a) and 3-NT protein (b) in the LV myocardium. Pre-treatment with telmisartan significantly inhibited CIH-induced MDA and 3-NT expression in the LV myocardium. (I) Normoxic, (II) normoxic + telmisartan, (III) hypoxia, and (IV) hypoxia + telmisartan. Results represent mean ± SD with means compared using one-way ANOVA. A value of P < 0.05 is considered statistically significant. ★P < 0.05 vs normoxia; ★★P < 0.05 vs hypoxia

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Treatment with telmisartan suppressed CIH-induced overexpression of inflammatory mediators

CIH can activate inflammation involved in the pathophysiological processes of CIH-induced tissue injury. In this study, we examined the plasma levels of two typical inflammatory cytokines, CRP and IL-6, using the ELISA method. Plasma levels of CRP (P < 0.05; Fig. 3a) and IL-6 (P < 0.05; Fig. 3b) were increased in rats exposed to CIH as compared to the control group. Treatment with telmisartan suppressed plasma CIH-induced overexpression of CRP and IL-6.

Fig. 3
figure 3

Inflammatory cytokines in the circulating blood. Chronic intermittent hypoxia (CIH) significantly increased expression of C-reactive protein (CRP) (a) and interleukin 6 (IL-6) (b) in the circulating blood. Pre-treatment with telmisartan significantly inhibited CIH-induced expression of these cytokines in the circulating blood. Results represent mean ± SD with means compared using one-way ANOVA. A value of P < 0.05 is considered statistically significant. ★P < 0.05 vs normoxia; ★★P < 0.05 vs hypoxia

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Telmisartan significantly reduced CIH-induced myocardial apoptosis in the left ventricle of rats

Little apoptosis were found in the left ventricle of control rats, but apoptosis was significantly increased in the left ventricle of rats exposed to CIH (P < 0.05; Fig. 4). Treatment with telmisartan effectively attenuated CIH-induced myocardial apoptosis in the left ventricle of rats exposed to CIH.

Fig. 4
figure 4

Chronic intermittent hypoxia (CIH) caused myocardial apoptosis. After 8 weeks of CIH, male SD rats showed clear evidence of myocardial apoptosis as compared to animals exposed to normal oxygen concentrations. Pre-treatment with telmisartan effectively inhibited CIH-induced myocardial apoptosis. Representative photomicrographs of TUNEL assay (×400). Apoptotic cardiomyocyte nuclei appear brown stained (red arrows), whereas TUNEL-negative nuclei appear blue. a Normoxic. b Normoxic + telmisartan. c Hypoxia. d Hypoxia + telmisartan. Results represent mean ± SD with means compared using one-way ANOVA. A value of P < 0.05 is considered statistically significant. ★P < 0.05 vs normoxia; ★★P < 0.05 vs hypoxia

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Discussion

Cardiovascular damage is the most common complication of OSAS. The precise pathophysiological mechanisms involved in OSAS-induced cardiovascular damage are poorly understood. There is no doubt that the pathogenesis of OSAS-related cardiovascular damage is multifactorial. In the current study, we used a rodent model of CIH to demonstrate that inflammatory processes and oxidative/nitration stress were involved in the pathophysiological myocardial apoptosis induced by CIH. More importantly, we found that CIH altered the activity of NO synthase and disrupted NO metabolism. Because treatment with telmisartan attenuated these pathological changes, these results support the potential of telmisartan in the treatment of OSAS-induced cardiovascular complications as an alternative to continuous positive airway pressure (CPAP). In addition, these results serve as a valuable reference for OSAS patients who need to take antihypertensive drugs.

Inflammatory processes and oxidative stress are considered to play key roles in the development of CIH-induced cardiovascular complications [13]. Similarly, in our current study, we found that CIH induced inflammation and expression of oxidative stress products in circulating blood and myocardial tissue. In addition, NO is believed to play a key role in the pathological mechanisms underlying myocardial damage induced by hypoxia/ischemia [7]. NO is an important signaling molecule that is involved in the regulation of almost all aspects of cellular function, including gene transcription [14] and apoptosis [15]. In humans and other mammals, NO is produced from l-arginine by the three enzymes of the NOS family: eNOS, nNOS, and iNOS. NO signaling also acts to maintain normal functioning of the cardiovascular system. Under normal physiological conditions, the level of NO in the cardiovascular system is stable, and it is mainly produced by eNOS and nNOS, which are constitutively expressed in cardiac myocytes [16] and vascular endothelial cells [17], and are known as constitutive NOSs. However, in pathological conditions such as ischemia and hypoxia, constitutive NOS activity is disrupted, NO homeostasis is disturbed, and, more importantly, iNOS is activated, which plays a significant role in the manifestation of harmful effects.

NO/NOS have been studied extensively in the context of cardiovascular diseases, but the precise role of NO/NOS in cardiovascular diseases is still unclear. The source of NO and the NO content in local tissue are of decisive significance for the ultimate effect of NO. eNOS, which is considered to be the most important NO synthase in myocardial tissue, maintains vascular tone and produces anti-thrombotic and anti-inflammatory effects [18]. When myocardial tissue is exposed to ischemia/hypoxia, eNOS activity is inhibited and tissue damage occurs [19]. Correspondingly, preservation of eNOS activity has become a focus of studies aimed at protecting myocardial tissue against hypoxic/ischemic damage [20–22]. In our study, we found that CIH significantly inhibited eNOS expression in myocardial tissue, and that telmisartan preserved the activity of eNOS and attenuated CIH-induced myocardial apoptosis. Therefore, we speculate that preservation of eNOS activity is a crucial mechanism by which telmisartan ameliorates myocardial injury induced by CIH.

nNOS is considered to mediate hypoxic/ischemic myocardial injury [7]. However, in our study, we found that there was no significant change in nNOS. In our test model, various reasons may explain why intermittent hypoxia did not cause any change in nNOS expression in myocardial tissue. However, in our study, intermittent hypoxia lasted for 8 weeks, which was longer than in most similar studies concerned with tissue injuries caused by intermittent hypoxia. Therefore, we hypothesize that in our study, nNOS in the myocardial tissue of the rat might have adapted to the intermittent hypoxic environment; therefore, there was no change in nNOS expression.

Low levels of NO are necessary to maintain normal cardiovascular function, and excess NO is harmful to cardiovascular tissue. When myocardial tissue was exposed to ischemia/hypoxia, NO levels significantly increased, and this excess NO was mainly produced by the activated iNOS [23, 24]. Excess NO can damage cardiac tissue through a variety of mechanisms such as the induction of apoptosis by changing the balance between the apoptosis mediators Bak and BCL-2 [25]. Most importantly, excess NO reacts with superoxide anion (O−· 2) to form the potent oxidant peroxynitrite (ONOO2 −), which causes oxidative damage, nitration, and S-nitrosylation of biomolecules such as proteins, lipids, and DNA [26]. Similarly, in our studies, high levels of NO were found in myocardial tissue exposed to CIH, which were accompanied by increased expression of iNOS, while eNOS expression decreased and nNOS expression remained constant. These results showed that elevated NO was a consequence of activated iNOS. In addition, the level of 3-NT was increased in the left ventricle after exposure to CIH. We speculate that 3-NT was produced because of reactions between the excessive NO and products of CIH-induced oxidative stress. Our results suggest that together, all these factors, namely inflammatory processes, high levels of NO, and oxidative/nitration stress, lead to the CIH-induced myocardial injury.

Telmisartan, an angiotensin II type 1 receptor blocker, is mainly used to reduce blood pressure. Many studies have demonstrated the therapeutic potential of ARB in the treatment of hypoxic/ischemic organ damage [27, 28]. In comparison with other ARBs, telmisartan has unique advantages, including better fat solubility that allows it to more easily penetrate cell membranes [29]. The protective effect of telmisartan against ischemic myocardial injury has been confirmed [11]. In our study, the protective effect of telmisartan on CIH-induced myocardial injury was demonstrated, and this effect was found to be mediated by regulation of NOS activity, inhibition of excessive NO synthesis, and suppression of oxidation/nitration stress and the inflammatory response.

In summary, this study provides the first evidence that telmisartan attenuates CIH-induced myocardial apoptosis, in part by preserving eNOS levels, inhibiting iNOS expression and excessive NO generation, and suppressing oxidation/nitration stress and the inflammatory response. The present findings provide novel insight into the mechanisms through which OSAS induces cardiovascular complications. Meanwhile, considering the extensive use of telmisartan in the clinic and the high incidence of OSAS, there will likely be more meticulous and in-depth studies performed in the future. Further research into the relationship between the dosage of telmisartan and its cardiovascular protective effect, and the relationship between the protective effect of telmisartan and the severity of OSAS, is required. However, it should be noted that our study had some limitations. The use of NOS or specific NOS inhibitors would help to clarify the role of NO/NOS in protective effects in this setting, and the relationship between NO/NOS, oxidative stress, and the inflammatory response should be demonstrated in future studies.