SAS is closely linked to the development of cardiovascular disease, in which CIH plays an important role. In our experiment, each intermittent hypoxia cycle lasted approximately two minutes, mimicking 30 times intermittent hypoxia per hour, which was equivalent to severe SAS. Chen et al.  studied a CIH animal model and concluded that the lowest oxygen saturation was approximately 70%, equivalent to severe SAS. In another model, Song et al.  also demonstrated that, owing to the gas constant exchange in the box, there was no carbon dioxide accumulation. The partial pressure of carbon dioxide in the blood of mice had no correlation with the intermittent hypoxia cycle, which confirmed that the mice did not rebreathe. These findings eliminated the interference of carbon dioxide retention.
CIH can stimulate hypoxic chemoreceptors in the carotid body, thus activating the systemic sympathetic nervous system and RAS , but how CIH affects local RAS and subsequently contributes to the development of CVD remains unknown. In our study, we found that ACE expression was highest in the CIH group (Fig. 1a, b), suggesting that CIH could facilitate the expression of ACE in mouse cardiac myocytes in vivo. ACE is the key enzyme of RAS and participates in the systemic and local effects of RAS through endocrine, autocrine and paracrine secretion. The most prominent physiological activity of ACE is to cleave Ang I to Ang II and inactivate bradykinin. Ang II is the product of ACE [11, 12], and ACE can damage the cardiovascular system through production of Ang II. Our study also demonstrated that Ang II expression was higher in the CIH group (Fig. 2a, b) than in the blank control and air control groups (Fig. 2e-h), indicating that CIH induced the expression of ACE and its product Ang II which is potentially implicated in CIH-linked CVD. AngII can act on the angiotensin type 1 receptor, and increase inflammation, oxidative stress, apoptosis, and cardiac remodeling, ultimately leading to heart failure.  As Ang II can up-regulate the expression of ACE , we believe that there was a positive feedback between Ang II and ACE.
ACE 2 is the homolog of ACE, which shares 42% sequence homology with ACE. The main function of ACE 2 is to cleave Ang I to Ang (1–9) and Ang II to Ang (1–7). The catalytic activity of cleaving Ang II to Ang (1–7) is 400 times higher than cleaving Ang I to Ang (1–9) . Therefore, the main product of ACE 2 is Ang (1–7). Ang (1–7) exhibits anti-RAS activity, as well as functioning as a vasodilatation and anti-proliferative agent, suggesting that ACE 2 is the key enzyme in balancing the vasoconstrictive and proliferative effects of Ang II, as well as the anti-proliferative effect of Ang (1–7). In our study, we observed higher expression of ACE 2 in the myocardium of mice from CIH group (Fig. 3a, b) than that from blank and air control groups (Fig. 3e-h). Since increased ACE 2 levels offer benefits to cardiac structure and function, we speculate that CIH-induced expression of ACE 2 was an adaptive response. Indeed, Zhang R  demonstrated that ACE 2 mRNA and protein expression increased in the early stages of hypoxia in pulmonary artery smooth muscle cells, but decreased at a later stage, which was accompanied by the accumulation of hypoxia inducible factor-1α (HIF-1α) and Ang II. Thus, it is possible that HIF-1α downregulates ACE 2 expression through Ang II. In our experiments, CIH lasted for 12 weeks, and ACE 2 expression remained higher in the CIH group than in blank and air control groups. Whether local accumulation of HIF-1α and Ang II at later hypoxic stages was responsible for the downregulation of ACE 2 remains unclear, and requires further study. This may include examining the correlation of the expression levels of myocardial ACE 2 with HIF1α and Ang II in hypoxic heart tissue for a prolonged CIH time more than 12 weeks.
In TUNEL assays, we also observed that myocardial cell apoptosis was the most severe in CIH group. Under electron microscopy, the apoptosis of cardiomyocyte and capillary endothelial cells were the most serious in CIH group. We also observed the ultrastructural changes of mitochondria and cristae, indicating that CIH can also cause mitochondrial damage. Chen et al. reported that the damage of CIH to mitochondria was related to myocardial apoptosis . Mitochondrial damage is one of the primary mechanisms leading to apoptosis, and hyperactivity of RAS can produce apoptosis in early stages of cardiac disease . Activated RAS can also exacerbate mitochondrial damage and increase apoptosis in endothelial cells, and mitochondria-ROS acts as a central regulator of RAS-mediated cell damage .We surmise that CIH can lead to myocardial damage by elevating the expression of ACE and Ang II, while the higher expression of ACE2 presents insufficiency of compensation.
Our study also showed that Ang II expression was highest in ARB group (Fig. 2c, d). ARBs can protect the cardiovascular system by blocking the activation of angiotensin type 1 receptor by Ang II, therefore, inhibit the negative feedback for the release of renin. Finally, ARBs increase renin, Ang I, Ang II and Ang (1–7) [20, 21]. Increased Ang II concentrations can interact with the angiotensin type 2 receptor. It is believed that this is mediated by bradykinin and nitric oxide (NO) , and the effect of this combination of mediators is both vasodilatory and anti-proliferative, which protects the cardiovascular system. Ang II has been shown to up-regulate the expression of ACE , which can be blocked by angiotensin type 1 receptor blocker (ARB) but not by angiotensin-2 receptor blockers, indicating that Ang II-induced expression of ACE was mainly achieved through angiotensin type 1 receptor- (AT-1) activation. In the present study, treatment with TERT, an ARB, reduced the level of ACE induced by CIH, which might alleviate the myocardial damage of CIH. Although this reduction did not reach statistical significance compared with the CIH group, maximal pharmacological effects of TERT were observed between four to eight treatment weeks, and we only treated mice with TERT for four weeks, which may account for this observation. Therefore, longer treatment with TERT is needed to further clarify if TERT could confer protection against CIH-induced cardiac injury via efficiently suppressing an increase in ACE.
In our study, we also observed that TERT treatment significantly elevated the expression of ACE 2 in the heart, suggesting that this may be one of the mechanisms by which TERT protects against hypoxia-induced heart injury. Previously, Koka et al.  showed that Ang II down-regulated ACE 2 expression in HK-2 cell line, which was blocked by the angiotensin type 1 receptor blocker losartan an extracellular signal-regulated kinase (ERK1/2) and p38 MAP kinase-specific antagonist, but not by the angiotensin type 2 receptor blocker PD123319, suggesting that Ang II suppressed ACE 2 expression through angiotensin type 1 receptor-ERK1/2 and p38 MAP kinase signaling. In the present study, Ang II expression in the hearts was increased in the CIH group, and this increase was further elevated by TERT treatment. These observations were in line with the TERT-induced increase in ACE 2. But increased Ang II expression did not suppress the expression of ACE 2 in mouse CIH hearts with TERT treatment, which was most likely due to the blockage of angiotensin type 1 receptor- by TERT.
Under electron microscopy and through TUNEL assay, we observed that the apoptosis of cardiomyocytes and capillary endothelial cells were rare in the ARB group. The ultrastructural changes of mitochondria and cristae induced by CIH were also alleviated by ARB treatment. We hypothesize that the blockade of the angiotensin type 1 receptor (AT1R) by ARB treatment can elevate the expression of ACE2, thus enhancing the protective effects of ACE 2 and alleviating the myocardial damage induced by ACE and AngII.
Furthermore, TERT, acting as an ARB, can block the effects of Ang II more thoroughly than ACEI, such as Ang II generated from chymotrypsin or other non-ACE pathways. Therefore, TERT may replace ACEI in long-term clinical use to avoid “AngII inhibition escape” phenomenon.