The heart is very sensitive to hypoxia due to its property of high oxygen uptake [15]. Exposure to hypobaric hypoxia can consequently result in exaggerated arterial hypoxemia and lead to cardiac dysfunction including SCD [2, 19]. In this study, we showed that RCE significantly normalized the decrease in phosphorylation of eNOS produced by hypoxia. RCE also reduced Arg-1 expression level and oxidative stress markers in heart tissues under hypoxic conditions and abolished hypoxia-induced cardiac apoptosis. Taken together, these findings indicate that RCE treatment exerts cardioprotective effects in hypoxic animals by reducing hypoxia-induced oxidative stress, repressing arginase activity, and regulation of cardiac NO metabolism (Fig. 5).
The proposed mechanism of RCE against hypoxia-induced cardiac apoptosis
The expression of eNOS and NO production in the vascular endothelium is crucial for the maintenance of vascular tone and cardiovascular physiology [20] including in the pulmonary vasculature [10]. This pathway is an oxygen-dependent process and limited by hypoxia. We showed that hypoxia decreased the expression of phosphorylated eNOS in the heart, as expected. However, this phenomenon was reversed by RCE treatment. Furthermore, the contents of cardiac nitrite and cGMP, which are markers of NO activity in biological systems [4], were restored by RCE treatment. These results indicated that RCE treatment shifted the consumption of L-arginine from Arg-1 to eNOS, increasing NO formation and its downstream effectors in the heart.
We also showed that RCE treatment affected upstream eNOS regulatory signaling by enhancing the p-AKT level under hypoxic conditions (Fig. 4f, g, and h). This is consistent with the fact that eNOS activity is regulated by protein kinase AKT in vascular endothelial cells [21] and hypoxic lung tissues [22]. Based on these findings, RCE may act to restore the expression of phosphorylated eNOS via the PI3K/AKT-associated signaling pathway in cardiomyocytes. However, this hypothesis requires further investigation. A recent study using RNA microarray technology showed that Rhodiola extract significantly regulates the eNOS pathway in T98G human neuroglial cells [23]. In our study, we demonstrated that RCE treatment increased both eNOS expression and NO signaling in hypoxic animals. These results might provide preliminary evidence that RCE is able to restore eNOS signaling in vitro and in vivo and that this may contribute to its ability to limit hypoxic cardiac failure.
Hypoxia has been reported to increase the expression and activity of cardiac arginase, which is highly correlated with the severity of several cardiac and vascular disorders, such as heart failure and myocardial ischemia [8]. Arginase inhibition has been shown to both improve NO availability by shifting L-arginine utilization from arginase to eNOS and to decrease oxidative stress in hypoxic heart tissue [9]. Taken together, these observations suggest that inhibition of arginase activity is a potential therapeutic strategy in hypoxia-associated heart disorders.
We showed that RCE treatment significantly decreased both the expression and activity of arginase in hypoxic heart tissue and restored NO signaling. These findings may indicate that RCE restored NO signaling partially by suppression of arginase activity, which is regulated by various upstream factors including ROS, inflammatory cytokines, and mitogen-activated protein kinase (MAPK) pathways [8].
Until now, the exact regulatory mechanism of RCE on arginase was unclear. It was reported previously that hypoxia contributed to arginase activation induced by the ROS burst through c-Jun and AP-1 interaction [9]. We have shown that RCE treatment is able to attenuate ROS intensity in both hypoxic lung [16] and heart tissues. These results suggest that RCE could inhibit arginase activity by limiting hypoxia-induced oxidative stress. Considering that arginase has been shown to be associated with many other cardiac diseases such as atherosclerosis and heart failure [8], it would be interesting to investigate the efficacy of RCE against these disorders.
Hypoxia increased cardiac oxidative stress and resulted in the impairment of NO signaling. Hypoxia also decreased the bioavailability of NO by increasing NO destruction through peroxynitrite formation as well as promoting uncoupling of eNOS [7]. These events all contribute to the pathological progression of cardiac failure [24].
Oxidative stress directly contributes to impaired cardiac-muscle contraction by modifying proteins with high levels of protein carbonyl groups [25]. In contrast, overexpression of antioxidant enzymes can improve oxidative stress-mediated cardiac defects [26]. In the present study, RCE did not change the antioxidant system under normoxia. However, in hypoxia, where SOD2 and GPx2 are decreased, RCE treatment restored expression of these fundamental cardiomyocyte anti-oxidant pathways [11, 27]. These results are also consistent with the marked radical scavenging activity of RCE reported in a previous study [28]. Together, these results suggest that RCE could decrease hypoxia-induced oxidative stress via the nonenzymatic pathway, but not by directly increasing the expression of antioxidant enzymes.
In a rodent model of exhaustive exercise, salidroside, a major bioactive compound of RCE, exerted a cardioprotective effect via its antioxidant activity [15]. In this study, we have shown that RCE reduces biomarkers of cardiac oxidative stress (ROS, MDA, and protein carbonyl) in rat myocardium. This suggests that the protective effect of RCE is associated with its antioxidant potential. In addition, growing evidence shows that obstructive sleep apnea is associated with excessive ROS production induced by hypoxia. These ROS directly contribute to decreased NO availability and eNOS uncoupling [29] as well as heart problems in the elderly [30]. It would be interesting to investigate whether RCE has beneficial effects for those populations.
Hypoxia triggers many different signaling pathways in the heart. Among them, MAPK pathways, such as ERK and p38MAPK, are of importance [31, 32]. Most studies concur that activation of p38MAPK and ERK signaling is positive for cell survival [31]. In the present study, RCE treatment significantly promoted the expression of phosphorylated p38MAPK. However, no change was observed in the level of p-ERK in our model. These results indicated that RCE promotes cardiomyocyte survival under acute hypobaric hypoxia exposure by p38MAPK, but not ERK, signaling pathway in heart tissue. These results are similar to those observed in the acute exhaustive rodent model where salidroside regulates MAPK signaling pathways, through modification of ROS generation [15]. Thus, the regulatory effect of RCE on MAPK pathways might again be due to the antioxidant property of RCE.
A recent study of note showed that oxidative stress is associated with coronary vascular tone through the AMPK-eNOS-NO pathway. This suggests a possible role of AMPK in the vascular endothelium under the hypoxic condition [30]. In our previous studies, RCE regulated both hepatic and pulmonary function via the AMPK pathway [17, 33]. It would be interesting to clarify the role of AMPK in hypoxia-treated animals in the future.
Myocyte apoptosis is a crucial modulator in the development of cardiac failure [34]. Based on this idea, a growing number of anti-apoptosis interventions for heart failure are now under investigation. The anti-cardiac apoptotic effects of RCE on chronic intermittent hypoxia in mice have been recently confirmed by Lai et al. [13, 14]. In the present study, the protective effect of RCE on acute and hypobaric hypoxia was investigated. We showed that RCE not only regulated members of the Bcl-2 family (Bcl-2, Bax, and Bcl-xL), but also the downstream markers of apoptosis, such as caspase 3 (Fig. 4). These findings indicate that RCE promotes an anti-apoptotic effect on hypoxia-treated animals, suggesting a cardioprotective effect of RCE for both acute and chronic hypobaric hypoxia exposures. It should be noted that the PI3K/AKT signaling pathway is a survival signal in response to hypoxia [35]. Thus, the increased p-AKT level conferred by RCE is consistent with the anti-apoptotic effect of RCE occurring through the activation of PI3K/AKT signaling pathway.
In addition, there are some limitations in this study. We did not monitor the blood pressure and heart rate due to the limitations of a simulated hypoxia chamber. It would be further investigated in the future study.