Discussion
Myocardial hypoxia is a negative prognostic marker associated with myocardial injury and a poor patient outcome. It is also a primary component of CHD microenvironment[13]. Recent work supports the idea that miRNAs are involved in the adaptive response to myocardial hypoxia in cardiovascular diseases. Liu et al. suggested that miR-130a participated in hypoxia/reoxygenation (H/R) induced injuries by accelerating autophagy and relieving apoptosis in cardiomyocytes[14]. Moreover, the silencing of miR-122 alleviated cardiomyocyte H/R injury by promoting GATA-4 expression [15]. Li et al. reported that miR-7a/b protected H9C2 cells from hypoxia-induced injury and promoted cardiac remodelling by decreasing fibrosis and apoptosis[16]. In particular, for the hypoxic response, all published literature agrees that miR-210 is a robust target of hypoxia-inducible factors. It is well known that the upregulation of miR-210 is a hypoxic signature in vivo[17]. Hence, miR-210-mediated myocardial hypoxia may represent an attractive target for therapeutic intervention in CHD. However, the mechanisms involved in miR-210 repair of myocardial hypoxia remains to be elucidated. Our study was performed to identify the role of miR-210 in a hypoxic H9C2 model, which might contribute to the development of potential diagnostic and therapeutic approaches in CHD. First, we determined that a 48 h hypoxia treatment duration in H9C2 cardiomyocytes induced myocardial injury. Also, miR-210 significantly inhibited cell apoptosis and autophagy in hypoxic H9C2 cells. Since Caspase8ap2 was a putative target of miR-210, miR-210 likely mediated apoptosis and autophagy of H9C2 cells via suppressing Caspase8ap2. Taken together, the miR-210/Caspase8ap2 pathway alleviated the effects of CoCl2 induced injury in H9C2 cells by regulating apoptosis and autophagy. There are few published articles on the effects of miR-210 on the other two apoptotic signaling pathways in cardiomyocytes. Besides of death receptor/ Caspase8 pathway, mitochondria/Caspase9 and ER stress/Caspase12 pathways are also major signaling pathways in apoptosis[18, 19]. Further studies on the association between miR-210 and other potential target apoptotic genes in cardiomyocytes with hypoxia need to be conducted.
In the miRNA field, extensive evidence consistently points to a dominant role for miR-210 in the hypoxic stress response[20]. The induction of apoptosis in cells subjected to hypoxia has been of great importance to cell survival and tissue homeostasis. Numerous previous reports have supported an anti-apoptotic role of miR-210 during hypoxia. For example, miR-210 exhibited cytoprotective effects from apoptosis[10, 21], while the downregulation of miR-210 induced apoptosis[22, 23]. Similar results were verified in this study in which the overexpression of miR-210 inhibited apoptosis, whereas silencing miR-210 strikingly enhanced apoptosis in hypoxic H9C2 cells. Further, Caspase8ap2 has been predicted as a downstream target of miR-210[24]. Caspase8ap2 was initially identified as a member of the death signalling complex joining Fas and tumour necrosis factor-α (TNF-α) during apoptosis[12]. Its interaction with death effectors promotes the delivery of caspase-8 from the apoptosis pathway to activate the downstream caspase cascade. Because of its role in apoptosis, we validated Caspase8ap2 for its functional involvement in H9C2 apoptosis under hypoxic conditions using the dual-luciferase reporter assay. Western blot analysis demonstrated that miR-210 overexpression reduced the protein level of Caspase8ap2, cleaved caspase 8 and caspase 3 upon exposure to hypoxia, suggesting that miR-210 inhibited cardiomyocyte apoptosis by modulating the levels of Caspase8ap2.
The double-edged sword biological function of autophagy in myocardial hypoxia injury was demonstrated by recent studies. Moderate autophagy contributes to ATP generation and cellular homeostasis when cells are exposed to non-serious injury. However, uncontrolled excessive autophagy could be induced by serious or long-term injury. Excessive autophagy may contribute to autophagic cardiomyocyte death[25, 26]. In our study, CoCl2 treatment for 48 h significantly inhibited cell survival. Hence, under this serious injury condition, we assumed that excessive autophagy was induced, and it may have contributed to cell death. Autophagy and apoptosis are considered to be highly interconnected through mechanisms that involve Beclin-1[27]. Beclin-1 is necessary for autophagy to occur, it can direct autophagy-related proteins to phagocytes, and can regulate autophagy formation and maturation with other proteins. Although it is widely accepted that autophagy can promote survival in response to milder stress, excessive and/or long-term autophagy can also cause cell death by promoting the excessive self-digestion of essential organelles and proteins. In the current study, both the upregulation of adapter protein p62 and downregulation of autophagy indicators LC3 II/I and Beclin-1 showed that miR-210 may protect H9C2 cells from excessive autophagy in response to hypoxia.
In conclusion, miR-210 collectively exerts anti-apoptosis and anti-autophagy effects, which alleviates myocardial injury in response to hypoxia. This study suggests that the miR-210/Caspase8ap2 pathway is essential to protect cells from hypoxia. Future studies aimed at further characterizing the underlying mechanism of miR-210 in other apoptosis-associated pathways should be conducted.