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.