3.1 The bioinformation related to apelin.
Elabela is considered as a peptide hormone that is derived from the placenta of mammalian animals. The human Elabela gene is located on chromosome 4 GRCh 38.p12, which contains three exons. The precursor protein encoded by this gene composed of 54 amino acids (aa) peptide, which is cut by the Golgi apparatus to a mature form with 32 aa. Elabela protein contains two bi-arginine sequences that are recognized and cleaved into different sizes. Apelin, the first endogenous ligand of ELA, to be discovered expressed widely in human body. We found that apelin and its family are named according to the length of their sequence. (Fig. 1). Hence, the biological function and mechanism need to be clarified.
3.2 The effect of doxorubicin and cobalt chloride on cell viability and death.
To investigate the effect of doxorubicin (DOX) on rat primary cell viability, the proportion of viable cells was determined by trypan blue assay. The results indicated that DOX (0.1, 0.5, 1, 2, 5uM/L) affected cell death in dose-dependent manner and 1 uM/L DOX significantly increased cell death rate (Fig. 2A). CCK-8 assay was performed to evaluate the cell viability of doxorubicin, and the 1 uM DOX administration reduced cell survival index dramatically (Fig. 2B). To determine the optimal interaction time, cells were incubated for different times and evaluated using Trypan blue and CCK-8 assays. The exposure of doxorubicin for 12 h significantly increased the rate of cell death and decreased cell viability in rat primary cardiomyocytes (Fig. 2C and 2D). The oxidative stress induced by cobalt chloride (CoCl2) was served as a complementary model. As shown in Fig. 2E, the cell viability was significantly decreased upon 800uM CoCl2 treatment. To determine the optimal working time points, the primary cells were incubated for 0, 6, 12, 18, 24, 30, and 36 h. Our results demonstrated that treatment with CoCl2 for 24 h significantly decreased cell survival rate (Fig. 2F).
3.3 Apelin-13 inhibits apoptosis by attenuating doxorubicin-induced cardiotoxicity.
To figure out the biological function of apelin-13 in DOX induced cardiotoxicity in rat cardiomyocytes, pretreatment with apelin-13 at different concentrations (0.1, 0.5, 1, 2 and 5uM) for 1 h and 1uM apelin-13 dramatically reduced the mortality of DOX-treated primary cardiomyocytes (Fig. 3A). To verify the cardioprotective effect of apelin-13, we assessed cell viability and LDH release. Compared with that of the scramble group, apelin-13 significantly enhanced cell viability and reduced LDH release (Fig. 3B and 3C), which means apelin-13 could resist DOX induced cell damage. The western results showed that apelin-13 reduced DOX-induced cell death by inhibiting apoptosis (Fig. 3D). The cleaved caspase3 and PARP was alleviated in the apelin-13 group. Next, we found that the number of apoptotic cells increased in DOX induced group and apelin-13 reduced apoptotic cell rate significantly by TUNEL assay (Fig. 3E). Changes in mitochondrial membrane potential can also reflect the state of early cell apoptosis. The result indicated that apelin-13 reduced the number of JC-1 monomers formation compared with DOX treatment (Fig. 3F). Therefore, our results indicated that apelin-13 possess a protective role in DOX induced cardiotoxicity.
3.4 The effect of apelin-13 in CoCl 2 induced oxidative stress.
To test whether apelin-13 possessed function in oxidative stress, we performed comprehensive functional analysis. The results demonstrated that treatment of apelin-13 decreased cell death rates (Fig. 4A). Then, CCK-8 result suggested that apelin-13 increased cell viability compared with scramble peptide (Fig. 4B). The result of LDH release showed that apelin-13 significantly lowered LDH release (Fig. 4C). To investigate whether apelin-13 could inhibit CoCl2 induced cell death, our result revealed that CoCl2 increased apoptotic cell rates and apelin-13 alleviated cell apoptosis (Fig. 4D and 4E). The expression of cleaved-PARP and caspase-3 proteins was decreased in apelin-13 treated group (Fig. 4F). These findings suggested that apelin-13 reduced the apoptosis and cell damage induced by CoCl2.
3.5 Apelin-13 significantly resists DOX-induced cardiotoxicity in vivo.
We next tested whether apelin-13 has a protective effect on rodent hearts in vivo. Male C57BL/6J mice were intraperitoneally injected with doxorubicin (5 mg/kg) for five weeks consecutively and apelin-13 was injected daily i.p. Then they were subjected to echocardiography and sacrificed the following week (Fig. 5A). The release of CKMB and LDH in peripheral blood were decreased significantly in apelin-13 administrated group (Fig. 5B). The EF and FS rates of were increased, whereas the LVED values were decreased significantly by echocardiography when treated with apelin-13 (Fig. 5C). The evidence from Sirius red staining suggested that fibrosis was significantly reduced by apelin-13 (Fig. 5D). Accordingly, HE staining showed that apelin-13 treatment alleviated the collagenous fiber induced by DOX in heart tissues (Fig. 5E). Therefore, these results suggested that apelin-13 ameliorated the cardiotoxicity induced by DOX in mice.
3.6 Apelin-13 inhibits cardiotoxicity through activating ERK/MAPK and PI3K/AKT signaling pathways.
ERK/MAPK and PI3K/AKT are important pathways that protect the physiological function of the heart by inhibiting apoptosis. To verify the molecular mechanism by which apelin-13 protects primary cardiomyocytes against apoptosis, a western blot analysis was performed. The results showed the expression of the phosphorylated ERK protein was activated upon apelin-13 treatment compared with DOX treatment interference (Fig. 6A). To validate the role of apelin-13 in PI3K/AKT signaling pathways, we found that apelin-13 activated the expression of the phosphorylated AKT and PI3K protein (Fig. 6B). When the action of CoCl2 was blunted, the expression of the phosphorylated ERK protein was upregulated (Fig. 6C). Meanwhile, apelin-13 activated the expression of the both phosphorylated proteins after CoCl2 treatment in rat primary cells (Fig. 6D). These results revealed that apelin-13 protects cardiomyocytes from apoptosis through ERK/MAPK and PI3K/AKT signaling pathways.
3.7 Apelin-13 protects cardiac function by binding APJ.
To determine the role of ML-221, treatment with ML-221 to detect its interaction with apelin-13 by western blot. We found ML-221 inhibited the effect of apelin-13 on apoptosis of cardiomyocytes with DOX administration (Fig. 7A). To determine whether ML-221 was involved in ERK/MAPK and PI3K/AKT signaling pathways when treated with DOX, we found ML-221 significantly increased the expression of the phosphorylated AKT, PI3K and ERK protein compared with apelin-13 (Fig. 7B and 7C). Furthermore, we found ML-221 can also inhibit apoptosis in CoCl2 induced apoptosis. The phosphorylated AKT, PI3K and ERK protein expressions were significantly upregulated after cell treatment with ML-221 (Fig. 7E and 7F). These results suggested that apelin-13 inhibits apoptosis by binding to APJ. In brief, we plotted the mechanism of apelin-13 according our research (Figure.S1) .