APMCG-1 attenuates ER and mitochondrial dysfunction in cardiomyocytes and skeletal muscles
ER is the main location for the storage of calcium ions in cardiomyocytes, and the apoptosis pathway mediated by ER is bound to be related to calcium ions (Groenendyk et al., 2021). As shown in Fig. 3A, the four indicated concentrations of APMCG-1 reduced PA-induced intracellular Ca2+ levels in H9c2 cells, which reflected the reduction of Ca2+ levels in the cytoplasm. The key signals of ER stress pathway, which involvs to GRP78, p-PERK, p-eIF2ɑ, ATF4 and CHOP in PA-induced H9c2 cells significantly increased. However, when the cells were pretreated with APMCG-1, the levels of these signal proteins all decreased (Fig. 3B). Hoechst 33342 and PI staining showed abnormal morphology and nuclear condensation in PA-induced H9c2 cells. With the increased concentration of APMCG-1, the fluorescence intensity gradually decreased, implying that APMCG-1 can repair PA-induced nuclear condensation (Fig. 3C, 3D). Excessive ER stress causes the release of Ca2+ from the ER to specific structures of mitochondria, which initiates the caspase cascade and ultimately leads to apoptosis. Thus, a rhodamine 123 fluorescent probe was used to evaluate mitochondrial dysfunction. Visualization of untreated H9c2 cells by a fluorescence microscope showed a bright green fluorescence around the nucleus. The bright green fluorescence in PA-induced H9c2 cells significantly decreased, indicating the loss of MMP. However, the fluorescence intensity increased after the pretreatment with APMCG-1 (Fig. 3E). In flow cytometry analysis of PA-induced H9c2 cells, APMCG-1 significantly reduced the proportion of early apoptotic cells against PA-induced mitochondrial apoptosis (Fig. 3F). The caspase family plays a key role in cell apoptosis and regulates cell apoptosis together with a variety of protein factors. The expressions of Bax, Bcl2 and Cleaved-Caspase3 of H9c2 cells were significantly decreased in APMCG-1-treated PA-induced H9c2 cells (Fig. 3G). These results illustrate that the Bax family signaling pathway is required for APMCG-1 to enhance ER and mitochondrial function in the cardiomyocytes.
We collected each group of cells and treated them with 2.5% glutaraldehyde fixed solution at room temperature in order to examine the ultra-structure of the cells using a transmission electron microscope (HT7700, HITACHI, Japan) in order to assess the ER stress and mitochondrial apoptosis in H9c2 cell (Jung & Mun, 2019). The findings demonstrated that the control group had healthy cardiomyocytes with intact mitochondria and ER. The model group’s myocardial cells displayed evident damage, swollen and vacuolated mitochondria, and expanded ER. The amount of healthy mitochondria and ER in H9c2 cells gradually increased as APMCG-1 concentration rose (Fig. 3H).
Meanwhile, APMCG-1 exhibited a strong protective property against in mitochondrial dysfunction in skeletal muscles. We characterized the morphological changes in PA-induced C2C12 cells (Fig. 3I and 3J). The morphology of cell death observed by fluorescence microscopy implied the APMCG-1 protection against PA-induced C2C12 cell damage. Further, the results showed that APMCG-1 up-regulated MMP (Fig. 3K) and down-regulated the number of apoptotic cells (Fig. 3L). In addition, APMCG-1 reduced the expression levels of Cleaved-Caspase3, Cleaved-Caspase9, and Bax/Bcl2 in PA-induced C2C12 cells, suggesting the action of APMCG-1 against PA-induced endogenous apoptosis (Fig. 3M). On the basis of the above information, we concluded that APMCG-1 enhances mitochondrial biosynthesis in skeletal myoblasts.