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.