Shengmai Injection Suppresses Angiotensin II-Induced Cardiomyocyte Hypertrophy and Apoptosis via Activation of the AMPK Signaling Pathway Through Energy-Dependent Mechanisms

Shengmai injection (SMI), a traditional Chinese herbal medicine extracted from Panax ginseng C.A. Mey., Ophiopogon japonicus (Thunb.) Ker Gawl., and Schisandra chinensis (Turcz.) Baill., has been used to treat acute and chronic heart failure. This study aimed to further clarify the effects of SMI on energy metabolism. SMI could improve cell-survival rate and also reduce myocardial cell hypertrophy and apoptosis. Mitochondria are important sites of cellular energy metabolism, and SMI protects mitochondrial function which was evaluated by mitochondrial ultrastructure, mitochondrial respiratory control ratio (RCR), and mitochondrial membrane potential (ΔΨm) in this study. The expression levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and phosphocreatine (PCr) increased. The expression levels of free fatty acid oxidation [carnitine palmitoyltransferase-1 (CPT-1)], glucose oxidation [glucose transporter-4 (GLUT-4)], and mitochondrial biogenesis-related genes (peroxisome proliferator-activated receptor-γ coactivator-1α [PGC-1α]) were upregulated after SMI treatment. AMP-activated protein kinase (AMPK) is an important signaling pathway regulating energy metabolism and also can regulate the above-mentioned indicators. In the present study, SMI was found to promote phosphorylation of AMPK. However, the effects of SMI on fatty acid, glucose oxidation, mitochondrial biogenesis, as well as inhibiting apoptosis of hypertrophic cardiomyocytes were partly blocked by AMPK inhibitor–compound C. Moreover, decreased myocardial hypertrophy and apoptosis treated by SMI were inhibited by AMPK knockdown with shAMPK to a certain degree and AMPK knockdown almost abolished the SMI-induced increase in the expression of GLUT-4, CPT-1, and PGC-1α. These data suggest that SMI suppressed Ang II–induced cardiomyocyte hypertrophy and apoptosis via activation of the AMPK signaling pathway through energy-dependent mechanisms.


INTRODUCTION
Heart failure (HF) is a major public health concern, affecting more than 6.2 million people in the USA (Benjamin et al., 2019) and more than 23 million people worldwide (Go et al., 2013). The pathogenesis of HF is pathological cardiac remodeling, manifested as left ventricular dilatation, myocardial cell hypertrophy, and interstitial fibrosis (Opie et al., 2006;Shah et al., 2012). Among them, cardiomyocyte hypertrophy is the main cause of decreased myocardial cell contractility and decreased blood supply capacity (Roman et al., 1997). A previous study confirmed the activation of the neuroendocrine system, especially the renin-angiotensinaldosterone system (RAAS), which is a key mechanism leading to cardiomyocyte hypertrophy. Cutting off RAAS to reduce cardiomyocyte hypertrophy has become the basis for preventing and treating of HF (Tamura et al., 2000). Classical HF treatments, in particular angiotensin-converting enzyme indicators/angiotensin receptor blockers and beta-blockers, have been successfully conducted. However, the 1-year mortality rate of HF has only slightly declined, and its 5-year mortality rate has not declined during the last 10 years (Benjamin et al., 2019).
Alterations in cardiac energy metabolism contribute to the severity of HF and cardiac hypertrophy. Myocardial oxygen consumption is very high in HF and cardiac hypertrophy, exhibiting an energy deficit (containing 30-40% less ATP compared with a healthy heart) due to altered energy substrate availability and impaired mitochondrial oxidative metabolism (De Jong and Lopaschuk, 2017). At the same time, important factors are involved in fatty acid metabolism and ATP synthesis, such as carnitine palmitoyl transferase 1 (CPT-1), peroxisome proliferator-activated receptorγ coactivator1α (PGC-1α), and malonyl-CoA decarboxylase, which were abnormally expressed during cardiac hypertrophy (Haynes and Campbell, 2014;Torrens et al., 2017). Among them, CPT-1 controls the rate-limiting step of mitochondrial fatty acid oxidation, and PGC-1α controls mitochondrial biosynthesis and the entire pathway of ATP synthesis (Hoppel et al., 2009). In addition to fatty acid oxidation, glycolysis is an alternate source of ATP production. Glucose is taken up by cardiomyocytes via glucose transporter1 (GLUT-1) and glucose transporter4 (GLUT-4); GLUT-4 is primarily responsible for the insulin-dependent uptake of glucose (Fan et al., 2011;Dolinsky et al., 2016;De Jong and Lopaschuk, 2017). After knocking out the glucose carrier GLUT-4 in mice, the heart gradually develops into a hypertrophic phenotype (Luo et al., 2018). Consequently, metabolic agents may be a complementary effective strategy for treating HF (Rosano and Vitale, 2018).
Shengmai is a traditional Chinese herbal medicine. It is a mixture extracted from three herbs: Panax ginseng C.A. Mey., Ophiopogon japonicus (Thunb.) Ker Gawl., and Schisandra chinensis (Turcz.) Baill. It is the most important rescue agent for patients before wide prescription of Western medicine in China. Nowadays, Shengmai is typically produced as a patented drug on the basis of a standardized formula in different forms, including capsule, powder, oral liquid, and injection. It is also typically prescribed in China as a complementary treatment compared with Western medicine recommended for acute HF and chronic HF. Although the poor quality of trials compromised the reliability of the evidence, the efficacy of Shengmai against HF has been demonstrated in several studies (Zhou et al., 2014). A recent clinical trial further confirmed the efficacy that the patients with CHF and coronary artery disease (CAD) could benefit from the integrative treatments with standard medicines plus Shengmai injection (SMI) with respect to the New York Heart Association (NYHA) functional classification, 6-min walking distance (6MWD), 36-item short-form health survey (SF-36) (SF-36) score, and traditional Chinese medicine (TCM) syndrome score (Xian et al., 2016). Previous basic studies have proved that SMI protected myocardial ischemia-reperfusion injury and doxorubicin-induced cardiomyopathy related to improving myocardial energy metabolism (Quaini et al., 2015;Zhan et al., 2015). However, whether it can improve the cardiac function via improving the mitochondrial energy metabolism of cardiac hypertrophy remains elusive.
Cardiac hypertrophy is characterized by increased cardiomyocyte size, a higher degree of sarcomere organization, and enhanced gene transcription and translation levels, all of which are closely associated with energy metabolism (Tham et al., 2015). This study established the model of cardiomyocyte hypertrophy induced by Angiotensin (Ang) II to investigate the protective effects of SMI on myocardial energy metabolism and the underlying mechanism, so as to provide a new complementary target for the energy metabolism of HF.
To prove that SMI regulates glycolipid metabolism and mitochondrial synthesis through the AMPK pathway, we later divided the cardiomyocytes into five groups: blank, Ang II (0.8 µM), compound C (Ang II 0.8 µM + compound C 0.1uM), SMI (Ang II 0.8 µM + SMI 1/4,000), and SMI + compound C (Ang II 0.8 µM + SMI 1/4,000 + compound C 0.1 µM). Also, the levels of proteins related to energy metabolism were examined, besides flow cytometry analysis of cell apoptosis and apoptosis-related proteins.

Preparation and Quality Control Standard of SMI
The quality control standard of SMI conformed to the National Drug Standard of the Ministry of Health of China, which specifies that the total amount of ginsenoside Rg1 and ginsenoside Re should not be lower than 0.08 and 0.04mg in a 1 ml injection respectively, as analyzed by high-performance liquid chromatography (HPLC).

Isolation of Neonatal Cardiomyocyte and Cell Models for Hypertrophy
Neonatal rat ventricular myocytes were isolated and cultured from the ventricles of postnatal days 1-3 SD rats. The hearts were removed from mice aseptically, and large vessels and atria were discarded. The ventricles were washed, cut into small pieces in Dulbecco's modified Eagle's medium (DMEM), and then digested in 0.25% trypsin in a CO 2 incubator at 37°C for 15 min. To enrich cardiomyocytes and deplete nonmyocytes, the cells were centrifuged and cultured in DMEM for 2 h, allowing the attachment of nonmyocytes. After the purification process, the cardiomyocytes were resuspended and cultured in DMEM supplemented with 15% fetal bovine serum. The culture medium was renewed after 48 h, and the cardiomyocytes were further cultured for 24 h. Next, the culture medium was changed with serum-free DMEM, and the cardiomyocytes were pretreated with PBS, SMI, or compound C (both diluted with PBS) for 1 h and then stimulated with Ang II (0.8 µM) for 48 h.

Cell Viability Assay
The CellTiter-Glo Luminescent Cell Viability Assay Kit (C0068M; Beyotime Institute of Biotechnology, Shanghai, China) was used to test the cell viability according to manufacturer's protocol. Briefly, the cells were seeded on 96-well plates at 1 × 10 5 cells/well overnight. The reagent was reconstituted and added to the cells. After mixing on an orbital shaker for 2 min to lyse the cells, the plate was incubated at room temperature for 10 min, and then the luminescent signals were recorded.

Determination of Cardiomyocyte Area
The cells with a satisfactory growth rate were digested and re-suspended the previous day. They were inoculated in a six-well plate and treated in different groups for 48 h after the liquid exchange. Then, 1 ml of carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) (C0051, Beyotime Institute of Biotechnology, Shanghai, China) was added; stock solution (2×) was added for 10 min at 37°C. The cells were washed one to two times with 1× PBS. After adding 1-2 ml of complete cell culture medium and incubating for 5 min at 37°C, the cells washed one to two times again with 1× PBS. Further 4% paraformaldehyde (1 ml) was added to fix for 30 min, and the cells were washed with 2× PBS two to three times. The cell nuclei were stained with Hoechst 33342 (C1022; Beyotime Institute of Biotechnology, Shanghai, China) solution (final concentration of 1 µg/ml) at room temperature for 5-10 min and washed two to three times with 1× PBS. Next, 1 ml of 1× PBS was added to each well, and the cells were observed and photographed under a fluorescence microscope; Image-Pro Plus software was used to calculate the total area and number of cardiomyocytes in the same field of view and the number of cardiomyocytes in the same field of view.
Average cell area in the same field of view = (total area of the same field of view)/(the number of core cells in the same field of view).

Detecting Protein Content of Cardiomyocytes by Bicinchoninic Acid Assay
Depending on the number of samples, 50 parts of bicinchoninic acid (BCA) reagent A plus 1 volume of BCA reagent (50:1, P0012, Beyotime Institute of Biotechnology) were mixed to configure the appropriate amount of BCA working solution. Then, 10 µl of protein standard diluted with 100 µl of PBS to a final concentration of 0.5 mg/ml. The standard was added to a 96-well plate and diluted to 20 µl. The sample to be tested was added to the 96-well plate, and the standard was diluted to 20 µl. Further, 200 µl of BCA working solution was added to each well and placed at 37°C for 30 min. The optical density (OD) value at 562 nm. The protein concentration was calculated from the standard curve.

Bromodeoxyuridine (BrdU) Incorporation
The cells were incubated with culture media containing 10µM 5-bromo-20-deoxybromouridine (BrdU) (Cat. No. B23151, Invitrogen, USA), for 6 h at 37°C; fixed with 3.7% formaldehyde for 15 min; and treated according to the BrdU experimental protocol. Briefly, the medium was removed, and the cells were washed with PBS. The PBS was replaced with 1 ml of Triton X-100 permeabilization buffer for 20 min at room temperature. The permeabilization buffer was removed, and 1 ml of 1N HCl was added, which was incubated for 10 min on ice. The cells were then incubated with 2N HCl for 10 min at room temperature. The acid solution was removed, and the cells were washed three times with Triton X-100 permeabilization buffer. The buffer was then removed and replaced with the staining solution. After an incubation period of 24 h, the anti-BrdU primary antibody (Cat. No. B35130, Invitrogen, USA) was removed, and the cells were washed three times with Triton X-100 permeabilization buffer. Each well was incubated with fluorescently labeled secondary antibody (Cat. No. A-11001, Invitrogen, USA) for 1 h at room temperature. Finally, the cells were imaged with appropriate filters.

Mitochondria Extraction and Determination of Oxidative Respiratory Function
Genmed medium (2.5 ml) was added into the reaction glass tank, mixed, and sealed. Further, mitochondria, state IV substrate solution, and state III substrate solution were added in a proper sequence following the manufacturer's protocol. Mitochondrial state III and state IV respiration rates in the closed reaction system were determined using dissolved oxygen electrolytic solution (Orion 4-tar; Thermo Fisher Scientific, MA, USA). The mitochondrial respiratory control ratio (RCR) was calculated according to the following formula: RCR, state III respiration rate/state IV respiration rate.

Transmission Electron Microscopy
The adherent cells were directly scraped off, and the cell suspension was centrifuged (1,000 rpm, 5 min). The supernatant was discarded, washed twice with PBS, and pelleted by centrifugation. Further, 2.5% glutaraldehyde was added for fixation at 4°C overnight. After washing with 0.1M PBS three times, the supernatant was incubated with 1% citric acid (Pelco, CA, USA) at 4°C for 3 h, washed with buffer three times, and dehydrated with ethanol step by step, followed by the replacement of propylene oxide. The Spurr resin (SPI-Chem, Structure Probe, Inc., PA, USA) was impregnated and embedded. Finally, it was polymerized in an oven at 70°C. The embedded blocks of different materials were placed on an ultrathin slicer (EM UC6; Leica, Wetzlar, Germany) for sectioning. The ultrathin section was 70 nm thick; it was stained with uranyl acetate (SPI-Chem, Structure Probe, Inc. A) and lead citrate (SPI-Chem, Structure Probe, Inc.) and then observed under a transmission electron microscope (JEM1230; JEOL Ltd., Tokyo, Japan).

JC-1 Stain for Mitochondrial Membrane Potential (Δψm)
Neonatal mouse ventricular myocytes in three groups were cultured in 24-well plates and treated as mentioned earlier.
The cells were washed with PBS once, and 0.25 ml of fresh medium was added into each well. JC-1 dye (0.25 ml, C2006; Beyotime Institute of Biotechnology) was added to each well and mixed well. The cells were then incubated in an incubator at 37°C for 20 min. At the end of incubation, the supernatant was discarded, and the cells were washed. The cell culture medium (0.5 ml) was added, and the cells were observed under a fluorescence microscope. Integrated OD (IOD), as calculated by multiplying the area and average density of the fluorescence, was evaluated using Image-Pro Plus 7 software (Media Cybernetics Inc., MD, USA).

Flow Cytometry Analysis of Cell Apoptosis
Five groups as mentioned earlier (see "Chemicals and Treatments" section) were investigated. Apoptotic rates were examined 48 h after the treatment. In accordance with the Annexin V/Propidium Iodide (PI) Apoptosis Kit (BioVision, CA, USA), 5 × 10 5 cells were collected in each tube and 1 ml of annexin V binding buffer was added, followed by thorough mixing. Subsequently, 5 ml of annexin V-fluorescein isothiocyanate and 10 ml of PI were added. After mixing, the tube was incubated in the dark at 37°C for 15 min. For the early apoptotic cells, the membrane phosphatidylserine was exposed and combined with annexin V, without PI. For the late apoptotic cells, the membranes were permeable to PI and the cells were stained with annexin V and PI. The dead cells were stained only with PI. The samples were analyzed using a FACScan flow cytometer (BD Biosciences, NJ, USA) within 1 h. Flow September 2019 | Volume 10 | Article 1095 Frontiers in Pharmacology | www.frontiersin.org cytometry (BD FACSAria; BD Biosciences) was performed to detect cell apoptosis.

Statistical Analysis
The experimental results were presented as mean ± standard error. One-way analysis of variance was used to compare differences among the three groups, followed by the Bonferroni post hoc test for multiple comparisons. P values <0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism 7.0 software (GraphPad Software Inc., CA, USA).

Selection of SMI Concentration and Its Effects on Cell Viability
Consistent with the findings of previous studies, cardiac hypertrophy could be induced by Ang II (Patrucco et al., 2014;Liu et al., 2015;Dong et al., 2017). RT-qPCR was used to quantify the RNA levels of the hypertrophic markers, such as ANP, BNP, and β-MHC, in cardiomyocytes to determine the concentrations of Ang II and SMI. For this purpose, the effects of Ang II (0.4, 0.8, and 1.2 µM) on the expression levels of these proteins in cardiomyocytes were first investigated. In addition, Ang II (0.8 µM) caused the maximum increase in the mRNA expression of ANP, BNP, and β-MHC compared with the blank group (Figure 1A-a). In contrast, 1/4,000 SMI significantly attenuated Ang II-induced expression levels of ANP, BNP, and β-MHC (Figure 1A-b). A sharp drop in NPPA after treatment with Ang II (1,200 nM) might be related to the cytotoxicity of high-dose Ang II, and NPPA was more sensitive.
To assess the effects of SMI on the viability of cardiomyocytes at different time points and concentrations, a CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, WI, USA) was used. The results showed that 1/4,000 SMI had the best cardiomyocyte viability after incubating with Ang II for 48 h (Figure 1B).

SMI Attenuated Ang II-Induced Cardiac Hypertrophy in Rat Neonatal Cardiomyocytes
To investigate whether SMI could attenuate Ang II-induced cardiac hypertrophy, neonatal mouse cardiomyocytes were treated with SMI (1/4,000) and/or Ang II (0.8 µM) as described in the Materials and Methods section. Ang II markedly increased the area of cardiomyocytes (P < 0.05), and SMI could reverse the effects of Ang II (P < 0.05) (Figure 2A).
To further investigate the effects of SMI on cardiac hypertrophy, the effects of SMI on the average protein content of cardiac hypertrophy were examined with BCA. Ang II significantly increased the average protein content of cardiomyocytes (P < 0.05), and SMI could reverse the effects of Ang II (P < 0.05) ( Figure 2B). Nascent DNA synthesis in replicating cells is traditionally measured with synthetic thymidine analogs such as BrdU. BrdU antibody staining showed a significant difference between BrdU nonproliferating cells and BrdU proliferating cells. Compared with the blank group, the proportion of BrdU + in the Ang II group decreased significantly (P < 0.05). However, the SMI treatment group showed an improved increase (P < 0.05) ( Figure 2C). Collectively, these observations indicated that the decreased average protein content of cardiomyocytes in the SMI group was not related to the inhibition of cardiomyocyte proliferation. Thus, SMI attenuated the hypertrophy of cardiomyocytes induced by Ang II.

SMI Attenuated Mitochondria Dysfunction
Next, the effects of SMI on the mitochondrial structure and function of myocardial cells were assessed. Ultrastructural changes were induced in cardiomyocytes and mitochondria in hypertrophic cardiomyocytes by Ang II treatment for 48 h, as detected by transmission electron microscopy. Compared with the blank group, the myocardial cell structure of the Ang II group was severely damaged, with several vacuole-like structures. The mitochondria became rounded and swollen, the sputum ruptured or even disappeared, the matrix became deep, some mitochondria dissolved and ruptured, and the membrane disappeared. The SMI group might improve the damage in mitochondrial structure to a certain degree ( Figure 3A).
The mitochondrial membrane potential acts as an electrochemical gradient necessary to maintain mitochondrial structure and function and is capable of reflecting the mitochondrial functional status, which is also an indication of early apoptosis in cardiomyocytes. Thus, fluorescence was used to observe the effects of SMI on mitochondrial membrane potential. Figure 3B, when the heart function was normal, the mitochondrial membrane potential was relatively stable, with mostly red fluorescence. The myocardial cell mitochondria were damaged in the Ang II group, and the mitochondrial membrane potential notably decreased (P < 0.05). SMI played a significant role in stabilizing mitochondrial membrane potential (P < 0.05) ( Figure 3B).

As shown in
The RCR is one of the parameters reflecting mitochondrial function that can be used to measure mitochondria (Fatini et al., 2010). The myocardial mitochondrial RCR values were found to be significantly lower than those after HF, suggesting that the mitochondria's ability to make use of oxygen was reduced. Compared with the blank group, the myocardial mitochondrial membrane potential declined in the Ang II group (P < 0.05). The SMI treatment group showed an improved mitochondrial respiratory function and increased RCR (P < 0.05) (Figure 3C).

Changes in Expression Levels of ADP, ATP AMP, and PCr in Hypertrophic Cardiomyocytes
The continuous production of ATP by heart is a prerequisite for maintaining its own pump function, and ATP production is achieved mainly by oxidative phosphorylation in the mitochondrial electron transport chain. The energy in the cells is stored mainly in the form of PCr (Chen et al., 2012). Therefore, the energy level of cardiomyocytes and the function of mitochondria were measured by detecting the expression levels of ADP, ATP, AMP, and PCr via HPLC. Compared with the blank group, the expression levels of ATP, ADP, and PCr in the Ang II group were significantly lower (P < 0.05), revealing a disturbance in the supply of energy for cardiomyocytes in the Ang II group. Compared with the model group, the levels of ATP, ADP, and PCr remarkably increased in the SMI intervention group (P < 0.05). No significant difference was found in the expression level between the three groups (P > 0.05) (Figures 4A-D).

SMI Regulates the Levels of Fatty Acid
Metabolism, Glucose Oxidation, and Mitochondrial Biogenesis With CPT-1, GLUT-4, and PPARγ/PGC-1α Through the AMPK Signaling Pathway SMI was found to regulate the expression level of p-AMPK consistent with the expression trends of CPT-1, GLUT4, and PGC-1α (Supplementary Figure 1). Previous studies showed that AMPK could regulate the expression level of CPT-1 (Kim et al., 2017), GLUT-4 (Manna et al., 2017;Niu et al., 2017), and PGC-1α (Guo et al., 2018). Therefore, it was speculated that SMI regulated the levels of fatty acid metabolism, glucose oxidation, and mitochondrial synthesis probably by activating the AMPK signaling pathway. The effects of AMPK inhibitor compound C on cardiomyocyte viability were evaluated at different concentrations. When the concentration of compound C was 0.1µM, the cell viability was the worst after incubation with Ang II for 48 h ( Figure 5A).
As illustrated in Figure 5B, after adding Ang II into cardiomyocytes, the expression level of CPT-1 protein significantly decreased compared with the control group (P < 0.05). During the process of Ang II-induced cardiac hypertrophy, the expression level of CPT-1 protein increased in the SMI group compared with the Ang II group. Compound C could reverse the effects of SMI (P < 0.05), indicating that SMI regulated CPT-1 via activating the AMPK signaling pathway. Consistent with fatty acid metabolism, the expression levels of GLUT-4 and PPARγ/ PGC-1α proteins significantly decreased (P < 0.05) during Ang II-induced cardiomyocyte hypertrophy, but when SMI was added, these expression levels increased (P < 0.05). However, treating cells with AMPK inhibitor compound C notably decreased the expression levels of GLUT-4 and PPARγ/PGC-1α proteins (P < 0.05). These data indicated that SMI regulated the expression levels of CPT-1, GLUT-4, and PPARγ/PGC-1α via activating the AMPK signaling pathway.

SMI Inhibited Apoptosis of Hypertrophic Cardiomyocytes via the AMPK Signaling Pathway
Apoptosis is a key feature of the progression of heart diseases, and the elimination of pro-apoptotic stimulation or inhibition of the apoptotic cascade can rescue damaged myocardium and prevent the progression of adverse remodeling and HF (Abbate and Narula, 2012). Cardiomyocyte apoptosis may be the cause of cardiac hypertrophy to HF (Abbate et al., 2003). SMI could increase mitochondrial membrane potential (ΔΨm). However, the decline or disappearance of ΔΨm is an early event of myocardial cell apoptosis (Wlodkowic et al., 2012). Therefore, the effects of SMI on cardiomyocyte apoptosis were evaluated. Ang II markedly decreased the rate of apoptosis (P < 0.05), and compound C could reverse the protective effects of SMI on cardiomyocyte apoptosis partly (P < 0.05) (Figure 6A).
To further reveal the role of SMI in inhibiting cardiomyocyte apoptosis, the levels of apoptosis-related proteins were examined by Western blot analysis. As displayed in Figure 6B, compared with the blank group, the expression levels of Bax and caspase-3 proteins in the Ang II group were significantly higher, while the expression level of Bcl-2 was lower (P < 0.05), demonstrating that cardiomyocyte apoptosis occurred in the Ang II-induced cardiomyocyte hypertrophy group. Compared with the model group, the expression levels of Bax and caspase-3 increased and the expression level of Bcl-2 decreased in the SMI intervention group (P < 0.05). Compound C could reverse the effects of SMI on the expression levels of Bax and Bcl-2 (P < 0.05) (Figure 6B).

Effect of AMPK on SMI-Mediated Cardiomyocyte Hypertrophy and Apoptosis
To further validate the effects of AMPK in inhibiting cardiomyocyte hypertrophy and apoptosis treated with SMI, AMPK knockdown experiments were performed using shRNA. Thus, lentivirus vectors expressing shRNA to both AMPK alpha1 and alpha2 were constructed (Tangeman et al., 2012) (Supplementary Figure 4). Decreased myocardial hypertrophy and apoptosis treated with SMI were partly inhibited by AMPK knockdown (Figures 7A, B). AMPK knockdown almost abolished the SMI-induced increase in the expression of GLUT-4, CPT-1, and PGC-1α ( Figure 7C). The mechanism underlying increased levels of fatty acid metabolism, glucose oxidation, and mitochondrial biogenesis with CPT-1, GLUT-4, and PGC-1α was dependent on an SMI-mediated increase in AMPK expression. These observations corresponded with the experiments on AMPK inhibitor compound C.

DISCUSSION
The present study demonstrated that SMI not only reduced the cardiomyocyte hypertrophy and cell apoptosis induced by Ang II but also protected the mitochondrial function in hypertrophic cardiomyocytes. In addition, SMI improved energy metabolism by activating the expression levels of related factors in Ang IIinduced cardiomyocyte hypertrophy, such as energy metabolism (AMPK), fatty acid metabolism (CPT-1), mitochondrial biogenesis factor (PGC-1α), and glucose oxidation . These data suggested the phosphorylation of AMPK as a metabolic switch reconstituting fatty acid metabolism, glucose metabolism, and mitochondrial biogenesis to prevent cardiomyocyte hypertrophy and cell apoptosis. A previous study found that cardiac hypertrophy was both an intermediate step and a determinant of HF (Meijs et al., 2007). This was of great significance to identify alternative therapeutic approaches for HF.
The main source of normal adult myocardial energy is FFA oxidation. FFA oxidation disorder and increased glucose utilization occur in hypertrophic and failing myocardium, in which the direct consequence of this transformation is the reduction of fatty acid utilization. Although the oxidation of glucose needs less oxygen, it produces much less energy compared with FFA oxidation, causing the heart to remain in an energy-starved state, ultimately leading to a lack of myocardial energy. Due to the high energy demand of the heart, a fine balance in energy substrate (glucose and fatty acid) use is crucial in maintaining metabolic flexibility (Karwi et al., 2018). A key metabolic regulator is AMPK; a heterotrimeric protein is centrally involved in controlling cellular energy homeostasis. Its activation through metabolic stress stimulates energy-generating processes and has been shown to regulate hypertrophy-regulating genes in several models. The activation of the AMPK signaling pathway increases FFA oxidation by phosphorylating ACC, in turn decreasing the malonyl-CoA content and activating CPT-1. AMPK stimulates GLUT-4 translocation and increases the whole-body use of glucose (Yamaguchi et al., 2005). However, knowledge about the influence of Ang II on cardiac energy metabolism is limited. In the present study, when the action time reached 48 h, the expression levels of p-AMPK, CPT-1, and GLUT-4 decreased, suggesting that glucose metabolism and fatty acid metabolism were weakened. To validate further the effects of AMPK in SMImediated energy metabolism, AMPK knockdown experiments were performed using shRNA. The findings were consistent with the results of previous studies (Liu et al., 2018). Mitochondrial biogenesis is regulated by a complex regulatory system. PGC-1α, a transcription coactivator of nuclear receptors and master regulator of metabolism, plays a pivotal role in mediating metabolic alterations in both physiological and pathological hypertrophies. PGC-1α is also involved in mitochondrial FIGURE 5 | SMI regulates expression levels of CPT-1, GLUT-4, and PPAR-γ/PGC-1α through activating AMPK signaling pathway (n = 3). (A) Selection of AMPK concentration. *, compared with the blank group, # P < 0.05; compared with compound C (1 nM) group, & p < 0.05; compared with compound C (10 nM) group, P < 0.05. (B) SMI regulates protein expression levels of CPT-1, GLUT-4, and PPAR-γ/PGC-1α through activating AMPK signaling pathway. *, compared with the blank group, # P < 0.05; compared with Ang II group, & p < 0.05; compared with compound C group,^P < 0.05; compared with SMI group, P < 0.05. biogenesis, which is vital for cell survival (Kulikova et al., 2018). The present study confirmed that SMI activated the AMPK signaling pathway and upregulated the expression levels of PGC-1α, CPT-1, and GLUT-4 compared with the model group. Thus, SMI regulated energy metabolism by activating the AMPK/ PPARγ/PGC-1α signaling pathway. Specifically, SMI regulated fatty acid and glucose metabolism via activating the AMPK signaling pathway.
The energy metabolism in cardiomyocytes is closely associated with the status of mitochondrial function. Mitochondrial dysfunction can be caused by the intermediate metabolite accumulation in HF. In the present study, SMI was found to reduce mitochondrial structural damage, decrease respiratory function, and reduce membrane potential, which were induced by Ang II induction. The cardiac hypertrophic program was accompanied by progressive mitochondrial remodeling, and particularly by mitochondrial permeability transition pore opening (Javadov and Karmazyn, 2007;Javadov et al., 2009). When the membrane potential decreased the uncoupling of oxidative phosphorylation, ATP was depleted, and the number of oxygen radicals increased, FIGURE 6 | SMI inhibited the apoptosis of hypertrophic cardiomyocytes (n = 3). (A) Effects of SMI on cardiomyocyte apoptosis in hypertrophic cardiomyocytes. 3 Control group; (b) Ang II group; (c) compound C group; (d) SMI group; and (e) SMI + compound C group. Dead cells were labeled with annexin V (-) propidium iodide (PI) (+) and are shown in the Q1 area; late apoptotic cells were labeled with annexin V (+) PI (+) and are shown in the Q2 area; early apoptotic cells were labeled with annexin V (+) PI (-) and are shown in the Q3 area; live cells were labeled with annexin V (-) PI (-) and are shown in the Q4 area. Summarized data for TMRE fluorescence intensity measured with confocal microscopy. (B) Effects of SMI on apoptosis-related proteins in hypertrophic cardiomyocytes. *P < 0.05, compared with the blank group; # P < 0.05, compared with the Ang II group; & P < 0.05, compared with the compound C group; ^P < 0.05, compared with the SMI group.
the expression level of Bcl-2 decreased in the Ang II group, thus justifying the increase in the rate of apoptosis; this result was consistent with the finding of a previous study (Hutchinson et al., 2008;Day et al., 2011).
ATP is produced in mitochondria, and it needs to be continuously produced to maintain their function. Studies have shown that mitochondria in cardiomyocytes account for about one third of the total cardiomyocytes (Ventura-Clapier et al., 2011), indirectly indicating that the supply of myocardial energy is inseparable from mitochondria with normal function and structure. Mitochondrial dysfunction may affect the function of myocardium (Guzun et al., 2011). When cardiomyocytes were induced by Ang II, the mitochondrial structure, respiratory function, and membrane potential were notably damaged. Therefore, a decline in the ATP level was noted in the Ang II group. A study suggested that an increased intracellular AMP/ATP ratio was the primary mechanism for the activation of AMPK (Oakhill et al., 2011). Recent studies have shown other sensitive AMPK activators besides the activation of AMPK signaling pathway. Also, the deletion and decline in FDP can directly activate AMPK rather than being dependent on AMP (Kemp and Oakhill, 2017). The present study revealed for the first time that FBP was associated with the activation mechanism of AMPK, with no dependence on AMP and ADP. The AMP content did not change in Ang II-induced hypertrophic cardiomyocytes compared with the blank group, and SMI did not increase the expression level of AMP. Thus, it was suggested that SMI might directly activate the AMPK signaling pathway to increase the expression levels of ATP, ADP, and PCr in hypertrophic cardiomyocytes.
However, this study had many limitations. It did not deeply investigate the specific mechanism by which SMI regulated the mitochondrial function, glucose metabolism, and fatty acid metabolism. No animal experiments were conducted to verify the results. SMI itself is a compound preparation of TCM, which is extracted from three herbs: Panax ginseng C.A. Mey, Ophiopogon japonicus (Thunb.) Ker Gawl., and Schisandra chinensis (Turcz.) Baill. More than 28 ginsenosides have been extracted from ginseng, which could delay cardiac mitochondrial impairment (Scott et al., 2001;Wang and Ren, 2002). SMI was found to have more than 10 components using HPLC in the present study, including ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rh1, ginsenoside Rd, schisandrin, ginsenoside Rg5 (Rk1), and ginsenoside Rh3. Which specific component of SMI regulated energy metabolism was not studied. These issues will be addressed in future studies.