Protective efficacy of Shenge San (参蛤散) on mitochondria in H9c2 cardiomyocytes

Abstract

OBJECTIVE:

To investigate whether peroxisome proliferator-activated receptor γ-coactivator-1α/nuclear respiratory factor 1 (PGC-1α/NRF1) activity can protect mitochondrial function in the setting of cardiac hypertrophy and improve cardiomyocyte energy metabolism.

METHODS:

Cardiac hypertrophy was modeled in H9c2 cells treated with isoproterenol (ISO) to assess the effects of Shenge San (参蛤散, SGS) on cell viability and mitochondrial membrane potential. We assessed mitochondrial complex mRNA levels and mitochondrial oxidative phosphorylation factor mRNA and protein levels.

RESULTS:

Compared with the 100 μM ISO group, cell size was significantly decreased in the 0.3 mg/mL SGS and 20 μM ZLN005 (PGC-1α activator) groups (P < 0.01). Compared with the SGS (0.3) +ISO group, we observed lower phosphorylated adenosine monophosphate-activated kinase (AMPK) protein levels in the ISO and ZLN005+SGS+ISO groups (P < 0.01). Compared with the compound C group, SGS significantly increased PGC-1α expression in ISO-induced cardiac hypertrophy cells (P < 0.01), and this was inhibited by compound C pretreatment (P < 0.05). Compared with the ISO group, the mitochondrial red-green fluorescence ratio increased in the 0.3 mg/mL SGS group (P < 0.05). mRNA levels of cytochrome c oxidase subunit 1 (CO1) in the ISO and compound C groups were lower than those in control group (P < 0.01), and the mRNA levels of CO1 and ATP8 were significantly lower in the ISO and compound C groups versus control (P < 0.01). Compared with the SGS (0.3) +ISO group, ATP synthetase subunit 8 (ATP8) mRNA was significantly decreased in the ISO group (P < 0.01) and compound C+SGS+ISO group (P < 0.05). Compared with the SGS (0.3) +ISO group, NRF1 mRNA levels were significantly decreased (P < 0.05) in the ISO and compound C+SGS+ISO groups.

CONCLUSIONS:

SGS can attenuate ISO-induced cardiomyocyte hypertrophy, restore the decrease in mitochondrial membrane potential, and upregulate PGC-1α/NRF1 levels. Notably, these effects can be blocked by AMPK inhibitor-compound C.

1. INTRODUCTION

Cardiac hypertrophy is a major pathogenic factor for heart failure, which has a high morbidity rate. Hypertrophy refers to increased volume of the myocardium and heart cavity, which also enhances energy demands.¹ In the early stage of heart failure, the nuclei and mitochondrial are compensatorily larger; however, the performance of the mitochondria is diminished and the morphology is altered.²

Mitochondria account for one-third of the total cell volume in cardiomyocytes, and 90% of energy required by the normal human myocardium is provided by mitochondrial oxidative phosphorylation (OXPHOS).³ This process depends on the biosynthetic function of mitochondria, which refers to the proliferation of mitochondria and the process of mitochondrial synthesis, which is an important mechanism for cells to achieve self-renewal and regulation.⁴ Abnormalities in mitochondrial biosynthesis that affect mitochondrial electron chain transmission and OXPHOS impair cardiac structure and function, aggravating load cardiomyopathy, in a process termed metabolic remodeling.⁵ Therefore, promoting mitochondrial biosynthesis and pathway regulation of metabolic substrates to fachieve optimal energy use could improve cardiac function and protect mitochondria.

Heart failure can cause palpitations, asthma, phlegm, and edema in Traditional Chinese Medicine (TCM). Heart failure is attributed to the heart itself and associated with lesions in other organs such as the lung, kidney, and liver. A lack of heart, unable to encourage blood flow, leads to stasis of blood, water, sputum, and other pathologies that further damage the heart Ying and Yang, creating a vicious circle. Shenge San (参蛤散, SGS) was first described in the Northern Song Dynasty as "Sheng Ji Zong Lu (General Medical Collection of Royal Benevolence)," formerly known as the Du Sheng Bing Zi, attributable to Chapter lung and edema, to treat "cough, swelling, and limb floating".⁶ In cases of chronic heart and kidney illness, kidney Yang failure is difficult to regulate with water metabolism; water evil attacks the heart and lung; leading to cough and asthma hardly. Ginseng tastes sweet and benefits Qi, while Gecko is warm, salty, and calms Qi. Both benefit the heart Qi and warm the lung and kidney.

Based on previous studies, peroxisome proliferator-activated receptor γ-coactivator-1α (PGC-1α) regulates mitochondrial biosynthesis through NRFs (or TFAMs), thereby affecting myocardial hypertrophy and metabolic remodeling. In this study, we assessed whether regulation of protein and gene expression was associated with metabolic remodeling and mitochondrial function and biosynthesis in H9c2 cells. We measured the effects of SGS on PGC-1α, nuclear respiratory factor 1 (NRF1), transcription factor A mitochondrial (TFAM), and mitochondrial OXPHOS complex-related genes in the presence of adenosine monophosphate-activated kinase (AMPK) inhibition or PGC-1α activation.

2. METHODS

2.1. Cell culture

H9c2 embryonic rat cardiomyocytes were provided by Professor Chan SW (Hong Kong Polytechnic University). Briefly, cells were cultured in medium containing Dulbecco’s minimum essential medium (Gibco, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 1% penicillin and streptomycin, pH 7.1, and maintained in 95% air and 5% CO₂ at 37 ℃.

2.2. Cell viability

The cells were seeded in 96-well plates at 1 × 10⁴ per well, they were treated with different concentrations of SGS (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 mg/mL), and cell viability was determined by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Similarly, when the cells were grown to 70%, they were treated with isoproterenol (ISO) (0, 100, 200, 400 μM) for 24 h in the presence or absence of 0.3 mg/mL SGS and 20 μM ZLN005 pre-treatment. Next, 1 mg/mL MTT was added to each well, the plate was incubated at 37°C for 4 h, and then the optical density value was measured at a wavelength of 490 nm by a microplate reader (CLARIOstar, BMG, Cary, NC, USA).

2.3. Cell size measurement

The cells were seeded in a 6-well plate at 5 × 10⁵ per well and grown to a density of 50%. The model groups were treated with 30 or 100 μM ISO for 24 h, the treatment group was pretreated with 0.3 mg/mL SGS and 20 μM ZLN005 for 1 h before adding 100 μM ISO. After 24 h, cells were viewed using an Olympus inverted microscope, equipped with fully automatic microscope digital camera system (DP2-BSW, Tokyo, Japan). Five random photographs were taken from each well, and at least 50 individual cell size measurements were made for each group.

2.4. Mitochondrial membrane potential (MMP)

The cells were plated in black 96-well culture plate and incubated for 30 min with the membrane potential-sensitive dye JC-1 (5 μM). The fluorescence intensity was immediately measured using a full-wavelength fluorescence scanning microplate reader (CLARIOstar, BMG, Cary, NC, USA) at 530/590 nm for emission and 488/535 nm for excitation. For confocal microscopy, cells plated on glass-bottom dishes were loaded with JC-1 (2 μM) for 30 min. at 37 °C, and images were captured using a laser confocal microscope (DMi8, Leica, Wetzlar, Germany).

2.5. Experimental protocol

Cells were treated with 100 μM ISO (I2760, Sigma; St. Louis, MO, USA) for 24 h or 6 h in the presence or absence of 0.1 mg/mL SGS, 0.3 mg/mL SGS, 20 μM ZLN005 (Selleckchem, Houston, TX, USA), 5 μM compound C (Selleckchem, Houston, TX, USA). All drugs were added to the culture medium 45 min before ISO administration.

2.6. Western blotting

Following the treatments, cells were washed twice with cold phosphate-buffered saline and lysed in radio-immunoprecipitation assay buffer. Phenylmethy-lsulfonyl fluoride was added to the cell lysates and placed on ice for 30 min, then transferred to 1.5-mL Eppendorf tubes, homogenized, and centrifuged at 12 000 ×g for 10 min at 4 °C. The supernatant was transferred to a fresh tube, and protein concentration was determined using protein dye (Bio-Rad, Hercules, CA, USA). Equal amounts of protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.2-μM polyvinylidene fluoride membranes (Roche, Basel, Switzerland). The membranes were immunoblotted with p-AMPK (ab133448, 64 kDa), PGC-1α (ab54481, 92 kDa), PPARα (ab8934, 52 kDa), NRF1 (ab175932, 54 kDa), TFAM (ab131607, 28 kDa) (all Abcam, Cambridge, UK), actin (4970, 45 kDa), and cyclic adenosine mono-phosphate (cAMP) response element binding protein (CREB) (9197, 43 kDa) (all CST, Danvers, MA, USA) antibodies, followed by appropriate secondary antibodies. The signals were visualized using enhanced chemil-uminescent reagents (Bio-Rad) at Azure c200 Gel Imaging System (Azure Biosystems, Dublin, CA, USA).

2.7. RNA extraction and real-time polymerase chain reaction (PCR)

Total RNA from cultured H9c2 cells was isolated using TRIzol (Sigma, St. Louis, MO, USA) and separated on 1.0% agarose gels to assess RNA integrity. Reverse transcription (RT) was carried out with 2 µg total RNA and primers with High Capacity cDNA Reverse Transcription Kits and Veriti Thermal Cycler (both ABI, Foster City, CA, USA). PCR reactions were performed with GoTaq qPCR Master Mix (A6002, Promega, Madison, WI, USA) and QuantStudio (TM) 7 Flex System (ABI, Foster City, CA, USA)). The expression levels of each gene were normalized to that the control gene (β-actin) and were calculated by the 2 ^-△△Ct method.

Primer sequences are as follow: PGC-1α 5'-AAAC-TTGCTAGCGGTCCTCA-3' and 5'-TTTCT-GTGGG-TTTGGTGTGA-3' (Sangon Biotech, Shanghai, China); NRF1 5'-AAGACAGGGTTGGGTTTGG-3' and 5'-CGAAAGAGACAGCAGACACG-3' (Sangon Biotech, Shanghai, China) ; TFAM 5'-TGAAGCTTGT-AAA-TCAGGCTTGGA-3' and 5'-GAGATCACTT-CGCCC-AACTTCAG-3' (TaKaRa, Kyoto, Japan); β-actin 5'-GGAGATTACTGCCCTGGCTCCTA-3' and 5'-GACT-CATCGTACTCCTGCTTGCTG-3 (TaKaRa, Kyoto, Japan); cytochrome c oxidase subunit 1 (CO1) 5'-GAGGCTTCGGAAACTGAC-3' and 5'-GCTTATG-TTATTTATTCGTGGG-3' (Invitrogen, Carlsbad, CA, USA); cytochrome c oxidase subunit 2 (CO2) 5'-GAAGTTGA-TAATCGGGTAG-3' and 5'-TAGTGA-AGGGATGG-CTC-3 (Invitrogen, Carlsbad, CA, USA); cytochrome c oxidase subunit 3 (CO3) 5'-GCC-CATC-ACAGCCTAATA-3' and 5'-GAAATCCCGT-TGCTA-TGA-3' (Invitrogen, Carlsbad, CA, USA); ATP synthe-tase subunit 6 (ATP6) 5'-TTTGCCTCTTTC-ATTACC-3' and 5'-TGAGTG-TAGTCGGTTGCT-3' (Invitrogen, Carlsbad, CA, USA); and ATP synthetase subunit 8 (ATP8) 5'-CTTCCCAAACCTTTCCTG-3' and 5'-GGTAATGAAAGAGGCAAATAGA-3' (Invitrogen, Carlsbad, CA, USA).

2.8. Statistical analysis

Statistical analysis and mapping were performed using SPSS 21.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism6 (GraphPad Inc., San Diego, CA, USA) software. Data are presented as mean ± standard deviation, and statistical comparisons were made using one-way analyses of variance. Differences were con-sidered statistically significant at P < 0.05.

3. RESULTS

3.1. Screening of drug concentrations

H9c2 cells were treated with different concentrations of SGS, and cell viability was determined by MTT assays. It was found that 0.3 mg/mL SGS had no obvious toxicity to cell within 48 h, and cell viability decreased significantly in 1 mg/mL compared to the 0-0.3 mg/mL concentration (P < 0.01).

3.2. SGS alleviated ISO-induced H9c2 cardiomyocyte hypertrophy

H9c2 cells were treated with 30 or 100 μM ISO for 24 h, and cell diameters were observed (Figure 1). Compared with the control and 30 μM ISO groups, the diameter of H9c2 cells in the 100 μM ISO group was significantly increased (P < 0.01). After treatment with 0.3 mg/mL SGS and 20 μM ZLN005 combined with 100 μM ISO for 24 h, the cell diameter was increased compared with the control group (P < 0.01). Compared with the 100 μM ISO group, the cell diameters of both treatment groups decreased significantly (P < 0.01).

Figure 1. Comparison of cell size (× 20).

A: control group; B: 30 μM ISO for 24 h; C: 100 μM ISO for 24 h; D: 0.3 mg/mL SGS for 1h combined with 100 μM ISO; E: 20 μM ZLN005 for 1 h combined with 100 μM ISO. ISO: isoproterenol; SGS: Shenge San.

3.3. SGS increased the survival rate in ISO-induced H9c2 cells

To observe the effect of SGS on cell survival rate, 100, 200, or 400 μΜ ISO and 0.3 mg/mL SGS were given together for 24 h. The results showed that ISO-induced cell damage was concentration dependent. SGS decreased the death rate, but this effect was only significant rate when the ISO concentration was 400 μM (P < 0.01).

3.4. SGS ameliorated compound C-induced down-regulation of PGC-1α

The cells were pretreated with 0.1 or 0.3 mg/mL SGS and 20 μM ZLN005 for 1 h, followed by 100 μM ISO for 24 h. PGC-1α protein expression levels in the control and activator ZLN005 groups were significantly higher than in other groups. (P < 0.01). The ZLN005 + SGS + ISO group was not significantly different from the SGS + ISO group. pAMPK protein levels were significantly lower in the control and ZLN005 groups compared to the other groups (P < 0.01). Compared with the SGS (0.3) + ISO group, pAMPK protein levels in the ISO and the ZLN005 + SGS + ISO groups were significantly decreased (P < 0.01, Figure S1).

There were no significant differences in NRF1, TFAM, or PPARα levels among groups, but the protein expression of CREB was decreased in the ZLN005 + SGS + ISO group (P < 0.05, Figure S1).

The cells were treated with 5 μM compound C, 100 μM ISO, and 0.3 mg/mL SGS for 6 h. pAMPK protein expression was not significantly different from the control group (Figure S2).

The effect of SGS on PGC-1α was observed after the addition the AMPK inhibitor compound C (Figure S3). PGC-1α expression was significantly decreased after treatment with 5 μM compound C (P < 0.05). Compared with the compound C group, SGS significantly increased PGC-1α levels in ISO-induced hypertrophic cardiomyocytes (P < 0.01), and this activation was inhibited by compound C pretreatment (P < 0.05). There was no increased PGC-1α expression in the ISO group.

Compared with the control group, ISO significantly down-regulated PPARα expression (P < 0.05), and SGS significantly up-regulated PPARα expression in ISO-induced hypertrophic cardiomyocytes (P < 0.01). Compared with the control group, ISO significantly down-regulated NRF1 expression (P < 0.01). SGS increased NRF1 expression (P < 0.01), and this effect was inhibited by compound C (P < 0.05). Compared with the ISO group, SGS also up-regulated the protein expression of TFAM in ISO-induced hypertrophic cardiomyocytes (P < 0.05).

3.5. SGS protected MMP and increase the mRNA expression of mitochondrial complex and related nuclear factor in OXPHOS

3.5.1. MMP

Figure 2 shows that under laser confocal microscopy, the JC-1 monomer was green fluorescent, the polymer was red, and Hoechst stained the nucleus. The mitochondrial red-green fluorescence ratio of the 0.3 mg/mL SGS group was increased compared with the ISO group (P < 0.05).

Figure 2. Effect of SGS on MMP.

A1: JC-1 monomer of control group; A2: polymer of control group; A3: merge of control group; B1: JC-1 monomer of ISO (100 μM, 24 h) group; B2: polymer of ISO group; B3: merge of ISO group; C1: JC-1 monomer of ISO + SGS (0.3 mg/mL, 6 h) group; C2: polymer of ISO + SGS group; C3: merge of ISO + SGS group. ISO: isoproterenol; SGS: Shenge San; MMP: mitochondrial membrane potential.

3.5.2. Effect of SGS on mRNA of five subunits in mitochondrial complex IV and V of H9c2 cardiomyocyte

The mRNA levels of mitochondrial electronic respiratory chain complex subunits CO2, CO3, and ATP8 (encoded by mitochondrial DNA [mtDNA]) were significantly lower in the ISO and compound C groups compared to the control group (P < 0.01). The mRNA level of CO1 in the ISO and compound C groups were also decreased versus control (P < 0.05). ATP6 mRNA was decreased in the ISO group (P < 0.05), and the expression of CO3 and ATP8 mRNAs in the SGS group also exhibited various degrees of decline. The mRNA expression of ATP8 in the ISO group was significantly decreased compared to the SGS (0.3) + ISO group (P < 0.01), and ATP8 mRNA levels in the compound C and compound C + SGS + ISO groups were also down-regulated (P < 0.05) (Table 1).

Table 1.

Comparison of mRNA expressions of five subunits in mitochondrial complex IV and V between groups

Group	n	CO1	CO2	CO3	ATP6	ATP8
CTRL	10	1.085±0.165	1.067±0.138	1.087±0.154	1.063±0.103	1.113±0.076
Compound C	10	0.703±0.086a	0.530±0.097b	0.622±0.110b	0.778±0.149	0.577±0.092bc
ISO	10	0.683±0.047a	0.587±0.064b	0.567±0.049b	0.684±0.090a	0.542±0.070bd
SGS (0.3) + ISO	10	0.969±0.173	0.783±0.105	0.785±0.136a	0.943±0.166	0.865±0.126a
compound C + SGS + ISO	10	0.815±0.039	0.670±0.169a	0.628±0.023b	0.681±0.047a	0.600±0.024bc

Notes: comparison of mRNA expression of five subunits in mitochondria complexes of H9c2 cardiomyocytes (^aP < 0.05, ^bP < 0.01 vs control; ^cP < 0.05, ^dP < 0.01 vs SGS (0.3) + ISO). CTRL: control group; ISO: isoproterenol (100 μM, 24 h); SGS: Shenge San (0.3 mg/mL, 6 h); compound C: 5 μM for 6 h; mDNA: mitochondrial DNA; complex IV: CO1, CO2, CO3, complex V: ATP6 and ATP8. CO: cytochrome c oxidase.

3.5.3. Effect of SGS on the mRNA expression of nuclear regulatory genes involved in mitochondrial OXPHOS

The mRNA levels of three nuclear regulatory genes (PGC-1α, NRF1, and TFAM) in mitochondrial OXPHOS were quantified. Each treatment group had different degrees of decline versus control. Compared with the SGS (0.3) + ISO group, NRF1 levels in the ISO and compound C + SGS + ISO groups were significantly decreased (P < 0.05) (Table 2).

Table 2.

Comparison of mRNA expressions of PGC-1α, NRF1, and TFAM between groups

Group	n	PGC-1α	NRF1	TFAM
CTRL	10	1.17±0.20	1.09±0.11	1.11±0.19
Compound C	10	0.62±0.18a	0.68±0.05a	0.69±0.14b
ISO	10	0.66±0.08b	0.65±0.0827ac	0.72±0.09b
SGS (0.3) + ISO	10	0.67±0.07b	0.93±0.16	0.89±0.10
compound C + SGS + ISO	10	0.58±0.15a	0.65±0.06ac	0.68±0.04b

Notes: comparison of mRNA expression of PGC-1α, NRF1, and TFAM in H9c2 (^aP < 0.01, ^bP < 0.05 vs control; ^cP < 0.05 vs SGS (0.3) + ISO). CTRL: control group; ISO: isoproterenol (100 μM, 24 h); SGS: Shenge San (0.3 mg/mL, 6 h); compound C: 5 μM for 6 h; mDNA: mitochondrial DNA; PGC-1α: peroxisome proliferator-activated receptor γ-coactivator-1α; NRF1: nuclear respiration factor 1; TFAM: transcription factor A mitochondrial.

4. DISCUSSION

4.1. "Cardiopulmonary Co-treatment" theory on the significance of heart failure

In TCM, "heart failure" early heart (lung) Qi deficiency can develop into heart and kidney Yang deficiency and blood and water stasis in the vessels. Stress, diet, and fatigue can aggravate this condition. Heart failure often involves more than the heart; it can heart often affect the lungs, manifesting as cough, asthma, shortness of breath, and other lung symptoms.

Based on the "Cardiopulmonary Co-treatment" theory of TCM, Professor Zhou used SGS in the treatment of chronic heart failure, especially for patients of heart and kidney Yang deficiency, and achieved good clinical effects. "Du Sheng Bing Zi" was first used 900 years ago, and this prescription has been continuously used.

4.2. Mitochondrial structure and functional impairment are the core of metabolic remodeling in heart failure

Eukaryotic mitochondria are the only organelles that perform a process involving co-coding of mtDNA and nuclear DNA (nDNA). Mammalian mitochondria are assembled by ~1500 proteins, most of which are encoded by nDNA and transport into the mitochondria after synthesis in the ribosomes. MtDNA is a 16 569-bp closed-loop double-stranded DNA molecule that is inherited by maternal and independent of nDNA. Energy metabolism in mtDNA contains limited genes including coding and noncoding regions. The coding region encodes 37 genes including 13 subunits associated with mitochondrial OXPHOS production, 2 ribosomal RNAs, and 22 transgenic RNAs.⁷ Among the 13 protein polypeptides encoded by mtDNA, 7 are subunits of respiratory chain complexⅠ(NADH2CoQ reductase), 1 is a subunit (Cytb) of respiratory chain complex

Ⅲ(CoQ2CytC reductase), 3 are subunits of the respiratory chain complex Ⅳ (cytochrome c oxidase, COX), and 2 are the complex Ⅴ (ATP synthetase) subunit. These proteins are located in the inner mitochondrial membrane and form the basic mitochondrial electronic respiratory chain involved in OXPHOS.

Mitochondrial-derived ATP is delivered to the cytoplasm to provide energy for cellular processes including cardiomyocyte contraction and electrophysiological activity. Mitochondrial dysfunction drives the development of metabolic disorders and cardiovascular disease. When the body is exerted or subjected to cold/heat stimulation or oxidative stress, mitochondria begin to split and differentiate, accompanied by changes in their size, quantity, and quality. These changes may be associated with "abnormal proliferation" or "functional mutation". In addition, mitochondrial damage can increase the production of reactive oxygen species, cause permeability changes in the mitochondrial inner membrane, imbalance ion concentration, and contribute to other factors in heart failure.⁸ These events further affect transfer in the mitochondrial electron chain and phosphorylation, leading to the decreased ATPase activity and reduced ATP synthesis.In heart failure, the metablic pathway and oxidative mode change, the use of free fatty acids is significantly reduced,⁹ and cells mainly rely on the glycolysis. In this case, the heart devolves to the "embryonic type" in which pyruvate reduces to lactic acid in the hypoxic state. This produces lactic acid, free fatty acid, and uric acid that lead to further myocardial injury.

4.3. Protective effect of SGS on mitochondrial function

4.3.1. PGC-1α is an important regulator of mitochondrial biosynthesis

PGC-1α is an important nuclear factor involved in mitochondrial OXPHOS regulation. PGC-1α is mainly found in tissues and organs with high oxidative activity, such as the heart, brown adipose tissue, skeletal muscle, and the kidney. It is widely involved in the regulation of nDNA and mtDNA, which are related to the energy metabolism. PGC-1α in the heart is susceptible to dietary effects and skeletal muscle exercise intensity.¹⁰ The metabotropic receptor adenylate kinase AMPK (ADP/ ATP level), nitric oxide, calmodulin, and PGC-1α participate in self-feedback; oxidative stress, hypoxia, and hormones (insulin, thyroxine, estrogen) also affect PGC-1α expression.¹¹ PGC-1α has many biological effects it is involved in metabolic regulation and mitochondrial biosynthesis, glucose utilization, and fatty acid oxidation by regulating downstream NRFs, estrogen-related receptor families, and PPARs.^12,13

Compared with the normal myocardium, ATP drops up to 30% in the setting of heart failure. The increases in ADP, AMP, and Pi give AMPK a "low oil" signal that activates AMPK to increase ATP synthesis.¹⁴ When ATP levels decrease, AMPK can rapidly activate downstream PGC-1α, regulating its transcriptional activity.¹⁵ In the heart, the main role of PGC-1α is related to mitochondrial function and fatty acid oxidation, and a shortage leads decreased cardiac function during exercise, making recovery of cardiac function difficult under stress loading.^16,17 Conversely, PGC-1α overexpression leads to abnormal mitochondrial proliferation and sarcomere disorder, triggering cardiac hypertrophy and heart failure.¹⁸ Enhancing PGC-1α expression in retrovirus-mediated white adipocytes can significantly increase mitochondria numbers and the expression of respiratory chain-related genes such as ATP synthase and complex IV.¹⁹ PGC-1α knockout mice show decreased expression of fatty acid oxidation-related proteins, and ATP is decreased by 20% in end-stage heart failure; increasing PGC-1α can increase ATP synthesis and reverse myocardial systolic dysfunction.²⁰ The coactivator PGC-1α is involved regulating mitochondrial biosynthesis and ATP synthesis.

4.3.2. PGC-1α/NRF1 is a common pathway for SGS to protect mitochondrial function and regulate intracellular energy metabolism

PGC-1α regulates mitochondrial OXPHOS, mitochondrial DNA transcription and replication, and mitochondrial biosynthesis through NRFs (or TFAMs).²¹ Electrical stimulation to myocardial cells of neonatal NRF1-overexpressing mice was shown to increase the number of mitochondria in the myocardium, confirming that NRF1 is associated with myocardial mitochondria formation. In contrast, loss of the NRF1 gene may affect mtDNA stability.²² TFAM is a major regulator of mitochondrial transcriptional and translational activity. Deletion of the cardiac-specific TFAM gene results in decreased mitochondrial levels, impaired respiratory chain function, cardiac hypertrophy, and progressive cardiomyopathy.^23,24 PGC-1α is coupled to mitochondria through the action of nDNA (NRF1 and TFAM) and promotes mtDNA express, thereby affecting mitoch-ondrial biosynthesis.²⁵

In this study, we used the H9c2 cell line to study the role of SGS in ISO-induced cardiac hypertrophy. The results showed that 0.3 mg/mL SGS could improve cardiac hypertrophy induced by 100 μM ISO within 24 h. After pre-treatment with SGS and the PGC-1α agonist ZLN005, we cultured H9c2 with ISO for 24 h. PGC-1α protein expression in the control and ZLN005 groups was significantly higher than other groups, and there was no significant difference between the ZLN005 + SGS + ISO and SGS + ISO groups. The effect of SGS on PGC-1α was observed after adding the AMPK inhibitor compound C. PGC-1α was significantly decreased after 6 h incubation with compound C (P < 0.05). Compared with the compound C group, the SGS group showed significantly increased PGC-1α expression in ISO-induced H9c2 cells (P < 0.01), and this was inhibited by pretreatment with compound C (P < 0.05). Conversely, there was no increase in the ISO group. The expressions of downstream PPARα, NRF1, and TFAM also showed corresponding trends.

The MMP directly reflects mitochondrial function and affects energy productivity. Decreased MMP is an important marker of inner mitochondrial membrane damage.²⁶ It occurs before the appearance of nuclear apoptosis, eventually causing mitochondrial expansion and rupture.²⁷ MMP decreased significant after 24-h incubation with 100 μM ISO. SGS can significantly attenuate the decline of MMP, protect mitochondrial function, and prevent apoptosis. The mRNA expression of three nuclear regulatory genes (PGC-1α, NRF1, and TFAM) involved in mitochondrial OXPHOS and several mtDNA-encoded complex subunits (CO1, CO2, CO3, ATP6, and ATP8) were decreased in different treatment groups. Compared with the SGS pretreatment group, mRNA levels of ATP8 were significantly decreased in the ISO, compound C, and compound C + SGS + ISO groups. Compared to the ISO and compound C + SGS + ISO groups, NRF1 mRNA levels were significantly higher in the SGS pretreatment group.

We found that after treating cells with compound C, ISO, and SGS for 6 h, the expression of pAMPK was not significantly different from control. Compound C, as an AMPK inhibitor used to study the effect of AMPK activity on its upstream and downstream pathways. Compound C binds free AMPK and suppresses its activity.²⁸ We observed that AMPK/PGC-1α signaling was significantly inhibited in the compound C group. ZLN005 can increase the ADP/ATP ratio by mildly uncoupling mitochondria, thereby activating AMPK, leading to positive feedback of downstream PGC-1α.²⁹ Indeed, we significantly higher levels of PGC-1α in the agonist ZLN005 group. The results of experiments using ZLN005 and compound C confirmed that SGS could up-regulate PGC-1α/NRF1 expression.

In conclusion, our team is guided by the "Cardio-pulmonary Co-treatment" theory of TCM. Mito-chondrial damage is one of the important events during metabolic remodeling in heart failure. Based on the previous basis and from the perspective of mito-chondria,^30,⇓-32 we assessed the scientific value of SGS in preventing and treating heart failure, which provided new ideas for the study of heart failure. The in vitro results showed that SGS could improve ISO-induced cardiomyocyte hypertrophy and alleviate the decrease in mitochondrial membrane potential in H9c2 cells, thus protecting organelle structure and function. We further confirmed that SGS could improve the down-regulation of PGC-1α induced by compound C, activate PGC-1α/NRF1 signaling, and up-regulate ATP8 mRNA to improve energy metabolism in hypertrophic cardiomyocytes. The mechanism by which SGS protects myocardial mitochondria and improving metabolic remodeling is related to PGC-1α/NRF1 signaling.

5. ACKNOWLEDGEMENTS

The authors are grateful for substantial support from Professor SW Chan and Daniel Mok, ABCT, PolyU.