All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee at Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional Chinese Medicine (approval no. 18922), according to the principles outlined in the National Institutes of Health Guidelines for Care and Use of Laboratory Animals.

Pregnant Sprague Dawley rats (n=2; female; 10 weeks old; 260-270 g) purchased from the Vital River Laboratory Animal Technology Co., Ltd. [animal license number, SCXK (Beijing)] were fed ad libitum in an animal facility room with a 12-h light/dark cycle at a temperature of 20-22˚C, with ~50% humidity. After pregnant rats gave birth, neonatal rats (within 24 h of birth; 5-6 g) were euthanized using CO₂ inhalation at a flow rate of 1.2 l/min, which displaces 30% of the cage volume per min, followed with further cervical dislocation.

Valsartan capsules (Novartis International AG) were dissolved in 100% DMSO as 10 mmol/l stock solutions and stored at -80˚C before use. The compound stock solution was diluted in cell culture medium (DMEM medium with 10% FBS and 1% double-antibiotic) to a final concentration of 10 µmol/l (20). HQQR, consisting of Salvia miltiorrhiza, Stone Cassia, Ligusticum chuanxiong, Uncaria angustifolia, corn whisker, mulberry parasite and hawthorn (5:10:3:5:10:5:5), was decocted and dried according to a common protocol (18).

Primary cardiomyocytes were isolated from the cardiac tissue of neonatal rats (within 24 h of birth) as previously described (21). Briefly, the hearts of the neonatal rats were collected, and the atrium of each heart was put into 5-ml sterile centrifuge tubes to be fully cut into small pieces. After full digestion with 0.4% Collagenase IV (cat. no. C4-BIOC; Sigma-Aldrich; Merck KGaA) and 0.05% trypsin-EDTA (cat. no. T1300; Beijing Solarbio Science & Technology Co., Ltd.) in 37˚C constant temperature water bath for 20 min, the cell suspension was centrifuged at room temperature for 10 min at 1,000 x g. After multiple digestion, the cell suspensions were filtered through a 100-mesh filter and mixed, and the cells were cultured at 37˚C in DMEM medium (cat. no. SH30243.01; Hyclone; Cytiva) with 10% FBS (cat. no. 16000-044, Gibco; Thermo Fisher Scientific, Inc.) and 1% double-antibiotic (penicillin-streptomycin; cat. no. P1400; Beijing Solarbio Science & Technology Co., Ltd.) in a 37˚C, 5% CO₂ incubator. Next, the isolated cells were subjected to flow cytometry detection of troponin I (troponin I, TnI; 1:50; cat. no. ab196384; Abcam) to identify the purity of primary cardiomyocytes. The morphology of cardiomyocytes was observed, and pictures were captured with a light microscope (magnification, x200).

Cardiomyocytes were divided into the following five groups (5x10⁵ cells; 6-well plate): i) Vehicle (solvent group, DMSO); ii) Ang II + vehicle; iii) Ang II + 0.2 mg/ml HQQR; iv) Ang II + 0.5 mg/ml HQQR; and v) Ang II + valsartan. Ang II (cat. no. 4474-91-3) was purchased from Beijing Solarbio Science & Technology Co., Ltd., and the corresponding concentration (1 µmol/l) was prepared according to the manufacturer's instructions. Ang II and HQQR were added at the same time.

Cell suspensions containing 1x10⁵ primary rat cardiomyocytes were added to a 24-well plate and treated with Ang II, Ang II + 0.2 mg/ml HQQR, Ang II + 0.5 mg/ml HQQR, Ang II + 10 µmol/l valsartan, or Ang II + ROS scavenger (1 mM; cat. no. S6205; Selleck Chemicals) for 24 h. Then, slides containing experimental primary cardiomyocytes were harvested, fixed with 4% formaldehyde for 30 min at room temperature, and permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. After blocking with 1% bovine serum albumin (BSA; cat. no. A8010; Beijing Solarbio Science & Technology Co., Ltd.) in PBS for 30 min at room temperature, the slides were incubated with anti-α-actinin antibody (1:200; cat. no. ab68194; Abcam) and anti-α-smooth muscle actin (α-SMA; 1:500; ab32575; Abcam) overnight at 4˚C in a wet-box. Subsequently, the slides were incubated with Alexa Fluor 555-labeled donkey anti-rabbit IgG (H + L) antibody (1:500; cat. no. A0453; Beyotime Institute of Biotechnology) for 1 h at room temperature in a wet-box before staining the nuclei with DAPI-containing media for 15 min at room temperature (1:500; cat. no. C1002; Beyotime Institute of Biotechnology). Pictures were taken using a fluorescence microscope (Nikon Corporation).

CCK-8 assay was performed in accordance with the manufacturer's kit protocol (Beyotime Institute of Biotechnology). Cell suspensions containing 3x10³ primary rat cardiomyocytes were added to a 96-well plate. The cells were treated with 1 µmol/l Ang II (22) and gradient concentrations of HQQR (0, 0.05, 0.1, 0.2, 0.5 and 1 mg/ml) or valsartan (10 µmol/l) after 24 h of incubation. Finally, 10 µl CCK-8 solution were added into each well, and the cells were incubated in an incubator at 37˚C with 5% CO₂ for 1 h. Cell proliferation at 0, 12, 24 and 48 h was detected by recording the optical density at 450 nm (OD₄₅₀).

Total RNA was extracted from primary rat cardiomyocytes using TRIzol^® reagent (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Complementary DNA was synthesized using the RevertAid First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and under the following conditions: 37˚C for 60 min; 85˚C for 5 min; 4˚C for 5 min. mRNA levels were quantified using SYBR Green qPCR Master Mix (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and under the following thermocycling conditions: 95˚C for 10 min; 40 cycles of 95˚C for 15 sec and 60˚C for 45 sec. Relative mRNA levels were obtained using the 2^-ΔΔCq method (23). GAPDH was used as an internal control for normalization. The primer sequences are listed in Table I.

The total protein of primary rat cardiomyocytes was extracted using RIPA buffer, which contained a protease and phosphatase inhibitor (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.). Protein was then quantified using a BCA kit (cat. no. PICPI23223; Thermo Fisher Scientific, Inc.). Subsequently, proteins (25 µg per lane) were separated using 10% SDS-PAGE and transferred to PVDF membranes. After blocking in 5% non-fat milk for 1 h at room temperature, the protein bands were incubated with primary antibodies overnight at 4˚C, followed by incubation with the corresponding secondary antibodies [anti-rabbit (cat. no. A0208; 1:1,000; Beyotime Institute of Biotechnology) or anti-mouse (cat. no. A0216; 1:1,000; Beyotime Institute of Biotechnology) labeled with HRP] at 37^°C for 1 h. After a 5-min incubation with Immobilon Western chemiluminescent HRP substrate (cat. no. WBKLS0100; MilliporeSigma) in the dark, the protein bands were visually measured using a chemiluminescent imaging system (Tanon-5200, Tanon Science and Technology Co., Ltd.), and then quantified using ImageJ software (v1.8.0.112; National Institutes of Health). Anti-SIRT1 (cat. no. ab110304; 1:1,000), anti-PGC-1α (cat. no. ab54481; 1:2,000), anti-nuclear respiratory factor 1 (NRF1; cat. no. ab175932; 1:5,000), anti-mitochondrial transcription factor A (Tfam; cat. no. ab131607; 1:2,000), anti-NADH:ubiquinone oxidoreductase subunit A13 (NDUFA13; cat. no. ab110240; 1:1,000), anti-succinate dehydrogenase complex iron sulfur subunit B (SDHB; cat. no. ab178423; 1:5,000), anti-cytochrome c oxidase subunit IV (COX IV; cat. no. ab16056; 1:500), anti-cyclooxygenase 1 (COX1; cat. no. ab695; 1:1,000), anti-ATPase 6 (cat. no. ab192423; 1:500), anti-brain natriuretic peptide (BNP; cat. no. ab239510; 1:2,000), anti-Bax (cat. no. ab32503; 1:5,000), anti-Bcl-2 (cat. no. ab196495; 1:1,000) antibodies were obtained from Abcam, anti-cleaved caspase 3 (cat. no. 9661; 1:1,000), anti-caspase 3 (cat. no. 9662; 1:1,000) were obtained from CST, anti-atrial natriuretic peptide (ANP; cat. no. RQ4453; 0.5 µg/ml) antibody was obtained from NSJ Bioreagents, anti-β-myosin heavy chain (β-MHC; cat. no. MA1-26180; 0.5 µg/ml) antibody was obtained from Invitrogen; Thermo Fisher Scientific, Inc., and anti-GAPDH as a loading control (cat. no. 60004-1-1G; 1:5,000) was obtained from ProteinTech Group, Inc. The antibodies were diluted in PBST (containing 0.05% Tween-20).

ROS accumulation in cardiomyocytes was quantified using the Active Oxygen Detection kit (cat. no. S0033; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. After treatment with 1 µmol/l Ang II and HQQR (0.2 and 0.5 mg/ml) or valsartan (10 µmol/l), DCFH-DA probe solution was prepared and used for incubation with the harvested cell samples at 37˚C for 30 min in the dark. The cells and solution were gently inverted every 10 min. After washing three times with DMEM without serum, the samples were examined via flow cytometry (Accuri C6; BD Biosciences) using FlowJo software (v10.0.7r2; Tree Star, Inc.).

Primary rat cardiomyocytes were treated with 1 µmol/l Ang II and HQQR (0.2 and 0.5 mg/ml) or valsartan (10 µmol/l) for 24 h, and then, the cells were collected, and the supernatant was kept. The levels of ATP and protein carbonyl, and the activities of glutathione peroxidase (GSH-PX), catalase (CAT), malondialdehyde (MDA) and superoxide dismutase (SOD) were respectively detected using the BCA (cat. no. A045-3), ATP (cat. no. A095), protein carbonyl (cat. no. A087), GSH-PX (cat. no. A005), CAT (cat. no. A007-1), MDA (cat. no. A003-2) and SOD (cat. no. A001-1) kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions, and measured using a visible spectrophotometer. The activities of the mitochondrial complexes I-IV and V were respectively detected using the mitochondrial complex I (cat. no. BC0515), II (cat. no. BC3235), III (cat. no. BC3245), IV (cat. no. BC0945) and V (cat. no. BC1445) Activity Test kits (Beijing Solarbio Science & Technology Co., Ltd.) and measured using a UV spectrophotometer.

Alterations in mitochondrial membrane potential were evaluated using the Mitochondrial Membrane Potential Detection kit with JC-1 (cat. no. C2006; Beyotime Institute of Biotechnology). According to the manufacturer's instructions, the cell suspension was prepared and detected using a flow cytometer (Accuri C6; BD Biosciences) via the software of Flow Jo (v10.0.7r2 version). JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential. In high mitochondrial membrane potential, JC-1 aggregates in the matrix of mitochondria and forms polymer (J-aggregates), which produces red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot aggregate in the matrix of mitochondria. At this time, JC-1 is a monomer and can produce green fluorescence.

Primary rat cardiomyocyte cells after 24 h treatment with Ang II, Ang II + 0.2 mg/ml HQQR, Ang II + 0.5 mg/ml HQQR, Ang II + 10 µmol/l valsartan were stained using Annexin V Apoptosis kit (C1062; Beyotime Institute of Biotechnology) according to the manufacturer's instructions and a Beckman CytoFLEX Flow Cytometer (Beckman Coulter, Inc.) with FlowJo software (v10.0.7r2; Tree Star, Inc.) was used to analyze apoptosis.

Statistical analysis was conducted using GraphPad Prism 7.0 software (GraphPad Software, Inc.). Data are presented as the mean ± SD. One-way ANOVA followed by Tukey's post hoc test was applied for comparisons between groups. Results are representative of at least three repeated experiments. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of HQQR on Ang II-induced cardiomyocyte hypertrophy, rat primary cardiomyocytes were isolated. After detection using TnI, the results of flow cytometry analysis confirmed that ~95% of the isolated cells were primary cardiomyocytes (Fig. 1A). Microscopic observations (light microscope) also indicated that the isolated cell morphology was consistent with the morphological characteristics of primary cardiomyocytes (Fig. 1B), including a short columnar form with generally only one nucleus being mostly located in the middle of the cell, while the cell shape is ellipse- or rectangle-like, and its long axis is in the same direction as in myofibrils. The cytotoxicity of HQQR was detected using a CCK-8 assay (Fig. S1), the results of which revealed that 0.2 and 0.5 mg/ml HQQR had no cytotoxicity and the 24 h after treatment had the best effect. Cell proliferation experiments revealed that, compared with normally cultured (medium without other treatment) primary cardiomyocytes, Ang II (1 µmol/l) treatment significantly inhibited the proliferation of primary cardiomyocytes, which was markedly abolished by incubation with valsartan (10 µmol/l), the antagonist of angiotensin receptor used as a positive control. The addition of HQQR solution ameliorated the inhibitory effect of Ang II on primary cardiomyocyte proliferation in a dose-dependent manner (Fig. 1C). On the basis of the present results, Ang II treatment for 24 h followed by 0.2 and 0.5 mg/ml HQQR concentrations were selected as treatment conditions for subsequent experiments. Valsartan was used as a positive control.

To further examine the role of HQQR in Ang II-induced cardiomyocyte hypertrophy, primary cardiomyocytes were divided into the following five groups: i) Vehicle; ii) Ang II (1 µmol/l) + vehicle; iii) Ang II (1 µmol/l) + HQQR (0.2 mg/ml); iv) Ang II (1 µmol/l) + HQQR (0.5 mg/ml); and v) valsartan + Ang II (1 µmol/l). The immunofluorescence of α-actinin revealed that, compared with that in the vehicle group, cardiomyocyte hypertrophy was observed in the Ang II-treated group, indicating that Ang II induced cardiomyocyte hypertrophy. However, treatment with HQQR could significantly inhibit Ang II-induced cardiomyocyte hypertrophy (Fig. 2A) and reduce the Ang II-induced expression of the myocardial hypertrophy markers ANP, BNP and β-MHC (Fig. 2B and C). Valsartan was used as a positive control.

Ang II treatment markedly enhanced ROS accumulation, whereas treatment with HQQR significantly inhibited ROS accumulation in Ang II-treated primary cardiomyocytes, in a dose-dependent manner (Fig. 3A). HQQR could also rescue the effect of Ang II treatment on the levels of MDA, SOD, ATP, CAT, GSH-Px and protein carbonyl in primary cardiomyocytes (Fig. 3B-G). Consistent with these results, the immunofluorescence of α-SMA revealed that ROS was directly involved in AngII-induced cardiomyocyte hypertrophy, and that the ROS scavenger alleviated AngII-induced cardiomyocyte hypertrophy (Fig. 3H). The present findings suggested that HQQR treatment could alleviate Ang II-induced cardiomyocyte hypertrophy, potentially by modulating ROS accumulation. Valsartan was used as a positive control.

Ang II treatment notably reduced mitochondrial membrane potential (Fig. 4A) and increased cell apoptosis (Fig. 4B). In Ang II-stimulated cardiomyocytes, treatment with HQQR significantly reversed the hypertrophy-induced membrane potential reduction and apoptosis in a dose-dependent manner. Moreover, Ang II induced the expression of apoptosis-related proteins Bax and cleaved caspase 3, which were significantly decreased upon HQQR treatment, while anti-apoptotic Bcl-2 was significantly decreased by Ang-II and increased by HQQR (Fig. S2). Valsartan was used as a positive control.

To further explore the mechanism by which HQQR affects Ang II-induced cardiomyocyte hypertrophy, the activities of the mitochondrial complexes I-IV and V were studied 24 h after treatment. As presented in Fig. 5A, Ang II markedly inhibited the activity of the mitochondrial complexes I-IV and V, whereas the addition of HQQR or valsartan partially rescued their activities. Furthermore, HQQR or valsartan treatment markedly inhibited Ang II-induced mtDNA leakage (Fig. 5B). The mRNA and protein expressions of genes encoding the mitochondrial complex and involved in mitochondrial biogenesis were subsequently evaluated. As indicated in Figs. 5C and S3, Ang II treatment reduced both mRNA and protein levels of SIRT1, PGC-1α, NRF1, Tfam, NDUFA13, COX IV, COX1 and ATPase 6, and elevated the expression of SDHB. These effects were notably rescued by treatment with HQQR or valsartan, which was used as a positive control.