The human myocardial cell line, AC16 (33), and the rat myocardial cell line, H9c2 (CRL-1446), were obtained from the American Type Culture Collection (ATCC). Dulbecco's modified Eagle's medium (DMEM; C11995500BT) and fetal bovine serum (FBS; 10270-106) were purchased from Gibco (Thermo Fisher Scientific, Inc.). Penicillin-streptomycin solution (PSS) (100X; E607011-0100) was purchased from Sangon Biotech (BBI Co., Ltd.). Iso (I5627) and PQQ (D7783) were purchased from Sigma-Aldrich (Merck KGaA). Anti-ANP antibody (GTX109255) was acquired from GeneTex, Inc. Anti-GAPDH antibody (10900R) was obtained from BIOSS. Anti-p65 (10745) antibody, which is an antibody against NF-κB, and anti-NF-κBIA (10268) were purchased from ProteinTech Group, Inc. Anti-histone-3 (H3; ab1791) antibody was obtained from Abcam. Horseradish-peroxidase (HRP)-conjugated secondary antibody (BA1054) was purchased from Wuhan Boster Biological Technology, Ltd. RIPA lysis buffer (P0013) was purchased from the Beyotime Institute of Biotechnology. The Cell Counting Kit-8 (CCK-8) kit (40203ES76) was purchased from Yeasen (Yeasen Biotech Co., Ltd.). Reverse transcription-quantitative (RT-q) PCR primers were synthesized by Thermo Fisher Scientific, Inc., while the PrimeScript™ RT Reagent kit (Perfect Real Time) (RR037Q) and TB Green™ Advantage^® qPCR Premix (639676) were acquired from Takara Bio, Inc. A bicinchoninic acid (BCA) protein quantitation kit (P0010), a reactive oxygen species assay kit (S0033) and the Actin-Tracker Green kit (C1033) were purchased from the Beyotime Institute of Biotechnology. All other chemicals used in the experiments were of analytical grade.

The animal procedures were approved by the Animal Care and Use Committee of Shanghai University of Medicine and Health Sciences (Shanghai, China; permit no. 210105197306201429-kjc) and were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH) (34). Eighteen C57BL/6 male mice (8 weeks old) were obtained from the Animal Resource Center of Shanghai University of Medicine and Health Sciences. All mice were housed in a constant environment with 55±10% humidity, a temperature of 20±5°C and a 12 h light/dark cycle. Adequate food and water were provided ad libitum. The health and behavior of the mice were monitored in real-time.

In this study, SPF grade C57 male mice (weighing 22-25 g) were used to establish the experimental model (35-38). First, 18 mice were randomly divided into 3 groups (6 mice per group). The treatment groups were as follows: The sham group in which mice received an intraperito-neal injection of normal saline (NS) 100 µl/time, and 3 h later, an intraperitoneal injection of physiological saline 100 µl/time; the Iso (30 mg/kg) group [the doses of Iso were in accordance with those of a previous study (39)] in which mice received an intraperitoneal injection of NS 100 µl/time, and 3 h later, an intraperitoneal injection of ISO dissolved in NS 100 µl/time; the PQQ (40 mg/kg) [the doses of PQQ were in accordance with those of a previous study (40)] + Iso (30 mg/kg) group in which PQQ dissolved in NS was intraperitoneally injected into the mice 100 µl/time, and 3 h later, ISO dissolved in NS was intraperitoneally injected into the mice 100 µl/time. The above-mentioned managements were performed once every 4 days, and the duration of the experiment was approximately 5 weeks. Following anesthesia by an intraperitoneal injection of 3% sodium pentobarbital at a dose of 40 mg/kg, the mice were examined to confirm that they were in a state of deep anesthesia; the adequacy of the anesthesia was detected by pinching the hindfoot to ensure there was no reflex, and the heart was then harvested.

The recovered heart tissue was rinsed with NS, fixed with 4% paraformaldehyde for 1 h at room temperature, and subsequently, H&E staining was performed at room temperature (hematoxylin, 7 min; eosin, 5 min) to observe the pathological changes of myocardial tissue and the images were observed using a digital pathological scanner (Precice 500; UNIC Technologies, Inc.) at ×200 magnification.

Either the AC16 cells or H9c2 cells were cultured in a 6-well plate with 2 ml DMEM containing 1 g/l glucose, 10% FBS and 1% PSS, and incubated at a constant temperature of 37°C in a humid atmosphere of 95% air and 5% CO₂. Once the cells had adhered to the wall, they were grouped as follows: i) The control group, which were treated with 2 ml DMEM without FBS (DMEMNF, DMEM NO FBS) as a blank control for 48 h; ii) the Iso group, which were treated with 2 ml DMEMNF for 24 h and then with 20 µM Iso in 2 ml DMEMNF for 24 h; iii) the low-dose PQQ group, which, after being cultured for 21 h in 2 ml DMEMNF, were treated with 1 µM PQQ in 2 ml DMEMNF for 3 h and then with 20 µM Iso in 2 ml DMEMNF for 24 h; iv) the mid-dose group, which, following 21 h of culture in 2 ml DMEMNF, were treated with 2.5 µM PQQ in 2 ml DMEMNF for 3 h and then with 20 µM Iso in 2 ml DMEMNF for 24 h; and v) the high-dose group, which, after 21 h of culture in 2 ml DMEMNF, were treated with 5 µM PQQ in 2 ml DMEMNF for 3 h and then with 20 µM Iso in 2 ml DMEMNF for 24 h (Fig. S1).

To explore the cytotoxicity of Iso and PQQ on the AC16 cells, cell viability was determined using a rapid and sensitive CCK-8 assay kit according to the manufacturer's instructions. The AC16 cells were seeded into 96-well plates at a density of 5×10³ cells per well. After 24 h, Iso was added to the 96-well plates at increasing concentrations (5, 10, 20, 40, 80 and 160 µM) and each concentration had 6 set parallel groups, and cultured in an incubator at 37°C with a humid atmosphere of 95% air and 5% CO₂ for 24 h. Moreover, PQQ was added to other 96-well plates at a concentration of 1, 5, 10, 25, 50, 100, 150 and 200 µM setting 6 parallel groups as well, and cultured for 12 h. The medium was then aspirated from the plates, which were rinsed gently with PBS before being filled with CCK-8 reagent and DMEM at a ratio of 1:10 in 100 ml. The plates were incubated for 2 h in an incubator at 37°C and the optical density (OD) value at a wavelength of 450 nm was measured using a microplate reader (SpectraMax M5; Molecular Devices, LLC).

To examine the effect of PQQ against Iso-induced hypertrophy, visual experimentation was carried out. First, either the AC16 cells or H9c2 cells were plated in 6-well plates, and cultured in a humid atmosphere of 95% air and 5% CO₂ at a constant temperature of 37°C. The cells were then treated as described above in the section entitled 'Cell culture and grouping'. The cells were then washed with PBS thrice (5 min each wash) before being fixed in a 4% paraformaldehyde solution. After 10 min, the 4% paraformaldehyde solution was aspirated, and 0.1% Triton X-100 (v/v in PBS) was used to wash the plates thrice for 5 min to flush any residual 4% paraformaldehyde. Subsequently, Actin-Tracker Green was diluted in PBS in a ratio of 1:40 as the working solution, which containing 5% FBS (v/v) and 1% Triton X-100 (v/v). Each well was supplemented with 300 µl working solution, and incubated at 37°C without light for 45 min. The working solution was then poured out and the excess solution containing PBS and 0.1% Triton X-100 (v/v) was removed by washing thrice (5 min each). Subsequently, either the AC16 cells or H9c2 cells were observed under a Nikon inverted microscope (Nikon Corp.) equipped with a Polaroid digital camera and photographed at ×200 magnification. A total of 5 random images from each well were captured, and 10 individual cell surface areas per photography were measured by ImageJ software (National Institutes of Health).

Total RNA was isolated from the AC16 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The concentration and purity of the total RNA were determined using a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, Inc.) and absorbance readings at 260 nm and 260/280 nm, respectively, which were used as indicators of RNA concentration and purity. First-strand complementary DNA (cDNA) synthesis was performed with the PrimeScript™ RT Reagent kit (Perfect Real Time) in the reaction system presented in Table I, and the reaction temperature was set as indicated in the manufacturer's protocol. Subsequently, using TB Green™ Advantage^® qPCR Premix, the obtained cDNA and the homologous primers [as presented in Table II (41)] were mixed at the ratio indicated in Table III) in 96-well optical reaction plates (04729692001, Roche) and analyzed on a LightCycler 96 system (05815916001, Roche Diagnostics) according to the manufacturer's protocol. The reaction conditions were set as presented in Table IV. Each gene was analyzed in 3 parallel wells in each experiment, and the difference in incorporation data (Cq values) from parallel wells should be <1. The results of ANP, BNP and β-MHC were normalized to those of GAPDH within the same sample and quantified by the formula 2^−ΔΔCq (42) relative to the control group.

Either the AC16 cells or H9c2 cells were lysed with RIPA lysis buffer and the total protein content was analyzed with a BCA kit. Western blot analysis was performed as previously described (43). Briefly, equal amounts of total protein were separated by 10-15% SDS-PAGE and then transferred onto PVDF membranes (EMD Millipore). The membranes were blocked with 5% non-fat milk solution for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against GAPDH (1:3,000), ANP (1:2,000), H3 (1:3,000), NF-κBIA (1:2,000), or p65 (1:2,000). The membranes were then washed 3 times with TBS-Tween-20 and further incubated with HRP-conjugated secondary antibodies (1:5,000) at room temperature for 1 h. The membranes with immune complexes were visualized by a chemiluminescence imaging system (ImageQuant LAS 4000 Mini; GE Healthcare Life Sciences). The intensity of the bands was measured by ImageJ software (National Institutes of Health) and analyzed with IBM SPSS Statistics 23.0 (IBM Corp.).

Using the Reactive Oxygen Species Assay kit, the intracellular ROS levels in the PQQ-pre-treated and/or Iso-treated groups were detected, and the changes in the mean fluorescence intensity were quantified by ImageJ software. The experimental procedure was performed according to the manufacturer's instructions. In brief, the cells were loaded with dichlorodihydro-fluorescein diacetate probes at a ratio of 1:1,000 (v/v) in 10 ml serum-free medium and were incubated at room temperature for 30 min. The groups were set as described above in the section entitled 'Cell culture and grouping'. The cells were then washed thrice for 15 min with serum-free medium. Images were obtained at ×200 magnification with a Nikon inverted microscope (Nikon Corp.) equipped with a charge-coupled device camera (Hamamatsu). Each well was photographed in 5 random fields and analyzed by ImageJ software as described above in the section entitled 'Cell morphometric analysis'. Moreover, the ROS levels were also analyzed by analytical flow cytometry (BD Biosciences) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, respectively. Flowjo software (Flowjo, LLC) was used to analyze the results of flow cytometry.

As JC-1 is an ideal fluorescent probe for detecting MMP, the change in fluorescent color by the JC-1 probe was obtained to detect the change in MMP (44). The JC-1 probe (Mitochondrial membrane potential assay kit with JC-1, C2006, Beyotime Institute of Biotechnology) was used to detect changes in MMP in the AC16 cells treated with Iso and/or PQQ pre-treatment. The experimental procedure was performed as previously described (45,46). The fluorescence images were obtained using a fluorescent microscope (Nikon Corp.).

IBM SPSS Statistics 23.0 (IBM Corp.) was used for statistical analysis. All data are presented as the means ± standard deviation. Differences between 2 groups were analyzed with an unpaired Student's t-test. Statistical analysis among various groups was conducted by one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The results obtained are presented in Fig. 1, which illustrates that the cell morphological changes in the mouse cardiac muscle in the Iso-treated C57 mice. The surface area increased significantly, while following pre-treatment with PQQ, the increase in the surface area was reduced (Fig. 1A and B). Moreover, the ratio of heart weight/body weight in the Iso group was higher than that in the control group. In the PQQ + Iso group, a decrease in the ratio of heart weight/body weight was observed compared to the Iso group (Fig. 1C). These results indicated that PQQ exerted an inhibitory effect on ISO-induced cardiac hypertrophy in vivo.

To examine the effects of Iso and PQQ on AC16 cells, cell viability was determined with a rapid and sensitive CCK-8 assay kit according to the manufacturer's instructions. The OD values, which directly reflect the survival status and viability of the AC16 cells, were 0.2-0.4 for the Iso and PQQ groups of AC16 cells (Fig. 2A and C). AC16 cell viability for the control well was set at 100%, and relatively, the other well percentages were calculated. The results of the CCK-8 assay demonstrated that concentrations of Iso from 5 to 160 µM and those of PQQ from 1 to 200 µM had no significant effect on AC16 cell viability (Fig. 2B and D). Thus, subsequent studies that could identify alterations in gene expression would not lead to changes in cell viability or cytotoxicity. Based on the above findings and those of previous studies (22,47), the concentration of 20 µM was selected as the therapeutic concentration of Iso.

The increase in AC16 cell size can directly reflect the progression of cardiac hypertrophy. This study found that the Iso-treated AC16 cells exhibited a marked change in morphology and their surface area increased significantly, while the increase in the surface area was reduced following pre-treatment of the AC16 cells with PQQ (Fig. 3A and B). With 1 µM PQQ pre-treatment, the surface area of Iso-induced cardiomyocyte hypertrophy was significantly reduced compared with that of the Iso group without PQQ pre-treatment, and the increased surface area was reduced by 29%. The 2.5 µM PQQ pre-treated group exhibited a 38% decrease in cell surface area compared with that of the Iso group, while the 5 µM PQQ pre-treated group exhibited a decrease of 53% in cell size compared with that of the Iso group, and the cell surface area was almost unaltered macroscopically compared with that of the control group (Fig. 3A and B). To further investigate the protective effects of PQQ on the heart, the effects of PQQ on H9c2 cells with Iso-induced hypertrophy were also detected (Fig. 3C and D), and the results revealed a consistent trend with what was observed with the AC16 cells.

The present study examined the mRNA expression levels of the hypertrophic markers, ANP, BNA and β-MHC, in AC16 cardiomyocytes relative to those of the blank control group in order to verify whether PQQ can protect against Iso-induced cardiac hypertrophy. The results (Cq value) of ANP, BNP and β-MHC were normalized to those of GAPDH in the same sample and were quantified by the formula 2^−ΔΔCq. The expression of ANP, BNP and β-MHC in the Iso-treated group increased by 1.42-, 1.71- and 1.74-fold vs. the control group at 24 h. Upon pre-treatment with PQQ, the expression of hypertrophic markers decreased. Following pre-treatment with 1 µM PQQ, the expression levels of ANP, BNP and β-MHC were downregulated, and the downregulation of BNP and β-MHC was statistically significant, while the difference in ANP was not statistically significant. Pre-treatment with a higher concentration of PQQ (i.e., 2.5 and 5 µM) led to a downregulation in the expression levels of ANP, BNP and β-MHC, which was more significant with increasing concentrations. The levels of these markers in the 5 µM PQQ-pre-treated group were similar to those of the control group (Fig. 4A).

To further examine the effect of PQQ on cardiac hypertrophy, the present study also detected the changes in ANP protein expression by western blot analysis. ANP expression in the Iso-treated group was significantly upregulated. Upon pre-treatment with various concentrations of PQQ (1, 2.5 and 5 µM), ANP protein expression was decreased compared with that of the Iso-treated group. Although the difference in the ANP protein level between the Iso-treated group and the 1 µM PQQ-pre-treated group was not statistically significant, the trend in the downregulation of ANP protein with the increasing PQQ concentration was consistent with the trend observed for ANP mRNA expression (Fig. 4B and C). Overall, these findings indicated that PQQ pre-treatment attenuated Iso-induced hypertrophy, and the protective effects of PQQ were more effective as the concentration increased from 1 to 5 µM. Furthermore, ANP expression was also detected in the H9c2 cells. The results revealed that ANP protein expression in the Iso-treated group was significantly upregulated, and the protective effects of PQQ were enhanced as the concentration increased, which was in accordance with the results obtained for the AC16 cells (Fig. 4D and E).

Under normal physiological conditions, NF-κB in the cytoplasm and NF-κB inhibitor (IκB) are combined and remain in an inactive state. However, under inflammatory or other stimulating conditions, the IκB kinase complex composed of IκB kinase α/β/γ is activated to promote serine phosphorylation at positions 32 and 36 of the IκBα protein, leading to the degradation of IκBα by ubiquitination and the release of free NF-κB. This free NF-κB can enter the nucleus binding with the specific site, and can subsequently mediate a series of cellular responses, such as inflammation through transcriptional activation of its target gene (48). NF-κBIA is one of the proteins of IκB that can inactivate NF-κB signaling by combining with NF-κB in the cytoplasm. The present study thus analyzed the expression of NF-κBIA and nuclear p65 by extracting nuclear proteins to determine the mechanism through which the NF-κB signaling pathway participates in cardiac hypertrophy induced by Iso or by treatment with PQQ.

As shown in Fig. 5, nuclear p65 expression in the Iso pre-treated group increased by 1.5-fold vs. the control group, while the expression of NF-κBIA decreased by 60%. Following pre-treatment with PQQ, the expression of nuclear p65 in the 1 µM PQQ group was decreased by 33% compared with that of the Iso pre-treated group, and it was significantly decreased with the increasing concentrations of PQQ. By contrast, NF-κBIA expression significantly increased with the increasing concentrations of PQQ (Fig. 5B). Following treatment with Iso, the NF-κB translocation to the nucleus significantly increased, while with the increasing concentrations of PQQ, the protective effects of PQQ were enhanced, as indicated by the significant proportionate decrease of NF-κB translocation to the nucleus (Fig. 5). The above-mentoined results indicated that, by inhibiting the NF-κB signaling pathway, PQQ may inhibit NF-κB from entering the nucleus, thus acting against Iso-induced hypertrophy in AC16 cells.

It has been reported that ROS accumulation is closely associated with inflammation (49), and PQQ effectively scavenges the production of intracellular ROS, as demonstrated in previous studies (50-52). The changes in ROS levels in AC16 cells with Iso or PQQ treatment were thus detected and photographed and quantified with ImageJ software (Fig. 6). As observed, the fluorescence intensity of the Iso pre-treated group was markedly stronger than that of the control and PQQ-treated groups (Fig. 6A). Upon treatment with Iso, the ROS levels were significantly increased by 2.2-fold relative to the control group, whereas the ROS levels of the group pre-treated with 1, 2.5 and 5 µM PQQ decreased by 18, 27 and 50% compared with those of the Iso pre-treated group (Fig. 6B). Moreover, the changes in the intracellular fluorescence intensity of the AC16 cells were detected by flow cytometry and the results revealed a consistent trend with the results of the images (Fig. 6C and D).

During the process of respiratory oxidation, the mitochondria store the energy generated by electrochemical potential energy in the mitochondrial inner membrane, causing asymmetric distribution of proton and other ion concentrations on both sides of the inner membrane to form MMP (53), which is a key parameter for evaluating mitochondrial function (54,55). The results obtained in this study for MMP are presented in Fig. 7. The red fluorescence intensity of JC-aggregates in the control group was significantly higher than that of JC-1monomers and the 'Merge' image synthesized by the two panels is dominated by the red fluorescence of JC-aggregates. The green fluorescence intensity of JC-1 monomers in the AC16 cells in the Iso group was significantly higher than that of the control group; the synthesized 'Merge' image is mainly compiled of green fluorescence. The red fluorescence of JC-aggregates in the group pre-treated with 1 µM PQQ was higher than that in the Iso group, and the 'Merge' image red fluorescence and the green fluorescence are almost the same, and the 'Merge' image is colored orange. The green fluorescence in the group pre-treated with 2.5 µM PQQ was weakened, and the red fluorescence in the synthesized 'Merge' image is greater than that in the Iso group. The green fluorescence in the group pre-treated with 5 µM PQQ was significantly weaker than that in the Iso group, and the red fluorescence in the 'Merge' image is greater than that in the Iso group, but slightly weaker than that in the control group (Fig. 7).