A novel traditional Chinese medicine ameliorates fatigue-induced cardiac hypertrophy and dysfunction via regulation of energy metabolism
Abstract
Prolonged exercise and exercise training can adversely affect cardiac function in some individuals. QiShenYiQi Pills (QSYQ), which are a compound Chinese medicine, have been previously shown to improve pressure overload-induced cardiac hypertrophy. We hypothesized that QSYQ can ameliorate as well the fatigue-induced cardiac hypertrophy. This study was to test this hypothesis and underlying mechanism with a focus on its role in energy regulation. Male Sprague-Dawley rats were used to establish exercise adaptation and fatigue model on a motorized rodent treadmill. Echocardiographic analysis and heart function test were performed to assess heart systolic function. Food-intake weight/body weight and heart weight/body weight were assessed, and hematoxylin and eosin staining and immunofluorescence staining of myocardium sections were performed. ATP synthase expression and activity and ATP, ADP, and AMP levels were assessed using Western blot and ELISA. Expression of proteins related to energy metabolism and IGF-1R signaling was determined using Western blot. QSYQ attenuated the food-intake weight/body weight decrease, improved myocardial structure and heart function, and restored the expression and distribution of myocardial connexin 43 after fatigue, concomitant with an increased ATP production and a restoration of metabolism-related protein expression. QSYQ upgraded the expression of IGF-1R, P-AMPK/AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α, nuclear respiratory factor-1, P-phosphatidylinositol 3-kinase (PI3K)/PI3K, and P-Akt/Akt thereby attenuated the dysregulation of IGF-1R signaling after fatigue. QSYQ relieved fatigue-induced cardiac hypertrophy and enhanced heart function, which is correlated with its potential to improve energy metabolism by regulating IGF-1R signaling.
NEW & NOTEWORTHY Prolonged exercise may impact some people leading to pathological cardiac hypertrophy. This study using an animal model of fatigue-induced cardiac hypertrophy provides evidence showing the potential of QiShenYiQi Pills, a novel traditional Chinese medicine, to prevent the cardiac adaptive hypertrophy from development to pathological hypertrophy and demonstrates that this effect is correlated with its capacity for regulating energy metabolism through interacting with insulin-like growth factor-1 receptor.
INTRODUCTION
Despite the fact that physical activity and exercise training benefit the population, prolonged exercise and exercise training can adversely affect cardiac function in some individuals. Exercise-induced cardiac fatigue has been reported in the literature (8), which is characterized by a reduction in left ventricular systolic and diastolic function subsequent to prolonged exercise in healthy humans. Prodigious amounts of exercise have been reported to result in increase of markers for (5, 17), and even the incidence of, cardiovascular disease (10). One large United States case series found that 44% of the deaths among young athletes were attributable to definite or possible hypertrophic cardiomyopathy (HCM) (23, 25). It is, however, difficult to distinguish mild forms of dilated cardiomyopathy from athlete’s heart (1, 11, 13), since the left ventricle ejection fraction (EF) augments normally (11) or becomes hyperdynamic (13) with exercise. This adaptation likely reflects increased cardiac output, as attained primarily through increase of stroke volume necessary to achieve the athletic strength of a competitive athlete. As a result, myocardial hypertrophy is a well-recognized concomitant of athletic training (31). Chamber enlargement with minor degrees of eccentric hypertrophy is typical of endurance athletes, and significant concentric hypertrophy without ventricular dilatation is observed in power athletes (21, 29). Most stimulation first induces single myocytes to grow in length and/or width to increase cardiac pump function and lower ventricular wall tension (3, 14). However, in the long term, cardiac hypertrophy makes individuals susceptible to heart failure, arrhythmia, and sudden death (3). Therefore, management is needed to protect the transformation of adaptive myocardial hypertrophy to HCM for the athletes.
QiShenYiQi Pills (QSYQ) are a compound Chinese medicine composing of Radix Astragali, Salvia miltiorrhiza, Panax notoginseng, and rosewood, which has been approved for treatment of coronary heart disease and angina by the Chinese State Food and Drug Administration. The efficiency of QSYQ in treatment of angina was documented and reviewed recently (4). Our previous study reported that QSYQ has the ability to attenuate energy metabolism deficiency and upregulate ATP 5D in cardiac iscehmia-reperfusion injury (20). QSYQ was also reported to be able to attenuate pressure-overload induced cardiac hypertrophy (6) and doxorubicin-induced myocardium injury in a multicomponent and multitarget manner (6, 36). We hypothesized that QSYQ may also benefit the cardiac hypertrophy caused by fatigue.
The present study intended to explore the potential effect of QSYQ on heart dysfunction caused by fatigue and the underlying mechanism, particularly focusing on the role of insulin-like growth factor-1 receptor (IGF-1R) in energy regulation.
MATERIALS AND METHODS
Animals.
Male Sprague-Dawley rats weighing 160–180 g were obtained from the Animal Center of Peking University [Certificate No. SCXK (Jing) 2006-0008]. Rats were raised in cages at temperature 22 ± 2°C and relative humidity 40 ± 5% under a 12-h:12-h light-dark cycle, with access to standard diet and water ad libitum. Rats were anesthetized with 2% pentobarbital (60 mg/kg) by peritoneal injection. Animals were euthanized by extracting blood from the abdominal aorta. All experimental procedures were approved by Peking University Biomedical Ethics Committee Experimental Animal Ethics Branch, complying with the “Guidelines for the Care and Use of Laboratory Animals,” published by the National Institutes of Health.
Regents.
QSYQ (batch no. 161208) was obtained from Tasly Pharmaceutical (Tianjin, China). QSYQ was dissolved in ultrapure water at the concentration of 0.2 g/ml (19, 36). ASIV was obtained from Fengshanjian Medicine Research (Kunming, Yunnan, China; purity ≥ 99.9%) and dissolved in ultrapure water at concentration of 0.132 mg/ml.
Experimental groups and treadmill running.
Rats were subjected to treadmill adaptation on a motorized rodent treadmill (YLS-15A; Dongguan BOZHI FAR Biotechnology Development, Guangdong, China) for 2 days, 60 min/day, 15 m/min, with treadmill being set at 0 incline. The animals showing adaptation to treadmill training were selected and randomized into sham group, sham + QSYQ group, fatigue group, and fatigue + QSYQ group, six rats in each. Rats in sham + QSYQ group and fatigue + QSYQ group were intragastrically administered with QSYQ daily at 0.8 g·kg−1·day−1 for 10 days. Animals in sham group and fatigue group received the same amount of ultrapure water (4 ml/kg body wt) the same way for 10 days. Animals in fatigue group and fatigue + QSYQ group experienced treadmill training for 10 days, 60 min/day, with the treadmill speed being set successively at 20, 20, 20, 25, 25, 25, 25, 30, 30, and 30 m/min. The last dose of QSYQ was administered on the day of fatigue protocol termination, while the echocardiographic analysis and euthanasia were conducted 24 h later. During the protocol, mild electric stimuli (1 mA, 3 Hz) were given from the back of the treadmill chamber to promote learning of running behavior. Animals in sham group and sham + QSYQ group were exempted from treadmill training after treadmill adaptation.
Cell culture.
H9c2 cells, a rat cardiac myoblast cell line (American Type Culture Collection), were used for determining underlying mechanisms (15). Cells were cultured in DMEM (Invitrogen, Grand Island, NY) containing 4 mM l-glutamine, 4.5 g/l glucose, and 10% FBS (Invitrogen) at 37°C in a humidified incubator with 95% air-5% CO2.
After medium was replaced with glucose-free DMEM with a half dose of FBS, two sets of experiments were conducted. In one set of experiments, H9c2 cells were cultured in the presence of QSYQ (1.25 mg/ml) for 48 h and collected at 0 min (T0), 5 min (T5), 10 min (T10), 20 min (T20), 30 min (T30), and 60 min (T60) of QSYQ treatment. In another set of experiments, confluent cells were divided into five groups: control, isoproterenol (ISO), ISO + compound C (CC), ISO + QSYQ, and ISO + CC + QSYQ. Cells were cultured for 48 h in a humidified atmosphere of 5% CO2 and 1% O2 (6) in the presence of ISO of 25 μM in all but control groups. In QSYQ groups, QSYQ (1.25 mg/ml) was added at 47 h and incubated for 1 h. In ISO + CC and ISO + CC + QSYQ groups, CC (10 μM) was added 1 h before treatment of QSYQ (9).
Food-intake weight.
Food-intake weight (FIW) of the animals was calculated by subtracting the food weight on the day from that on the previous day.
Echocardiographic analysis.
The left ventricle function was evaluated using a Vevo 770 High-Resolution Imaging Systems (Visual Sonics, Toronto, ON, Canada) with a 17.5-MHz linear array transducer. Two-dimensional cine loops and guided M-mode frames were recorded from the parasternal short and long axis (19). The following parameters were measured as indicators of cardiac function or remodeling: left ventricular posterior wall thickness at end diastole and systole (LVPWd and LVPWs), left ventricular internal diameter systole (LVIDs), EF, and fractional shortening (FS).
Heart function test.
Heart function was tested by a biofunction experiment system BL-420F (Chengdu Taimen Technology, Chengdu, Sichuan, China), which was connected to a cannulation inserted into left ventricle (LV) through right carotid artery. Left ventricular end diastolic pressure (LVEDP) and left ventricular maximum upstroke velocity (+dp/dtmax) were evaluated at the indicated time points (20).
Histology and immunofluorescent staining.
The hearts were fixed in 4% paraformaldehyde solution for 48 h and processed for paraffin section (5 μm). Sections were stained with hematoxylin and eosin, wheat germ agglutinin, rhodamine phalloidine (Invitrogen), and antibody against connexin (Cx43; Abcam, Cambridge, MA), The nuclei were labeled with Hoechest 33342. The sections were observed with a microscope (BX512DP70; Olympus, Tokyo, Japan) or laser scanning confocal microscope (TCS SP5; Leica, Mannheim, Germany). Five fields were randomly selected, wherein the cardiomyocyte cross-sectional area was determined on sections stained with wheat germ agglutinin, calculating the average of cross section areas of three to five cells in each using Image-Pro Plus 6.0 (Media Cybernetic, Bethesda, MD).
Protein extraction.
About 100 mg of myocardium tissue were harvested in animals (n = 6) from the same region of LV, which was 7 mm above apex cordis. Whole protein of tissues was extracted with a protein extraction kit (Applygen Technologies, Beijing, China), according to the manufacturer’s instruction.
Western blot analysis.
Whole protein was separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. To assess whether regulation of energy metabolism and IGF-1R signaling are involved in the effect of QSYQ on fatigue-induced cardiac hypertrophy, proteins related to the two processes were evaluated by Western blot (WB). For this, membranes were incubated overnight at 4°C with antibodies against GAPDH, cardiac troponin I (cTnI), ATP 5D, ATP synthase-α, ATP synthase-β, ENOα, ENOβ, enoyl coenzyme A hydratase 1 (ECH1), heat shock protein 70 (HSP70), phosphofructokinase-2 (PFK-2), carnitine palmitoyltransferase 1 (CPT1A), PDH, phosphatidylinositol 3-kinase (PI3K), P-PI3K, Akt, P-Akt, IGF-1R, P-AMPK, AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and nuclear respiratory factor-1 (Nrf1; 1:1,000; Abcam, Cambridge, MA). They were then incubated with secondary antibody for 1 h at room temperature, and the antibody binding was detected using enhanced chemiluminescence detection kit (applygene). Band intensities were quantified by densitometry and are expressed as mean area density using ImageJ (Bethesda, MD) software.
ELISA.
The content of ATP, ADP, and AMP in myocardium was determined by ELISA using a microplate reader (Beijing Huanya Biomedicine Technology, Beijing, China) according to the manufacturer’s instructions (20).
ATP synthase activity and quantity.
ATP synthase activity and quantity were determined using ATP Synthase Specific Activity Microplate Assay Kit (Abcam), with the setting of the plate in the MULTISKAN MK3 enzyme microplate reader (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Surface plasmon resonance.
Surface plasmon resonance (SPR) assay was used to analyze the binding capacity of AS-IV, one of the major components of QSYQ, to IGF-1R. A carboxymethylated 5 (CM5) sensor chip (GE Healthcare Life Sciences, London, UK) was docked into a Biacore T200 (Biacore, GE Healthcare, Uppsala, Sweden) and prepared as previously reported (12). Human IGF-1R full-length protein (Abcam) was immobilized on a CM5 sensor chip by injection of 40 μl of IGF-1R at a rate of 5 μl/min. AS-IV was dissolved by running buffer to different concentrations before injection. Ninety microliters of each AS-IV solution were injected and dissociated after 300 s. Equilibrium dissociation constant (Kd) was calculated by fitting a 1:1 Langmuir model using Biacore T200 evaluation software v2. 0 (Biacore, GE Healthcare, Sweden) (40).
Statistical analysis.
All data are expressed as means ± SD. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test or using two-way ANOVA followed by Bonferroni for multiple comparisons in body weight (BW) and FIW/BW. Data were analyzed using GraphPad Prism 7 software (GraphPad Software). P < 0.05 was considered to be statistically significant.
RESULTS
QSYQ prevents reduction in FIW/BW and attenuates myocardial injury after fatigue.
Figure 1, A and B, illustrates the change of BW and FIW/BW with time in different groups. As shown, fatigue induced a reduction in BW. QSYQ showed no effect on this reduction (Fig. 1A) but significantly attenuated the fatigue-induced reduction in FIW/BW (Fig. 1B).
Fig. 1.QiShenYiQi Pills (QSYQ) prevents reduction in food-intake weight (FIW)/body weight (BW) and attenuates myocardial injury after fatigue. A: the change of BW with time in different groups. BW was calculated once a day. B: the change of FIW/BW with time in different groups. The linear mixed effect models were analyzed for repeated measurement data, and least squares means were calculated between the groups of different time points. Statistical analysis was performed using two-way ANOVA followed by Bonferroni for multiple comparisons. Values are means ± SD from 6 animals. *P < 0. 05 vs. sham; #P < 0. 05 vs. fatigue. C: representative Western blot bands of cardiac troponin I (cTnI) in myocardium in different groups. GAPDH was used as a loading control. D: the semiquantitative analysis of cTnI in myocardium from different groups. Data are expressed as means ± SD; n = 6. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0. 05 vs. sham group; #P < 0.05 vs. fatigue group. E: images of heart macromorphology in different groups (e1–e4). Bar = 1 cm. F: representative histological image of myocardium tissue stained by hematoxylin and eosin in each group. Bar = 100 μm. G: representative F-actin staining photographs of rat myocardium in each group. F-actin are stained with red and nuclei blue. Bar = 50 μm. e1–e4, f1–f4, and g1–g4: sham, sham + QSYQ, fatigue, and fatigue + QSYQ group, respectively.
The level of cTnI in myocardium was determined to assess cardiomyocyte injury. The level of cTnI in myocardium decreased significantly in fatigue group compared with sham group (Fig. 1, C and D), which was significantly protected by QSYQ.
The histology of rat myocardium in sham groups showed normal tissue with continuous F-actin bundles. Tissue edema and myocardial fiber thickening and F-actin rupture appeared in the fatigue group, which were obviously attenuated by QSYQ (Fig. 1, F and G).
QSYQ improves fatigue-induced cardiac hypertrophy.
The distribution of Cx43 between cardiomyocytes in each group was determined by immunofluorescent staining (Fig. 2A), revealing a continuous distribution of Cx43 in sham groups, which, however, became dotted lines in fatigue group, concomitant with reduced immune staining, indicating fatigue-induced degradation of the gap junction (GJ) protein Cx43. QSYQ inhibited the breakdown of Cx43 notably. These results were supported by assessment of WB (Fig. 2, E and F).
Fig. 2.QiShenYiQi Pills (QSYQ) reduces heart weight (HW)/body weight (BW) and cardiac hypertrophy and attenuates downregulation of connexin 43 (Cx43) proteins after fatigue. A: representative immunofluorescence confocal images of Cx43 in different groups. Cx43 (green) localized at the gap junction of cardiomyocytes, which was marked with wheat germ agglutinin (WGA; red). Nuclei were stained with blue. a1, a2, a3, and a4: sham group, sham + QSYQ group, fatigue group, and fatigue + QSYQ group, respectively. The area within the rectangle in each picture in a1–a4 is enlarged and presented in b1–b4 correspondingly. Bar = 10 μm. B: representative rat heart cross sections in each group stained by WGA (red) to show demarcating cell boundaries. The heart sections were cut at the middle of heart vertical axis. Nuclei are stained blue. b1, b2, b3, and b4: sham group, sham + QSYQ group, fatigue group, and fatigue + QSYQ group, respectively. The area within the rectangle in each picture in a1–a4 is enlarged and presented in b1–b4 correspondingly. Bar = 25 μm. C: quantitative evaluation of BW. Data was expressed as means ± SD. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0.05 vs. sham; n = 6. D: quantitative evaluation of HW/BW. The data are presented as means ± SD. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0.05 vs. sham, #P < 0.05 vs. fatigue; n = 6. E: the representative WB bands of Cx43 in myocardium in different groups. GAPDH was used as a loading control. F: the semiquantitative analysis of Cx43. The samples derived from the same experiment, and gels were processed in parallel. Data are expressed as the means ± SD (n = 6). Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0.05 vs. sham group; #P < 0.05 vs. fatigue group. G: quantitative measurement of cardiomyocyte cross-sectional area. The data are presented as means ± SD. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0.05 vs. sham; #P < 0.05 vs. fatigue; n = 6.
Cardiac hypertrophy occurred obviously in fatigue rats as manifested an increase in cardiomyocyte size (Fig. 2B), whole heart cross-sectional area (Fig. 2G), and ratio of heart weight to BW (Fig. 2D). QSYQ significantly protected against all but BW the alterations.
QSYQ attenuates fatigue-induced ventricular wall thickening and heart dysfunction.
The representative images of M-mode echocardiograms from each group are presented in Fig. 3A. Compared with sham group, the hearts in fatigue group exhibited hypertrophy remarkably as shown by a significant increase in LVPWd and LVPWs accompanied with observably increased LVIDs (Fig. 3, B–D) and a reduction in left ventricle EF and FS (Fig. 3, E and F). QSYQ treatment had no effect on the change in LVPWd and LVPWs (Fig. 3, B and C) but showed strong efficacy in restoration of LVIDs, EF, and FS after fatigue (Fig. 3, E and F), suggesting the protection of QSYQ against systolic function impairment.
Fig. 3.QiShenYiQi Pills (QSYQ) improves heart function after fatigue. A: echocardiographic parameters in each group showing ventricle wall thickness during cardiac cycles. a1, a2, a3, and a4: sham, sham + QSYQ, fatigue, and fatigue + QSYQ, respectively. B–F: quantitative measurement of left ventricular posterior wall thickness at end diastole (LVPWd; B), left ventricular posterior wall thickness at end systole (LVPWs; C), left ventricular internal diameter systole (LVIDs; D), ejection fraction (EF; E), and fractional shortening (FS; F). Data are presented as means ± SD. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0.05 vs. sham; #P < 0.05 vs. fatigue. G and H: heart function shown as left ventricular end diastolic pressure (LVEDP; G) and left ventricular maximum upstroke velocity (+dp/dtmax; H) in different groups. Data are expressed as the means ± SD; n = 6. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0.05 vs. sham group; #P < 0.05 vs. fatigue group.
Further study found that in comparison with sham group, fatigue caused an apparent elevation in LVEDP (Fig. 3G) and a significant decline in +dp/dtmax (Fig. 3H), indicating an obvious impairment of heart function. QSYQ exhibited a significant protective role for +dp/dtmax (Fig. 3H) but not for LVEDP (Fig. 3G).
QSYQ attenuates fatigue-induced energy metabolism disorder.
The subunits of ATP synthase ATP 5D, ATP synthase-α, and ATP synthase-β were determined by WB to gain insight into the rational for fatigue-induced alterations. The results showed that fatigue did not affect the expression of ATP synthase-α and ATP synthase-β but decreased the expression of ATP 5D significantly, which was, however, prevented by QSYQ (Fig. 4, A–D).
Fig. 4.QiShenYiQi Pills (QSYQ) attenuates the dysregulated energy metabolism after fatigue. A: the representative Western blot bands of ATP 5D, ATP synthase-α, and ATP synthase-β in myocardium in different groups. GAPDH was used as a loading control; n = 6. B–D: the semiquantitative analysis of ATP 5D (B), ATP synthase-α (C), and ATP synthase-β (D). Data are expressed as the means ± SD; n = 6. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0.05 vs. sham group, #P < 0.05 vs. fatigue group. E–G: the activity (E), quantity (F), and activity/quantity (G) of ATP synthase in myocardial tissue from different groups. H–J: the level of ATP (H), ADP (I), and AMP (J) tested by ELISA in myocardium in each group. All data are expressed as means ± SD; n = 6. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0.05 vs. sham group; #P < 0.05 vs. fatigue group.
The activity and quantity of ATP synthase were investigated by ELISA. ATP synthase quantity remained unchanged among groups (Fig. 4F), while the activity (Fig. 4E) and activity/quantity of ATP synthase (Fig. 4G) diminished significantly after fatigue compared with sham group, which was considerably ameliorated by QSYQ.
The levels of ATP, ADP, and AMP were further determined in different conditions (Fig. 4, H and I). As compared with sham group, fatigue challenge had no effect on ATP level but increased the levels of ADP and decreased the level of AMP, suggesting a disorder of energy metabolism after fatigue. QSYQ elevated the level of ATP and decreased the level of ADP after fatigue significantly.
QSYQ regulates the expression of energy metabolism-related proteins after fatigue.
We next assessed by WB the expressions of the proteins that engage in energy metabolism. As shown in Fig. 5, fatigue challenge led to an increased expression of ENOα (the fetal form of ENO), PFK2, and HSP70, the three proteins that are implicated in glycolysis, and a decreased expression of ENOβ (the adult form of ENO), CPT1A, PDH, and ECH1, which are known to participate in regulation of oxidation of fatty acids and glucose. Of notice, QSYQ restored all the fatigue-induced alterations significantly.
Fig. 5.QiShenYiQi Pills (QSYQ) attenuates the alteration in the expression of energy metabolism-related proteins after fatigue. A: the representative Western blot bands of ENOα, ENOβ, enoyl coenzyme A hydratase 1 (ECH1), heat shock protein 70 (HSP70), phosphofructokinase-2 (PFK2), carnitine palmitoyltransferase 1 (CPT1A), and PDH in myocardium in different groups. GAPDH was used as a loading control; n = 6. B–H: the semiquantitative analysis of ENOα (B), ENOβ (C), ECH1 (D), HSP70 (E), PFK2 (F), CPT1A (G), and PDH (H). The samples derived from the same experiment and gels were processed in parallel. Data are expressed as means ± SD n = 6. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0. 05 vs. sham group; #P < 0. 05 vs. fatigue group.
The role of QSYQ implicates IGF-1R/Akt signaling.
The phosphorylation of PI3K and Akt is related to cell survival signaling, which showed an obvious reduction after fatigue but was restored by QSYQ (Fig. 6, A–C). The expressions of IGF-1R, P-AMPK/AMPK, PGC-1α, and Nrf1 in myocardium were further detected by WB. As shown in Fig. 6, D–H, the phosphorylation of AMPK and the expression of IGF-1R, PGC-1α, and NRF1 significantly decreased by fatigue, which were noticeably restored by QSYQ treatment. These results highlight the involvement of IGF-1R/Akt signaling in the beneficial role of QSYQ.
Fig. 6.QiShenYiQi Pills (QSYQ) regulates insulin-like growth factor-1 receptor (IGF-1R)/phosphatidylinositol 3-kinase (PI3K)/Akt and AMPK/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α)/nuclear respiratory factor-1 (Nrf1) signaling. A: the representative Western blottin (WB) bands of P-PI3K, PI3K, P-Akt, and Akt in myocardium in different groups. GAPDH was used as a loading control. n = 6. B and C: the semiquantitative analysis of P-PI3K/PI3K (B) and P-Akt/Akt (C). The samples were derived from the same experiment and gels were processed in parallel. Data are expressed as the means ± SD; n = 6. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0. 05 vs. sham group; #P < 0. 05 vs. fatigue group. D: the representative WB bands of IGF-1R, P-AMPK, AMPK, PGC-1α, and Nrf1 in myocardium in different groups. GAPDH was used as a loading control; n = 6. E–H: the semiquantitative analysis of IGF-1R (E), P-AMPK/AMPK (F), PGC-1α (G), and Nrf1 (H). The samples were derived from the same experiment and gels were processed in parallel. Data are expressed as the means ± SD; n = 6. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0. 05 vs. sham group, #P < 0. 05 vs. fatigue group.
QSYQ component AS-IV interacts with IGF-1R and QSYQ activates IGF-1R-AMPK-PGC-1α signaling.
As shown in Fig. 7B, the result of SPR indicated that AS-IV bound to IGF-1R in a dose-dependent manner. The equilibrium dissociation constant (Kd) of AS-IV binding to IGF-1R was 4.847e-6.
Fig. 7.QiShenYiQi Pills (QSYQ) component AS-IV interacts with insulin-like growth factor-1 receptor (IGF-1R) and QSYQ activates IGF-1R-AMPK-peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling. A: the chemical structure of AS-IV. B: AS-IV binding to IGF-1R tested by surface plasmon resonance (SPR). Shown are the representative sensorgrams obtained from the injections of AS-IV at concentrations of 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39 μM (curves from the top to the bottom). C: IGF-1R and AMPK were activated by QSYQ. The representative Western blot (WB) bands of P- IGF-1R, IGF-1R, P-AMPK, and AMPK in H9c2 cells in different groups are shown. GAPDH was used as a loading control; n = 3. D and E: the semiquantitative analysis of P-IGF-1R/IGF-1R and P-AMPK/AMPK is presented. The samples were derived from the same experiment and gels were processed in parallel. Data are expressed as the means ± SD; n = 3. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0. 05 vs. T0 group. F: the representative WB bands of P-AMPK and AMPK in H9c2 cells in different groups. GAPDH was used as a loading control; n = 3. CC, compound C. G and I: the semiquantitative analysis of P-AMPK/AMPK (G) and PGC-1α (I). The samples were derived from the same experiment and gels were processed in parallel. Data are expressed as the means ± SD; n = 3. H: the ATP level in H9c2 cells in different groups. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. *P < 0. 05 vs. control group; #P < 0. 05 vs. isoprotenol (ISO) group.
To further verify the effect of QSYQ on IGF-1R, H9c2 cells were used to assess the effect of QSYQ on the phosphorylation of IGF-1R and AMPK. The results shown on Fig. 7, C–E, suggest that IGF-1R was positively activated by QSYQ. In another experiment (Fig. 7, F–I), wherein ISO was used to stimulate AMPK while CC was used to inhibit AMPK, we observed that activation of AMPK by ISO was further potentiated by QSYQ, which, however, was abolished by CC. In addition, QSYQ increased the expression of PGC-1α, and this effect was prevented by CC as well, suggesting the critical role of AMPK in mediating the effect of QSYQ. Taken together, the results aforementioned highlight the involvement of IGF-1R-AMPK-PGC-1α signaling in QSYQ effect.
DISCUSSION
A number of clinically unsuspected cardiovascular diseases have been reported in young athletes, usually in association with physical exertion (22). We thus employed relatively young rats to establish fatigue model in the present study. In this model, the fatigued rats have messy and dull hair, decreased appetite, look tired, are insensitive to stimuli, have a reduction in exploration, and a slowed increase in body weight. With the use of this model, this study demonstrated the potential of QSYQ to protect against fatigue-induced cardiac hypertrophy, as shown by 1) the attenuation of the cardiomyocyte morphology and Cx43 distribution and expression, 2) the reduction of HW/BW and the increase in myocardium cTnI level, and 3) an improved heart function and an increase in FIW/BW. These results suggest QSYQ as a prophylaxis for the athletes at risk of pathological cardiac hypertrophy. Moreover, QSYQ has increased the level of ATP and ATP synthase activity and quantity compared with the fatigue group, implying the involvement of regulation of energy metabolism in the beneficial role of QSYQ.
Regular physical exercise can lead to physiological adaptation in cardiac dimensions, including increased left ventricular wall thickness and left ventricular cavity size, which may be reflected on electrocardiogram and echocardiography (24). In the present study, the rats in QSYQ + fatigue group showed thickening of the ventricular wall and enlarged ventricular cavity but a restored EF after fatigue. This result suggested that treatment with QSYQ results in a physiological cardiac hypertrophy after fatigue, although fatigue per se leads to pathological hypertrophy.
GJs provide connections and communication between cells, allowing the passage of ions and small molecules such as ATP, glutathione, cAMP, IP3, and glucose (30), which is crucial for synchronous contraction of cardiomyocytes. Cx43 is a type of GJs proteins expressed in cardiomyocyte and is known to redistribute along the cardiomyocyte surface with reduced expression when hypertrophy becomes prolonged and putatively maladaptive (37). Consistent with this, we observed a reduced expression and a disturbed distribution of Cx43 in the myocardium tissue after fatigue. QSYQ restored the alteration of Cx43 after fatigue, showing an attenuation in cardiac hypertrophy and an improvement of cardiac contraction. However, the rationale behind the role of QSYQ in Cx43 disorder is at present unknown.
An altered energy homeostasis has been revealed in patients with HCM (35). Evidence shows that the impaired energy metabolism seems to precede the development of hypertrophy (7) and contractile abnormalities (2). In line with these reports, we observed a dysregulated energy metabolism in myocardium of the rats submitted to fatigue, which manifested a decrease in ATP 5D expression; an increased glycolysis as shown by elevated expression of ENOα, PFK2, and HSP70; and a decreased oxidation of fatty acids and glucose presenting as decrease in the expression of ENOβ, CPT1A, PDH, and ECH1. Interestingly, despite of the dysregulated energy metabolism, fatigue rats still exhibited an EF > 60%, suggesting that fatigue-induced energy metabolism disorder resulted in myocardial compensatory hypertrophy, which can support heart function for a short time. QSYQ treatment ameliorated all the alterations in energy metabolism after fatigue, suggesting that this medicine must act at some target upstream these proteins.
Energy metabolism is regulated by an array of proteins. Exercise stimulates cardiomyocyte contractile activity, which induces mitochondrial biogenesis and increases glucose transport capacity as an adaptation. Adaptation reaction involves a multitude of events initiating with activation of AMPK due to increase in AMP level. The activated AMPK in turn induces PGC-1α and PGC-1β expression via NRF-1 (39) leading to mitochondrial biogenesis (18, 32). Moreover, IGF-1 has been shown to increase intracellular ATP levels, mitochondrial metabolism, mitochondrial Ca2+ uptake, and oxygen consumption by Akt/mammalian target of rapamycin (mTOR) and AMPK/mTOR axes (38). In the present study, we observed in the fatigue group a significantly reduction in the expression of all the proteins involved in adaptation suggesting failure in adaptation in the condition of fatigue. The exact reason for this failure is unclear. However, the fact that the ADP increased while AMP decreased in fatigue group indicates an inability of ADP to be hydrolyzed to AMP for some unknown reason. As a consequence, AMPK activation was inhibited as it was for all the events that followed. QSYQ attenuated the decline in all the proteins concerned and thus prevented maladaptation after fatigue. We speculated that QSYQ takes effect by targeting IGF-1R, because it is upstream of both AMPK-PGC-1α/NRF-1 and PI3K/Akt signaling. To test this speculation, we assessed the binding ability of AS-IV, one of the major components of QSYQ known to regulate cardiac energy metabolism (6) to IGF-1R by SPR (Fig. 7). The positive finding of this test provided support for our speculation.
Both physiological and pathological stimuli initially induce hypertrophy as an adaptive process, but pathological hypertrophy generally progresses to heart failure (27). The type of hypertrophic stimuli and the downstream signaling determine cardiac hypertrophy either physiological or pathological. Physiological cardiac hypertrophy in athletes is associated with an exercise-induced increase in IGF-1 levels in serum (28). AMPK was reported to inhibit mTORC1 attenuating angiotensin II-induced and pressure-overload-induced pathological hypertrophy and heart failure in mice (33, 34). AMPK increases mitochondrial biogenesis, promoting ATP production and inhibiting energy-consuming biosynthetic pathways by negatively regulating mTOR through phosphorylation (16, 26). AMPK inhibition exacerbates pathological hypertrophy and heart failure, whereas AMPK activation can be protective against pathological hypertrophy (27). In the present study, fatigue induced a decreasing level of IGF-1R and AMPK, suggesting that a pathological hypertrophy occurred. QSYQ increased the level of IGF-1R and AMPK, suggesting the physiological hypertrophy took place in response to QSYQ.
In conclusion, as shown in Fig. 8, the present study demonstrated the potential of QSYQ to prevent fatigue-induced cardiac hypertrophy, which is associated with counteracting maladaptation in energy metabolism via regulating IGF-1R signaling and promoting fatty acid oxidation and glucose oxidation of myocardium while reducing glycolysis. The result of the present study suggests QSYQ as a stratagem to protect the heart from pathological hypertrophy. Nevertheless, more study is needed for translation of the results to clinical situations.
Fig. 8.A diagrammatic sketch showing the pathways that lead to the various effects of QiShenYiQi Pills (QSYQ) on protection of fatigue-induced cardiac hypertrophy and dysfunction by modulating energy metabolism. Fatigue induced the decrease of the expression of Cx43, ATP 5D, and ATP synthase activity as well as the changes of enzymes involved in glucose oxidation and fatty acid oxidation, inactivated AMPK-peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α)/nuclear respiratory factor-1 (NRF-1) and phosphatidylinositol 3-kinase (PI3K)/Akt, which collectively result in cardiac hypertrophy and dysfunction. QSYQ regulated insulin-like growth factor-1 receptor (IGF-1R) signaling, activated AMPK-PGC-1α/NRF-1 and PI3K/Akt signaling, and increased ATP synthase activity, the expression of Cx43 and ATP 5D, and enzymes involved in glucose oxidation and fatty acid oxidation and thus attenuated the fatigue-induced insults. HSP70, heat shock protein 70; ECH1, enoyl coenzyme A hydratase 1; PFK2, phosphofructokinase-2; CPT1A, carnitine palmitoyltransferase 1.
GRANTS
This work was supported by the Production of New Medicine Program of Ministry of Science and Technology of China Grant 2013ZX09402202 and State Key Laboratory of Core Technology in Innovative Chinese Medicine Grant 20170034.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
J.-Y.H. conceived and designed research; R.H. and S.-Y.H. performed experiments; R.H., C.-S.P., and Q.L. analyzed data; R.H., Y.-C.C., X.-H.W., Q.L., and J.-Y.F. interpreted results of experiments; R.H. prepared figures; R.H. drafted manuscript; Y.-C.C., X.-H.W., and J.-Y.F. edited and revised manuscript; R.H., Y.-C.C., X.-H.W., C.-S.P., Q.L., J.-Y.F., and J.-Y.H. approved final version of manuscript.
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