Comparative metabonomics of Wenxin Keli and Verapamil reveals differential roles of gluconeogenesis and fatty acid β-oxidation in myocardial injury protection

Metabonomics/metabolomics is a rapid technology for comprehensive profiling of small molecule metabolites in cells, tissues, or whole organisms, the application of which has led to understanding pathophysiologic mechanisms of cardiometabolic diseases, defining predictive biomarkers for those diseases, and also assessing the efficacious effects of incident drugs. In this study, proton nuclear magnetic resonance (NMR)-based metabonomics was employed to identify the metabolic changes in rat plasma caused by myocardial ischemia-reperfusion injury (MIRI), and to compare the metabolic regulatory differences between traditional Chinese medicine Wenxin Keli (WXKL) and Western medicine verapamil. The results revealed that energy-substrate metabolism were significantly disturbed by ischemia-reperfusion (I/R) in myocardium and bulk of the key metabolites could be further modulated by verapamil and/or WXKL. Lipid metabolism and amino acid transamination occurred mainly following the treatment of verapamil, whereas glucose oxidation and BCAA degradation were prominently ameliorated by WXKL to content the energy demands of heart. Moreover, both WXKL and verapamil improved the secretions of taurine and ketone bodies to overcome the oxidative stress and the shortage of energy sources induced by ischemia-reperfusion.

Multivariate data analysis of metabolic changes. To explore the metabolic alterations that associated with clustering trends, NMR data of the Sham and MIRI, Ver and MIRI, WXKL and MIRI sets were subjected respectively to supervised PLS-DA analysis and the results of which were exported in both the score plots and VIP plots as shown in Fig. 4. Clear discriminations between Sham and MIRI, Ver and MIRI, WXKL and MIRI groups were observed in the PLS-DA score plots with the first principal component (PC1) explaining 39.7%, 47.4% and 55.2% of the total variances. The corresponding R 2 and Q 2 values yielded from cross-validation were 0.68 and 0.32, 0.77 and 0.59, 0.56 and 0.28, respectively, indicating good fit and classification of the models on PC1. Another fundamental ability of PLS-DA is to select meaningful variables on the basis of VIP values. With a selection criterion of VIP > 1, 13 metabolites were identified for contributing the discrimination between the Sham and MIRI groups, including lipids, three branched-chain amino acids (BCAAs: valine, isoleucine and leucine), three ketone bodies (HB, acetone and acetoacetate), alanine, lactate, glutamate, choline, taurine, and glucose. In the Ver vs. MIRI dataset, 9 of the abovementioned metabolites (except for BCAAs and choline) were selected using the same selection criterion. In the WXKL vs. MIRI dataset, 10 of the metabolites (except for lipids, glutamate and choline), together with glutamine and creatine phosphate, were extracted for clustering based on their VIP values.
Taking the univariate measures (t-test) into consideration, I/R injury caused significant elevations of lipids, isoleucine and leucine but reductions of lactate, alanine, and glutamate, compared to those of the control rats with p values less than 0.05. Lipids, lactate, alanine and glutamate were uniquely dominant when comparing the Ver to MIRI groups, whereas BCAAs, HB, glutamine, creatine phosphate and glucose were uniquely dominant when comparing the WXKL to MIRI groups. Besides these significantly altered metabolites, two outstanding ones that indicated cluster discrimination were also found in the Ver vs. MIRI and WXKL vs. MIRI datasets, namely: taurine and acetoacetate. The variation trends of these metabolites and the involved pathway were summarized and presented in Table 2 and Fig. 5.   Table 1.

Discussion
Traditional Chinese medicine (TCM), built on a foundation of more than 2,500 years of medical practice, has been widely used in China and is becoming increasingly accepted elsewhere in the world. However, it remains a mounting challenge deciphering the mechanisms of such complex medicine. Metabonomics offers an ideal platform and state-of-the-art approach for the scientific investigation of TCM. It has been successfully used to group TCM symptoms into clinically useful "patterns" for better diagnosis of diseases, and also to provide crucial insights into the holistic assessment of the quality and effectiveness of Chinese medicines [20][21][22] . In the present study, a systematic metabonomics approach based on 1 H-NMR technique was performed to compare the metabolic alterations in plasma responding to the modulations of verapamil and WXKL on the myocardial injury induced by I/R. Multivariate statistics analysis for NMR profiles revealed distinct metabolite changes between Sham and MIRI groups mainly associated with the disturbances of myocardial energy substrate metabolism, bulk of which could be further regulated by verapamil and/or WXKL (Fig. 6). The beneficial effect of WXKL pretreatment on cardiac functions induced by I/R was mainly evaluated by quantitative analysis of echocardiography and hemodynamic assessment. It revealed that an ischemia for 30 min followed by reperfusion for 2 hours in rats led to a decrease in LVEF and LVFS, a reduction of LVAWs and E/A, and an increase in LVIDs and LV Vols. In addition, I/R caused reduction of AV Peak and AoV VTI. All the I/R-induced alterations were evidently ameliorated by pre-administration of WXKL or verapamil for one week, suggesting the potential of WXKL to relieve I/R-induced cardiac insufficiency. After pretreatment for one week, LVDP and −dp/dt max of the verapamil group were not higher than those of the I/R group. Interestingly, one-week pretreatment by WXKL led to a significant increase in LVDP and −dp/dt max compared with the I/R group. These results showed that WXKL pretreatment could effectively improve cardiac hemodynamics, increase coronary blood flow, enhance myocardial contractility, and reduce myocardial reperfusion injury in a rat I/R model. Furthermore, WXKL and verapamil are equally protective for heart function by Echo, but WXKL is somehow better by hemodynamics. Previous reports 13,15,16 have also provided additional supporting evidences, including the measurements of infarct area and other crucial biochemical factors, to confirm the effects of WXKL on improving myocardial infarction, ventricular arrhythmias and heart failure.
We hypothesize that the above functional change may be correlated to the metabonomic differences as illustrated in Fig. 6. Circulating lipids and glucose are known to be the predominant energy providing substrates for normal cardiac contractility. Lipids account for the majority (60-80%) of oxidative energy metabolism, while carbohydrates accounts for those remaining 20-40% 23,24 . After the period of ischemia and following reperfusion, the level of lipids significantly increased, while the level of glucose had no significant change in plasma. In the first stage of ischemia, fatty acids compete with glucose and become the major source of energy with a high rate of uptake resulting in their preferential use in oxidative metabolism 25,26 . Within minutes of reperfusion, a large burst of ROS is produced 27 . It can induce membrane injury by lipid peroxide formation and damage on fatty acid β-oxidation, further leading to an excessive accumulation of lipids in MIRI plasma. The uptake of glucose from the bloodstream into the cardiomyocytes can be inhibited by high circulating lipid concentrations 28 , hence the level of glucose in MIRI-induced plasma was observed remaining relatively unchanged compared to that of the control group.
Pyruvate, as the last glycolytic intermediate, can further be reduced to lactate, transaminated to alanine, carboxylated to oxaloacetate or malate, or, most importantly, oxidized to acetyl-CoA 29 . Although we failed to detect pyruvate in the present work, lactate and alanine were determined with significantly decreased levels as a result, which indicates a depressed glycolysis in the cardiomyocytes induced by MIRI and leading to limited productions of pyruvate and its derivatives.  Amino acids also can serve as metabolic fuel in muscle and other tissues by the way of transamination. In the reaction, the amino group of an amino acid is transferred to α-ketoacid, typically catalyzed by a transaminase. A very common α-ketoacid is α-ketoglutarate, an intermediate in the citric acid cycle. Transamination of alanine or aspartate together with α-ketoglutarate can give the products of pyruvate or oxaloacetate and glutamate 30 . It is supposed that a decreased level of glutamate caused by MIRI possibly accompanies with a deficiency of pyruvate and oxaloacetate, which contribute as substrates or intermediates in fundamental processes such as glycolysis and the citric acid cycle for further ATP production.
In a worse situation of the MIRI rats, levels of other amino acid sources such as BCAAs (isoleucine and leucine) were elevated in plasma to compensate energy shortages. It suggests that the degradation of BCAAs are probably blocked involving the inhibition of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex, leading to a buildup of BCAAs (leucine and isoleucine) and their toxic by-products in the blood. By converting BCAAs either into acetyl-CoA or succinyl-CoA that enter the citric acid cycle, BCKDH complex is a key enzyme for serving BCAAs as energy fuel 31 . The above results reveal that the generations of acetyl-CoA Figure 5. Visualization of the involved pathway for the differential metabolites. The summarized pathway is based on KEGG database (http://www.kegg.jp/).The metabolites in blue were detected in the NMR spectra whereas those in black failed NMR detection. Box plots showed the normalized relative contents of these differential metabolites in the four treatment groups.
SCIenTIFIC REPORts | 7: 8739 | DOI:10.1038/s41598-017-09547-w derivatives from complementary energy sources are impaired by IR injury, which further leading to the accumulations of BCAAs in blood, and then failed to afford the greater energy demand.
Compared to the model group, verapamil-treated rats showed a significantly decreased level of lipids, while a fair change of plasma glucose. The results imply that the most beneficial effect of verapamil is related to amendment on mitochondrial function for accelerating the utilization of excessive lipids induced by MIRI. The elevated level of glutamate indicated an amelioration of amino acid transamination to afford relatively sufficient pyruvate and oxaloacetate after verapamil treatment. However, plasma levels of lactate and alanine were observed increasingly in Ver group, which indicate the completeness of glycolytic pathway in cardiac myocytes rather than further formations of acetyl-CoA or oxaloacetate from pyruvate entering the mitochondrion through a transporter.
On the other hand, the WXKL-treated group showed no significant change in the level of plasma lipids while the glucose level was obviously elevated compared with MIRI group. The results imply that WXKL contributes to the supplement of glucose from liver glycogen degradation as energy provision to support the function of the heart, an energy metabolic control distinct from that of verapamil. Previous studies have demonstrated that provision of glucose together with insulin and potassium improves contractile function in the acutely ischemic and reperfused myocardium 32 . Moreover, plasma BCAAs (isoleucine, leucine and valine) showed decreased levels in WXKL-treated rats, suggesting WXKL may also modulate the degradations of BCAAs for providing more Acyl-CoA derivatives to generate ATP.
The metabolic analysis revealed that both verapamil and WXKL could increase the levels of taurine and acetoacetate in plasma compared to those of MIRI group. Taurine is a major constituent of bile with various fundamental biological roles, such as conjugation of bile acids, antioxidation, osmoregulation, membrane stabilization, and modulation of calcium signaling 33 . It is reported essential for cardiovascular function with a specialty of increasing the force and effectiveness of heart-muscle contractions 34 . It also acts as an antioxidant and protects against toxicity of various substances 35 , and the supplementation with taurine has been shown to prevent oxidative stress induced by exercise 36 . Acetoacetate together with 3-hydroxybutyrate are known as ketone bodies that are produced by the liver from fatty acids. During the periods of extreme circumstance, these ketone bodies are readily picked up by the extra-hepatic tissues, and converted into acetyl-CoA which then enters the citric acid cycle and is oxidized in the mitochondria 37 . Pathological states could significantly alter energy metabolism. For example, during the development of heart failure, the capacity of heart to utilize fatty acids is diminished. Recent studies suggested a role of ketone bodies as an alternative fuel and myocardial ketone oxidation as a key metabolic adaptation in the failing human heart 38 . Interestingly, studies of relative oxidation in an isolated heart preparation using ex vivo nuclear magnetic resonance combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids 39 . Our results showed that both verapamil and WXKL could stimulate the generations of ketone bodies in liver and transport them through blood to heart for energy. With the elevating levels, verapamil and WXKL are supposed to stimulate bile secreting taurine and liver generating ketone bodies and transport them to heart through blood to resist excessive ROS induced by MIRI, regulate calcium signaling to enhance contractile function, as well as deliver fuels for energy supplements.
In summary, our 1 H-NMR-based metabonomics approach provides a promising technique to elucidate and compare the cardioprotective mechanisms of traditional Chinese medicine (WXKL) and Western medicine (verapamil) on ischemia-reperfusion injury. Several metabolites, involving some complementary energy sources, and their metabolic pathway were found closely related to the progression of ischemia-reperfusion on myocardium. A prominent mechanism of verapamil was the modulations on lipid metabolism and amino acid transamination in cardiomyocytes, whereas that of WXKL was mainly the regulation of glucose oxidation and BCAAs degradations to content the energy demands of heart. Besides these differences, both WXKL and verapamil can improve the secretions of taurine and ketone bodies to overcome the oxidative stress and the shortage of energy sources induced by ischemia-reperfusion. Although lipid metabolism fluctuates significantly, it is still a challenge to identify the individual lipid species in a serum sample by 1 H-NMR spectroscopy without any lipid-enriching step. A lipidomics research based on chromatography coupled with time of fight mass spectrometer (LC-MS) technique would be necessary and is in our subsequent plan to profile more detailed information of disturbance on lipid metabolism by MIRI and its therapeutic efficacy by WXKL.

Materials and Methods
Reagents and drugs. Potassium phosphate dibasic trihydrate (K 2 HPO 4 ·3H 2 O), sodium phosphate monobasic dehydrate (NaH 2 PO 4 ·2H 2 O), disodium fumarate and deuterium oxide (D 2 O, 99.9% in D) were purchased from Sigma-Aldrich (St. Louis., MO, USA). Sodium azide (NaN 3 ) in analytical grade was obtained from Tianjin Nankai Share Compounds Co., Ltd. (Tianjin, China). Chloral hydrate was purchased from Tianjin Kermel Chemical Reagent Co. Ltd. (Tianjin, China) and freshly prepared to 5% solution with saline before experiment. WXKL was obtained from Shanxi Buchang Pharmaceutical Co., Ltd (Shanxi, China) and dissolved in saline to a concentration of 0.9 g/mL for experiments. Verapamil was purchased from Tianjin Centralpharm Co., Ltd. Myocardial I/R model and drug administration. Rats were randomly divided into four groups, including Sham, MIRI, WXKL and Ver groups. Seven days before I/R surgery, the rats in treatment groups were administered with WXKL via gavages in saline at a dose of 9 g/kg and Ver via peritoneal injection at a dose of 20 mg/ kg once daily for 7 days. The animals in Sham and MIRI groups received saline at 10 ml/kg at the same time. For MIRI model preparation, rats were subjected to ligation on the proximal left anterior descending coronary artery for 30 min and then released according to the previous studies 40 . Rats were anesthetized with 5% chloral hydrate (300 mg/Kg) by peritoneal injection. Skin preparation was completed and placed in a supine position. After left thoracotomy was performed to expose the heart, the proximal left anterior descending coronary artery (LADCA) was ligated with a 5/0 silk. The suture silk was released after 30 min, allowing reperfusion to occur. Then the thorax was closed, and as soon as spontaneous respiration was sufficient, the rats were released and allowed to recover on an electric blanket. The animals in Sham group underwent the same procedure but without ligation of suture silk.
Echocardiographic assessment on left ventricular function. The left ventricle function was evaluated 2 h after I/R, using a Vevo 2100 Ultra-high resolution small animal ultrasound imaging system in real time (Visual Sonics Vevo 2100, Canada) with a MS-250 ultrasound scanning transduce. Two-dimensional cine loops and guided M-mode frames were recorded from the parasternal long axis by using 13-24 MHz variable frequency ultrasound scanning transducer. Left ventricular ejection fraction (LVEF), left ventricle fractional shortening (LVFS) and other indicators were measured to determine the left ventricular systolic function. All data were analyzed off-line at the end of the study with software resident on the ultrasound system. Hemodynamic evaluation of cardiac function. Two hours after reperfusion, rats were anesthetized with 5% chloral hydrate (peritoneal injection) and the left ventricle was cannulated through right carotid artery, which was connected to a bio-function experiment system MP100-CE (BIOPAC systems Inc., Santa Barbara, California, USA). Briefly, the right common carotid artery was dissected and separated from the connective tissue. A catheter was inserted into the left ventricle through the carotid artery. HR, LVEDP, LVSP, +dp/dt max , and −dp/dt max were measured at baseline.
Adequacy of anesthesia was controlled by monitoring corneal reflex and the lack of response to toe-pinching. Hearts were excised from rats for further investigation after terminal anesthesia by i.p. injection of 5% chloral hydrate at dose 300 mg/kg and heparin (100U). Euthanasia was performed by excessive inhalation of isoflurane. Death was monitored by the cardiac activity and respiration.
Sample preparation and NMR measurements. After fasting for 12 h, blood samples were collected by abdominal aortic method. Serum were obtained by the centrifugation of blood at 4 °C, and all the samples were snap frozen in liquid nitrogen and stored at −80 °C until required for analyses. Serum samples were thawed at room temperature and centrifuged at 14 000 rpm for 10 min. A 250-μl supernatant was transferred into 5 mm NMR tubes (Norell, USA) and blended with the same volume of 0.2 M phosphate buffer in D 2 O (pD 7.4) containing 0.1% of NaN 3 .
All NMR spectra were acquired at 298 K on a 600.25 MHz Bruker AVIII HD spectrometer (Bruker BioSpin, Germany) equipped with a 5 mm BBO H&F cryogenic probe. Automatic shimming and adjusting of 90° pulse length were performed for each sample. Standard Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was used to suppress the residual water signal and also filter the broad signals from the molecules with short T 2 relaxation times 41 . A total of 64 scans were collected into 32 K data points over a spectral width of 12019.2 Hz with a relaxation delay of 4 s and an acquisition time of 3.07 s. An exponential line-broadening of 0.3 Hz was applied to the free induction decay prior to Fourier transformation. Additional 1 H, 1 H-correlation spectroscopy (COSY) and 1 H, 13 C-heteronuclear single quantum correlation (HSQC) spectra were recorded on the selected samples for the purpose of resonance assignment.