Taurine detected using high-resolution magic angle spinning 1H nuclear magnetic resonance: A potential indicator of early myocardial infarction

Abstract

Magnetic resonance spectroscopy (MRS) is a unique non‑invasive method for detecting cardiac metabolic changes. However, MRS in cardiac diagnosis is limited due to insensitivity and low efficiency. Taurine (Tau) is the most abundant free amino acid in the myocardium. We hypothesized that Tau levels may indicate myocardial ischemia and early infarction. Sprague‑Dawley rats were divided into seven groups according to different time points during the course of myocardial ischemia, which was induced by left anterior descending coronary artery ligation. Infarcted myocardial tissue was obtained for high‑resolution magic angle spinning 1H nuclear magnetic resonance (NMR) analysis. Results were validated via high‑performance liquid chromatography. The Tau levels in the ischemic myocardial tissue were reduced significantly within 5 min compared with those in the control group (relative ratio from 20.27±6.48 to 8.81±0.04, P Introduction Cardiovascular diseases (CVD) are a significant cause of mortality worldwide and the main cause of CVD is acute myocardial infarction (AMI). In the United States, acute coronary syndrome (ACS) affects 1.3 million individuals (1). Clinically, early detection is essential for the treatment and prognosis of myocardial infarction. At present, the serum levels of cardiac troponins cTnI and cTnT, are used to diagnose early myocardial infarction (2). In addition, the uptake of radioactive tracers, such as exogenous glucose and acetate, is applied clinically to predict cardiac functional improvement (3). Ischemia and myocardial infarction are different stages in the progression of heart failure. Identifying the early changes of various metabolic parameters in myocardial ischemia is of clinical importance (4). The myocardium benefits from reperfusion-based therapeutic approaches if ischemia is detected in the initial period. Therefore, detecting early metabolic parameter changes in myocardial ischemia may have diagnostic value (5). Magnetic resonance spectroscopy (MRS) is a unique non-invasive method for detecting cardiac metabolic changes, which usually occur significantly earlier than irreversible organic lesions in myocardial infarction (6). This method detects biochemical processes and evaluates metabolism without blood sampling or the use of radionuclides (5). To date, cardiovascular magnetic resonance studies using 1H MRS have focused on its sensitivity and specificity in the study of myocardial ischemia (7,8). 31P-MRS is commonly used to study myocardial energy metabolism (9,10). In previous studies, 31P-MRS has been employed to measure indicators, including phosphocreatine (PCr)/ATP and PCr/inorganic phosphate (Pi) ratios, in humans to guide heart transplantation and myocardial infarction non-invasively (6,11,12). It is well recognized that protons have the highest magnetic moment of all biologically relevant nuclei, with a high concentration in organic molecules. These features make it possible to achieve enhanced spatial resolution by 1H MRS. Compared with 31P MRS, 1H MRS provides higher sensitivity and detects more metabolites, such as unphosphorylated creatine (3,13). 1H nuclear magnetic resonance (NMR) has been used to evaluate heart tissue ex vivo to produce a high-resolution metabolic profile in myocardial infarction animal models. However, cardiac motion, respiratory motion and epicardial fat make it difficult to apply 1H NMR clinically (5). Metabolomics is a rapidly evolving field that aims to identify and quantify the concentration changes of all metabolites in a model system. This approach involves large metabolite datasets and high-throughput techniques, including NMR spectroscopy or mass spectroscopy. High-resolution magic angle spinning (HRMAS) 1H NMR spectroscopy has been used to investigate cardiac metabolites in rodent models (14–17). Multivariate pattern recognition analysis coupled with the use of HRMAS was able to identify metabolic biomarkers of disease in intact tissue. The metabolic information obtained from HRMAS spectra may be transferred to the clinical environment (18,19). Taurine (Tau), a sulfur-containing β-amino acid, is the most abundant free amino acid in the myocardium (∼60%) (4). The Tau content is high in mammalian hearts, ranging between 5 and 40 μmol/g wet weight (20). It has been demonstrated that Tau has a number of functions, including the regulation of intracellular calcium balance, antioxidant and anti-inflammatory actions, immune regulation and cardiovascular protection (4,20–22). Cardiac muscle lacks the ability to synthesize Tau (23). This suggests that Tau originates from a transport process. It has been recognized that myocardial Tau synthesis is limited while the majority of the Tau in cardiac tissue is accumulated by uptake from the blood (20). As such, myocardial Tau levels may change when ischemia occurs. The Tau transporter, located on the cell membrane, is important in Tau metabolism in the myocardium. In Tau transporter knockout mice, the level of Tau was decreased by 98% in the myocardium, which indicates that the Tau uptake process is solely Tau transporter dependent without compensation from other transport systems (24,25). We hypothesize that changes in the Tau level in myocardial tissue may be a potential indicator of myocardial ischemia and early infarction. In the present study, a myocardial ischemia model was created in rats. Metabolic markers, including Tau, creatine (Cre), choline (Cho) and lactate (Lac), were analyzed at various time points ex vivo using HRMAS 1H NMR. The results were further confirmed by high performance liquid chromatography (HPLC). Materials and methods Animal model and cell culture The animals were provided by the Animal Center of Fudan University (Shanghai, China). All procedures were performed with approval from the Animal Care and Use Committee of Shanghai, China. Adult Sprague-Dawley (SD) rats (male, body weight 200–250 g) were anesthetized with pentobarbital sodium (30 mg/kg I.P.; Sigma, St. Louis, MO, USA; Lot No. P3761). The left anterior descending coronary artery (LAD) was ligated with a 6-0 suture (Jinhuan Medical, Shanghai, China; Lot No. 20F101) as previously reported (26). Sham-operated animals underwent the same procedure but the LAD was left untied. The animals were divided into seven groups according to the time points following ligation: 0 min (control, sham-operated), 5 min, 20 min, 30 min, 45 min, 1 h and 6 h. The heart was then excised. The infarction zone was isolated and then frozen in liquid nitrogen for further analysis. For three rats from the 6 h group, the rat LAD ligation model was verified by 1% triphenyltetrazolium chloride (TTC) and Evans Blue double staining, as previously described (27). The H9c2 rat cardiomyoblast cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Beijing, China). For the HPLC and apoptosis assay, the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Waltham, MA, USA) supplemented with 10% FBS, 100 U/ml penicillin G and 100 mg/ml streptomycin. The cells were cultured in standardized cell culture incubator conditions at 37°C in a humidified atmosphere containing 5% CO2. HRMAS 1H NMR For the HRMAS 1H NMR analysis, the tissue samples (from the control and myocardial ischemia groups, including the 5 min, 20 min, 30 min, 45 min, 1 h and 6 h groups, n≥3), weighing 25±2 mg each, were placed into a 25 μl zirconium oxide rotor with drops of D2O (deuterium lock reference). NMR tests were performed on a Bruker DRX-500 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometer (1H frequency at 500.13 MHz) at 300.0 K, with a rotor spin rate of 5 kHz. Carr Purcell Meiboom Gill (CPMG) pulse sequences were used with solvent presaturation during the relaxation delay of 2 sec. The NMR spectra were acquired with 256 scans collected into 64,000 data points with a spectral width of 15 kHz. The CPMG pulse sequence was applied to suppress signals from the molecules with short T2 values, such as macromolecules and lipids, using a total echo time (TE) of 320 msec. The stability of tissue samples was evaluated by repeating a one-dimensional NMR experiment following overall acquisition. No biochemical degradation was observed in any of the tissue samples. Spectral assignments were further confirmed by two-dimensional 1H-1H total correlation spectroscopy (TOCSY)and 1H-1H correlation spectroscopy (COSY; data not shown) with values obtained from the literature (14,28). Principal component analysis (PCA) Spectral data were phased and baseline-corrected using XWINNMR (Bruker Biospin GmbH). All free induction decays (FIDs) were multiplied by an exponential function equivalent to a 0.3-Hz line-broadening factor prior to Fourier transformation. Each HRMAS 1H NMR spectrum was segmented into 236 regions of equal width (0.04 ppm) over the region δ0.00–10.00 and the signal intensity in each region was integrated by AMIX (version 3.6, Bruker Biospin GmbH). The region δ4.40–5.00 was removed to eliminate the baseline effects of imperfect water saturation. Prior to PCA, each integral region was normalized by dividing by the sum of all integral regions for each spectrum (29,30). PCA was used to calculate a new, smaller set of orthogonal variables from linear combinations of the intensity variables while retaining the maximum variability present within the data. These new variables were the derived principal components and the distribution of their values (scores) permitted the simple visualization of separation or clustering between samples. The weightings (loadings) applied to each integral region in calculating the principal components allowed for the identification of those spectral regions having the greatest effects on separation and clustering and, hence, the deduction of the characteristic metabolites of myocardial ischemia. HPLC The ischemic myocardial tissue (250 mg) was homogenized by ultrasound (BILON96-II; Bilon Instruments, Shanghai, China) and mixed with 18% sulfosalicylic acid (SCRC, 250 μl/100 mg). The mixture was centrifuged at 13,000 rpm for 5 min. The supernatant was then taken and filtered through a 0.22 μm membrane. O-phthalaldehyde (OPA) precolumn derivatization was performed as previously described (31). After OPA derivatization, the sample extract was immediately detected by HPLC (Agilent 1100; Agilent, Santa Clara, CA, USA) with the parameters as follows: column, ZORBAX Eclipse XDB-C18 4.6x150 mm, 5 μm (Agilent); mobile phase A, methanol:acetonitrile:H2O = 45:45:10 (v/v/v); mobile phase B1, methanol (0.05 mol/l):sodium acetate buffer (pH 5.3):tetrahydrofuran = 42:57:1; mobile phase B2, 40 mM phosphate buffer (Na2HPO4, pH 7.8). Fluorescence detection was performed at 450 nm. L-norvaline (Agilent, Lot No.1103756) was added as the internal standard. Hypoxia treatment and apoptosis assay A cardiac myoblast cell line hypoxia model was established as described previously (32). Briefly, after washing with PBS, the cells were placed in serum- and glucose-free DMEM and incubated in a sealed, hypoxic anaerobic rectangular jar fitted with a catalyst (BioMérieux, Marcy l’Etoile, France) to scavenge free oxygen. For the Tau treated group, Tau was added to these cultures (final concentration 40 nM) and allowed to incubate for 3 h before hypoxia treatment was commenced. The myocardial cells were digested and collected by centrifuging. The myocardial cell single cell suspension was stained with an apoptosis assay kit (Annexin V-FITC and propidium iodide, KeyGene Biotech, Wageningen, the Netherlands) at room temperature for 15 min. The sample was then evaluated by flow cytometry (BD FACS Calibur) and analyzed using CellQuest (BD, version 5.1). Statistical analysis Statistical analysis was performed using the SPSS statistical program (version 11.0, SPSS Inc., Chicago, IL, USA). All values were expressed as the mean ± standard deviation (SD). Differences between groups were evaluated using a one-factor ANOVA test and P Figure 2. Taurine decreased most notably of the potential markers ex vivo. 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