Protein acetylation in skeletal muscle mitochondria is involved in impaired fatty acid oxidation and exercise intolerance in heart failure

Abstract Background Exercise intolerance is a common clinical feature and is linked to poor prognosis in patients with heart failure (HF). Skeletal muscle dysfunction, including impaired energy metabolism in the skeletal muscle, is suspected to play a central role in this intolerance, but the underlying mechanisms remain elusive. Lysine acetylation, a recently identified post‐translational modification, has emerged as a major contributor to the derangement of mitochondrial metabolism. We thus investigated whether mitochondrial protein acetylation is associated with impaired skeletal muscle metabolism and lowered exercise capacity in both basic and clinical settings of HF. Methods We first conducted a global metabolomic analysis to determine whether plasma acetyl‐lysine is a determinant factor for peak oxygen uptake (peak VO2) in HF patients. We then created a murine model of HF (n = 11) or sham‐operated (n = 11) mice with or without limited exercise capacity by ligating a coronary artery, and we tested the gastrocnemius tissues by using mass spectrometry‐based acetylomics. A causative relationship between acetylation and the activity of a metabolic enzyme was confirmed in in vitro studies. Results The metabolomic analysis verified that acetyl‐lysine was the most relevant metabolite that was negatively correlated with peak VO2 (r = −0.81, P < 0.01). At 4 weeks post‐myocardial infarction HF, a treadmill test showed lowered work (distance × body weight) and peak VO2 in the HF mice compared with the sham‐operated mice (11 ± 1 vs. 23 ± 1 J, P < 0.01; 143 ± 5 vs. 159 ± 3 mL/kg/min, P = 0.01; respectively). As noted, the protein acetylation of gastrocnemius mitochondria was 48% greater in the HF mice than the sham‐operated mice (P = 0.047). Acetylproteomics identified the mitochondrial enzymes involved in fatty acid β‐oxidation (FAO), the tricarboxylic acid cycle, and the electron transport chain as targets of acetylation. In parallel, the FAO enzyme (β‐hydroxyacyl CoA dehydrogenase) activity and fatty acid‐driven mitochondrial respiration were reduced in the HF mice. This alteration was associated with a decreased expression of mitochondrial deacetylase, Sirtuin 3, because silencing of Sirtuin 3 in cultured skeletal muscle cells resulted in increased mitochondrial acetylation and reduced β‐hydroxyacyl CoA dehydrogenase activity. Conclusions Enhanced mitochondrial protein acetylation is associated with impaired FAO in skeletal muscle and reduced exercise capacity in HF. Our results indicate that lysine acetylation is a crucial mechanism underlying deranged skeletal muscle metabolism, suggesting that its modulation is a potential approach for exercise intolerance in HF.

Introduction
Exercise intolerance, defined as dyspnea and fatigue on exertion, is a clinical hallmark of patients with heart failure (HF). As the objective assessment of exercise intolerance, a patient's peak oxygen uptake (peak VO 2 ) is determined by cardiopulmonary exercise testing (CPX) and is well known as an independent predictor of prognosis in HF patients. 1 Because exercise tolerance is not directly correlated with cardiac function and hemodynamics, the role of peripheral tissues, especially skeletal muscles, has been a focus of exercise-tolerance research for several decades. 2,3 The intrinsic skeletal muscle abnormalities in HF include factors such as mitochondrial dysfunction, 4 fibre-type transition, 5 and muscle atrophy, 6 but impaired energy metabolism 7 is considered the primary mechanism of the easy fatigability and exercise intolerance in HF settings. 8 Indeed, our previous studies using magnetic resonance spectroscopy demonstrated that an increased accumulation of intramyocellular lipid is associated with exercise intolerance. 9 This is in line with early findings of the reduced activity of diverse mitochondrial oxidative enzymes in biopsied skeletal muscle from HF patients. 10 However, the exact mechanisms underlying these enzymatic alterations remain to be clarified.
Lysine acetylation, a reversible post-translational modification, has gained much attention as a novel regulatory mechanism of mitochondrial energy metabolism. 11,12 The target proteins of acetylation include fatty acid β-oxidation enzymes, glycolytic enzymes, the tricarboxylic acid (TCA) cycle, and the electron transport chain, resulting in the inhibition or activation of their enzyme activities. 13 Of these, the acetylation of fatty acid β-oxidation was shown to be a contributing factor to the cardiac metabolic derangements in diabetes and HF. [14][15][16][17] Sirtuin 3 (SIRT3), a nicotinamide adenine dinucleotide (NAD + )-dependent deacetylase, is a major regulator of mitochondrial acetylation, and has been shown to decline in the failing heart. [18][19][20] Although there are controversies regarding the effect of lysine acetylation on myocardial fatty acid oxidation, 14,16 several lines of evidence indicate that an increased acetylation of fatty acid β-oxidation enzymes is linked to energy metabolic disturbances in the pathogenesis of exercise intolerance associated with HF. [21][22][23] In addition, hyperacetylation due to decreased SIRT3 has been reported to facilitate skeletal muscle insulin resistance in diabetic animals. 24, 25 We thus hypothesized that mitochondrial protein acetylation could contribute to impaired energy metabolism in the skeletal muscle that is primarily or secondarily linked to the development of exercise intolerance in HF.
Using a reverse translational approach, we first identified the circulating metabolites associated with peak VO 2 in HF patients by conducting a comprehensive metabolomic analysis. We then created a murine model of HF after myocardial infarction (MI) with limited exercise capacity and skeletal muscle abnormalities, in order to determine the role of acetylation in mitochondrial fatty acid oxidation and exercise capacity.

Human studies
Ten patients with stable HF (57 ± 10 years old, New York Heart Association [NYHA] functional class II or III) and five age-matched healthy controls (56 ± 3 years old) without underlying heart diseases were studied. HF was diagnosed on the basis of the Framingham criteria. All participants underwent CPX on an upright electromechanical bicycle ergometer (Aerobike 75XLII; CombiWellness, Tokyo) using a ramp protocol described in our previous studies. 26,27 Briefly, after 3 min of unloaded cycling, the exercise load was increased in 10 to 15 W increments every 1 min in HF patients and 25 W increments per min in the control subjects to symptom-limited maximal work. The patients stopped the exercise when they had severe leg fatigue and/or dyspnea. Although it is possible that a different protocol could have affected the value of peak exercise capacity, this protocol was necessary for the symptomatic HF patients to achieve a fatigue-limited exercise duration of 8-12 min based on the American Heart Association recommendation. 28 Oxygen uptake was measured at rest and throughout the exercise period using a 280E Aero-monitor (AeromonitorAE-300S; Minato Medical Science, Osaka, Japan). Peak VO 2 was defined as the maximal VO 2 attained during exercise, and the anaerobic threshold was determined by the V-slope method. 29 We measured the participants' minute ventilation (VE) and carbon dioxide production (VCO 2 ) responses throughout the exercise to calculate the VE/VCO 2 slope via least squares linear regression (y = mx + b; m = slope). In echocardiographic measurements, each participant's left ventricular (LV) end-diastolic dimension and LV end-systolic dimension were obtained from the parasternal long-axis view, and the LV ejection fraction was calculated based on a modification of Simpson's method.
Within 3 months after each participant's CPX was performed, blood samples were obtained from the participants after an overnight fast and were stored at À80°C until analysis. The interval between the blood sampling and the CPX was necessary to minimize the possibility that the metabolomic profile was a collective snapshot of all of the metabolic perturbations and therefore influenced by various confounding factors such as other disease states, acute illnesses, or medication use.
A 50 μL sample of plasma was mixed with 450 μL of methanol containing internal standards (20 μM of methionine sulfone and D-camphor-10-sulfonic acid), and then 500 μL of chloroform was added. The mixture was centrifuged at 2,300 g for 5 min at 4°C. Subsequently, 400 μL of the upper aqueous layer was centrifugally filtered through a 5-kDacutoff ultra-centrifugal filter unit (Ultrafree®-MC-PLHCC-HMT; MilliporeSigma, Burlington, MA) at 9,100 g for 2 h at 4°C. The filtrate was dried and re-suspended in 25 μL of Milli-Q water for a capillary electrophoresis-mass spectrometry (CE-MS) analysis.
Metabolomic measurements were carried out using an Agilent CE capillary electrophoresis system equipped with an Agilent 6210 Time-of-Flight (TOF) MS, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-electrospray ionization-MS sprayer kit (Agilent Technologies, Waldbronn, Germany) as described previously. 30 For the CE-TOFMS system control and data analysis, Agilent G2201AA ChemStation software ver. B.03.01 for CE (Agilent Technologies) was used. The CE-TOFMS datasets were processed using the automatic integration software MasterHands (Keio University, Tsuruoka, Japan) in order to obtain peak information including the m/z, the migration time for the CE-TOFMS measurement, and the peak area. 31 Signal peaks corresponding to isotopomers, adduct ions, and other product ions of known metabolites were excluded, and the remaining peaks were annotated with putative metabolites from the database of the Human Metabolome Technologies according to their migration time and m/z values determined by CE-TOFMS. This study was performed under protocols approved by the Medical Ethics Committee of Hokkaido University Hospital and in accord with the ethical principles in the Declaration of Helsinki (2013). Informed consent was obtained from all participants.

Experimental animals
Male C57BL6J mice (10-12 weeks old, body weight [BW] 23-25 g; CLEA Japan, Tokyo) were bred in a pathogen-free environment and housed in an animal room under controlled conditions on a 12 h light/dark cycle maintained at 23-25°C. Normal chow containing 4.2% fat and 54.6% carbohydrate (CE-2; CLEA Japan, Tokyo) and water were provided ad libitum. The post-MI HF model was established by ligating the left coronary artery as described previously. 32,33 The permanent coronary occlusion model is recommended as a model of acquired dilated cardiomyopathy and HF, which usually occur 4-8 weeks post-MI, according to a scientific statement from the American Heart Association. 34 A sham operation without ligation of the coronary artery was also performed; the experiments were performed 4 weeks after the operation in both the sham (n = 11) and HF (n = 11) groups. For each surgical procedure, the mice were anaesthetised with an intraperitoneal injection of a mixture of 0.3 mg/kg of medetomidine (Dorbene® Kyoritsuseiyaku, Tokyo), 4.0 mg/kg of midazolam (Dormicum®, Astellas Pharma, Tokyo), and 5.0 mg/kg of butorphanol (Vetorphale®, Meiji Seika Kaisha, Tokyo), and the adequacy of the anaesthesia was monitored based on the disappearance of the pedal withdrawal reflex.
All procedures and animal care were approved by our institutional animal research committee and conformed to the animal care guidelines for the Care and Use of Laboratory Animals at the Hokkaido University Graduate School of Medicine. The procedures and care were also in accord with the relevant national and international guidelines and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

Echocardiography, histological analysis, treadmill test, and physical activity measurement
Standard echocardiographic short-axis and long-axis views were obtained at the levels of the papillary muscles under light anaesthesia using tribromoethanol/amylene hydrate (avertin; 2.5% wt/vol, 8 μL/g BW, i.p.). We measured the LV endo-diastolic diameter, LV endo-systolic diameter, wall thickness, and LV fractional shortening from M-mode frames at a paper speed of 50 mm/s. For histological analysis, LV tissues were fixed in 6% formaldehyde, cut into three transverse sections-the apex, middle ring, and base-and stained with Masson's trichrome. We subjected the mice to a treadmill test with a gas analysis for the measurement of indexes defining whole body exercise capacity, as described previously. 35 In short, each mouse was forced to run on a motor-driven treadmill enclosed by a metabolic chamber with constant air flow (Oxymax 2; Columbus Instruments, Columbus, OH, USA) in which the O 2 and CO 2 gas fractions were monitored.
After basal measurements and following a 10 min warmup at 6 m/min at 0°inclination, the angle was fixed at 10°a nd the speed was incrementally increased by 2 m/min until the mouse reached exhaustion. Exhaustion was defined as spending more than 10 s on the shocker plate without attempting to re-engage the treadmill. Whole-body VO 2 and VCO 2 were automatically acquired every 10 s by taking the difference between the inlet and output gas flows. The respiratory exchange ratio was calculated as the ratio of VCO 2 to VO 2 . Work was defined as the product of the vertical running distance to exhaustion and BW.
Spontaneous physical activity was measured by an animal movement analysis system (ACTIMO System; Shintechno, Fukuoka, Japan) as described previously. 36

Study protocol
The study protocol is illustrated in Figure S1. Briefly, 4 weeks after the MI surgery, the spontaneous physical activity of the mice was measured and then the treadmill test was conducted. Within 3 days after the measurement, echocardiography was performed. The mice were then euthanized by cervical dislocation under deep anaesthesia using additional avertin (2.5% wt/vol, total 10 μL/g BW, i.p.). The blood, heart, lungs, and hindlimb skeletal muscles including the quadriceps, gastrocnemius, and soleus were immediately collected for further analysis. To avoid direct effects of the treadmill test on the muscle acetylome analysis, we used a ≥2 day interval between the treadmill test and the tissue sampling.

Western blotting
Forty milligrams of frozen gastrocnemius was homogenized with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland) and deacetylase inhibitors (10 mM nicotinamide, 10 μM trichostatin A, and 10 mM sodium butylate). Mitochondrial fractions were also collected using a mitochondrial isolation kit (Thermo Scientific). Twenty micrograms of denatured proteins was subjected to 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a polyvinylidene difluoride membrane.

Mass spectrometry-based acetylome analysis
For the acetylation analysis, gastrocnemius tissues were lysed with IP buffer (50 mM Tris HCl, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton-X) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland) and the earlierdescribed deacetylase inhibitors. A total of 500 μg of lysates was precleared with 40 μL of protein A/G-agarose beads. The lysates were then incubated with acetyl-lysine antibodies (3 μg/100 μg lysate; Cell Signaling Technology) overnight at 4°C, and 50 μL of protein A-agarose beads was added to each sample and incubated on a rotator for 6 h at 4°C as described previously. 17,37 After 6 h, the samples were washed three times and centrifuged at 16,000 g for 5 min. The bound acetylated proteins were boiled at 100°C for 15 min in 60 μL of 1.0035× LDS buffer.
Beads were pelleted and 50% of the eluates were processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 10% Bis-Tris NuPAGE gel (Invitrogen, Carlsbad, CA, USA) with the MES buffer system. The mobility region was excised into 10 equal-sized segments and subjected to in-gel trypsin digestion. The enriched lysineacetylated peptides were then analysed by nano-liquid chromatography-tandem mass spectrometry with a NanoAcquity HPLC system (Waters, Milford, MA, USA) interfaced with a ThermoFisher Q Exactive HF hybrid mass spectrometer (Thermo Scientific, Tokyo, Japan).
Mascot database search software (ver. 2.4, Matrix Science, London) was used to search for and identify lysine-acetylated peptides. Mascot DAT files were parsed into Scaffold (Proteome software) for validation and filtering and to create a non-redundant list per sample. Variable modifications included the acetylation of the ε-amino group of lysine, oxidation of methionine to methionine sulfoxide, N-terminal pyroglutamate, and the deamidation of asparagine residues; a search was also performed for carbamidomethylation of cysteine as a fixed modification. The peptide mass tolerance was set to 10 ppm for precursors and 0.02 Da for fragment ions.
The search results were filtered using a 1% protein and peptide false discovery rate with a requirement of at least two unique peptides per protein. To approximate the relative abundance of proteins within a given sample, we calculated the normalized spectral abundance factor using an in-house program as described previously. 38 A binary difference in proteins with a t-test P-value < 0.05 or with a 1.5-fold or more change in the average normalized spectral abundance factor values was defined as significant.

Measurement of nicotinamide adenine dinucleotide metabolites
Approximately 50 mg of frozen gastrocnemius tissues was added to methanol (500 μL) containing internal standards (H3304-1002; Human Metabolome Technologies, Tsuruoka, Japan), and the mixture was homogenized by a Polytron homogenizer. The lysate was further processed for the CE-MS analysis described earlier. Nicotinamide adenine dinucleotide (NAD + ) and its reduced form (NADH) were quantified using the CE-electrospray ionization-MS method. 39
After the addition of permeabilized fibres (approx. 1.5-3.0 mg of gastrocnemius muscle) to the chamber in the respirometer filled with 2 mL of MiR05 and the stabilization of baseline respiratory rates, the following respiratory substrates were added in the following order: (i) malate 2 mM, (ii) octanoyl-carnitine 0.15 mM, (iii) ADP 5 mM in each step, and (iv) cytochrome c 10 μM. Mitochondrial coupling states are distinguished as leak-state respiration (without ADP) and oxidative phosphorylation (saturating ADP). We tested the integrity of the outer mitochondrial membrane by adding cytochrome c, and the data were eliminated when the increase in the oxygen consumption rate was 10% as the sign of a damaged outer mitochondrial membrane. The respiratory rates (i.e. O 2 consumption rates) are expressed as the O 2 flux normalized to the muscle weight (pmoL/s/mg wet weight of muscle tissue).

Activity assay for citrate synthase and β-hydroxyacyl CoA dehydrogenase
For citrate synthase activity measurements, frozen quadriceps were homogenized in buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10% glycerol (wt/vol), 0.02% Brij-35 (wt/vol), 1 mM dithiothreitol with protease, and phosphatase inhibitor cocktails (Sigma-Aldrich). Thirty microlitre of homogenate (1 mg/mL) was used for the analysis, and the reaction was initiated by 0.2 mM acetyl-CoA and 0.5 mM oxaloacetate in 100 mM Tris buffer and 0.1 mM DTNB. Absorbance at 412 nm wavelength was followed for 3 min with a spectrophotometer.
β-hydroxyacyl CoA dehydrogenase (β-HAD) activity was assayed in whole lysates prepared from frozen tissues or cultured cells as described previously. 17 First, 40 μL of homogenate (1 mg/mL) was added to a cuvette, which was then was brought to a final volume of 990 μL with 930 μL of 50 mM imidazole (pH 7.4) and 10 μL of 1.5 mM NADH. The reaction was initiated by the addition of 10 μL of 2 mM acetoacetyl-CoA. Absorbance at 340 nm wavelength was followed for 5 min with a spectrophotometer.

Cell culture and transfection
To further examine the regulatory role of SIRT3 in fatty acid β-oxidative activity in the skeletal muscle, we conducted cell culture studies using mouse C2C12 myoblast cell lines purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco's Modified Eagle's Medium (4.5 g glucose/L; Sigma, St. Louis, MO, USA) containing 10% foetal bovine serum (Cell Culture Bioscience), 100 IU/mL penicillin, and 100 IU/mL streptomycin in a humidified incubator (Sanyo, Osaka, Japan) at 37°C with 5% CO 2 in air.
For transfection, C2C12 myoblasts were plated in a T75 flask at a density of 1.2 × 10 6 cells and then transfected 24 h later with a non-targeting pool of small interfering RNA against SIRT3 (Dharmacon, Brébières, France) using DharmaFECT reagent (Dharmacon). At 48 h after transfection, the cells were harvested and lysed with homogenization buffer containing 10 mM HEPES-KOH (pH 7.4), 220 mM mannitol, and 0.07 M sucrose with several deacetylase inhibitors. The mitochondrial fraction was also isolated by the sequential centrifugation method. Whole cell lysates or mitochondrial fractions were then subjected to western blotting and a β-HAD activity analysis.

Statistical analysis
Data are expressed as the mean ± SD or SEM for normally distributed variables, or the median and interquartile range for non-normally distributed variables. The statistical analyses were performed using Student's t-test for comparisons between two groups and a one-way ANOVA followed by Tukey's test for more than two groups with GraphPad Prism 6 software (GraphPad, San Diego, CA, USA). A P-value < 0.05 was considered significant. Correlations were examined by linear regression analysis using the least squares method. Significant acetylation changes were defined as a cutoff of ±1.5-fold change in the mean values of the HF group vs. the sham group. When acetylated protein was detected only in HF mice, the protein was also defined as a significant acetylation target.

The plasma acetyl-lysine levels correlate with exercise capacity in patients with HF
The characteristics of the 10 patients with HF and five controls are summarized in Table 1. The primary aetiology of their HF was dilated cardiomyopathy (n = 4), followed by ischemic heart disease (n = 3) and hypertensive heart disease (n = 3); this distribution reflects the real-world aetiology of HF in Japan. 40 The patients' exercise capacity was reduced as evidenced by the median peak VO 2 of 16.3 (interquartile range, 11.2-18.9) mL/kg/min. Our metabolomic analysis of the human plasma samples identified a total of 233 charged metabolites, in which significant increases were observed in N6-acetyl-lysine, arginine methylation, phospholipid metabolites, and urea cycle metabolites in the HF patients compared with controls.
In contrast, there was no association between the N6-acetyl-lysine level and the LV ejection fraction, log BNP, or thigh circumference in the HF patients, suggesting that acetylation played a specific role in exercise capacity rather than the cardiac function or congestive state, or skeletal muscle mass in HF ( Figure S2). Because N-acetyl-lysine is a metabolite of protein lysine acetylation, this increase may reflect the hyperacetylation status, especially in skeletal muscle, which is a determinant factor of exercise capacity in HF.

Functional impairment in muscle mitochondria in a murine HF model
To examine the derangements in mitochondrial oxidative respiration in the skeletal muscle seen in HF, we used a murine model of post-MI HF. Representative graphs of the mitochondrial respiratory capacity in permeabilized gastrocnemius tissues clearly demonstrated that the levels of complex I-linked and complex I + II-linked oxidative phosphorylation in the presence of octanoyl-L-carnitine, a fatty acid substrate, were lower in the HF mice compared with the sham-operated mice (Figure 2A). Moreover, the β-HAD activity was reduced in the quadriceps from the HF mice compared with the shamoperated mice ( Figure 2B). These results suggest that the acetylation of fatty acid oxidation enzymes inhibits the β-HAD activity, and this inhibition is associated with the impaired mitochondrial oxidation that accounts for the lowered exercise tolerance in HF.

Acetylation profile in skeletal muscle and blood in a murine model of HF
Based on the results of our study of the 10 patients with HF and five healthy controls (Figure 1), we examined the global lysine acetylation in the gastrocnemius tissues from shamoperated and HF mice. As expected, lysine-acetylated protein levels were higher in the mitochondrial fraction of gastrocnemius from the HF mice compared with that of the shamoperated mice, whereas the whole muscle lysates were not significantly different between the two groups ( Figure 3A,B). In conjunction with the increased mitochondrial acetylation, the protein expression of the mitochondrial deacetylase SIRT3 was decreased in the gastrocnemius from the HF mice compared with the sham group ( Figure 3D). As is observed in humans, the acetylated protein levels in the murine blood samples were higher in the HF mice than the sham-operated mice, in parallel with the increased acetylation of muscle mitochondrial proteins in the HF mice ( Figure 3C). Because SIRT3 activity is dependent on NAD + availability, we quantified the NAD + and NAD + /NADH ratio by conducting a CE-MS analysis. Neither the NAD + level nor the NAD + /NADH ratio differed significantly between the HF and sham groups ( Figure 3E). To further clarify the molecular functions of acetylated proteins, we performed a mass spectroscopy-based global analysis followed by enrichment of the acetyl lysine peptide in gastrocnemius tissues in the two groups. This approach enables the detection of low stoichiometry of acetylation and was established in previous studies. 41 A total of 151 proteins were identified as having at least one portion of acetyl-lysine residues, and 95 acetylated proteins were shared between the sham-operated and HF mice ( Figure 3F). Of these, 50 acetylated proteins were classified as mitochondrial enzymes and were all observed in HF muscle, whereas 26 of the proteins were derived from muscles of the sham-operated animals. This indicates that 24 of the mitochondrial acetylated proteins were unique to the HF mice and that mitochondria are important targets of the post-translational modifications in HF (Figure 3F). The hyperacetylated mitochondrial enzymes were linked to lipid metabolism, carbohydrate metabolism, the TCA cycle, branched amino acid (BCAA) metabolism, and the electron transport chain ( Figure 3G). Notably, the major targets of lysine acetylation were fatty acid β-oxidation enzymes, including long chain acyl-CoA dehydrogenase (LCAD), enoyl-CoA hydratase, trifunctional enzyme subunit α (HADHA), and 3-ketoacyl-CoA thiolase ( Figure 3H).
In sharp contrast to the difference in post-translational modification, there was no significant difference in the protein expression of peroxisome proliferator-activated receptor gamma coactivator 1-α, a master transcriptional regulator of mitochondrial biogenesis, between the HF mice and shamoperated mice ( Figure 4A). Consistently, the mitochondrially encoded cytochrome c protein expression and citrate synthase activity, both of which are surrogate markers for mitochondrial content, were comparable between the shamoperated and HF mice ( Figure 4B,C). Moreover, there were no significant differences in the various protein expressions related to fatty acid cellular uptake (CD36), mitochondrial transport (CPT1, CPT2), and fatty acid β-oxidation enzymes (ACADL, EHHAD) in the gastrocnemius from the HF mice compared with the sham-operated mice ( Figure 4D-H). Taken together, these results suggest that post-translational mechanisms are involved in the derangements in fatty acid oxidation during the development of the skeletal muscle dysfunction and exercise intolerance associated with HF. Figure 1 (A,B) Plasma metabolites that correlate with peak VO 2 , AT, or VE/VCO 2 in all subjects (n = 15), i.e. the heart failure patients (HF) (n = 10) plus the normal controls (n = 5) (A), and in only the 10 HF patients (B). The upper value indicates the correlation coefficient between peak VO 2 , AT, or VE/ VCO 2 and each metabolite. The lower value in parentheses indicates the P-value of each correlation. (C) Association between plasma acetyl-lysine levels and NYHA functional class in the controls, the NYHA I or II HF patients, and the NYHA III HF patients. (D-F) Correlation between plasma acetyl-lysine level and peak VO 2 (D), AT (E), and VE/VCO 2 (F) among the HF patients. Data are the mean ± SD. *P < 0.05 vs. controls. ADMA, asymmetric dimethylarginine; AT, anaerobic threshold; HF, heart failure; NYHA, New York Heart Association class; peak VO 2 , peak oxygen uptake; SDMA, symmetric dimethylarginine VE/VCO 2 , minute ventilation vs. carbon dioxide production. Table 2 shows the characteristics of the HF mice at 4 weeks after MI. The heart weight and lung weight/BW were significantly increased in the HF mice compared with the shamoperated mice. A representative echocardiographic image is shown in Figure 5A, indicating that the HF mice had greater LV diameters and lower LV systolic function than the shamoperated mice ( Table 2). The Masson's trichrome staining of ventricular tissues further confirmed the infarct formation ( Figure 5B). The mortality rates up to 4 weeks after the operation was significantly lower in the HF mice compared with the sham group (44% vs. 0%).

Post-infarct HF mice mimic exercise intolerance in humans
In the CPX measurements, the peak VO 2 as well as the work, run distance, and run time were significantly reduced in the HF mice compared with the sham-operated mice at maximal effort with equivalent respiratory exchange ratios (Figure 5C-E; Table 2). In contrast, the spontaneous physical activity was not significantly different between the HF and sham groups ( Figure 5F). The muscle weights including those of the quadriceps, gastrocnemius, and soleus were also comparable between the two groups (Table 2). Collectively, these results indicate that our model of post-MI HF sufficiently mimics the exercise intolerance seen in patients with HF, possibly due to the functional alterations in skeletal muscle.

The effect of SIRT3 knockdown on the acetylation and β-HAD activity in vitro
To further investigate whether the decrease in SIRT3 was closely associated with increased acetylation of skeletal muscle, thereby decreasing the activity of fatty acid β-oxidation Figure 2 (A) Representative graphs of mitochondrial respiratory capacity with fatty acid substrates in sham and HF gastrocnemius muscles. The mitochondrial respiratory capacity at each state with octanoyl-1-carnitine in the gastrocnemius was lowered in the HF compared with the sham group (n = 6 each). (B) The β-HAD enzyme activity in skeletal muscle from sham-operated and HF mice (n = 6 each). Data are the mean ± SEM. *P < 0.05 vs. sham. CI, complex I-linked substrates; CI + II, complex I + II-linked substrates; FAO, fatty acid oxidation; HF, heart failure; LEAK, leak-state respiration (non-ADP stimulated respiration); OXPHOS, oxidative phosphorylation capacity (ADP-stimulated respiration); ROX, residual oxygen consumption; β-HAD, β-hydroxyacyl CoA-dehydrogenase. enzymes, we conducted SIRT3-knockdown experiments in C2C12 mouse myotubes. The transfection of SIRT3 small interfering RNA in C2C12 cells reduced the SIRT3 protein expression by 70% compared with the non-targeting pool ( Figure 6A), in conjunction with a significant increase in the overall protein acetylation in the mitochondrial fraction ( Figure 6B), but not in the whole muscle lysates ( Figure 6A). More importantly, the β-HAD activity was significantly reduced following SIRT3 knockdown ( Figure 6C), supporting the concept that SIRT3 is a key molecule modulating the acetylation of muscle fatty acid β-oxidation enzymes in HF.

Discussion
The present study provides numerous novel observations. First, the mass spectroscopy-based metabolic profiling revealed that the plasma acetyl-lysine level was elevated in HF patients and was negatively correlated with their exercise capacities. Second, acetylation modification was enhanced in skeletal muscle mitochondria from our murine model of HF with lowered exercise capacity, which was attributed to a decreased level of a mitochondrial deacetylase, SIRT3. Third, label-free acetylproteomics identified key metabolic . Total acetylated proteins were normalized to GAPDH in whole lysates or COX IV in the mitochondrial fraction. (C) Total protein acetylation in blood samples normalized to the non-specific band of CBB-stained gel from sham-operated and HF mice (n = 6 each). (D) Protein expression of SIRT3 in gastrocnemius muscle from sham-operated and HF mice (n = 6 each). (E) The abundance of NAD + and NADH, and the ratio of NAD + to NADH in skeletal muscle tissues from sham-operated and HF mice (n = 5-6 each). Data are the mean ± SEM. *P < 0.05 vs. sham. (F) Venn diagram showing overlapping of lysine-acetylated peptides in total and mitochondrial proteins identified in sham and HF muscles (n = 3 each). Numbers in brackets represent the total number of acetylated proteins identified in each group. A total of 151 acetylated proteins were identified, and 95 acetylated proteins overlapped between the sham and HF muscle. Of these, 50 proteins belonged to mitochondria and were all derived from HF muscle, and 26 mitochondrial proteins were shared with the sham-operated mice, indicating that 24 proteins were unique to the HF mice. (G) Localization annotation (gene ontology) was assessed for 48 acetylpeptides that were increased significantly or at least 1.5-fold increase in the HF mice compared with the sham-operated mice. (H) Specific acetylation sites in the pathway map showing fatty acid β-oxidation, TCA cycle, and electron transport chain. The acetylation status is indicated by colour coding: proteins with increased acetylation (HF/sham) are in red; proteins with decreased or unaltered acetylation are in black. ACADL, long-chain specific acyl-CoA dehydrogenase; BCAA, branched-chain amino acid; CBB, Coomassie Brilliant Blue; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HADHA, trifunctional enzyme subunit α; HF, heart failure; NAD + , nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide (reduced form); SIRT3, Sirtuin 3; TCA, tricarboxylic acid. pathways for acetylation, including fatty acid oxidation, the TCA cycle, BCAA metabolism, and electron transport. Specifically, the acetylation of fatty acid β-oxidation enzymes was related to reduced activity and a further reduction in fatty acid-derived mitochondrial oxidative capacity in the skeletal muscle. We also observed that the knockdown of SIRT3 in cultured skeletal muscle cells resulted in enhanced mitochondrial acetylation, which in turn resulted in a reduction in the enzymatic activity of fatty acid oxidation. These findings suggest that enhanced acetylation is linked to the pathogenesis of exercise intolerance in HF, at least in part through impaired fatty acid metabolism in the skeletal muscle.
Prior studies have consistently demonstrated that cardiac function does not necessarily correlate with exercise capacity in HF 42 and that exercise training does reduce mortality in HF patients without any changes in cardiac function. 43 These findings have raised considerable interest in skeletal muscle dysfunction as a major contributor to the pathophysiological features and symptomatology of HF.
The peak VO 2 measured on CPX is a powerful prognostic indicator for HF patients, but a large clinical trial indicated that many HF patients are often unable to achieve maximal efforts due to their multiple comorbidities. 43 As a result, the prognostic value of peak VO 2 is suboptimal. 44,45 As an incremental risk stratification tool for HF, various biomarkers  (H) normalized to the GAPDH or non-specific band of CBB-stained gel in sham and HF gastrocnemius muscles (n = 6-10 each). CS activity in the gastrocnemius muscle from the shamoperated and the HF mice (n = 8 each). Data are the mean ± SEM. *P < 0.05 vs. sham. ACADL, long-chain specific acyl-CoA dehydrogenase; CBB, Coomassie Brilliant Blue; CD36, cluster of differentiation 36; CPT, carnitine palmitoyltransferase; CS, citrate synthase; EHHADH, enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HF, heart failure; MTCO1, mitochondrially encoded cytochrome c oxidase 1; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-α. have been suggested. 46 Notably, recent metabolomic analyses of plasma samples have indicated that the specific patterns of metabolites in HF patients offer substantial value for the prediction of prognosis. [47][48][49] This corroborates the concept that metabolic impairment is pronounced not only in the heart but also in the peripheral organs. 50 Until now, however, few studies have focused on metabolites associated with exercise capacity in HF. This study is the first to show that N6 acetyl-lysine is the most relevant metabolite that correlates with peak VO 2 , and that the increased levels of other metabolites such as asymmetric dimethylarginine, choline derivatives, and urea were also consistent with previous findings. 47,51 N6 acetyl-lysine is an amino acid derivative that may be released from protein lysine acetylation, which may reflect a systemic increase in this post-translational modification in the setting of HF. Although acetylation was originally reported to regulate gene transcription by neutralizing the positive charge of histone proteins, 52 acetylation was more recently recognized as a novel regulator of mitochondrial metabolic pathways. 41 Horton et al. reported that they observed a profound increase in the acetylation of mitochondrial proteins in human cardiac samples as well as murine hearts with pressure overload. 22 Aside from the failing heart, we found that the muscle mitochondria were also a focus of the acetylation modification in HF. Because approx. 95% of acetyl CoA, a primary substrate for protein acetylation, is localized to the mitochondria, 53 it is likely that the mitochondrion is a major targeted compartment for acetylation. It has been reported that increased acetylation of mitochondrial proteins is evident in failing or diabetic hearts. 14,22 In addition, studies of diet-induced diabetic animals have demonstrated that mitochondrial proteins in liver and skeletal muscle were also acetylated. 12,24 Given that HF is characterized by systemic insulin resistance that involves multiple organs, 50 we speculate that in HF, the acetylation of metabolic enzymes may occur in the mitochondria of organs other than skeletal muscle.
On the other hand, although it remains unclarified how much the acetyl lysine levels in human blood samples reflect acetylation alterations in the muscle mitochondrial compartment as was observed in the HF mice, we observed that the acetylated protein levels in the murine blood samples were higher in the HF mice than the sham-operated mice, in parallel with the increased acetylation of muscle mitochondrial proteins in the HF mice. This result suggests that the levels of blood acetylated proteins may reflect, at least to some extent, those in the muscle mitochondria. The summarized data assessed by exercise testing with expired gas analysis indicate the work (D) and peak VO 2 (E) (n = 8 each). Data are the mean ± SEM. *P < 0.05 vs. sham. AW, anterior wall; EDD, end-diastolic diameter; HF, heart failure; LV, left ventricular; peak VO 2 , peak oxygen uptake; PW, posterior wall. Intriguingly, our further functional profiling of acetylation revealed key fuel metabolism and energetic pathways, including fatty acid β-oxidation, the TCA cycle, BCAA metabolism, and the electron transport chain, which are in complete agreement with the patterns reported in the failing heart. 22 Alterations in mitochondrial lysine acetylation are known to be regulated by the balance between SIRT3 deacetylase activity due to NAD + availability and/or a nonenzymatic reaction of mitochondrial acetyl CoA to lysine. 54 In the present study, the reduction in SIRT3 expression rather than a reduction in the NAD + amount or the NAD + /NADH ratio appeared to account for the enhanced mitochondrial acetylation in the skeletal muscle from HF. However, an accurate determination of subcellular NAD + and acetyl CoA concentrations is technically difficult at this time. A more sophisticated assay could elucidate the role of the compartmentalization of those cofactors in pathological settings.
Previous studies have shown that a decrease in SIRT3 expression is responsible for the increased lysine acetylation and promotes a shift in substrate metabolism from fatty acids toward glucose in diabetic hearts 14,55 as well as in hypertrophied hearts. 16,21,56 This substrate alteration is thought to induce insulin resistance and therefore cardiac energetic inefficiency, leading to the development of HF. 57,58 Similar to the findings in diabetic or failing hearts, SIRT3 was also shown to regulate skeletal muscle function via alterations in mitochondrial oxidation and oxidative stress. 25 Collectively, these findings lead us to speculate that the common mechanism of SIRT3-mediated hyperacetylation between heart and skeletal muscle may predispose to energetic derangements due to alterations in substrate metabolism and hence the deterioration of exercise intolerance in HF.
In the present study, the acetylation of key fatty acid βoxidation enzymes was associated with a reduction in their activities and fatty acid-derived mitochondrial oxidation. In line with this finding, early studies using muscle biopsy samples showed that a variety of mitochondrial oxidative enzymes, including β-HAD, are decreased in patients with HF. 4,10 Our previous investigation using magnetic resonance spectroscopy revealed that intramyocellular lipid deposition was increased in HF patients, and its content was closely associated with muscle energy production and whole-body exercise capacity. 9 Similar muscular fat infiltration has also been observed in muscle dysfunction linked to muscle disuse and aging, as well as in patients with iron deficiency and patients with preserved ejection fraction (HFpEF). [59][60][61][62] Alternatively, it has been documented that higher exercise capacity is associated with increased fatty acid oxidation in skeletal muscle during exercise. 63,64 Combined, these observations have suggested that intrinsic deficits in fatty acid oxidative The efficacy of non-targeting pool or SIRT3 small interfering RNA on these protein levels in C2C12 cells (n = 4 each). (B) The SIRT3 knockdown with small interfering RNA increased the total protein acetylation in the mitochondrial fractions, but not in the whole cell lysates of C2C12 cells (n = 3-4 each). (C) The β-HAD activity was reduced in SIRT3-knockdown C2C12 cells (n = 3 each). Data are the mean ± SEM. *P < 0.05 vs. non-targeting pool. β-HAD, β-hydroxyacyl CoA-dehydrogenase; CBB, Coomassie Brilliant Blue; SIRT3, Sirtuin 3. metabolism in skeletal muscle may provoke energetic deprivation in response to increased energy demand during exercise, which may be partially responsible for the reduced exercise capacity in patients with HF. However, we cannot exclude the possibility that low cardiac output is a definitive factor for the exercise capacity in HF. In addition, the causal relationship between the decreased fatty acid β-oxidation activity and lowered exercise capacity observed in the in vivo experiments was not fully clarified in the present study.
The molecular process controlling fatty acid oxidation in the skeletal muscle has yet to be fully elucidated. Indeed, metabolic genes and related proteins were reported to remain unaltered, whereas the accumulation of intermediate metabolites was dramatically increased, in the process leading to decompensated HF. 65 That finding strongly suggests that the post-translational regulation of metabolic remodelling is an early and sensitive molecular signature of impaired energy metabolism in HF. A growing body of evidence has indicated that the acetylation of mitochondrial fatty acid oxidation enzymes can modulate their activities in a tissue-dependent manner. For instance, Hirschey et al. demonstrated that the hyperacetylation of a key fatty acid β-oxidation enzyme, LCAD, results in reduced enzymatic activity and hepatic lipid accumulation, 12 whereas Lopaschuk and his colleagues concluded that high rates of myocardial fatty acid oxidation are attributable to hyperacetylation and the subsequent activation of fatty acid β-oxidation enzymes. 14,17,37 As for the effects of acetylation modification on skeletal muscle proteins, it has been consistently reported that the acetylation of a fatty acid-related pathway negatively regulates lipid metabolism. 63,66 As evidence supporting this, our present findings demonstrated that enhanced acetylation due to SIRT3 knockdown resulted in a reduction of β-HAD activity in skeletal muscle cells. Therefore, at least in skeletal muscle, increased acetylation via a decrement of SIRT3 has inhibitory impacts on fatty acid β-oxidation enzymes, which in turn results in the impairment of mitochondrial oxidative capacity when fatty acids are used as a substrate. As a result, impaired fatty acid oxidation in the skeletal muscle in combination with the aforementioned insulin resistance may further exaggerate the mitochondrial energy perturbations and exercise intolerance seen in HF. Moreover, based on another study's finding that skeletal muscle from patients with mitochondrial disease exhibited enhanced ergoreflex sensitivity, we speculate that sympathetic activation and exercise intolerance due to ergoreflex hypersensitivity may also be involved in this phenomenon. 67

Limitations
Given the observational nature of this study, there are several limitations that should be noted. First, although our data presented in vivo are generally solid, cause-and-effect relationships between acetylation and impaired fatty acid oxidation or lowered exercise capacity remain to be determined. Specifically, the lack of any evidence regarding whether hyperacetylation via SIRT3 knockdown could ultimately affect mitochondrial respiration is a major limitation of our study. In addition, off-target effects of knockdown experiments will limit the experiments' translation into in vivo findings. We also failed to determine the correlation between acetylation and mitochondrial respiration in the skeletal muscle, due to a technical limitation. To gain direct evidence of the role of acetylation in the regulation of muscular fatty acid oxidation, further studies using skeletal muscle-specific SIRT3-deficient mice are necessary. Second, the number of patients in the HF group was twice the number of control subjects, due to the limited number of healthy control subjects. Our results regarding the metabolites identified herein should be verified in a future large-scale study. Third, the upstream mechanism by which the SIRT3 amount is reduced is yet to be elucidated, but it may be linked to altered expression in response to nutrient conditions. Finally, the functional effects of mitochondrial acetylation via SIRT3 other than energy metabolism remain to be determined. In particular, as our post-MI HF model was far from the cachectic state without any significant reduction in muscle weights, the roles of other factors such as Forkhead Box O family members and inflammatory signalling in the muscle dysfunction of HF have remained unexplored. 68,69

Conclusions
We observed that increased acetylation in skeletal muscle mitochondria was associated with reduced fatty acid oxidation and impaired exercise tolerance in HF. Novel approaches targeting the acetylation of skeletal muscle mitochondria would provide important clues for the development of an effective intervention for exercise intolerance in patients with HF.


s2fieldsofstudy: Biology Medicine

text:
Matrix Metalloproteinase-2 and -9 Expression in the Epiligament of the Medial Collateral and Anterior Cruciate Ligament in Human Knees: A Comparative Study

Aim Ninety percent of knee ligament injuries involve the medial collateral ligament (MCL) and the anterior cruciate ligament (ACL) of the knee joint. Matrix metalloproteinases (MMPs) are a large group of calcium- and zinc-dependent endopeptidases responsible for cleaving and rebuilding various connective tissue components. Previous studies showed that MMP-2 and 9 have a significant effect on the healing process of injured ligaments. Therefore, the aim of this study was to evaluate for the first time in literature the expression and localization of MMP-2 and 9 in the epiligament (EL) and the ligament tissue of the MCL and the ACL of the human knee joint in order to assess their role in ligament healing. Materials and methods For the present study, we used histological material from the mid-portion of the MCL and the ACL of 14 knee joints from fresh cadavers. For the purpose of the immunohistochemical analysis, we used primary polyclonal antibodies against MMP-2 and 9. The obtained results were evaluated semi-quantitatively through ImageJ. Results Immunoreactivity for MMP-2 was predominantly positive (2+) in the EL of the MCL and remained mostly negative (0) in the ligament tissue. The expression of MMP-9 was mostly low-positive (1+) in the EL of the MCL and almost entirely negative (0) in the ligament tissue. In the EL of the ACL, the immunohistochemical expression of MMP-2 was predominantly low-positive (1+) and that of the MMP-9 was read as mostly low-positive (1+). Expression of the two enzymes in the ligament tissue was similar to the MCL. Conclusion The present study is the first comparison of the expression of the aforementioned MMPs in the EL tissue of the MCL and the ACL in human knees, which may play a key role in physiological and pathophysiological processes such as tissue healing and repair and basement membrane degradation.

Introduction
The medial collateral ligament (MCL) of the knee joint is one of the most commonly injured ligaments of the knee [1][2]. Ninety percent of knee ligament injuries involve the MCL or the anterior cruciate ligament (ACL) [2][3]. The incidence of injuries of these ligaments has increased in recent years and presents a commonly encountered problem in modern sports medicine [3][4][5]. Moreover, the ACL has a poor healing ability, especially in comparison with the MCL, which can heal relatively well [6][7].
The thin connective tissue layer enveloping the ligament was first described as the 'epiligament' (EL) by Bray et al. [8]. In 2017, Georgiev et al. [4] hypothesised that the EL of the MCL has specific characteristics and suggested that the fibroblasts in particular, together with the abundant blood vessels are essential for its nutrition and healing. These data were in line with the previous studies on the MCL in rat model [9][10][11][12][13][14][15][16][17]. According to Georgiev and Vidinov [13][14][15] and Georgiev et al. [9][10][11][12] the EL of the MCL is a donor of fibroblasts, progenitor cells, and blood vessels during the process of ligament recovery. In contrast, the biological basis for failure of the human ACL to heal after rupture is still unknown.
Matrix metalloproteinases (MMPs), also known as matrixins, are a large group of calcium-and zinc-dependent endopeptidases responsible for cleaving and rebuilding connective tissue components such as collagen, elastin, gelatin, and casein [18][19][20][21]. These enzymes are produced by various connective tissue cells, such as fibroblasts, endothelial cells, osteoblasts, macrophages and others [16]. According to literature data, MMP-2 and 9 are involved in complex processes, such as tissue remodeling and repair, basement membrane degradation, healing of acute MCL and ACL tears, tumour invasion, angiogenesis, etc. [4,[22][23]. Taking into consideration the difference in the healing potential between the MCL and the ACL, we were interested to evaluate the expression of MMPs in the EL of the two ligaments, in order to determine whether there were any variations. Therefore, the aim of this study was to evaluate for the first time in literature the expression and localization of MMP-2 and 9 in the EL and the ligament tissue of the MCL and ACL in the human knee joint in order to assess their role in ligament healing.

Tissue preparation
For the present study, we used 14 knee joints from fresh cadavers from the autopsy material available at the Department of Anatomy, Histology and Embryology at the Medical University of Sofia. Eight specimens were female and six were male with a mean age at death of 64.5 years old. No medical or surgical history for trauma of the knees of the cadavers was available. The study was approved by the Medical Legal Office, the Local Ethics Committee, and the Institutional Review Board (No. 4866).
After skin incision, the underlying subcutaneous tissue was dissected to expose the MCL of the knee joint. The MCL and the external surface of the surrounding EL were precisely dissected, the points of insertion of the ligament were excised, and the mid portion of the MCL was immediately fixed in 10% formalin solution (Merck Catalog No. 1040031000, Merck KGgA, Darmstadt, Germany) for 24 hours and were then dehydrated in increasing concentrations of ethanol (70%, 80%, 95%, 100%) (Merck Catalog No. 1009835000). After opening the capsule of the knee joint, the mid portion of the ACL was processed and fixed in the previously described way. Next, ethanol was removed using cedar oil until the samples became translucent. The samples were then rinsed in xylene (Merck Catalog No. 1082984000) and embedded in paraffin (Merck Catalog No. 1071511000).

Immunohistochemistry protocol
Each paraffin block (n = 28, 14 of each ligament) was cut on a microtome (Leica, Wetzlar, Germany) into 5 µm thick sections which were mounted on slides previously coated with chrome-gelatin. Next, we randomly selected 10 slides per paraffin block, thus obtaining a total number of 140 slides of each ligament. Sections were deparaffinized, rehydrated with ethanol (100%, 95%, 80%, 70%) ( . Sections used as controls were incubated in the way previously described, but omitting the primary or secondary antibody. All controls were negative. The immunohistochemical staining of all sections was conducted under the same conditions. Photomicrographs of representative fields of the immunohistochemical staining were obtained using an Olympus CX 21 microscope fitted with an Olympus C5050Z digital camera (Olympus Optical Co., Ltd., Tokyo, Japan).

Semi-quantitative analysis
For semi-quantitative analysis of the expression of MMP-2 and -9, we used the software ImageJ 1.52a. The intensity of staining was assessed through the IHC Profiler plugin, according to the well-established protocol. As indicated above, we used 140 slides per ligament and analyzed at least 10 randomly selected visual fields on each slide. The IHC Profiler assigned a score to each visual field in a four tier system-high positive (3+), positive (2+), low positive (1+), and negative (0). The immunohistochemical expression in the EL and the ligament tissue of the MCL and the ACL were presented as percentages of the respective scores as calculated by the IHC Profiler.

Immunohistochemical analysis of MMP-2 and -9 expression in the MCL
The immunohistochemical analysis of the expression of MMP-2 and 9 revealed a heterogeneous distribution of the enzymatic activity of the two enzymes in the EL and in the ligament tissue of the MCL. The positive staining for MMP-2 was distributed along the entire thickness and was best expressed in the adventitia of blood vessels in the EL, as well as in the perivascular zone (Figures 1a-1b). The semi-quantitative analysis revealed positive (2+) expression in 70% of visual fields and low-positive (1+) expression in the remaining 30%. Perhaps an interesting finding was that some fibroblasts in the ligament tissue also stained positive for MMP-2. Immunoreactivity was calculated as negative (0) in 76% of visual fields and as low positive (+) in the remaining 24%. The enzymatic activity of MMP-9 in the EL was read as low positive (+) in 63% of visual fields, positive (2+) in 28%, and negative (0) in 9% of visual fields. As in the case of MMP-2, staining was best expressed in the adventitia of blood vessels and in the perivascular zone ( Figures  2a-2b). In the ligament tissue, the expression of MMP-9 was calculated as negative (0) in 88% of visual fields and low positive (1+) in 12% of visual fields, where it was observed mostly in the fibroblasts and in the adventitia of the few blood vessels.

Immunohistochemical analysis of MMP-2 and -9 expression in the ACL
In the EL of the ACL, we observed MMP-2 expression in fibroblasts, fat tissue cells, and the adventitia of blood vessels (Figures 3a-3b). In 74% of visual fields, immunoreactivity was read as low positive (1+), while in the remaining 26%, it was calculated as positive (2+). In the ligament tissue, enzymatic activity was predominantly negative (92% of visual fields). In certain areas, it was calculated as low positive (1+) (8% of visual fields). This low-positive reaction was observed in some fibroblasts located close to the border between the EL and the ligament tissue. The distribution of the immunoreactivity of MMP-9 was similar to that of MMP-2. In the EL, it was reported in the adventitia of blood vessels and the perivascular space and in the adipose tissue cells (Figures  4a-4b). The semi-quantitative analysis revealed a low-positive reaction (1+) in 83% of visual fields and negative reaction (0) in 17% of visual fields. In the ligament tissue, enzymatic activity was read as negative (0) in 94% of visual fields and low positive (1+) in the remaining 6% of visual fields. This enzymatic expression in the ligament tissue was only noted in individual fibroblasts located close to the border between the ligament tissue and the EL.

Discussion
The MMPs are a family of calcium-and zinc-dependent endopeptidases which play a key role in the breakdown and removal of extracellular matrix molecules from the tissue, a process that takes place during developmental stages such as growth and morphogenesis [19,24]. MMPs are also responsible for cleaving and rebuilding of collagen, elastin, gelatin, and casein and are thus involved in tissue-remodeling and wound healing [19,[24][25]. Apart from physiological processes, MMPs have also been implicated in pathological conditions and diseases such as Alzheimer's disease and other disorders of the central nervous system, atherosclerosis, arthritis, liver cirrhosis, tumors and their metastases [21].
After comparing the expression of different MMPs in the MCL and the ACL, Zhou et al. [27] concluded that numerous MMPs could be associated with the differences in healing potential. They reported that MMP-2 activity was higher in the injured ACL than in the MCL, which could present one potential reason of ACL healing failure. Tang et al. [7] described a 6.3fold increase in MMP-2 expression in fibroblasts in injured ACL in comparison to injured MCL. MMP-9, on the other hand, was up-regulated in the injured MCL but to a much lesser degree than in the injured ACL. Ishiguro et al. [28] reported the presence of promatrix metalloproteinase-9 positive cells in the perivascular area in the ruptured ACL and promatrix metalloproteinase-2 positive cells between irregular collagen bundles in stumps of this ligament. According to these authors the positive reaction of these MMPs could not be determined whether it was due to rapid degradation or as a result of the degradative changes. Zhang et al. [29] reported a higher expression of MMP-2 in MCL fibroblasts than in ACL fibroblasts. Furthermore, they suggested that the differential expression of MMPs in the MCL and in the ACL may be involved in the differential healing potential of the two ligaments. Previous studies by Georgiev et al. [9] and Iliev et al. [16][17] observed the distribution and expression of MMP-2 and MMP-9 in normal rat tissue and also during the process of healing after acute injury. They showed that fibroblasts in the EL of the MCL normally generate low levels of MMP-2 and 9. These studies also noted that there was a more intensive reaction of MMP-2 in comparison to MMP-9. During ligament healing, after grade III injury, high levels of MMP-2 were expressed during all early ligament repairs [17]. In contrast, MMP-9 expression diminished and on the 30th day after injury, it was observed predominantly on the border between the EL and the MCL and in the perivascular zones in the EL [17].
In the present study, we compared the immunohistochemical expression of MMP-2 and 9 in the EL and ligament tissue of the MCL and the ACL. Our data supported the findings of Zhang et al. [29], who reported stronger expression of MMP-2 in the MCL than in the ACL. On the other hand, Tang et al. [7] described a much stronger expression of MMP-2 in the ACL as opposed to the MCL after injury. The discrepancy in observations could stem from the fact that these authors studied the expression and distribution of the enzyme after traumatic damage to the ligament. The differential expression of MMP-9 between the two ligaments has not been specifically studied in the literature; most often, authors have chosen to assess a large number of other enzymes of the MMP family [7,19,21]. Herein, we reported that the enzymatic activity of MMP-9 was stronger in the MCL than in the ACL and was observed predominantly in the EL tissue.
Limitations of the current study existed and should be outlined. The visual quantification of immunohistochemical images is associated with significant inter-and intra-observer variation. We attempted to resolve this issue by using the IHC Profiler plugin for ImageJ software, which eliminates inter-observer visual perception bias. In fact, 88.6% of scoring determined by the IHC Profiler coincides with blinded manual scoring determined by trained pathologists (P < 0.0001, confidence interval, CI = 95%) [30].

Conclusions
This study represents the first report on the immunohistochemical localization and distribution of the enzymes MMP-2 and 9 in the EL of the MCL and the ACL in humans. Furthermore, we demonstrated and compared the enzymatic activity of MMP-2 and 9. These findings confirm our previous observations in rat model and are thus a further piece of evidence of the important role of the enzymes of the MMP group for the normal function of the MCL and the ACL and their role for the difference in the healing potential between the two ligaments.

Additional Information Disclosures
Human subjects: Consent was obtained by all participants in this study. The study was approved by the Medical Legal Office, the Local Ethics Committee and the Institutional Review Board (No. 4866) issued approval 4866. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.


s2fieldsofstudy: Biology Chemistry

text:
Molecular Docking and Pharmacophore-Based Virtual Screening of Novel Inhibitors for HCV NS5B RNA-Dependent RNA Polymerase Enzyme from Crude Sesame Essential Oil

Concern has been expressed worldwide about the rising prevalence of HCV induced acute hepatitis and chronic liver diseases with associated cirrhosis and liver cancers. However, the available synthetic drugs are ineffective for all the HCV genotypes especially in genotype-1 patients with about 40% viral response rates and numerous side effects. Besides, the availability of veritable bioinformatics tools which includes molecular docking and virtual screening studies have shown that computational generated models nowadays assists in modern drug design and development of novel and more potent inhibitors through the understanding of protein (receptors) -ligand (drugs) interaction mechanisms. Non-structural proteins especially the 5B (NS5B) is an RNA-dependent RNA polymerase implicated in the synthesis and replication of the HCV RNA; and has been a potential target for its inhibitory activities. Due to the paucity of knowledge, we aimed to determine the differential inhibitory activity of essential oils present in the crude Sesame leaves extracts on HCV-NS5B RNA dependent RNA polymerase. Using in-silico studiesa Microsoft pharmacophore-based virtual screening and molecular docking tools on the iGEMDOCK vs 2.0 software was used to dock the essential oil ligands on the generated HCV NS5B (PDB ID: 4EO6) RNA-dependent RNA polymerase protein. GC-MS of the leaves confirmed carboxylic acids and phenolic groups in the essential oils especially some potent antioxidants like alpha-linolenic acid, linoleic acid, oleic acid, etc. Moreover, Alpha-Linolenic acid/ALA (-102.2/-103.4 kcal/mol) and Linoleic acid/LA (-94.8/-109.8 kcal/mol) showed higher inhibitory impacts among the six top different docked ligands, selected based on their high differential binding affinity and pharmacological interaction energy profiles against HCV NS5B RNA polymerase activities, by forming more H-bond interactions than the NS5B co-crystallized ligand. ADMET showed that ALA is well tolerated without any apparent toxicity in the body. Hence, ALA having the highest inhibitory impacts against the HCV NS5B goes to confirm the beneficial impacts of carboxylic acids from Sesame plant in maintaining liver cellular integrity and as a natural inhibitor of HCV NS5B RNA dependent RNA Polymerase enzyme activities. Lukeman A.J Shittu 1, , Abiodun M. Jinadu , Remilekun K. Shittu , Solomon A. Shittu Department of Anatomical Sciences, University of Abuja, Gwagwalada, Abuja, Nigeria. Research and Development Department, Jireh Laboratories International LLC, P. O. Box 1813, Alief Texas, 77411, USA. Department of Microbiology, Jireh International Laboratory, P. O. Box 882, Gwagwalada, Abuja, Nigeria. ABSTRACT

Introduction
Primarily the crackdown of the entire human genome about a few decades ago has positively impacted on the enhanced roles of computational tools applications in modern medicine. No doubt, computer applications in the whole drug discovery and drug development pipeline system have further helped pharmaceutical companies to save millions of US dollars and time, usually lost as a result of so many failed clinical drug trials in bringing drugs to reality. Besides, Computer-Aided-Drug-Discovery (CADD) approaches are now extensively been used in the drug investigation to enhance the efficiency of the entire drug discovery process [1].
The liver is considered the largest organ in the human body, weighing on the average, about 1.44-1.66 kg (3.2-3.7 lb) for both sexes [2]; as a result, it is tasked with enormous responsibilities which include metabolism and detoxifications of ingested substances in the body. Which further predisposed the liver to suffer reversible or irreversible damages due to several mechanisms caused by toxins, infectious agents; although, these are usually insidious in several conditions like hepatitis. One of such implicated agents is viral hepatitis caused by different type of viruses, such as hepatitis A, B, C, D, and E. In addition, jaundice is usually one of the most apparent characteristic features of viral hepatitis. As such, early detection of viral hepatitis and its treatment will indeed have a significant impact on the disease progression and prognosis. Hence, there is the need for a rapid and proper diagnosis of viral hepatitis through serological testing of the patient's blood for antiviral antibodies detection [3] with characteristic features of liver disease. Since, over 60% of the acute sufferers progress to the chronic stage of the infection [4].
Moreover, in recent times, hepatitis C virus (HCV) liver infection has become a major health concerns with significant economic and medical impacts, affecting humans with about 170 million chronic sufferers estimated worldwide [5,6]. Also, it is considered as one of the primary causes of cirrhosis and hepatocellular carcinoma (HCC); and a leading cause for liver transplantation worldwide [5][6][7]. In addition, HCV belongs to the family-Flaviviridae and genus-hepacivirus [8]. The HCV genome contains about 9.6 kilobase pairs in length that encode a polyprotein with over 3000 amino acids, which is cleaved by HCV genome proteases to 10 structural (S) and nonstructural (NS) proteins [8,9]. The HCV nonstructural (NS) protein especially, the NS5B polymerase, is an RNA-directed/dependent RNA polymerase and is also responsible for the complete replicate of the HCV RNA genome that is, it can catalyze the synthesis of both the positive (genomic) and negative (template) strand HCV RNA. However, it has no functional equivalent in mammalian cells. In addition, clinical evidence has shown that NS5B is essential for HCV replication [10] and can be detected with biochemical testing [8].
Recently, a dual therapy is said to be more effective than the usual single vaccine treatment approach, for example, the combination of pegylated interferon and ribavirin vaccines yielded a higher response rate against the HCV than a separate individual treatment [9,11]. However, both vaccines are still characterized with severe side effects such as hemolytic anemia, renal failure, etc. [11]. In addition, studies have shown that there have been no effective vaccines or therapy without any significant side effects [6,9,11].
Moreover, the past two decades have witnessed several strategies used to inhibit HCV NS -viral proteins with NS5B RNAdependent RNA polymerase emerging as one of the most attractive target choices for most anti-HCV drugs development for obvious reasons as already stated [8,10]. Furthermore, a growing number of small molecules have been reported as allosteric inhibitors of NS5B with only a very few of them still active in clinical trials in recent times [5][6][7]. However, limited studies are available on medicinal small molecules, as HCV NS5B RNA-dependent, RNA polymerase inhibitors from other plants like Acacia concinna [9], Phyllanthus niruri [10].
Hence, recently expressed is the searching for ideal natural medicinal antimicrobial agents with effective antiviral activity against the HCV NS5B RNA-directed RNA polymerase with minimal or no side effects as compared to the available synthetic agents in the markets [12][13][14][15][16]. Interestingly, phytoestrogens are one of such natural plant-based estrogenic agents that have recently attracted so much attention during the last decade because of their essential health benefits to humans. They include four broad classes of phytochemicals namely the lignans (sesame seed and flaxseed), isoflavonoids (soybeans), stilbenes (peanuts and grape) and coumestans (mung beans and clover) [17][18][19][20]. Moreover, phytoestrogens tend to mimic endogenous estrogens. Depending on their concentrations in the body, they act either as agonists or antagonists by displaying the endogenous estrogens from their estrogens receptors (Era and Erb) binding sites among their other known mechanisms of action [15,18,19,21].
Moreover, all parts of the sesame plant such as the seed, oil, leaves and roots are of great folkloric medicinal values locally. Also, subsistence farmers in some areas of Nigeria (Northern/NN, South-west/SWN and Middle-belt/MBN) consumed sesame as staple food sources. In addition, sesame bears different local names based on the areas of cultivations. Like ekuku-gogoro (SWN-for black-seed), yanmoti (SWN-for white-seed), ridi (NN); and Beni (MBN/English) or gingelly (English) in Asia and Africa [12,14,15,18,19,29]. Also, sesame bioavailability in the body after its oral ingestion in man revealed that the lignans usually undergo extensive metabolism in the intestine depending on the characteristics of the individuals' intestinal microflora to produce mammalian enterolignans, enterodiol and enterolactone [14,22].
More so, molecular docking is a software technique that fits a small molecule into a target's binding sites and is used to study the binding affinity modes of protein (large molecule) with ligands and inhibitors (usually small molecules) [30,31]. Thus, docking tools can be used to predict the structure of the intermolecular complex formed between two or more molecules including matching the molecules with overall minimum energy. In addition, it gives the prediction of absolute ligand binding affinities, as well as the binding orientation of ligands [31,32]. Hence, its application in drug discovery and drug design process, since drugs-receptor interactions are also highly specific and complimentary. Similarly, studies have shown that the predicted binding affinities of the ligands from the dry laboratory are usually in excellent agreement with the wet laboratory/ experimental values with an average deviation of 0.61 +/-0.4 kcal/mol [32].
Hence, we aim to study the inhibitory impact of the different natural active essential oil (small molecules) extracted from sesame plants (Sesamum radiatum-black) against the HCV NS5B RNA polymerase enzyme (belonging to transferase/transferase inhibitor enzyme class) using in-silico studies (virtual screening and molecular docking) on the iGEMDOCK computational suite [31]. In addition, to confirm the folkloric claim of sesame leaves effectiveness as an anti-viral agent.

Plant-source and preparation
The leaves from the sesame plant species (Sesamum radiatum, Schum and Thonn, Pedaliacaea family) purchased from a local vendor in SW Nigeria were processed accordingly. The extraction was allowed to proceed for five days (120 h) in the Refrigerator at 4°C for the stability of the essential oils. The crude extracts solution obtained was regarded as the full concentration with the crude extract later reconstituted for further (in-vitro) studies as stated in previous studies [12,13,15,16,18,19].
Phytochemical screening using gas chromatography-mass spectral (GC/MS) Crude methanolic extracts of Sesame leaves were analyzed by GC/MS. The GC analyses were performed using a Hewlett Packard gas chromatograph (model 6890) equipped with a flame ionization detector and injector MS transfer line temperature of 230°C respectively. Also, a fused silica capillary column HP-InnoWax (30 in x 0.25 mm, film thickness 0.25 μm) was used.
The oven temperature was set at 50 °C for 5 minutes holding time and then gradually raised from 50-230°C at a rate of 2 °C /min. Helium is the carrier gas used at a flow rate of 22cm/sec. One milliliter of extract mixed with methanol (80%), at a split ratio of 1:30 was later injected into the system [12,13,15,16,33].
GC/MS analyses were carried out on an Agilent Technologies Network mass spectrometer (model 5973) coupled to H.P gas chromatograph (model 6890) equipped with NBS 75K Library Software data.
The capillary column and GC conditions were as stated above and helium was used as the carrier gas with a flow rate of 22cm/s. The mass spectra were recorded at 70 eV/200°C at a scanning rate of 1scan/sec with a run time of 90 minutes as reflected in previous studies [12,13,15,16]. Compound identification carried out by comparing the obtained GC relative retention times and mass spectra to those of authentic substances analyzed under the same conditions; using their retention indices (RI) and by comparison to reference compound [12,13,15,16]. Preparation of the protein structure Protein structure retrieval-The Fasta sequence obtained from the HCV NS5B protein was subjected to sequence similarity search by using the BLASTP program [34] and the protein molecule having 100% sequence identity structure was then retrieved from the Brookhaven RCSB Protein Data Bank (https://www.rscb.org) [35]. Thus, the threedimensional (3D) structure for the target HCV NS5B protein, an RNA-dependent RNA polymerase protein, obtained at a resolution of 1.79Ǻ root mean square deviations (RMSD); and corresponding to the PDB ID-4EO6 was downloaded in pdf format. The HCV NS5B protein was later uploaded into the iGEMDOCK software [31] for further analysis and docking studies.

Protein structure visualization
The three-3-dimension (3D), PDB ID-4EO6 protein structure that was retrieved from RCSB PDB was visualized using the iGEMDOCK inbuilt RasMol v 2.7.5 software, flexible, robust molecular visualization software (http://rasmol.org/) [36]. Also, the bound ligand in the protein structure was visualized and removed before in-silico analysis.

Active binding site preparation
Previous studies have shown that the HCV NS5B protein has catalytic and allosteric active binding sites for its enzymatic activity [9] as also predicted in this present study using the CASTp webserver [37]. This tool helps identify ligand binding sites, using energy interaction scheme between the protein and a simple Van der Waal's probe to locate energetically favorable binding sites of a ligand. Here, the CASTP showed the binding site-amino acid residues, participating in the active site of the macromolecule (protein), including calculation of the binding active site's area and volume, by using the probe with a radius of 4Ǻ. The obtained active site residues were then mapped on to the HCV NS5B structure to check exactly where the residues are present.
Moreover, the iGEMDOCK software [31] has its own in-built protein binding site prediction, which centered on the protein's ligand (0S2) in itself at any given radius (3-30Ǻ) for docking reference. The binding site of the ligand-0S2 in the HCV NS5B protein was set at a radius of 4.0 A for the present study=docking reference. The platform also showed a RasMol displayed of the referenced, centered ligand (0S2) in its active binding sites on the protein as reflected in figure 1. In addition, the binding site for the target protein -NS5B was prepared at a probe radius of 4 Ǻ for each platform and the top 6 compounds with higher binding energy and scores were selected in the present study. Moreover, the PDBsum analysis of the HCV NS5B 4EO6 protein-ligand (0S2) binding sites was carried out to validate information on the binding site of the protein in the present study (http://www.ebi.ac.uk/thorntonsrv/databases/pdbsum/) [38].

Ligands/Compounds preparation
The ligand molecules used for the present docking process were the natural essential oil compounds extracted and analyzed from the leaves of Sesame plants using Gas Chromatography-Mass Spectroscopy [ [12,13,15,16]. The extracted compounds were then subjected to extensive database/library survey, especially, with the PubChem library (https://pubchem.ncbi.nlm.nih.gov) [39] to identify their structures, properties and uses further.
In addition, the 3D structure of each essential oil compound downloaded from the PubChem database in SDF format was then loaded into the Chimera 1.11.2 software (http://www.rbvi.ucsf.edu/chimera) [40]. Which was further built through a geometric energy minimization process (assigning bond, hydrogen atoms, charges and flexible torsion) to be converted into pdf format and saved as a MOL file for further study.
The Molecular docking process This was performed by docking each ligand separately into all the potential active sites detected on NS5B-RNA-dependent RNApolymerase enzyme. At first, the molecules were prepared and bonds, with bond orders, explicit hydrogen's atoms, charges, flexible torsions assigned to both the protein and ligands.
Furthermore, all the prepared volatile essential oils compounds saved in pdf format (MOL files) were uploaded into the iGEMDOCK software [31] using the following set parameters by default: Population size: 200, Generations: 70, Number of solutions: 3. The output path was set for data retrieval with the 'Start docking' option clicked; and, when docking was completed, a post-screening/analysis of the docked ligands was done.
The iGEMDOCK is unique because it measures the ligand docked fitness as the total energy (Etot) of a predicted pose in the binding site as dissected into the following terms below: where Ebind is the empirical binding energy used during the molecular docking; Epharma is the energy of binding-site pharmacophores; Eligpre is a penalty value if the ligand unsatisfied the ligand preferences.
In addition, the Epharma and Eligpre were used to improve the number of true positives by discriminating active compounds from hundreds of thousands of non-active compounds. S/N Natural Essential Oil Compounds. IUPAC NAMES PubChem CID 1.
Moreover, iGEMDOCK has an empirical scoring function for each ligand posed obtained during docking, which is estimated as: Fitness = vdW + Hbond + Elec. Where vdW represents van der Waal energy, while, Hbond and Elect stood for hydrogen bonding energy and the electro statistic energy respectively.
Thus, the docking process resulted in binding affinities in kcal/mol and docking run time due to energy-related conformational changes involving the receptor-protein and ligands with the surrounding solution. Moreover, the virtual drug screening scores for the top five phytochemicals-natural essential oil exhibiting higher binding affinity and pharmacological interaction are obtained from iGEMDOCK [31]. The highly negative energy score represents the stronger binding affinity and positive score represents poor or no effective binding between the HCV NS5B -4EO6 protein receptor and the natural essential oil small molecule. However, the molecule with the lowest binding energy is considered as the best inhibitor for the present study.

Molecular
properties like membrane permeability and bioavailability of leading compounds are always associated with some fundamental molecular descriptor such as logP (partition coefficient), molecular weight (MWt), or the number of hydrogen bond acceptors and donors in a molecule, logP (partition coefficient) [42]. These molecular properties were useful in formulating "rule of five" [43]. Lipinski's rule states that molecule with good membrane permeability must have molecular weight value ≤ 500, Hydrogen Bond Donor ≤ 5, Hydrogen Bond Acceptor ≤ 10 and partition coefficient (LogP) value ≤ 5. Therefore, Lipinski's Rule of Five [43] was used to test the bioavailability characteristics such as Adsorption, Distribution, Metabolism and Elimination (ADME) of the ligands used in this study.

Prediction of Compound Toxicity
The AdmetSAR database was also used to predict the adverse effects and other properties such as Absorption, Distribution, Metabolism, Excretion and the Toxicity of the natural essential oil compounds -ADMET profiles (http://www.admetexp.org) [43] in the present study.

Results
The CASTP of HCV NS5B -4EO6 protein showed that there are 15 binding pockets with pocket # -13 having the largest area and volume of 962.8 Ǻ 2 and 1345.1 Ǻ 3 respectively; and, calculated at a radius of 4.0Ǻ. In addition, the iGEMDOCK's platform showed a RasMol display of centered ligand (0S2) in its active binding sites on the protein as reflected in figure 1a.
The HCV NS5B structure has double chain such as chain-A represented in blue color, chain-B represented in white with different atoms constituting various colors; its N-terminal indicating the starting point of the structure and C-terminal indicating the end point of the structure. Also, RasMol image of the whole HCV NS5B protein showing both double chains A and B with its centered ligands as prepared from the iGEMDOCK suite [31] is shown in figure 2a The GC-MS finding of the extracted natural essential oil (small molecules) from the Sesame plant with their respective chemical ID numbers as retrieved from the PubChem library are shown in table 1. Therefore, the following natural essential oil extracted from the GC-MS analysis of Sesame leaves includes: (ZZZ)-9,12,15 octadecatrienoic acid, (ZZ)-9,12 octadecadienoic acid, (Z)-9 octadecenoic acid,   (Z)-9 hexadecenoic acid, octadecanoic acid, eicosanoic acid, tetradecanoic acid, decanoic acid, dodecanoic acid, methyl salicylate acid, etc.
The essential oil molecules extracted from the Sesame plant were docked using iGEMDOCK software [31] with their respective docked scores, total binding energy, Van-der Waal energy, electrostatic and hydrogen bonding profiles are reflected in table 2.  Table 4 showed the result of the different amino acid residues involved in the various pharmacological bonding interactions between the different carboxylic acids ligands and the HCV NS5B RNA dependent RNA polymerase enzyme.
The bioactivity and druggableness of ALA as described and calculated using specific vital molecular descriptors. Such as molecular weight, polar surface area volume, hydrogen bond donor, hydrogen bond acceptor, LogP, polarizability, lipophilicity and lipophobicity are all summed up in the Lipinski rule of five as shown in table 5. The LogP is an estimate of the overall lipophilicity of a molecule, a factor that influences the behavior of such molecule in a wide range of biological membranes, hepatic clearance, selectivity and non-specific toxicity [42]. As such, based on the rule of five [43], ALA has a Lipinski's score of 2.
ADMET profile was evaluated using the admetSAR for predicted classification and regression values were calculated for alinolenic acid/ALA (with the highest binding energy); by using different types of models such as blood-brain-barrier, human intestinal absorption, Caco2 permeability. They all showed positive values indicating that the ALA molecule passes all the tested models with no significant side effects on absorption as shown in table 6. In addition, various Cytochrome P450 (CYP) substrate and inhibitor models were calculated in the case of metabolism and the result showed that they are non-substrate and non-inhibitor except CYP450 1A2 Inhibitor. Moreover, for toxicity study, ALA was found to be Ames non-toxic and non-carcinogenic as reflected in table 6.
The finding of the sequence alignment of the NS5B protein with 100% identity is displayed in table 7. In addition, the PDBsum findings of the HCV NS5B 4EO6 protein-co-crystallized ligand (0S2) binding sites as analyzed are shown in table 8.

Discussion
Undoubtedly, humanity is increasingly exposed to evolving viral strains, bacterial multidrug resistance, undruggable targets, epidemics and accelerated lifestyle-associated risks, since the dawn of civilization. However, the demand for smart and robust technologies that are capable of handling the generated biological big data as par the 5 Vs': volume, velocity, variety, veracity and value, cannot be overemphasized [45]. Thus, unlike the traditional wet laboratory system, molecular docking technique and other bioinformatics tools (important techniques in Computer-Aided Drug Design -CADD) operate in the dry laboratory system. CADD is an evolving area in structure-based drug design that has the computational power to screen million of chemical libraries in a matter of minutes; as such they are less labor-intensive, very convenient and efficient time-saving processes. In addition, molecular docking has served as one of the most critical objectives in structure-based drug design and molecular biology, which modeled and comprehend molecular interactions between protein and ligands [45]. Hence, having a CADD platform that is standalone, fast/sharp, effective, flexible, accurate, reproducible and multi-tasking are some of the critical factors that matter for high validity and reliability of obtained predictive values.
Molecular dynamic (MD) stimulation is combined with linear interaction energy approximation (LIE) in the present study. This is based on the assumption that free energy binding (∆G) is linearly/directly proportional to changes in the van der Waals and electrostatic energy of the system, as previously proposed [32,46]. Which, has hitherto provided a good model for further prediction of absolute binding affinities and binding orientation of ligands as well [32].
Moreover, the iGEMDOCK v2.1 suite [31] was found to execute flexible docking for each ligand by generating protein-ligand interaction profiles of electrostatic (E), hydrogen-bonding (H), and Van der Waal's (V) interactions based on the LIE model. This is very interactive and has automatically integrated virtual screening (VS) environment with different phases from preparation through molecular docking and post-docking/screening analysis with pharmacological interactions studies between the different docked ligands and the protein. It also provides the interactive interfaces to prepare both the target protein (using the specified coordinates obtained from the PDB) and the screening ligands/compounds database carried out using the atom coordinates gotten from the compound library to infer the ligand binding site area, atom formal charge and atom types for each ligand studied [31,46]. Furthermore, iGEMDOCK [31,47] infers the pharmacological interactions and clusters the screening compounds for the postscreening analysis based on these profiles and the screening compound structures. Finally, it re-ranks and visualizes the screening compounds by combining the pharmacological interactions and energy-based scoring function of GEMDOCK [31,46]. Besides, other studies have shown that iGEMDOCK has a better overall performance in docking simulations compared to other docking software [31,47,48]. Thus, the iGEMDOCK v2.1 suite [31,46] was adopted for the present in-silico studies.
The result of the GC-MS analysis done on sesame plant (black Sesame) were similar to what obtained in previous studies on sesame leaves [12,13,15,16]. These findings were also similar to that of a recent study that showed Sesame is very rich in carboxylic acids in a relatively balanced content, which includes saturated (SFA)-23.05%, monounsaturated (MUFA)-33.65% and polyunsaturated fatty acids (PUFA)-39.7% in the seed-oil [28]. Moreover, other study has shown that the PUFA content mainly the Linoleic acid/LA (n-6) and ALA (n-3) are relatively similar depending on the varieties of sesame being analyzed [23]. Henceforth, virtual screening and molecular docking were done by taking the 4EO6 as a target for HCV NS5B RNA-dependent RNA polymerase enzyme inhibitory activity against the various extracted natural essential oil compounds using the iGEMDOCK v2.1 suite [31,46].
Based on the result, Alpha-Linolenic acid (ALA) was found to be the top natural essential oil compounds with the highest binding energy affinity (∆G score -most negative dock score) of -102.2 kcal/mol against NS5B RNA-directed   The total binding energy (∆G) and pharmacological interaction energy (E-pharma) of the protein-ligand (drug) interactions were considered in the present study as shown in table 3. This is based on the fact that hierarchical clustering and the profiling derived from the pharmacological scoring function (Epharma) are different significantly from those derived from the energy-based scoring function (∆G) alone [47]. Hence, compounds with high pharmacological scores tend to be structurally and chemically similar to the active compounds, in describing how fit the natural essential oil binds into the binding pockets of HCV NS5B RNA polymerase. In addition, the E-pharma for each docked ligand was found to be higher than the ∆G, indicating that pharmacological interactions are more useful for identifying active compounds, which showed active biological interaction and binding of the HCV NS5B protein molecule. Thus, palmitoleic acid/POA was found to have the highest Epharma of 126.1 kcal/mol of all the docked ligands, despite being ranked 5th based on the free binding energy profile as reflected in table 3.
Conversely, in another similar NS5B RNAdirected RNA polymerase study; Palmitic acid/PA (SFA) was found as the essential oil ligand with the highest ∆G score among the other docked ligands and may be attributed to its relatively higher percentage (about 23% PA) in the Acacia concinna pod [9,49]. Thus, in comparison, Alpha-Linolenic acid/ALA was found to have the highest ∆G score among the other docked essential oil ligands. This may be due to PUFA (including Linoleic acid/LA) found to have a relatively higher percentage (of about 37.4%) among the fatty acid contents in Sesame [28]. Also, ALA content in the Sesame leaves tends to be more than that in the seeds [Shittu, unpublished work]. More so, PUFA contents in sesame plant are relatively similar depending on the cultivated varieties [23].
In addition, the major fatty acids in sesame seeds such as Linoleic acid methyl ester/LA (37.40%)-PUFA and oleic acid methyl ester/ OA-MUFA (29.09%) [28], were found to have similar major impact on the HCV NS5B RNA dependent RNA polymerase enzyme inhibitory activity, ranked 2nd and 3rd molecules respectively as seen in table 2. Moreover, a recent study has revealed that intake of unsaturated fats like oleic acid (richly available in Sesame plant) is more beneficial to human health, in that it allows for better flexibility and fluidity of the cell membrane functional structures, especially the endoplasmic reticulum (ER) components; thereby, prompting their proper functioning such as easy transport of substances in and out of the ER compared to the stiff, solid-like patches obtained in the palmitic acid/PA (SFA) treated-ER membrane [50,51]. In addition, Oleate/OA supplementation was found to reverse palmitate/PA-induced ER stress and lipotoxicity in the ER of primary hepatocytes and the H4IIEC3 hepatoma cell line [51]. Although, palmitate is reported to be the most abundant fatty acid found in the circulation [52] that helps to promotes phospholipid synthesis and its accumulation in the ER [51,53].
Based on the results, the ADMET properties predicted showed that ALA is a readily biodegradable, druggable molecule with no significant side effects on absorption, metabolism and toxicity in the body and thus, fit for human consumption. This further confirms the beneficial roles of carboxylic acids especially the omega-6 and omega-3 played in maintaining the integrity of the liver cells in general.
To the best of our knowledge, after extensive and thorough literature search, this is the first in-silico studies done on the natural essential oil compounds of Sesame species against HCV NS5B polymerase-protein.

Conclusion
No doubt, protein-ligand interaction plays a significant role in structure-based drug designing. As such, the use of iGEMDOCK tool has demonstrated its efficacy in the present study in successfully elucidating the novel inhibitory anti-viral (hepatitis) agents from Sesame plant.
Based on the present study, Apha-Linolenic acid/ALA (-102.2) is considered the molecule with the highest binding energy/score and to be a very effective natural potential inhibitor of HCV NS5B RNA dependent RNA polymerase with no significant side effects. Moreover, the study reflects the importance of volatile natural essential oil compounds extracted from Sesame plant and further confirms their folkloric uses as natural anti-viral agent as demonstrated.