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Quantitative Real Time Polymerase Chain Reaction (RT-qPCR)

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All primer sequences were designed using the online tool Primer 3-BLAST (NCBI) and the primers were obtained from Sigma Aldrich, Bangalore, India. Relative expression of transforming growth factor beta (TGF- β), myosin heavy chain beta (β-MHC), endothelial nitric oxide synthase (eNOS) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was studied. Forward and reverse primers for the above genes were used for amplification.

Table 5. PCR Primer details


Primer sequence

TGF- β

Forward primer: 5′- ATG ACA TGA ACC GAC CCT TC-3′

Reverse primer : 5′- GTA GTT GGT ATC CAG GGC TCT C-3′


Forward primer: 5′- GTA GAC AAG GGC AAA GGC AA-3′

Reverse primer : 5′- GGA TGA TGC AGC GTA CAA AG-3′


Forward primer: 5′- CTG GCA AGA CCG ATT ACA CGA-3′

Reverse primer : 5′- CGC AAT GTG AGT CCG AAA ATG-3′


Forward primer: 5′- ACC ACA GTC CAT GCC ATC AC-3′

Reverse primer : 5′- TCC ACC ACC CTG TTG CTG TA-3′

RNA isolation

All glasswares were rinsed with diethyl-pyrocarbonate (DEPC) treated water to inhibit RNases. Total RNA was isolated using guanidium thiocynate-chloroform-phenol method of Chomczynski and Sacchi (1987). Total RNA isolation kit (BioUltra, Sigma Aldrich,USA) was utilized for this study

After cleaning with saline, heart and aorta tissues were homogenized in denaturing solution with freshly added β-mercaptoethanol. After homogenization 2M sodium acetate solution (pH. 4.0), water saturated phenol and chloroform: isoamyl alcohol (49:1) was added. The mixture was shaked vigorously and allowed to cool on ice for 15 minutes. The mixture was centrifuged at 10,000 × g for 20 minutes at 4 oC. The aqueous phase was transferred in a fresh tube and an equal volume of ice cold isopropanol was added. RNA was precipitated by placing the sample at -20 oC for one hour. Then the mixture was centrifuged at 10,000 × g for 20 minutes at 4 oC. The pellet was washed with 70% ethanol and RNA was stored in DEPC water at -80 oC. RNA quality and quantity was assessed by nano-drop spectrometer.

Real time PCR amplification

SYBR Green Quantitative RT-qPCR Kit was used in this study and the PCR experiment was carried out in eppendorff realplex mastercycler. 1µg RNA was reverse transcribed by using Molone murine leukemia virus (M-MuLV) reverse transcriptase as per manufactures instructions. Then the amplification program (94 oC – 45 seconds, annealing – 45 seconds, extension 72 oC- 1 minute) was applied with specific annealing temperature. The annealing temperatures of TGF-β, β-MHC, eNOS and GAPDH were 58, 52, 55, and 55 oC, respectively. The specificity of the primers was confirmed by resolving the PCR products in 1.5% agarose gel electrophoresis. The relative fold change of expression was calculated by normalized the expression with GAPDH.

The RT-qPCR results were quantified using the ‘threshold line’ and the ‘cycle threshold’. The ‘threshold line’ is the point at which the reaction reaches a fluorescent intensity above background. The cycles at which the samples reach this level is called the ‘cycle threshold’ (Ct). The statistical analysis of the RT-qPCR results was calculated by using the ∆Ct = (Ct value of gene of interest – Ct value of GAPDH). Relative gene expression was obtained by ∆∆Ct methods (∆Ct sample – ∆Ct of control), with the use of the control group as a calibrator for comparison of all unknown sample gene expression levels. The relative gene expression fold change was derived from 2–∆∆Ct (Schmittgen and Livak, 2008).


Immunohistochemistry (IHC)

IHC was performed as described by Rocha et al., (2009) using Super Sensitive Polymer-HRP Detection System kit, from Biogenex, USA. The Super Sensitive Polymer-HRP Detection System is a atypical detection system using a non-biotin polymeric technology that makes use of two major components: a Poly-HRP reagent and super Enhancer™. As the system is not based on the biotin-avidin system, the problems associated with endogenous biotin are completely eliminated. The detection of antigens in tissues by immunostaining is a two-step process. The first step involves the binding of an antibody to the antigen of interest and the second step involves the detection and visualization of bound antibody by one of a variety of enzyme chromogenic systems. The choice of detection system will dramatically impact the sensitivity, utility and ease-of-use of the method.


Paraffin-embedded tissue was cut to obtain sections of about 4 µm thickness. The mounted paraffin-embedded slices are deparaffinized in xylene and rehydrated using an ethanol/H2O gradient. Heat mediated antigen retrieval step was carried out for 10 min and then the slides were allowed to cool to room temperature for another 20 min. This was followed by peroxidase block treatment (to block endogenous peroxidase enzyme activity) for 10-15 min and then power block treatment (to block non-specific binding of antibodies to highly charged sites) for another 15 min. The sections were incubated with the concerned diluted primary antibody solution (for 2 h (1:200)) followed by treatment with the super enhancer solution (for 30 min) and super sensitive Poly-HRP solution (for 30 mins). After colour development with DAB and counterstaining with haematoxylin, the sections were observed under the microscope and photographs were taken.


The ultrastructure of the heart specimen was examined by Transmission Electron Microscopy (TEM) according to the method of Lang (1987), by the technique of thin sectioning.


  1. Glutaraldehyde solution: 3%
  2. Osmium tetroxide: 2% osmium tetroxide in 10 mM sodium phosphate buffer, pH -7.4
  3. Ethanol: 75%, 95% and 100%
  4. Uranyl acetate: 1%
  5. Lead citrate: 3%
  6. Sodium phosphate buffer: 0.1 M, pH 7.4


Immediately after the sacrifice, the heart tissues were dissected and fixed with a solution of 3% glutaraldehyde for 2 hours at room temperature and washed thrice with phosphate buffer to remove glutaraldehyde. Post-fixation was done by a solution containing 2% osmium tetroxide in 10mM sodium phosphate buffer and left overnight. Then, the osmium tetroxide solution was removed and replaced with 75% ethanol. This reduces the remaining osmium tetroxide to osmium dioxide, which forms a precipitate in the alcohol. After 10 minutes, the alcohol was replaced with a few ml of 75% ethanol. After 30 minutes, the alcohol was replaced with 95% ethanol and left for 30 minutes. This solution was replaced with 100% ethanol and washed thrice and then dried in acetone.

After dehydration, the tissues were equilibrated for 30 minutes in 1:1 mixture of epoxy propane and the embedding medium, epon 812 (also called epikote resin-812). A mixture of the resin and two hardening agents, dodecyl succinic anhydride and methyl anhydride were used. A diamine catalyst generally N-benzyl-N-diethylamine was added just before use. The 1:1 mixture was poured off and replaced with full strength resin. This step was repeated several times to ensure full infiltration of the embedding medium. The tissue was then transferred to a beam capsule with a wooden stick and the capsule was filled with fresh resin mixture. The wooden stick was used to tease the specimen down to the center of the bottom of the capsule. Next, the block holder was placed with the specimen in hot air oven at 60°C for 48 hours to polymerize the resin completely. Once the blocks are hardened, they are ready for sectioning. The ends of the specimen blocks were trimmed using glass knives and ultra thin sections were cut using an LKBUM4 ultramicrotome. The sections were picked upon carbon grids and post-stained with combined uranyl and lead stain and rinsed with distilled water and dried. After drying, the grids were examined under a Philips EM201C transmission electron microscope (Philips, Eindhoven, Netherlands).


Western blotting was performed to analyze the expression pattern of eNOS in the aorta and reperfused hearts according to method of Laemmli (1970).


Following the protein estimation, the samples were separated using SDS-PAGE gel electrophoresis and the separated molecules are blotted onto a polyvinylidene fluoride (PVDF) membrane. After blocking, the primary antibody was added and allowed to bind to the protein followed by washing (which removes non specifically bound antibody); then an enzyme-labeled secondary antibody was added, to detect the primary antibody. The location of the secondary antibody was determined by adding an appropriate substrate for the enzyme conjugated to the secondary antibody.


  1. Acrylamide stock: 30% acrylamide, 0.8% N,N′-methylene bisacrylamide
  2. Separating gel buffer: 1.5 M Tris, pH 8.8
  3. Sample buffer: 0.5 M Tris, pH 6.8
  4. Sodium dodecylsulfate (SDS): 10%
  5. Ammonium per sulfate (APS): (10%)
  6. N,N,N',N'-tetramethylethylenediamine (TEMED)
  7. Separating gel overlaying solution: Water-saturated isobutanol
  8. Sample Buffer:

Tris (0.5M, pH 6.8)-2.5 mL

SDS (10%)-4.0 mL

Glycerol (100%)-2.0 mL

β-Mercaptoethanol-0.8 mL (or 1 M DDT-0.5 mL)

Bromophenol Blue (0.1%)-300 µL

Distilled water (400 µl) to 10.0 mL

  1. Running gel buffer

Tris-6.05 g

Glycine: 28.80 g

10% SDS: 10.0 mL or (1.0 g)

Distilled water to 1000 mL

  1. Staining solution

Coomassie brilliant blue R250- 300 g

Methanol-80 mL

Acetic acid-20 mL

Distilled water-100 mL

  1. Destainning solution

Acetic acid-100 mL

Methanol-300 mL

Distilled water: 1000 mL


The aortic tissues were homogenized in an ice-cold radio immuno precipitation buffer (RIPA) (1% Triton, 0.1% SDS, 0.5% deoxycholate, 1 mM/L EDTA, 20 mM/L Tris (pH 7.4), 150 mM/L NaCl, 10 mM/L NaF, and 0.1 mM/L phenylmethylsulfonyl fluoride (PMSF)). The homogenate was centrifuged at 10,000 × g for 20 min at 4°C to remove debris and the supernatant was used to determine the protein concentration of the lysates using the BCA protein assay kit (Merck, India).

Transfer of proteins to membrane

Samples containing 50 μg of total cellular proteins were loaded and separated using 10% SDS polyacrylamide gel electrophoresis. Following electrophoresis, the proteins were transferred from the gel to a membrane by using semi-dry blotting system (AA Hoefer, SEMIDRY BLOTER, USA). Before assembling the transfer system, soaked PVDF membrane in methanol for 10 minutes and blotting papers in cold transfer buffer. Prepared sandwich, blotting paper, membrane, gel and blotting paper, were placed in the transfer apparatus and few drops of transfer buffer was added and subjected to an electric current 20 V for 1 h under cold condition. After the transfer, the sandwich was removed from the transfer system. Membrane was stained with 0.5% ponceau in 1% acetic acid to confirm equal loading and then washed with distilled water.

The PVDF membrane were blocked with 5% blocking solution (containing 5% BSA in 0.5 M Tris-buffered saline, pH 7.5) for 2 h to reduce the non-specific protein binding sites and then incubated with primary antibody (anti-eNOS), in blocking solution with gentle shaking overnight at 4°C. After this, the membranes were washed with TBST (Tris-buffered saline and 0.05% Tween-20 (TBST)) thrice for 10 minutes interval and then incubated with respective secondary antibody anti-mouse IgG (Sigma-Aldrich, USA) conjugated to horseradish peroxidase. Then the membranes were washed with TBST thrice for 10 minutes interval. The reaction was developed with a DAB detection system (Merck, India). Bands were scanned using a scanner and quantitated by Image J, a public domain Java image processing software, Wayne Rasband, NIH, Bethesda, MD, USA.

H9c2 cardiomyoblast cell culture

Rat embryonic cardiomyoblast derived H9c2 cells was obtained from National Centre for Cell Science (NCCS), Pune, India. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and a combination of penicillin-streptomycin (1%) in a humidified 5% CO2atmosphere at 37°C. The assay was performed by seeding H9c2 cells in the concentration of 1×104 cells/well in 96-well plate.

In vitro oxidative stress and mitochondrial transmembrane potential study

In order to evaluate the cytotoxic effect, viability was checked with MTT assay on D-carvone (25–100 µM) treated H9c2 cells. For assessment of protective potential of D-carvone against oxidative stress, different concentrations of D-carvone (0.1, 1 and 10 µM) were incubated with H9c2 cells for 2 h, and then co-incubated with 500 µM/L H2O2 for further 18 h (Jia et al., 2012; Zhang et al., 2011). For viability analysis, MTT solution (5 mg/mL) was added to each well, and incubated for 4 h at 37°C. After incubation, optical density (OD) was measured on a microplate reader at 570nm. With the 10 µM dose of D-carvone, the level of intracellular ROS formation was quantified with fluorimetry using redox-sensitive fluorescent probe 2, 7-dichlorodihydrofluorescin diacetate (DCFH-DA). Further, to examine mitochondrial membrane permeability transition (MPT), H9c2 cells were incubated with 5 mg/mL Rhodamine 123 (Rh123) at 37°C for 30 minutes (Park et al., 2003). The images were acquired using the Olympus IX71 inverted fluorescence microscope.

Ischemia/reperfusion (I/R) protocol

D-carvone was dissolved in 1% DMSO (vehicle) and administered orally to rats using an intragastric tube daily for 7 days. The rats were randomly divided into four groups of six rats per group: (i) control group pre-treated with vehicle alone for 7 days (isolated rat hearts subjected to continuous perfusion). Isolated rat hearts obtained from the following three groups were perfused with a modified Krebs buffer solution for 10 minutes to stabilize the cardiac functions and then subjected to 30 minutes of global ischemia, followed by 60 min of reperfusion: (ii) I/R hearts pre-treated with vehicle alone for 7 days (Control (I/R)); (iii) I/R hearts pre-treated with D-carvone (I/R + D-C 10 mg/kg body weight); (iv) I/R hearts pre-treated with D-carvone (I/R + D-C 20 mg/kg body weight).

Langendorff isolated heart preparation

The animals were anaesthetized with an intramuscular injection of ketamine (75 mg/kg body weight). After thoractomy, the hearts were rapidly excised and placed in cooled (4°C) Krebs Henseleit bicarbonate solution [composition (in mM): 118 sodium chloride (NaCl), 4.7 potassium chloride (KCl), 1.2 magnesium sulphate (MgSO4), 1.2 potassium dihydrogen orthophosphate (KH2PO4), 2.3 calcium chloride (CaCl2), 25.0 sodium bicarbonate (NaHCO3), 11.0 glucose].

composition (in mM): 118 sodium chloride (NaCl), 4.7 potassium chloride (KCl), 1.2 magnesium sulphate (MgSO4), 1.2 potassium dihydrogen orthophosphate (KH2PO4), 2.3 calcium chloride (CaCl2), 25.0 sodium bicarbonate (NaHCO3), 11.0 glucose. The heart was then attached to the cannula through aorta and retrogradely perfused with the Krebs solution maintained at 37°C and continuously gassed with a mixture of 95% O2 - 5% CO2. Perfusion pressure was kept constant at 80 mmHg. The ischemia and reperfusion protocol was followed as described previously (Khan et al., 2006; Senthamizhselvan et al., 2014).

An elastic water-filled balloon was introduced into the left ventricle through a left atrial incision and connected to a Pressure Transducer (AD Instruments) linked with a PowerLab data acquisition unit (AD Instruments). The balloon volume was adjusted to achieve a stable left ventricular end-diastolic pressure (LVEDP) of 5-10 mmHg. The percentage rate-pressure product [RPP = (LVSP-LVEDP) ×HR] and percentage coronary flow was assessed as described previously (Esterhuyse et al., 2005; Ferrera et al., 2009; Swaminathan et al., 2010). Coronary effluent was collected for the estimation of LDH activity.

Macroscopic enzyme mapping of infarcted myocardium (Triphenyl Tetrazolium Chloride test)

TTC (triphenyl tetrazolium chloride test) test used for a section of the heart tissue. Lie et al. (1975) method was used for the triphenyl tetrazolium chloride test (TTC) analysis acclimated for the macroscopic enzyme mapping appraisal of the infarcted myocardium was completed. A freshly prepared solution of 1% TTC in phosphate buffer was prewarmed at 37-40°C for 30 minutes in a darkened glass. To remove the excess blood, the heart tissues were washed rapidly in cold water without macerating the tissue. After removing epicardial fat, the left ventricle was taken separately.

To obtain slices not more than 0.1-0.2 mm in thickness, the heart was transversely cut across the left ventricles. The heart tissue slices were kept in the covered, darkened glass dish containing prewarmed solution of TTC and the dish was kept in an incubator and heated to 37-40°C for 45 minutes. The heart slices were turned over thrice and made certain that it remains fully immersed in the TTC solution. At the end of the incubation period, kept the heart slice in fixing solution to fix the tissue. Colour photographs of slices were obtained by a camera with macro lens. The expected reaction of the TTC test was as follows: normal myocardium (LDH enzyme active) turned to bright red, infarcted myocardium (LDH enzyme deficient) turned to uncolored white.

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