4.1 Protein Distribution of Carbonic Anhydrase Isoenzymes in the Heart and Protein-Protein Interaction Analysis
CAIX is one of carbonic anhydrase isoenzymes. Isoenzymes have the same catalytic function, but there are differences in immunological properties, tissue and organ distribution among them. We searched and compared with these isoenzymes by various databases. The databases include Uniprot Database (https://www.uniprot.org), Bgee Database (https://bgee.org), Pathway Commons Database (http://apps.pathwaycommons.org), String Database (https://string-db.org). The Universal Protein Resource (UniProt) is a comprehensive resource for protein sequence and annotation data. Bgee is a database to retrieve and compare gene expression patterns in multiple animal species based exclusively on curated "normal", healthy, expression data. This allows comparisons of expression patterns between species. Pathway Commons is an access to biological pathway information collected from public pathway databases. String is a database of known and predicted protein-protein interactions. The interactions include direct (physical) and indirect (functional) associations; they stem from computational prediction, from knowledge transfer between organisms, and from interactions aggregated from other (primary) databases.22
4.2 Palmitic Acid (PA) Simulated Hypoxia Modeling
The variation trend of HIF-1α and CAIX protein expression was studied by western blot (WB). The correlation between myocardial CAIX and HIF-1α could be found. In detail, H9c2 cell hypoxia model was simulated by PA with different concentrations (0−100 μM). Protein was extracted from cells with ice-cold radioimmunoprecipitation assay (RIPA) buffer containing 1 mmol/L phenylmethanesulfonyl fluoride (PMSF). The lysates were centrifuged at 12000 g for 15 min at 4 °C. The protein concentration was quantified by Bicinchoninic Acid Protein Assay kit (Biosky Biotechnology Corporation, Nanjing, China). Western blot assay was used for protein expression analysis with primary antibody (anti-HIF-1α, proteintech, 1:500, 20960-1-AP; anti-CAIX, proteintech, 1:500, 11071-1-AP; anti-GAPDH, Bioworld Technology, 1:500, AP0063) and HRP-conjugated secondary antibody (Goat Anti-Rabbit IgG (H+L) HRP, 1:3000, Bioworld Technology, BS13278, or Rabbit Anti-Goat IgG (H+L)-HRP, 1:3000, Bioworld Technology, BS30503). Equal loading was verified by incubation with anti-GAPDH. The blots were visualized using ECL, and the signals were quantified by densitometry (Image-Pro Plus 6.0).
4.3 HIF-1α siRNA Transfection
H9c2 cells were transfected with HIF-1α siRNA (sc-45919) (Santa Cruz, CA, USA) in 6-well plates using siRNA transfection reagent (sc-29528) (Santa Cruz). 1 mL pre-diluted mixture containing siRNA (HIF-1α) and transfection reagent was incubated at 37 °C for 5−7h, and the culture was continued for 48h with twice liquid exchange. Some transfection groups were subjected to 100 μmol/L palmitic acid (PA) for additional 2h. The cell proteins were obtained for WB analysis to analyze HIF-1α and CAIX expressions. The detailed WB method was the same as in 4.2.
4.4 Overexpression Plasmid Construction
H9c2 cells were transfected with pcDNA3.1-HIF-1α (GenePharma, Shanghai, China) in 6-well plates using Hieff Trans™ Liposomal Transfection Reagent (Yeasen, Shanghai, China). pcDNA3.1-HIF-1α and transfection reagent were mixed and incubated at r.t. for 20 min to form a complex of DNA-liposomes. The mixed solution was added to each well of the cell culture plate, and after continuous culture for 48h, the cell proteins in each group were extracted. WB assay was performed to determine the protein expression level and the correlation between HIF-1α and CAIX. The detailed WB method was the same as in 4.2.
4.5 Synthesis of Compound AcAs
Reagents for compound synthesis such as acetazolamide (Ac), aspirin (As), 6-heptyl acetylenic acid, 2-bromine ethanol, triethylamine, sodium azide, copper acetate, ascorbic acid, concentrated hydrochloric acid, sodium bicarbonate were of analytical grade and used without further purification. 1H-NMR spectra were performed on a Bruker 600 MHz Ultra shield spectrometer (Avance Av-500) and reported as parts per million (ppm) from TMS. The final product AcAs was also characterized by 150 MHz 13C-NMR spectra. High-resolution mass spectrum was measured by an Agilent 6224 ESI/TOF MS instrument.
The compound was prepared as follows.
5-Amino-1,3,4-thiadiazole-2-sulfonamide (Intermediate 2): To a solution of acetazolamide (22.5 mM, 5.0 g) in ethanol (30 mL), concentrated hydrochloric acid (5 mL) was added at room temperature, then the mixture was kept stirring and refluxing until the reaction completed. After the solution was concentrated in vacuum, a small amount of saturated sodium bicarbonate solution was added to adjust pH=9. The resulting solution was extracted by ethyl acetate, and the organic phase was concentrated to produce white powder. Yield: 3.5g, 86.4 %. 1H NMR (600 MHz, DMSO-d6) δ 8.06 (s, 2H), 7.81 (s, 2H).
N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)hept-6-ynamide (Intermediate 3): Under ice bath, 2 eq oxaloyl chloride and a drop of DMF was added to a solution of 6-heptylic acid (277 mg) in dichloromethane (DCM) (6 mL). After stirring for 6–8 h, it was concentrated to remove DCM by a rota-vapour. Intermediate 2 (360 mg) and pyridine (316 mg) were dissolved in DMF (5 mL) under ice bath, then the above 6-heptylenyl chloride in DMF was added for reaction which was monitored by TCL. The solution was concentrated in vacuum and the residue was isolated by silica gel column chromatography (DCM: MeOH= 40:1, V:V). The product was obtained as white powder. Yield: 0.42g, 73.7%. 1H NMR (600 MHz, DMSO-d6) δ 13.0 (s, 1H), 8.32 (s, 2H), 2.78 (t, J = 2.4 Hz, 1H), 2.55 (t, J = 7.2 Hz, 2H), 2.20–2.17 (m, 2H), 1.73–1.68 (m, 2H), 1.50–1.45 (m, 2H).
2-Bromoethyl 2-acetoxybenzoate (Intermediate 5): Aspirin (360 mg), 2 eq oxaloyl chloride and a drop of DMF were mixed in DCM (6 mL) in an ice bath. The mixture was kept stirring for 6–8 h, then DCM was removed by a rota-vapour. To a solution of 2-bromoethanol (250 mg) and trimethylamine (303 mg) in DCM (6 mL) in an ice bath the above solution of aspirinyl chloride was added. The reaction continued stirring at 0 °C for 6 h, DCM was used for extraction, the resulting organic phase was concentrated in vacuum and the residue was isolated by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1, V:V) to obtain yellow oil. Yield: 0.40 g, 70.2 %.
2-Azidoethyl 2-acetoxybenzoate (Intermediate 6): A mixture of intermediate 5 and NaN3 (130 mg) in DMF (6 mL) was heated to 50 °C and kept stirring overnight. The solution was concentrated and the resulting residue was isolated by silica gel column chromatography to get yellow oil. Yield: 190 mg, 76.3%. 1H NMR (600 MHz, CDCl3) δ 8.04 (d, J = 7.8 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.12 (d, J = 7.8 Hz, 1H), 4.43 (d, J = 4.8 Hz, 1H), 3.59 (d, J = 4.8 Hz, 1H), 2.37 (s, 3H).
Compound AcAs: To a stirring solution of intermediate 3 (60 mg) in methanol (8 mL) copper acetate (8 mg) and intermediate 6 (52 mg) were subsequently added. 15 mg of ascorbic acid was added 5 min later. The reaction maintained stirring for 4h, then the solution was concentrated, white solid powder was obtained by silica gel column chromatography. Yield: 80 mg, 71.4%. Purity is 98.9% detected by HPLC. 1H NMR (600 MHz, DMSO-d6) δ 13.00 (s, 1H), 8.32 (s, 2H), 7.93 (s, 1H), 7.82 (d, J = 6.9 Hz, 1H), 7.69–7.60 (m, 1H), 7.42–7.32 (m, 1H), 7.21 (d, J = 7.6 Hz, 1H), 4.69 (s, 2H), 4.61 (s, 2H), 2.69–2.59 (m, 2H), 2.59–2.50 (m, 2H), 2.20 (s, 3H), 1.70–1.54 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 172.19, 169.10, 164.26, 163.40, 161.13, 150.16, 146.66, 134.52, 131.16, 126.24, 124.10, 122.50, 122.34, 63.27, 48.28, 34.55, 28.35, 24.67, 23.92, 20.65. Ms: Found m/z: 538.11157 (calculated for C20H23N7O7S2, 537.1).
4.6 Cell Culture and Modelling
Cardiac myoblasts H9c2 were cultured in Dulbecco’s Minimum Essential Medium (DMEM) containing 10% fetal bovine serum (FBS). Meanwhile, Primary cultured neonatal rat myocardial cells were used in some experiments. These cells were extracted from the hearts of 10 neonatal rats born 1-2 days old and cultured with DMEM containing 10% FBS. Blank group cells were common cultured under normal oxygen. Model group cells were cultured under hypoxia or given simulated hypoxia modeling reagent palmitic acid (PA). For hypoxia treatment, cells were starved 2h in DMEM without FBS and glucose in cell incubator (37 °C, 21% O2, 5% CO2), treated with or without drugs (10 μm) for 2h and finally subjected to hypoxia conditions (37 °C, 1% O2, 5% CO2) for 4h, then cells were placed on ice to stop the cell activities. It has been reported that PA oxidation leads to local hypoxia in myocardial cells, so PA can be used as a simulated hypoxia modeling agent.23 0.0513g PA was dissolved in 1 ml anhydrous ethanol to get 200 mM mother liquor, which was diluted at 1:19 (10% BSA) to 10 mM working liquor and further diluted 100 times to 100 μM as modeling reagent.
4.7 Cell proliferation Assay
Primary cultured neonatal rat myocardial cells were used. After cells culturing with aspirin (As), acetazolamide (Ac), AcAs or a combined use of Ac and As (Ac&As) under hypoxia or normoxia treatment, cell proliferation was detected with Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology, Shanghai, China). The CCK-8 kit contains 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsodiumsalt (WST-8). In the presence of the electron carrier 1-methoxy phenazine methosulfate (1-methoxy PMS), WST-8 is reduced by intracellular dehydrogenase to generate water-soluble orange-yellow formazan which can be dissolved in the tissue culture medium, and the amount of formazan is proportional to the number of living cells.24 Cells were harvested and stained in a microplate with 96 wells with working solution WST-8. The absorbance intensity at 450 nm was measured with a microplate reader. We calculated the ratio of the absorbance value between the administration group and the blank control group for graphical analysis.
4.8 Cell Membrane Integrity Assay
The integrity of cell membrane can be used to distinguish normal cells from necrosis. In this study, apoptosis and necrosis assay kit was used to detect the improvement effect of aspirin (As), acetazolamide (Ac), AcAs or a combined use of Ac and As (Ac&As) on apoptosis or necrosis caused by hypoxia. H9c2 cells were cultured in a six-well plate overnight. After the cells adhered, As, Ac, AcAs (10 μmol/L) or Ac&As (10 μmol/L each for Ac and As) were added, respectively, and then incubated for 4h under hypoxia condition. Cells were washed with PBS for 3 times and resuspended in 1 mL cell staining buffer, added Hoechst 33342 (5 μL), PI (5 μL) and incubated at 4 °C for 30 minutes, and then detected by flow cytometry. The results of cell double staining test were analyzed with FCS Express V3 software.
4.9 ROS Level
DCFH-DA was used as a fluorescent probe to detect the content of ROS in cells. DCFH-DA is not fluorescent. It easily penetrates into the cell, and is hydrolyzed to DCFH by the esterase. Non-fluorescent DCFH can no longer penetrate the cell membrane, so DCFH-DA is easily load fluorescence probe. ROS in cells can oxidize DCFH to fluorescent DCF, and the fluorescence intensity can indicate the level of ROS content in cells. Cell grouping and drug administration methods were the same as described in 4.8. DCFH-DA was added into DMEM (containing 10% FBS) to incubate for at least 30 min. ROS level was then detected by fluorescence spectrophotometer and flow cytometry.
4.10 ATP Level
ATP production was measured using ATP Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer’s instructions. The kit is based on the fact that firefly luciferase catalyzes the fluorescence production of fluorescein, which requires ATP to provide energy, and the fluorescence is proportional to the concentration of ATP, thereby detecting ATP concentration.24 After hypoxia treatment, the culture dish was placed on ice rapidly, and all metabolic activities of the cells were stopped. The culture solution was aspirated and the cell lysate was added, cell lysis solution was collected, centrifuged at 12000 g for 5 min under 4 °C, and the supernatant was taken for detection. The RLU value (The fluorescence intensity) was measured by fluorescence spectrophotometer. Solutions were evaluated in triplicate.
4.11 Western Blot to Study the Interaction between Compounds and CAIX
After hypoxia or normoxia cell culture with aspirin (As), acetazolamide (Ac) or AcAs, CAIX expression level in myocardial cells was studied by WB assay. The detailed WB method was the same as in 4.2.
4.12 Extracellular pH Regulation by Compounds
The cells were inoculated at the density of 1×105 cells/well in a 24-well plate. After cells were attached to the well, the supermatamt was replaced with DMED containing compounds and incubated in a normoxic (21% O2) or hypoxic (1% O2) incubator. At the beginning and end of each experiment, we inserted the pH probe into the medium to measure the extracellular pH and △pH was calculated. 3 replicate wells were set for each group.
4.13 Docking
Molecular modelling was operated to exhibit the detail binding mode between the compounds and CAIX by docking program AutoDock 4.2.6.25, 26 The 3D structure of CAIX (PDB ID: 6FE2) was downloaded from NCBI database (http://www.ncbi.nlm.nih.gov/). The structure of compounds AcAs and acetazolamide was extracted from Chem3D Ultra Pro 14.0 (Table S3). The size of the docking box was 60 Å × 60 Å × 60 Å, which was centered at the enzyme active pocket. The dimension grid box with a certain grid spacing (0.375 Å) was large enough to enclose the active pocket. The protein structure was kept fixed during molecular docking. For each complex, the top ranked docking pose was optimized in the binding pocket, and then used as the geometry for the binding mode analysis. All the water molecules except coordinated water were removed and charges were assigned to the molecule file. The remaining genetic algorithm (GA) parameters were set as default values, except the genetic algorithm adjusted to run at 100.27
4.14 Myocardial Ischemia Injury Mouse Modeling and Evaluation of Drug Efficacy
Kunming male mice (4–6 weeks) were purchased from Shanghai Jiesijie Laboratory Animal Co., LTD. The animal care and experimental procedures were approved by Animal Ethics Committee of Southeast University (0990200).
Male kunming mice were randomly divided into four groups, six in each group, respectively Blank group (normal feeding), Model group (ISO modeling), two Medicated groups (20 mg/kg As or AcAs and ISO). The hypoxia modeling method was as follows: In the pre-experimental stage, both high and low concentrations were used for modelling, results showed that 20 mg/kg continuous administration for three days could not cause significant myocardial hypoxia injury, and 85mg/kg showed obvious effect. 85 mg/kg isoproterenol (ISO) was injected subcutaneously once a day for three consecutive days. ISO is a β-adrenergic agonist that could induces severe stress to cardiomyocyte, which can lead to the loss of myocardial integrity through oxygen deficit.28 ISO-induced myocardial injury serves as a well standardized model to study the beneficial effects of drugs and cardiac function.29 In the blank group, an equal amount of 0.5% sodium carboxymethyl cellulose was injected. In the medicated groups, drugs were given 20 mg/kg 2h before the injection of ISO. After the ISO injection on the third day for 1h, the mice were sacrificed by cervical dislocation method. The heart was quickly removed after the euthanasia, detected by TTC and HE staining, or stored at −80 °C for subsequent western blot experiments, or placed in 4% paraformaldehyde for Immunohistochemical (IHC) detection. Myocardial tissue protein was attracted for WB detection. Blood was taken for the detection of myocardial enzymes.
4.15 TTC Staining of the Mouse Heart
The mice hearts were isolated followed by 1% triphenyltetrazolium chloride (TTC) staining to test infarct size. TTC is a proton receptor of the pyridine-nucleoside structure enzyme system in the respiratory chain. It reacts with dehydrogenases in normal tissues and turns red, while dehydrogenase activity in ischemic tissues decreases and turns white. The heart was washed by PBS and cut into 5 pieces, and then stained in TTC solution with a water bath at 37 °C for 15−30 min. Pictures were taken under the microscope to calculate the percentage of myocardial infarction area.
4.16 HE Staining of the Mouse Heart
Hematoxylin-eosin (HE) staining was used to observe the pathological changes of myocardial tissue. The hearts of mice were fixed by soaking in 4% paraformaldehyde, and paraffin sections were prepared for staining. They were observed by light microscope and images were collected for analysis.
4.17 IHC Assay of the Mouse Heart
The mouse hearts of each group were isolated and fixed with 4% paraformaldehyde. After dehydration, the hearts were embedded in paraffin and sliced. The slices were pasted on glass slides and put in the fixative. After blocking with 3% peroxy methanol for 25 min at r.t., the primary antibody (anti-HIF-1-inhibitor, proteintech, 1:50, 20960-1-AP; anti-CAIX, proteintech, 1:50, 11071-1-AP) was added to incubate overnight at 4 °C, then incubated with HRP-labeled broad-spectrum secondary antibody for 1h at r.t.. The nucleus was stained with DAPI. After dehydration stabilized, the staining was detected with NanoZoomer 2.0-RS (Hamamatsu, Japan).
4.18 WB Assay of the Myocardial Tissue Protein
Myocardial tissue protein preparation: After the end of the perfusion experiment, quickly remove the heart, cut out the ventricular tissue, weigh a certain amount of tissue and cut it, and add the lysate containing the protease inhibitor at a ratio of 1:9 (mg:μL). Grind on ice for 40 times using a glass homogenizer. The homogenate was collected, centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was collected. A small amount of the supernatant was collected for protein quantification according to the BCA protein content determination kit. Add the remaining supernatant to 1/2 volume of 6× loading buffer, mix well, boil in boiling water for 10 min, cool it for WB detection, or put it in −80 °C refrigerator for future use.
4.19 Myocardial Enzyme Level in Mouse Serum
Creatine kinase MB isoenzyme (CK-MB) is one of the most important indicators of diagnosing myocardial injury through detecting enzyme levels in serum. Cardiac troponin I (cTNI) is a specific antigen of cardiomyocytes, which is degraded from the myocardial fiber when the cardiomyocytes are injured, and the content significantly increases. cTNI is another marker of myocardial injury. The contents of CK-MB and cTNI were detected by the corresponding kit and the automatic biochemical instrument.
4.20 Statistical Analysis
The results were expressed as the mean±S.D.. The significance of differences was analyzed by one-way ANOVA followed by the Bonferroni correction. A value of P < 0.05 was considered statistically significant.