Antibodies and reagents
Cell Signaling Technologies was the source of primary antibodies against mTOR (2983), CaMKKβ (16810), AMPK (2532), p-CaMKKβ (12716), p-AMPK (2535), p-mTOR (5536), Beclin1 (3495), LC3B (3868), and GAPDH (5174). Alomone provided the primary antibodies against TRPC6 (ACC-017). Proteintech provided the main antibody against Bax (50599-2-Ig). Primary antibodies against Bcl-2 (ET1702-53) and SQSTM1/P62 (R1309-8) were purchased from Hangzhou HuaAn Biotechnology Company. The secondary antibody HRP-linked anti-rabbit IgG (7074) was acquired from Cell Signaling Technologies. We bought the secondary antibody, CY3 anti-rabbit IgG (BA1032), from Boster Biological Technology Co., Ltd. We bought 3-methyladenine (HY-19312) and SAR7334 hydrochloride (HY-15699A) from MedChemExpress.
Mice
At the Comparative Medicine Branch (CMB) of the National Institute of Environmental Health Sciences (NIEHS), in North Carolina, USA, mice lacking TRPC6 (TRPC6-/-) were produced on a 129SvEv background (21, 45). Additionally, WT 129SvEv mice were imported from NIEHS to function as the KO mice's controls. For every study, age-matched male KO mice with WT controls were employed. The National Academy of Science's Guide for the Care and Use of Laboratory Animals was followed when treating the animals. The animals were housed in a temperature-controlled environment with a 12-hour light-dark cycle and unlimited access to food and water. The Animal Care and Utilization Committee of Zunyi Medical University gave its approval to all animal investigations.
In vivo I/R
Mouse model of I/R injury.
Male TRPC6-/- and WT mice, aged eight to twelve weeks, were given anesthesia and put to sleep on a heating pad at 37℃. A MiniVent mouse ventilator was used to ventilate the animals with ambient air after they were intubated with a 20G stump needle. The left anterior descending coronary artery was seen under a dissecting microscope (OLYMPUS) and ligated with a sterile 8 − 0 Prolene suture after a left thoracotomy between the third and fourth ribs. A snare was used to run a suture into and around the coronary artery, which is located 2 mm below the left atrium. Tying off the snare allowed the left coronary artery to become ligated. The left ventricle wall's apparent paleness served as visual confirmation that the ligation was done correctly. The heart was allowed to reperfuse (by removing the snare and suturing the incision) after 30 minutes of regional ischaemia, which resulted in the disappearance of the myocardium's discoloration distal to the blockage. The identical treatment was performed on animals used as sham patients, except the left anterior descending coronary artery was not blocked. The Center of Institutional Animal Care and Use Committee at Zunyi Medical University gave its approval to all procedures (12).
Infarct size
The animals were reanesthetized after a 24-hour period of reperfusion, the left anterior descending coronary artery was once more blocked, and the heart was stopped at the diastolic phase by an injection of KCl (20 mM). The ascending aorta was cannulated in reverse direction in order to demarcate the ischemic AAR, and the coronary arteries were perfused with 0.2 mL of 1% (wt/vol) Evans blue. The heart was removed right away, and each heart was usually divided into five 1-mm-thick slices that were cut perpendicular to the long axis of the heart. The 1% (wt/vol) 2,3,5-triphenyltetrazolium chloride (TTC) from Sigma was used to counterstain the left ventricle. The counterstaining process was carried out at 37C for 15 minutes. The analysis of the images was done using Image-Pro Plus from Media Cybernetics. In order to determine the left ventricle AAR and IS after I/R,measurements were taken for the pale areas (IS),blue areas (area not at risk,ANAR),and nonblue areas (AAR). The percentages of IS/(AAR + ANAR) and AAR/(AAR + ANAR) were then calculated (12).
In vivo functional analysis
To examine the hearts of mice after the ischemia/reperfusion (I/R) procedure,echocardiography (ECHO) was conducted using a Vevo1100 imaging system and an MS400 transducer specifically designed for rats and mice (VisualSonics). In order to perform the ECHO procedure,the mice were anesthetized with 2% isoflurane. The hearts were visualized in a cross-section between the two papillary muscles and analyzed using M-mode. Various parameters,such as the end-diastolic diameter,end-systolic diameter,posterior wall thickness,and septal wall thickness,were measured offline with the Vevo software to assess changes in cardiac morphology. Additionally,the ejection fraction (EF),heart rate,and fractional shortening (FS) were determined (12).
Cell culture and treatment
The H9c2 cell line was purchased from the National Collection of Authenticated Cell Cultures and cultured in DMEM/High Glucose (Gibco, CA, USA) supplemented with 10% (v/v) FBS (Gibco, Australia), 100 IU/ml penicillin, and 100 µg/ml streptomycin (both from Gibco, CA, USA). The cells were cultured to 80–90% confluence in culture bottles with humidified air (5% CO2) at 37℃ in a normal incubator (Thermo Fisher Scientific, MA, USA). The culture medium was replaced every 2 days.
In order to replicate the effects of myocardial ischemia/reperfusion injury in a laboratory setting,H9c2 rat cardiomyoblasts were exposed to hypoxia and subsequent reoxygenation. To achieve hypoxia,the cardiomyocyte cells were placed in a hypoxic incubator with an atmosphere consisting of 5% CO2 and 95% N2. The cells were cultublue in a serum-free and glucose-free HEPES-buffeblue medium containing specific concentrations of various components. These included NaH2PO4 (0.9 mmol/L),NaHCO3 (6.0 mmol/L),CaCl2 (1.0 mmol/L),MgSO4 (1.2 mmol/L),sodium lactate (40 mmol/L),HEPES (20 mmol/L),NaCl (98.5 mmol/L),and KCl (10 mmol/L),with a pH of 6.8. The cells were incubated at 37℃ to simulate hypoxic conditions. Then,the reoxygenation phase was simulated by transferring the cells to a normoxic H9c2 culture medium. The cells were exposed to 9 hours of hypoxia to mimic ischemia,followed by 6 hours of reoxygenation to mimic reperfusion. Control cells,cultured in the normoxic H9c2 culture medium,were prepablue alongside the experimental conditions. These control cells were used for comparison purposes. For the treatment of SAR7334 hydrochloride (SAR7334) and 3-methyladenine (3-MA),H9c2 cells were stimulated with a concentration of 1 M SAR7334 and 5 mM 3-MA,which were diluted in the medium used for incubation.
Cell viability assay
In 96-well plates, H9c2 cells were seeded at a density of 1×104 cells/well, covered with 100 µl media for 12 hours, and then subjected to various treatments based on group assignment. Using the Cell Counting Kit-8 (CCK-8, CK04; Dojindo, Tokyo, Japan), cell viability was assessed at 450 nm using an iMarkTM Microplate Reader (Bio-Rad, USA).
LDH assay
Using commercially available kits and following the manufacturer's instructions, the levels of LDH in the H9c2 cell supernatants were determined (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). In a nutshell, cell supernatants were collected, combined with the LDH test kit's ingredients, and incubated at room temperature for five minutes. The absorbance was measured at 450 nm using a microplate reader. The formula included in the assay kit is used to express the LDH level in units/litre.
Enzyme-linked immunosorbent assay
For twenty-four hours, H9c2 cells (5 × 105) were sown in culture plates. The levels of the isoenzymes cardiac troponin-T (cTn-T) and creatine kinase MB (CK-MB) in the cell supernatant were used to assess myocardial damage following various therapies. The protocol for the enzyme-linked immunosorbent test (ELISA) kits (Shanghai Jianglai Biological Technology Co., Ltd., Shanghai, China) was followed.
Tandem mCherry-eGFP-LC3 fluorescence microscopy
By transfecting cells with a tandem mCherry-eGFP-LC3 plasmid and monitoring the green and red signals using fluorescence microscopy, autophagic flow was examined. Before autophagosomes fuse with lysosomes, this plasmid exhibits both green and red signals; only red signals are seen following fusion, since the acidic lysosome environment quenches green fluorescence (49).For 12 hours, H9c2 cells (1×105) were sown in culture plates. Following various treatments, each group's cells were fixed for 15 minutes with 4% paraformaldehyde before being twice washed with PBS. Under a confocal microscope (Zeiss, Heidenheim, Germany), cells were mounted and viewed. Several confocal microscopy photos were selected at random, and the yellow and red dots—which stand for autophagosomes and autolysosomes, respectively—were studied in order to measure the amount of autophagy.
Transmission electron microscopy
Animals that received an I/R for 30 minutes or 24 hours were killed and separated in preparation for electron microscope examination. The relevant therapy was applied when cultivating H9c2 cells. 2.5% glutaraldehyde was used to fix the cells for two hours at 4℃. After scraping the cells, they were centrifuged at 1000 rpm for 15 minutes at 4℃ to pellet them, and then they were fixed for 2 hours at 4℃ in a solution containing 2.5% glutaraldehyde and 1% osmium tetroxide. Samples were dehydrated and embedded in Epon-812 resin in accordance with the protocol. After that, slices of 60–70 nm were cut with an ultramicrotome and stained with lead citrate and uranyl acetate. Finally, using a transmission electron microscope (Hitachi, Tokyo, Japan), autophagosomes were detected.
Immunofluorescence staining
H9c2 cells were cultivated onto coverslips on a 6-well plate, incubated for 12 hours, and then exposed to the conditioned media for the designated durations. After removing the medium, PBS was used to clean the cells. The cells were permeabilized with 0.03% Triton X-100 for 15 minutes following their fixation with 4% paraformaldehyde. For localization and detection, the cells were subsequently treated with LC3B anti-rabbit IgG (1:100, CST, USA) antibody for an entire night. After washing with PBS, the samples were treated for 1 hour at 37℃ with anti-rabbit IgG-(H + L) (1:150, Boster, China). After that, nuclei were counterstained for five minutes using 5 µg/mL DAPI (Solarbio, China). Using an Olympus microscope (Olympus IX73, Japan), fluorescent pictures were captured and then digitalized.
Western blot analysis
Standard procedures were followed for doing a western blot analysis. For thirty minutes on ice, the cells were lysed using lysis buffer. The supernatant was collected following 15 minutes at 4℃ of centrifugation at 12,000 rpm. Following the addition of the protein sample loading buffer, the samples were boiled for five to ten minutes at 95℃. The protein extracts (10–30 µg) were transferred to a PVDF membrane (Merck Millipore, Ltd., Darmstadt, Germany) after being separated by 4–15% SDS–PAGE. After blocking the membranes for two hours with 5% (w/v) skim milk in PBS Tween-20 (PBST; 0.1%), the primary antibodies (1:1000 in PBST) were added, and the membranes were left to overnight at 4℃. The PVDF membranes were subjected to three PBST washes before being incubated for one hour at room temperature with the corresponding HRP-linked secondary antibodies (1:10,000 in PBST). Using ECL (7Sea Biotech Co., Ltd., Shanghai, China) chemiluminescence reagents, the signals were analyzed using the Bio-Rad ChemiDocTM Touch Imaging System (Bio-Rad, Hercules, CA, USA). The outcomes were then analyzed using Bio-Rad Image Lab software (Bio-Rad, Hercules, CA, USA).
Flow cytometric apoptosis assay
Flow cytometry analysis was used to measure apoptosis. Using an AnnexinV-APC/7-AAD apoptosis kit (Tianjin Sungene Biotech Co., Ltd.), H9c2 cells were stained with fluorescein isothiocyanate-conjugated annexin-V protein with APC-labelled (Annexin V-APC) and 7-aminoactinomycin D (7-AAD). In culture dishes, 5 × 105 cells were planted and left for 12 hours. Each group's cells underwent various treatments before being collected, centrifuged, cleaned, and resuspended in PBS. Following the manufacturer's instructions, the cells were combined with Annexin V-APC and 7-AAD, and a CytoFLEX flow cytometer cell sorter (Beckman Coulter, Brea, California, USA) was used for analysis. Flow Jo software was used to analyze the data.
TUNEL assay
Following the mice's death, the heart samples were separated, preserved for 24 hours in 10% phosphate-buffered formalin, embedded in paraffin, and cut into 3- to 5-micrometer slices. Deoxyuridine triphosphate nick-end labeling (TUNEL) was carried out by terminal deoxynucleotidyl transferase-mediated method utilizing a commercially available kit (In Situ Cell Death Detection Kit, Roche, USA). according to the manufacturer's guidelines. Total cardiomyocyte nuclei were labeled with DAPI, while apoptotic nuclei were labeled with green fluorescein labeling. The heart tissues were analyzed using an Olympus IX73 microscope (Japan). The ratio of TUNEL-positive nuclei to DAPI-stained nuclei indicates the rate of apoptosis.