Animals
Our study is approved by the Animal Care and Use Committee of the Wenzhou Medical University(wydw2021-0277).The animal operations involved in this study comply with "Guidelines for Care and Use of Laboratory Animals"(NIH Publication No.85 − 23,revised 1996) published by the US National Institutes of Health(Bethesda, MD).The mice used in this study(C57BL/6,male,6–8 weeks and 20–25 g) were purchased from the Zhejiang Vital River Laboratory Animal Technology Co.,Ltd through the Experimental Animal Center of Wenzhou Medical University.
Myocardial Infarction Model
Firstly,we used isoflurane to anesthetize the mice,and then conducted tracheal intubation.The heart was opened between the third and fourth intercostal space.We used a 7 − 0 silk suture to ligate the left anterior descending coronary artery(LAD) with left atrial appendage as reference.Then the heart was placed back in situ,followed by discharging the air in the anocelia and suturing the skin incision with a 4 − 0 suture.After the operation,the mice were closely observed with being placed on an electric blanket for recovery.The mice in the sham operation group were treated with the same operation without ligation.Mice were sacrificed 7 days after MI for further experimentation.
Animal Grouping And Pretreatment
All C57BL/6 mice(male,6–8 weeks and 20–25 g) were assigned randomly to three groups:(1)Sham group.The mice in the sham group were subjected to sham operation;(2)MI group.These mice were subjected to MI intervention;(3)MI + BaP group.These mice were pretreated with BaP(Sigma,USA,10 mg/kg/week) for 4 weeks and then subjected to MI intervention.BaP was dissolved with medium chain triglycerides and injected intraperitoneally(Supplemental Fig. S1A,B) and the dose was determined based on the 7-day survival rate after MI(Supplemental Fig. S2).
Cardiac Functional Assessment
One week after the induction of MI,the mice from sham,MI and MI + BaP groups underwent ultrasonic examination to measure the following parameters:left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),left ventricular internal diameter in diastole(LVIDd) and left ventricular internal dimension in systole(LVIDs).The parameters of each group were analyzed and compared.
Myocardial Infarction Size Detection
The size of myocardial infarction area was detected by triphenyltetrazolium chloride(TTC,Sigma,USA) staining.After we sacrificed the mouse,removed the heart as soon as possible,extruded residual blood,embedded it in OCT and sliced the heart.The heart slices were incubated with 1% TTC staining solution in a 37℃ thermostatic water bath for 15 min and then fixed with 4% paraformaldehyde for 24 h.The infarct area was measured with ImageJ software(NIH,USA).
Serum Biochemical Analysis
After we sacrificed the mouse,immediately taken blood,and the serum was separated from the blood in centrifuge at 3,000 rpm for 15 min.The creatine kinase MB(CK-MB) isoenzyme assay kit(Nanjing jiancheng,China) was used to measure the CK-MB in the serum.The Lactate dehydrogenase(LDH) assay kit(Nanjingjiancheng, China) was used to measure the LDH activity in the serum.And the enzyme-linked immunosorbent assay(ELISA) measurement of Troponin I kit(Elabscience, E-EL-M0086c) was used to measure the TnI concentration in the serum.
Histological Analysis
The hearts were fixed in the 4% paraformaldehyde,and further prepared for paraffin sectioning.Serial sections were cut and were used for hematoxylin-eosin(HE) staining and masson staining.Sections were stained using the HE staining kit(Solarbio,China) and the masson's trichrome stain kit(Solarbio,China) according to the manufacturer’s instructions.Images were taken under an Olympus microscope.
Oxygen And Glucose Deprivation(Ogd) Model
The H9C2 cells were used to simulate OGD model.We conducted 24-hour hypoxia simulation in a hypoxia incubator with the concentration of 94% N2,1% O2 and 5% CO2 in the medium deprived of serum and glucose.For in vitro experiments,BaP(Sigma,USA) was administered for 24 h at 20µM,CsA(GlpBio,China) was administered for 25 h at 1 µM(NavaneethaKrishnan 2020) and CH223191(GlpBio,China) was administered for 25 h at 10 µM(Melhem 2021) in advance.Before putting into the hypoxic incubator,replace with a new medium(Supplemental Fig. S1C).
Cell Viability Assay
The 96-well plate was used to culture the H9C2 cells.After BaP administration and OGD treatment,the Cell Counting Kit-8(Beyotime,China) was used to measure the number of living cells in each group according to the manufacturer’s instructions.
Hoechst 33342/pi Staining Assay
The 6-well plate was used to culture the H9C2 cells.After BaP administration and OGD treatment,the cells were washed with phosphate buffered saline(PBS),followed by adding Hoechst 33342 and PI working solution according to the manufacturer’s protocol(Beyotime,China).
Western Blot Analysis (Wb)
Western blot analysis (WB)
Myocardial tissue/cell protein samples were extracted with a RIPA buffer containing phosphatase and protease inhibitor.The concentration determination kits and SDS-PAGE sample loading buffer are from Beyotime(Shanghai,China).Proteins with different molecular masses were separated on SDS-PAGE gels,and then transferred to 0.22 µm PVDF membrane.Proteins transferred to PVDF membrane were blocked with skimmed milk powder for at least 2 hours and then incubated with primary antibodies of NLRP3(A12694,ABclonal),ASC(abs135824,absin),Caspase-1(22915-1-AP,proteintech),IL-18(57058S,CST),IL-1β(A1112,ABclonal),GSDMD(AF4012,affinity),P62(23214S,CST),LC3B(A19665,ABclonal),Parkin(14060-1-AP,proteintech),PINK1(DF7742,affinity),and GAPDH(10494-1-AP,proteintech) at 4℃ overnight.The following day,after incubation of the protein bands with the corresponding secondary antibody,the ChemiDoc MP device(Bio-Rad,Hercules,CA,USA) was used to scan and ImageJ software(NIH,USA) was used for analysis.
Myocardial Immunohistochemistry And Immunofluorescence
For immunohistochemistry,paraffin-embedded slices were incubated with the respective primary antibody targeting GSDMD(AF4012,affinity),Parkin(14060-1-AP,proteintech),LC3B(A19665,ABclonal) and then incubated with horseradish peroxidase and colored with 3,3’-diaminobenzidine(DAB).
For immunofluorescence,cells fixed with 4% paraformaldehyde or frozen tissue sections were used.Incubation with 0.3% Triton for 10 min to rupture the cell membrane,and incubation with 5% BSA for 1 hour to block.Then the primary antibodies of NLRP3(A12694,ABclonal),GSDMD(AF4012,affinity),Parkin(14060-1-AP,proteintech),LC3B(A19665,ABclonal) were used to incubate at 4℃ overnight.The next day,add corresponding secondary antibodies and incubate for 1 hour away from light,followed by adding DAPI to stain the nucleus.
Images were detected with Olympus microscope.
Mitophagosome Formation Determination
After the treatment of each group,Lyso-Tracker Green(LTG) and Mito-Tracker Red(MTR) were used to stain the cardiomyocytes according to the manufacturer’s instructions(Beyotime,China),followed by imaging of mitochondria,lysosomes,and colocalization of both markers is an indicator of mitophagosome formation.Fluorescent images were detected with Olympus microscope.Quantitative analysis of Mito-Tracker and Lyso-Tracker colocalization was presented by the merged spots.
Mptp Opening Detection
Calcein fluorescence was measured with the mitochondrial permeability transition pore assay kit(Beyotime,China) as per the manufacturer’s instructions.Briefly,cells were loaded with calcein AM treated with or without CoCl2 in PBS,and incubated with a CO2 concentration of 5% at 37℃ for 30 min.Images were detected with Olympus microscope.
Ros Detection
ROS fluorescence was measured using the DHE/DCFH-DA staining(Beyotime,China).After processing,add the DHE/DCFH-DA working solution according to the manufacturer’s protocol.Then put the cells into the incubator for 30 min at 37℃ under 5% CO2.Images were finally taken under an Olympus microscope.
Statistical Analysis
Data were presented as mean ± SD,and were analyzed using Graph-Pad Prism 8 (GraphPad Software,LLC,San Diego,CA,USA).All the data we used were normally distributed.A two-tailed unpaired Student’s t-test was used for the comparison of two experimental groups or one way analysis of variance(ANOVA) followed by Tukey’s post hoc test was conducted to compare more than two groups.P < 0.05 were considered as statistically significant.