Cohort study
In the period from May, 2017 to May, 2019, 115 patients diagnosed as STEMI with the culprit vessel of LAD at Shaanxi Provincial People’s Hospital were initially included. The final cohort consisted of 101 STEMI patients. 3 patients were excluded due to identification of malignant cancer; 1 patient was excluded due to diagnosis of systemic lupus erythematosus; 10 patients were excluded because of rapid development of cardiac systolic dysfunction which were eventually supported by extra-corporeal membrane oxygenation (ECMO) or left ventricular assist devices. STEMI was diagnosed by the current guideline[14]. Culprit vessel was determined by ECG manifestation and coronary angiography. Diabetes was diagnosed according to current guideline[15]. Post-MI VAs were recorded by Holter ECG. Frequent premature ventricular contractions (PVCs) was defined as ventricular premature beats take ≥ 10% of total cardiac beats during 24 hour- Holter ECG recording[16]. The exclusion criteria were: age below 18 years or above 80 years; pregnant women; previous myocardial infarction; previous PCI/CABG history; New York Heart Association (NYHA) functional class III or IV, renal failure, hepatic dysfunction, known history of cancer, immune-mediated disorders, mental disorders, recent/current use of anti-arrhythmic drugs. The peripheral venous blood samples were collected in a fasting state. Serum AGEs concentrations were determined by using a Human AGEs ELISA kit (CUSABIO, China) per the protocols provided by the manufacturer. All of the patients gave informed consent to participate in this study, which was approved by the ethics committee of Shaanxi Provincial Peoples’ Hospital. Specifically, patients signed the informed consent and agreed the collection and research purposes of medical records for our investigation.
AGEs-BSA preparation
The protocol was in accordance with our previous investigations[17]. Briefly, 0.1 mmol/L glyceraldehydes (Sigma) and bovine serum albumin (BSA) were incubated in 0.2 mmol/L NaPO4 buffer solution (pH = 7.4) at 37℃ at sterile condition for 7 days. BSA prepared without glyceraldehydes by the same protocol was used as control.
MI model establishment and animal treatments
SPF class Sprague-Dawley (SD) rats (9-week old, male/femal = 1, weighted 255 ± 6 g) were provided by Animal Experimental Center of Xi’an Jiaotong University. Animals were maintained in independent polypropylene cages under controlled conditions (12 h/12 h artificial light/dark cycle, humidity at 56%±4%, temperature at 25℃±1℃). Animals were accessible to standard chow and sterilized water freely. Animals were accommodated 1 week prior to experiments.
Rats were anaesthetized by inhalation of isoflurane (2% for introducing and 4% for continuous anesthesia) in oxygen at 0.6L/min. MI model was induced by ligation of LAD following the protocol adopted from our previous investigation [18]. Before establishment of MI model, AGEs-BSA and/or anti-RAGE IgG3 (Abcam) were administrated to animals via remaining needles implemented in tail veins. The dosages anti-RAGE antibody were selected according to previous reports[19, 20]. Treatments of each group were listed in Table 1.
Table 1
Animal treatments in each group
Groups (n = 10) | Treatment1 | | Treatment2 | |
| Reagent | Description | Reagent | Description |
Control (sham operation) | BSA | chronic tail vein injection for consecutive 8 weeks (1 injection/d) | Physiological saline | chronic tail vein injection for 8 weeks (3 injection/w) |
Myocardial infarction (MI) model | BSA | chronic tail vein injection for consecutive 8 weeks (1 injection/d) | Physiological saline | chronic tail vein injection for 8 weeks (3 injection/w) |
MI model and AGEs treatments | AGEs-BSA (100 mg/Kg) | chronic tail vein injection for consecutive 8 weeks (1 injection/d) | Physiological saline | chronic tail vein injection for 8 weeks (3 injection/w) |
MI model, AGEs exposure and RAGE antibody treatments | AGEs-BSA (100 mg/Kg) | chronic tail vein injection for consecutive 8 weeks (1 injection/d) | monoclonal anti-RAGE IgG3 (1 mg/Kg) | chronic tail vein injection for 8 weeks (3 injection/w) |
Table 1
Baseline clinical characteristics of the study patients
| Total (n = 101) | DM(-) | DM(+) | P value |
(n = 67) | (n = 34) |
Age (year) | 53.54 ± 4.64 | 54.21 ± 4.36 | 52.21 ± 4.93 | 0.052 |
Male (%) | 62(61.4) | 42(62.7) | 20(58.8) | 0.829 |
BMI (kg/m2) | 24.12 ± 1.42 | 24.12 ± 1.45 | 24.10 ± 1.37 | 0.963 |
Smoking history (%) | 38 (37.6) | 27 (40.3) | 11(32.4) | 0.517 |
Mean heart rate/24hours (bpm) | 69.56 ± 9.93 | 70.21 ± 9.15 | 68.29 ± 11.34 | 0.314 |
CRE (µmol/L) | 60.60 ± 7.30 | 61.12 ± 7.06 | 59.56 ± 7.77 | 0.291 |
UA (µmol/L) | 282.75 ± 41.31 | 288.16 ± 43.18 | 272.09 ± 35.58 | 0.071 |
ALT (U/L) | 26.84 ± 11.11 | 27.53 ± 11.40 | 25.47 ± 10.55 | 0.495 |
AST (U/L) | 81.39 ± 14.97 | 80.69 ± 13.98 | 82.76 ± 16.88 | 0.490 |
LDL (mmol/L) | 1.98 ± 0.74 | 2.08 ± 0.74 | 1.78 ± 0.73 | 0.097 |
K+ (mmol/L) | 4.26 ± 0.31 | 4.25 ± 0.33 | 4.29 ± 0.26 | 0.433 |
Na+(mmol/L) | 138.10 ± 2.50 | 138.01 ± 2.30 | 138.26 ± 2.90 | 0.977 |
BNP(pg/ml) | 68.15 ± 30.06 | 66.07 ± 31.63 | 72.24 ± 26.69 | 0.163 |
Troponin I(ng/ml) | 5.36 ± 3.31 | 5.20 ± 3.02 | 5.67 ± 3.85 | 0.736 |
QTc interval (ms) | 426.42 ± 11.26 | 426.51 ± 10.47 | 426.24 ± 12.84 | 0.838 |
AGEs (µg/mL) | 18.45 ± 2.00 | 15.30 ± 0.96 | 24.60 ± 1.92 | < 0.001 |
PVCs/24 hours | 5685.62 ± 2940.11 | 4638.40 ± 2419.98 | 7749.27 ± 2804.95 | < 0.001 |
Continuous variables are presented as mean ± SD; categorical variables are presented as numbers or percentages. |
Abbreviations: ALT, glutamic-pyruvic transaminase; AST, glutamic-oxalacetic transaminease. BMI, body mass index; BNP, brain natriuretic peptide; CRE, creatinine; LDL, low density lipoprotein; UA, uric acid; AGEs, advanced glycation end products; PVCs, premature ventricular contractions. |
Electrocardiography (ECG) evaluation
Anasthetized rats were fixed in a supine position, the standard limb leads were attached to the upper and lower limbs of the rats and connected to Powerlab 4/25 Biological Analysis System (AD Instruments) which recorded the ECG. Occurring amount of VAs, including ventricular premature beats and tachycardia were recorded.
Primary myocytes isolation
The primary myocytes were isolated from the hearts harvested from the rats in accordance with the protocol described in our previous study[2]. Specifically, the myocytes were carefully extracted from the adjacent area around infracted region in the anterior left ventricular wall under a dissecting microscope. Myocytes were maintained in Dulbecco Modified Eagle’s Medium (DMEM) supplemented fetal bovine serum (15%, FBS, Hyclone) and antibiotic mix (Sigma) at 37℃ in an atmosphere composed of 95% fresh air and 5% CO2. When cell populations reached confluence at 50%-60%, the cells were used for subsequent experiments.
Immunofluorescent staining
The protocols were carried out in accordance with our previous investigation[10]. Cultured primary myocytes were fixed by 4% paraformaldehyde for 15 min and permeabilized by 0.2% Triton on a cover glass. After incubation with blocking buffer (Abcam), fixed cells were incubated with primary antibody against RyR2 (Abcam, 1:200) and primary antibody against FKBP12.6 (Abcam, 1:100) at 4℃ for 12 hours. Nuclei were tagged by 4,6-Diamidino-2-phenylindole (DAPI, Sigma Aldrich). After washing, Alexa-488 conjugated secondary antibody (Invitrogen) and Alexa-555 conjugated secondary antibody (Invitrogen) were used to tag RyR2 and FKBP12.6 respectively. Fluorescence quenching was alleviated by using Antifade Kit (Molecular Probes). Axio Imager 2 inverted microscope (Zeiss) was used to capture the fluorescent images after excited at 488 nm and 594 nm respectively. Zeiss Physiological software (Zeiss, ver3.2) was used to analyze the co-localizations of RyR2 and FKBP12.6 based on these images.
Calcium spark detection
The calcium spark was detected in isolated myocytes by confocal optical calcium imaging according to the methods described previsouly[21]. Isolated myocytes were loaded with 10 µmol/l Fluo3/AM (Sigma-Aldrich) for 30 minutes at 37℃ in a humidified dark chamber. After washed by PBS, calcium sparks were observed with a SP8 STED confocal microscope (Leica) equipped with an argon laser at wave length at 488 nm. Liner scan was used to acquire the line scan images (512 pixels/line) at sampling rate of 2 ms/line. The scanning frequency was 600 Hz. Calcium sparks were analyzed by Image J with SparkMaster Plugin according to previous described method[22].
Cytosol and ER membrane fraction preparation and Western blotting
Subfraction isolation protocols were carried out according to ours and others’ previous descriptions[10, 23]. With Protein Extraction kits (Beyotime), proteins were extracted from cytosol preparation and ER membrane preparation according to the protocol provided by the manufacturer respectively. A BCA kit (Beyotime) was used to determine the protein concentrations of the samples which were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were then transferred to polyvinylidene fluoride (PVDF) membranes. After treated with blocking buffer (Abcam), primary antibodies against GRP78 (Abcam, 1:4000), phosphorylated PERK (p-PERK, Cell Signaling Tech, 1:2000), PERK (Cell Signaling Tech, 1:2000), FKBP12.6 (Invitrogen, 1:2000), Sigma receptor 1 (SigmaR1, Invitrogen, 1:1000) and GAPDH (Abcam, 1:1000) were used to incubate the membranes at 4℃ for 12 hours. After TBST washing, the membranes were incubated by corresponding HRP conjugated secondary antibodies (Abcam) at room temperature for 2 h. After developed with Super Signal West Pico chemiluminescence reagent (Thermo Scientific), the immunobands were visualized on X-ray films and then analyzed with ImageJ2x software (Rawak Software).
[H3]-ryanodine bind assay
[H3]-ryanodine bind assay was used to assess the channel activity of RyR2 according to the protocol described previously[10]. Cell lysate were incubated with [H3]-ryanodine solution (PerkinElmer) at final concentration of 20 nmol/L in binding buffer (25 mmol/L Tris, 50 mmol/L HEPES, 100 µmol/L CaCl2, 1 mmol/Lbenzamidine, 0.5 mmol/L phenylmethanesulfonyl fluoride, 2 µg/mlpepstain A, 2 µg/ml leupeptin and 2 µg/ml aprotinin) at 37℃ for 3 h. After washed with washing buffer (25 mmol/L Tris and 250 mmol/L KCl) and filtered with washing buffer- soaked membrane filter (Millipore), the radioactivity of the filter was detected by liquid scintillation counter (Bioscan) which represented the binding [3H]-ryanodine.
Calcineurin (CaN) activity detection
The CaN enzymatic activity was determined in total protein samples with a colorimetric method according to the protocol described previously[10]. A Calcineurin Activity Assay kit (Merck) was used per manufacturer’s instructions.
Statistics
Data acquired in this study were presented in (mean ± standard deviations) or percentage manner. Number of independent experiments carried out was indicated as n. NSK tests were performed as post-hoc tests. Participants were divided into 2 groups (diabetes and non-diabetes). The baseline characteristics among the 2 groups were analyzed by t test for parametric variables, the Mann-Whitney U test for nonparametric variables, and the chi-square test for categorical variables. The Spearman rank correlation coefficient was computed to assess correlation between continuous variables. The association between serum AGEs and frequent PVCs were estimated with univariate and multivariate logistic regression models. Model 1 was unadjusted. Model 2 was adjusted for age, sex, body mass index, cigarette smoking. Model 3 was adjusted for age, sex, body mass index, cigarette smoking, creatinine, UA, low density lipoprotein, K+, Na+, troponin I, BNP, QTC interval. All statistical testing was 2-sided. Results were considered statistically significant at a level of P < 0.05. All analyses were performed with PASW Statistics 20.0 software.