Animals
A total of 48 adult male Sprague-Dawley rats, weighing 250–300 g, were purchased from Suzhou Xishan Biotechnology Co., LTD. [License No: scxk (Xiang) 2019-0014]. All rats were housed in a temperature-controlled environment with a 12:12 h light-dark cycle, with free access to food and water. The rats were sacrificed with an intravenous injection of 10% chloral hydrate at 3 mL/kg after the experiments. The animal experiments were approved by the Animal Experimentation Committee of the Institutional Review Board of Guizhou Medical University and complied with the guidelines of Guizhou Medical University for the care and use of animals.
Myocardial ischemia -reperfusion protocol
The rats were anesthetized with chloral hydrate (3 mL/kg) and an intraperitoneal heparin injection. When the rats were mechanically ventilated after endotracheal intubation, the tidal volume was 1.0 mL per min, and the breathing rate was 100 breaths per minute. A left-sided chest opening was performed at the fourth intercostal space, and the pericardium was opened. An 8 − 0 filament gently crossed the LAD 2/3 of the way around the LAD, located between the starting points near the pulmonary cones. Occlusion of the LAD caused epicardial cyanosis with local hypokinesis and typical ECG changes of acute myocardial infarction (marked ST-segment elevation with T-wave changes). LAD was ligated for 30 min, following which the ligation wires were released, and reperfusion was performed for three days[18]. A total of 48 adult male Sprague-Dawley rats were randomly divided into three experimental groups using the methodology of random number table, as follows: (I) the control group in which the rats were subjected to the same manipulation but without LAD ligation (n = 16); (II) the ischemia/reperfusion (I/R) group (n = 10); and (III) the I/R + siRNA groups, where the left ventricular wall was injected with siRNA (1 µg/10 g) at multiple points, using an insulin needle, began ischemia-reperfusion. For three days, the rats were cared after the chest was closed, and they began to recover. The sequence of rat-derived PCSK9 siRNA was as follows: sense 5′-GGAGGUGUAUCUCUUAGAUTT-3′ and antisense 5′-AUCUAAGAGAUACACCUCCTT-3′. The sequence of rat-derived scrambled siRNA was as follows: sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense 5′-ACGUGACACGUUCGGAGAATT-3′.
Assessment of the size of the myocardial infarct
Infarct size was estimated using Evans blue (EB, Beijing Solarbio Science & Technology Co., Ltd., China)/2,3,5-triphenyltetrazolium staining (TTC, Beijing Solarbio Science & Technology Co., Ltd., China). Following reperfusion, the rats were re-anesthetized and the LAD re-ligated; they were then injected with 2 ml 2% Evans blue via the tail vein. After the skin of the lips and distal limbs was stained blue, the hearts were removed, rinsed with 4 ℃ phosphate-buffered saline (PBS), and frozen at -80 ℃ for 30 min before being cut into 5–7 slices. The sections were immersed in 1% TTC buffer (pH = 7.5) for 30 min at 37◦C. The area at risk (AAR) was defined as the area not stained by Evans blue, and the infarct area (IA) was defined as the area not stained by TTC. Images of the stained slices were captured with a digital camera, quantified using Image-Pro Plus, and presented as a percentage[19].
Echocardiographic assessment of LV function
A VINNO6 high-resolution ultrasound system was used to perform an echocardiographic analysis on day 3 after IRI to assess cardiac function in anesthetized rats. Ejection fraction (EF) and shortening fraction (FS) measurements were performed to assess the left ventricular (LV) systolic function of the heart. The average of three consecutive cardiac cycles was used for each measurement.
Cell Culture、Simulated ischemia and Transfection of siRNA
AC16 cells (American Tissue Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and streptomycin. The cells were maintained in a humidified 95% air/5% CO2 incubator at 37°C. Briefly, cardiomyocytes were exposed to a glucose-free, serum-free medium and transferred to a hypoxic modular incubator for 10 h at 37°C with 5% CO2 and 95% N2. After hypoxia, the medium was replaced with a fresh oxygenated normal or high glucose medium, and the dishes were transferred to a normoxic incubator (95% air/5% CO2) for 8 h of reoxygenation. All cells were starved for 12 h with serum-free media before being subjected to normoxia or hypoxia.
siRNA duplexes corresponding to human-derived PCSK9 were purchased from RiboBio Biotechnology (Guangzhou, CHN). The sequence of overexpressed human-derived PCSK9 siRNA1(Ps1) was CCCATGTCGACTACATCGA, and for silenced siRNA2(Ps2), it was GGTCACCGACTTCGAGAAT. Cardiomyocytes were transfected with 50 nM of each siRNA for 48 h using siRNA transfection reagent to overexpress and inhibit PCSK9 gene expression. The medium was replaced with a normal medium, and the cells were subsequently exposed to hypoxia for the times specified. As a control, cells were transfected with sequence-disordered siRNA control. To confirm the efficiency of protein knockdown using siRNA, cell lysates were used for Q-PCR or Western Blot analysis.
Western blot analysis
Human-derived cardiomyocytes and infarcted tissue in the left ventricular of the rat were extracted with RIPA lysis buffer (Beyotime, China) and PMSF (Roche, USA) to extract total protein. Protein content was measured using the BCA protein assay and protein samples were separated by electrophoresis on SDS-PAGE and transferred to a polyvinylidene difluoride membrane.
After 2 hours of blocking with 5% skim milk, the membranes were incubated overnight at 4°C with the primary antibody at a dilution of 1:1000. After washing with PBS containing 0.2% Tween, the membrane was incubated with a secondary antibody for 1 h at room temperature. Then, signals were detected with Pierce ECL Western Blotting Substrate. Intensity quantitation of the bands was captured using Image J software and normalized to β-actin.
Antibodies directed at PCSK9 (ab31762、ab84041), BNip3 (ab109362), Beclin-1 (ab210498) were purchased from Abcam (San Francisco, CA), LC3 (CST#4599) was purchased from Cell signaling (Danvers, MA).
Real time Q-PCR
The infarcted area of the left ventricle of the rats was removed and homogenized. Total RNA was extracted with TRizol reagent (Thermofisher). cDNA was synthesized with Hiscript III-RT Supermix for the qPCR kit (Vazyme) following the manufacturer's instructions. Relative β-actin levels were quantified for each sample based on Ct (amplification cycle threshold), normalized to a value of 1 as an endogenous mRNA standard and expression relative to pseudo-ischemia. Real-time quantitative PCR using gene-specific primers was employed, as demonstrated in Table 1.
Table 1
List of primers used in this study
Gene Species
|
Forward
|
Reverse
|
PCSK9 rat
|
TGGAACCTGGAGCGGATTAC
|
TTCCCGGTGGTCACTCTGTA
|
BNIP3 rat
|
GGGCTCCTGGGTAGAACT
|
AGACGGAAGCTGGAACG
|
Beclin-1 rat
|
GCGTCAGCTCTCGTCAA
|
GCCCGGTCTTCAGCTAC
|
LC3 rat
|
GGAGTCCTGTGTCTACGG
|
AAAAGCTGGGGTGTTCCT
|
β-actin rat
|
CACCCGCGAGTACAACCTC
|
CCCATACCCACCATCACACC
|
PCSK9 human
|
GCTGTGCCTTGGTTTCCT
|
TGTGAAGTAGGGGTGCGA
|
BNIP3 human
Beclin-1 human
LC3 human
β-actin human
IL-1β rat
NLRP3 rat
|
CCAGCCTCGGTTTCTATTT
GGATGGTGTCTCTCGCA
GGCACCAACCCACCTACTC
CTCGCTTCGGCAGCACA
CCCTTGACTTGGGCTGT
TGTTGTCAGGATCTCGCA
|
TATCTTGGTGTCTGCGA
CAGTCTTCGGCTGAGGTT
ATCCCACCAGCCAGCAC
AACGCTTCACGAAATTGCGT
CGAGATGCTGCTGTGAGA
AGTGAAGTAAGGCCGGAAT
|
Histopathological change
Hematoxylin-eosin (H&E) staining and Masson trichrome staining were used to examine myocardial tissue's pathological and morphological changes. Light microscopy was used to examine paraformaldehyde-fixed, paraffin-embedded myocardial tissue slices stained with hematoxylin for 5 minutes, eosin for 2 minutes, or the Masson trichrome staining kit (Beijing Sola Biotechnology Co., Ltd., China).
Immunohistochemistry (IHC)
The left ventricular infarct tissue in the rats was prepared into 5-µm thick paraffin sections, dewaxed, and antigenically repaired in 1 mM EDTA (pH 9.0) for 15 min. The slides were then incubated with 10% goat serum for 1 h at room temperature, with primary antibody overnight at 4°C and secondary antibody for 30 min. Hematoxylin was used to re-stain the nuclei for 2 min, and a differentiation solution was employed to differentiate the cells for 5 s before ammonia was then utilized to return the blue. Finally, using alcohol, the eluate was dehydrated and clear, and the neutral resin was used to seal the slides for fixation, microscopic observation (Lycra), and analysis using Image Pro6.0.
Measurement of cTnT, CK-MB
Myocardial injury was evaluated by measuring the plasma concentrations of cardiac troponin T (cTnT) creatine kinase-MB (CK-MB). At the end of the reperfusion period, blood was collected and centrifuged at 1,500 rpm for 10 min to obtain plasma. CKMB and cTnT levels and activity were measured using the appropriate ELISA kits (Biosino Bio-Technology and Science Inc., China) according to the manufacturer's protocols, respectively.
Statistical analysis
All experiments were repeated, and data were obtained by repeating them with similar quality. All data were analyzed using GraphPad Prism 7.0 software (CA, USA). Comparisons between groups were made via the t-test or one-way ANOVA test for continuous numerical variables. Values were expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant.