Roles of HDAC3-orchestrated circadian clock gene oscillations in diabetic rats following myocardial ischaemia/reperfusion injury

Roles of HDAC3-orchestrated circadian clock gene oscillations in diabetic rats following myocardial ischaemia/reperfusion injury

Experimental animalsSPF healthy male Sprague-Dawley (SD) rats weighing 200–220 g, 6–8 weeks of age, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Rats were kept at the Animal Experimental Center of Renmin Hospital of Wuhan University under constant temperature and humidity with a strick 12-h light/dark cycle regime and free access to water and food. The light time is 7 a.m.–7 p.m. (zeitgeber time (ZT) 0–ZT12), and the dark time is 7 p.m.–7 a.m. (ZT12–ZT24). Experimental protocols were implemented after being reviewed and approved by the Laboratory Animal Welfare & Ethics Committee (IACUC) of Renmin Hospital of Wuhan University. All animal procedures have conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes or the NIH Guide for the Care and Use of Laboratory Animals.Establishment of type 1 diabetic rat modelAfter fasted 12 h, rats were administrated by intraperitoneal injection 60 mg/kg streptozotocin (STZ) (Sigma, USA) to establish diabetes model as described previously4,5,6. The fasting blood glucose (after fasting for 6 h) was measured after 72 h. The hallmarks of successful establishment of diabetes model are blood glucose levels ≥16.7 mmol/L with increased consumption of food and water and increased urination of rats. After that, all rats are continuously raised for 8 weeks. With the premise of statistical significance and repeatability, we use the smallest sample size for all animal experiments. There are six successful modelling samples for each related indicators in each group of diabetic rats or non-diabetic rats, respectively.AAV9 infectionTo examine the effects of HDAC3 gene knockdown in MI/R‐stimulated diabetic rats, we used recombinant adeno‐associated virus serotype 9 (AAV9) vectors which carry a CMV promoter with GFP reporter (HBAAV9-HDAC3 shRNA1-GFP) or HBAAV9-GFP NC which were produced by Hanbio Biotechnology Co., to knock down HDAC3 gene expression or as control. AAV-HDAC3 was given via tail vein injection at a dose of 2 × 1012 vg/kg 3 weeks before I/R insult.Experimental model of MI/RIThe model of MI/RI was established as previously described4,5,6. Briefly, after given 1% pentobarbital sodium formulated with normal saline by intraperitoneal injection at a dose of 60 mg/kg to anaesthetize rats, the rats were received mechanical ventilation after endotracheal intubation with ECG monitoring. At the fourth intercostal space of left subclavian midline, we opened the chest of rats to expose the heart; then the left descending coronary artery (LAD) was occluded for 30 min followed by reperfusion for 2 h. Sham control group rats underwent same operation without LAD ligation. The non-diabetes group (N) and the diabetes group (D) were divided into sham (S) (n = 6 each group) operation group and ischaemia/reperfusion (I/R) (n = 12 each group) group at ZT0, 6, 12, and 18, respectively, according to the random number table method (Fig. 1A).Fig. 1: Circadian clock gene rhythmicity was altered with attenuated autophagy in the hearts from STZ-induced diabetic rats.A Schematic diagram of experimental programme. B Cardiac function was assessed by echocardiography in non-diabetic and diabetic rats. Scale bar: 2 mm. C–E Expression levels of Rev-erbα (C), BMAL1 (D), and C/EBPβ (E) mRNA were measured by qPCR over time after reperfusion. F–J The protein levels of Rev-erbα (G), BMAL1 (H), C/EBPβ (I) and LC3 II/I (J) were detected by western blotting in the myocardial tissues of non-diabetes and diabetes. n = 6 per group. *P < 0.05 versus NS within ZT; #P < 0.05 versus ZT0 within NS; &P < 0.05 versus ZT0 within DS.Cardiac functional assessmentCardiac function was monitored by animal ultrasound system with rat ultrasound by measuring the left ventricular EF and FS% recorded on a polygraph (RM-6240C; Chengdu Instrument Co., Ltd, China) when rats were anaesthetized by 40 mg/kg pentobarbital sodium. The measurements of two-dimensional and M-mode echocardiographic were analysed with a GE vivid 7 high-resolution in vivo-imaging system (VisualSonics, Toronto, ON, Canada).Infarct size measurementAt the end of reperfusion, six rats were taken from each group to detect myocardial infarct size by ligating LAD again with 0.3% evans blue dye (Sigma, USA) with 1% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, USA) staining. Briefly, 3% Evans blue stain was slowly injected through the femoral vein until a clearly distinguish between the blue stained and non-blue stained areas of the myocardium, and then the heart was quickly obtained. After being frozen at −20 °C for half an hour, it was cut into 2 mm thick heart slices into 4–5 slices. Slices were incubated with 1% TTC solution at 37 °C. Then 4% paraformaldehyde was used to fix slices for 15–20 min. The area of red, white, and blue in myocardial tissue were detected with a scanner (Epson, v30, Japan), and myocardial infarct size were analysed by an image analysis system software Image-ProPlus as described previously4,5,6.Measurement of serum troponin-ISerum troponin-I was used to evaluate myocardial injury. After reperfusion, we used assay kit (Jiancheng, Nanjing, China) to analyse the level of serum troponin-I by collecting arterial blood samples through the carotid artery.Transmission electron microscopy (TEM)After reperfusion, 1 mm3 tissue of the left ventricle in hearts isolated from rats were collected and fixed within 2.5% glutaraldehyde for 24 h. The tissue samples were washed, fixed, dehydrated, embedded, and cured with buffer solution, then were cut into ultrathin sections by an ultra-thin microtome. The TEM micrographs of ultrathin sections were detected by TEM (Tecnai G2 20 TWIN, USA) under the guidance of professional teachers of the Core Facility of Wuhan Institute of Virology.Histological examination (HE)After reperfusion, tissues from the heart apical region were isolated and rinsed with phosphate buffered saline and then fixed with 4% buffered paraformaldehyde embedded in paraffin. HE staining and the images of cross sections from the heart were obtained by upright Metallurgical Microscope (Canon, Tokyo, Japan).In vitro experimental protocol and hypoxia/reoxygenation modelPrimary neonatal rat cardiomyocytes were obtained from newborn SD suckling rats at 1–3 days. Briefly, newly born SD rats were disinfected with 75% alcohol, then chest was opened and the heart was harvested. The heart tissue fragments were digested in ADS buffer solution containing 1% collagenase from clostridium histolyticum (Sigma, USA) and 0.75% pancreatin from porcine pancreas (Sigma, USA) for six times, 20 min each time at 37 °C, 70 rpm in constant temperature shaker. Cell suspension was then centrifuged for 3 min at 2200 rpm, and the supernatant was discarded. Resuspend the obtained cell pellet into the top of the percoll gradient, centrifuge at 3000 rpm for 30 min at room temperature. Collect the cardiac cells and then was centrifuged at 2200 rpm for 3 min. Then the cells were cultured in 6-well and 96-wells plates for experiments under low glucose (LG) (5.5 mM glucose concentration) DMEM (Gibco, USA) containing 10% foetal bovine serum (Gibco, USA) and 1% penicillin and 1% streptomycin in a cell culture incubator at 37 °C within 5% CO2. HDAC3 siRNA and Rev-erbα siRNA (Ribobio, China) transfection were performed by using lipofectamineTM 2000 (Invitrogen, USA). After 24 h post-transfection, the cells were exposed to high glucose (HG) DMEM at a concentration of 30 mM glucose for 24 h treated at non-toxic concentrations. Subsequently, neonatal cardiomyocytes were subjected to hypoxic conditions (0.9% O2/94.1% N2/5% CO2) for 6 h, followed 2 h normal condition for reoxygenation.Cell viability assayCCK-8 assay kit (Jiancheng, Nanjing, China) was used to measure cell viability. After stimulation, the cultured cells in 96-well plates were given 10 μl CCK-8 reagent for each well and then incubated for 3 h in darkness. Perkin Elmer Microplate reader (PerkinElmer Victor 1420, USA) was used to analyse the absorbance at 450 nm.Mitochondrial ROS measurementMitoTracker Red CMXRos assays (YEASEN, China) was used to measure the mitochondrial ROS production. 500 nM MitoTracker Red CMXRos working liquid was added to the cells and incubated at 37 °C for 30 min in darkness. After that, cold PBS was used to wash the cells twice. The fluorescence intensity of mitochondrial ROS was recorded by using fluorescence microscopy (Olympus IX51, Japan).Assessment of mitochondrial membrane potential (MMP)MMP was detected by JC-1 (Beyotime, China) staining according to manufacturer’s instructions. Briefly, at the end of experimental stimulation, cells were incubated with JC-1 dye working liquid at 37 °C for 20 min in darkness. Subsequently, JC-1 buffer was used to wash the cells twice. Images of cells were obtained immediately and analysed by using a fluorescence microscope (Olympus IX51, Japan). When the MMP is high, JC-1 aggregates into the matrix of the mitochondria to form a polymer (J-aggregates) which can produce red fluorescence. When the MMP is low, JC-1 cannot aggregate mitochondria in the matrix and JC-1 is a monomer at this time, and then green fluorescence can be produced.Measurement of autophagic fluxTo measure the autophagic flux of cardiomyocytes, we used the tandem fluorescent mRFP-GFP-LC3 adenovirus (MOI = 100) to transfect cultured cells. GFP and mRFP in mRFP-GFP-LC3 adenovirus were used to label and track LC3. 24 h after adenoviral transfection, cells were washed with PBS, fixed with 4% paraformaldehyde, mounted with a reagent containing DAPI (Sigma, USA). The expression of GFP and mRFP was detected with Olympus FV1200 laser scanning confocal microscope (Olympus, Japan). Attenuation of GFP indicate that lysosomes and autophagosomes fuse to form autophagosomes, which red fluorescence can only be detected at this time. Yellow (merge of GFP signal and RFP signal) puncta represented early autophagosomes, while red (RFP signal alone) puncta indicate late autolysosomes. The autophagic flux was evaluated by counting the spots of different colours.Mitophagosome formation determinationAfter H/R stimulation, MitoTracker Green (MTG) and LysoTracker Red (LTR) were used to stain the cardiomyocytes, followed by confocal imaging of mitochondria, lysosomes, and colocalization of both markers is an indicator of mitophagosome formation. Cardiomyocytes were incubated with 300 nM MTG for 30 min, followed by 150 nM LTR for 45 min at 37 °C in darkness. Fluorescent images were detected with Olympus FV1200 laser scanning confocal microscope (Olympus, Japan). Quantitative analysis of MitoTracker-LysoTracker colocalization was presented by the merged spots.Gene expressionFollowing the manufacturer’s instructions, total RNA was extracted from myocardium and cardiomyocytes by trizol reagent (Invitrogen, USA), then reversely transcribed 1 μg total RNA into cDNA by using a reverse transcription kit (Takara, China). The mRNA levels of HDAC3, Rev-erbα, BMAL1, C/EBPβ were performed by quantitative RT-PCR using Bio-Rad CFX Connect Real-Time PCR Detection System (Bio-Rad, USA) with the following primers: HDAC3 (forward): 5′-ACCGTGGCGTATTTCTACGAC-3′; HDAC3 (reverse): 5′-CCTGGTAAGGCTTGAAGACGA-3′; Rev-erbα (forward): 5′-AAGGTTGTCCCACATACTTCCC-3′; Rev-erbα (reverse): 5′- CAGTAGCACCATGCCGTTAAG-3′; BMAL1 (forward): 5′-ACACCTAATTCTCAGGGCAGC-3′; BMAL1 (reverse): 5′-GAAGTCCAGTCTTCGCATCG-3′; C/EBPβ (forward): 5′-CGGTGGACAAGCTGAGCGACGAGTA-3′; C/EBPβ (reverse): 5′- GTTCCGCAGCGTGCTGAGCTCTC-3′; GAPDH (forward): 5′- CGCTAACATCAAATGGGGTG-3′; GAPDH (reverse): 5′-TTGCTGACAATCTTGAGGGAG-3′. The levels of mRNA were normalized relative to GAPDH. The expression of genes was analysed by using the 2−ΔΔCT method.Western blottingWestern blot analysis was performed as described previously20 using antibodies against HDAC3 (1:1000, CST, #3949), Rev-erbα (1:1000, CST, #13418), BMAL1 (1:1000, CST, #14020), C/EBPβ (1:1000, abcam, ab32358), BNIP3 (1:1000, CST, #3769), Atg4b (1:1000, CST, #13507), p62 (1:1000, CST, #23214), LC3B (1:1000, CST, #3868), and GAPDH (1:1000, CST, #5174). Myocardial proteins were lysed in ice-cold radio immunoprecipitation assay buffer, and then centrifuged at 12,000 rpm at 4 °C for 15 min to collect supernatants. Protein lysates were loaded into an 5% to 10–12% SDS–PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane, and then incubated with specific primary antibodies at 4 °C overnight. Subsequently incubated with fluorescent secondary antibody for 1 h at room temperature. The protein bands were obtained by using odyssey colour infrared laser scan-imaging instrument (Li-Cor, USA).Statistical analysisAll data are expressed as the mean ± standard deviation. GraphPad Prism version 8.0 (GraphPad Software, USA) was used to statistical software analysis. Differences among experimental groups were analysed by ANOVA followed by post-assay/test method by Bonferroni correction for post hoc t-test. P values < 0.05 were considered to be statistically significant. All data analysis were performed by observers blinded to the experimental groups.

Via Source link