CARDIOVASCULAR JOURNAL OF AFRICA • Volume 33, No 6, November/December 2022 AFRICA 305 To date, there has not been much research on the specific effects of VRP on acute pressure overload-induced ventricular arrhythmias in both diseased and normal hearts. Therefore, the underlying mechanism of action of VRP in acute pressure overload-induced ventricular arrhythmias needs further characterisation and understanding. We studied gene expression profiles involved in stretchinduced arrhythmias and detected potential biomarkers related to the anti-arrhythmic effects of VRP during five-minute transverse aortic constriction (TAC) surgery. This study identified common signalling pathway alterations between the VRP-pretreated model group (group 4) and the untreated model group (group 2). Next, we investigated the potential molecular mechanisms fundamental to the anti-arrhythmic effects of VRP. The results of this study will assist in further understanding the mechanisms underlying the preventative effects of VRP on pressure overloadinduced ventricular arrhythmias. Methods VRP was purchased from Shanghai Harvest Pharmaceutical Co, Ltd (Shanghai, China) and was initially dissolved in 0.9% saline solution. TRIzol reagent and RNAlater™ were purchased from Thermo Fisher Scientific Co, Ltd (USA). In this study, 40 male Wistar rats (234.68 ± 13.86 g body weight) were randomly divided into four groups: group 1 was the sham surgery group (control group); group 2 underwent fiveminute TAC surgery (model group, acute LV pressure overload) without treatment; and groups 3 and 4 were administered 0.5 and 1 mg/kg VRP before TAC surgery, respectively. VRP was administered intravenously via the tail vein over a 10-minute period before TAC surgery in the VRP-pretreated groups (groups 3 and 4). The dosage regimen of VRP was based on a previously published study.9 All animal experiments in this study were approved by the Animal Ethics Committee of Peking University People’s Hospital [approval number: 2013 (65)] and performed according to the instructions of our institute. For the transverse aortic constriction procedure, male Wistar rats were intraperitoneally anaesthetised with chloral hydrate (10%) at a dose of 3 ml/kg. The hair of the animals was removed with a mechanical shaver after the application of a depilatory cream. Left ventricular (LV) pressure overload was induced surgically by narrowing the ascending aorta as previously described.12 The sham-operated animals underwent the same surgical procedure except for the ligation of the aorta. Electrocardiograms (ECGs) of anesthetised rats were obtained by inserting needle electrodes subcutaneously into each limb. ECG signal recordings were displayed in real time and analysed using the ECG module of the LabChart 7 software (AD Instruments, Dunedin, New Zealand). To assess the haemodynamic changes that occur during aortic constriction, a polyethylene catheter (PE-50) was inserted into the ascending aorta via the right carotid artery and introduced into the left ventricle. Haemodynamic signals were recorded by PowerLab using the LabChart 7 acquisition system and were used to confirm the placement of the PE-50 catheter in the left ventricle. The LV systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP) were continuously recorded and stored for analysis from baseline to five minutes after the beginning of aortic constriction. ECG recordings were analysed for ventricular arrhythmias during the ligation of the aorta. Ventricular arrhythmias in animal models were defined and analysed based on the Lambeth Conventions criteria published by Walker et al. in 1988.13 The arrhythmia scores were evaluated according to the principles of Yang et al.,14 ensuring that the ventricular arrhythmia quantitative analysis scores were comparative. The ventricular arrhythmias were categorised into five groups and assigned the following point values: no arrhythmias, zero points; occasional premature ventricular beats, one point; frequent premature ventricular beats (three or more premature ventricular premature contractions within one minute), two points; ventricular tachycardia (one or two episodes), three points; ventricular tachycardia (three to five episodes), four points; and ventricular tachycardia (more than five episodes), five points. The most serious score of ventricular arrhythmias observed during TAC surgery was assigned as one appropriate point. A total of six LV apex samples of VRP-pretreated (1 mg/ kg) and saline-pretreated model groups (groups 2 and 4, n = 3) were used for RNA sequencing. Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc) according to the manufacturer’s instructions. Total RNA purity was estimated using ND-1000 NanoDrop (Thermo Fisher Scientific, Inc). RNA concentration and integrity were evaluated using an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Only high-quality RNA samples with an RNA integrity number greater than or equal to seven were used to construct the sequencing library using NEBNext Ultra RNA Library Prep Kit for Illumina. RNA sequencing was performed using an Illumina HiSeq 2500 sequencing system (Illumina, San Diego, CA, USA). Prior to read mapping, the raw sequencing data were analysed with FAST-QC by removing adaptors at both ends of the raw reads, eliminating low-quality reads, and cleaning up the pollutant. Subsequently, the gene expression level was normalised to reads per kilobase per million mapped reads to compare transcription levels among samples.15 Differentially expressed genes (DEGs) were identified as significantly different using normalised fold change values [log 2(fold change)] = log 2[mean expression ratio (group 4/group 2)] > 1 and Q-values (correction of the p-value) < 0.05 between groups 2 and 4. A volcano plot was drawn according to the analysis of the DEGs.16 Pathway-enrichment analyses of DEGs were performed using the DAVID system (DAVID V6.7; https://david-d.ncifcrf.gov/).17 Gene ontology (GO) annotation analysis via comprehensive categorical data was performed for the screened DEGs.18 After Bonferroni correction, p-values were calculated to identify GO terms signifcantly enriched among DEGs, with a corrected p-value < 0.05 as the threshold. Thus, cellular component, biological process and molecular function of DEGs can be predicted by GO functional significance enrichment analysis. We also used the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to perform pathway-enrichment analysis of DEGs and generate a report for DEGs; p-values < 0.05 of pathways were considered significantly enriched. Identifcation of enriched pathways can assist prediction of the signalling pathways in which DEGs participate. The ratio of the number of DEGs annotated in a given pathway to the number of all genes annotated in the pathway term was defined as rich factor. The highest rich factor indicates the greatest intensity.
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