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MicroRNA (miRNA, miR) serves as an important regulator in cardiac hypertrophy. The present study aims to investigate the differential expression profile of miRNAs between young and aging spontaneously hypertensive rats (SHRs) and how these profiles are related to the pathogenesis of heart failure with preserved ejection fraction (HFpEF).
3-month-old and 12-month-old SHRs (n=3 for each) were subjected to echocardiographic study, histopathological analysis, CD31 immunohistochemical staining and dihydroethidium (DHE) staining. Subsequently, small RNA sequencing and data processing was executed to identify differentially expressed miRNAs between these two groups. Eight significantly up-regulated miRNAs were validated by real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR), followed by in silico target genes prediction. Functional annotation analysis of the predicted targets were performed by Gene Ontology and KEGG databases.
Compared with 3-month-old SHRs, a significantly impaired left ventricular diastolic function was observed in 12-month-old SHRs with declined E/A ratio (1.72±0.23 vs. 2.25±0.07, p<0.05), E’/A’ ratio (0.78±0.02 vs. 1.64±0.26, p<0.01) and increased E/E’ (38.2±2.0 vs. 26.0±2.5, p<0.01). Histological studies revealed significantly increased myocyte cross-sectional area (864.0±69.1μm2 vs. 461.7±40.4μm2, p<0.01), higher percentage of fibrosis area (10.4±1.3% vs.7.1±1.1%, p<0.05), increased reactive oxygen species (ROS) production (DHE fluorescent intensity 0.055±0.009 vs. 0.045±0.004, p<0.05), and reduced microvessels density (14.2±3.3 vs. 26.3±4.9, p<0.01) in hearts of 12-month-old SHRs. Compared with 3-month-old SHRs, 21 miRNAs were significantly up-regulated and 5 miRNAs were down-regulated in 12-month-old ones (p<0.05). qRT-PCR results were consistent with small RNA sequencing data on eight up-regulated miRNAs, including rno-miR-132-3p, rno-miR-182, rno-miR-208b-3p, rno-miR-212-3p, rno-miR-214-3p, rno-miR-218a-5p, rno-miR-221-3p and rno-miR-222-3p, which were submitted to bioinformatics analysis. Target genes were significantly enriched in 688 GO terms and 39 KEGG pathways, including regulation of peptidyl−tyrosine phosphorylation, regulation of protein serine/threonine kinase activity, adrenergic signaling in cardiomyocytes, ErbB signaling pathway, mTOR signaling pathway, FoxO signaling pathway, Ras signaling pathway, insulin secretion, adipocytokine signaling pathway, HIF-1 signaling pathway, Rap1 signaling pathway, VEGF signaling pathway, chemokine signaling pathway, TNF signaling pathway, etc.
Our data reported a dysregulated miRNA profile in aging SHRs, indicating the critical role of miRNAs in the process of HFpEF. Target genes of these differentially expressed miRNAs were involved in multiple signaling pathways associated with cardiac hypertrophy, autophagy, insulin metabolism, angiogenesis and inflammatory response.