Abstract
Study of microRNA (miRNAs) using sheep models is limited due to lack of miRNA information. We therefore investigated oar-miRNAs and their regulation in an ovine model of heart failure (HF). Left ventricular (LV) tissue was collected from normal (Cont), HF (LV pacing @ ~220bpm for 13-days) and HF-recovery sheep (HF-R, 26-days after pacing cessation). MiRNA expression was profiled using next-generation sequencing (NGS) and miRNA array, and validated by stem-loop qPCR. Detected sequences were mapped against the ovine genome (Oar v4.0) and aligned with known miRNAs (miRBase v21). A total of 36,438,340 raw reads were obtained with a peak distribution of 18–23 nt. Of these, 637 miRNAs were detected by NGS and mapped to the ovine genome. With cut-off at 10 counts, 275 novel miRNAs were identified (with 186 showing 100% alignment and 89 showing 70–99% alignment with human/mouse and/or rat miRNAs, respectively), and 78 known oar-miRNAs. Cardiac-enriched miRNA-1, -133a, -208a/b and -499 were highly expressed in the LV. With HF induction, miRNA-133b-3p, -208b-3p, -125a-5p, -125b-5p, -126-3p, -21-5p, -210-3p, -29a-3p, -320a and -494-3p were significantly up-regulated relative to Cont and tended to return to normal levels following HF-recovery. This study has expanded the sheep miRNA database, and demonstrated HF-induced regulation of miRNAs.
Highlights
Since the first discovery of microRNA lin-4 in 19931, it has become clear that miRNAs play key roles in regulating both normal development and the response to injury in cardiovascular health and disease
Thirteen days of rapid left ventricular (LV) pacing produced the hemodynamic and neurohumoral hallmarks of established heart failure (HF), with marked decreases in cardiac output (CO) and mean blood pressure (MBP), and rises in atrial pressure and plasma atrial and B-type natriuertic peptides (ANP and B-type natriuretic peptide (BNP), resepectively). These changes returned to baseline levels following 26 days recovery from HF, with the exception of still slightly reduced MBP and elevated plasma atrial natriuretic peptide (ANP)
They were further classified into three categories (Fig. 2A): (1) Sequences of 53.5% counts which were novel oar-miRNAs aligning with hsa, mmu- and rno-miRNAs. (2) Sequences of 20.9% counts which were known oar-miRNAs aligning with oar-miRNAs. (3) Sequences of 25.6% counts which were not aligned with any known miRNAs
Summary
Since the first discovery of microRNA (miRNA) lin-4 in 19931, it has become clear that miRNAs play key roles in regulating both normal development and the response to injury in cardiovascular health and disease. The ovine rapid ventricular pacing model of HF, in particular, has been utilized extensively in the study of biological pathways involved in HF, especially those relating to neurohormonal systems, and in the development and refinement of HF therapies[7,8,9]. This model closely replicates the hemodynamic, endocrine and metabolic characteristics of severe low-cardiac output HF in humans[10], with the generation of cardiac and kidney dysfunction and activation of neurohormonal systems occurring in a time-dependent and highly predictable fashion. We attempted to expand the oar-miRNA database by examining sheep left ventricular (LV) tissue using NGS, miRNA array and qPCR techniques, and to investigate cardiac miRNA regulation in the well-established sheep rapid-pacing model of HF
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