Mapping of the human sinus node with emphasis on the expression and function of key microRNAs

UoM administered thesis: Phd

  • Authors:
  • Maria Petkova

Abstract

The sinus node (SN) is the primary pacemaker of the heart. Its unique function depends on the expression of ion channels, connexins, and Ca2+-handling proteins, involved in the pacemaker mechanisms, and regulated by small non-coding RNAs (microRNAs) and transcription factors. The aims of my work were 1) to map the most abundant microRNAs in the healthy human SN in comparison with the right atrial (RA) working myocardium, 2) to study their role in the SN pacemaker activity, and 3) to map the transcription factors that potentially regulate the pacemaking mechanism of the healthy human adult SN compared to RA. To address the first aim I used tissue from six human SN/RA preparations that were characterised via histology and immunolabelling/immunoblotting for SN-specific ion channels and Tbx18. Using quantitative polymerase chain reaction (qPCR) and RealTime Statminer software, I identified 66 microRNAs that were significantly more or less expressed in the human SN compared to the RA. miR-1-3p, miR-10b-5p, miR-30c-5p, miR-133a-3p, miR-153-3p, miR-198, miR-204, miR-215-5p, miR-371-3p, miR-422a, miR-429, miR-486-3p, miR-512-5p, miR-938 and miR-1225-3p were predicted to target genes regulating the pacemaking mechanisms using Ingenuity Pathways Analysis (IPA) software. TarBase database, miRecords, TargetScan, and RNA22 online softwares were used to predict that 7 of these microRNAs are conserved in human and rat, and were injected in cultured rat SN preparations for 24 hours. Extracellular recording showed miR- 486-3p and miR-429 significantly reduced the beating rate of the SN preparations. Luciferase reporter gene assay in H9C2 cells demonstrated that miR-486-3p binds to the HCN4 3-untranslated region (UTR). Immunolabelling and qPCR demonstrated that HCN4 protein expression and mRNA levels were significantly reduced in the presence of miR- 486-3p in isolated rat SN preparations. miR-133a-3p transfected cardiac induced pluripotent stem cells (iPSCs) exhibited significant downregulation of HCN4 mRNA levels. Transcription factors expression in human SN vs. RA were measured via next generation sequencing (NGS), which identified 68 transcription factors significantly more expressed in the human SN, and 60 transcription factors significantly less expressed in the SN compared to the RA. IPA software narrowed down the list of cardiac specific transcription factors to Isl1, Tbx1, Shox2, Tbx3 and Tbx18, which were upregulated, and Tbx5 and Nkx2-5, which were downregulated in the human adult SN. This study demonstrated the complex regulatory machinery of the human SN, which is based on a precise balancing of microRNAs and transcription factors.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award date1 Aug 2019