miR-363-5p and IL-34 Mediated Modulation of Pacemaker Channel, HCN4, on iPSC-CM; Translation into the Human Sinus Node Microenvironment

Background

The human sinus node (SN) contains cardiac fibroblasts and resident macrophages, with microRNAs (miRNAs) and interleukins as regulators of SN function. However, the mechanisms how they influence heart rate (HR) remain unclear.

Objectives

This study aims to investigate the SN microenvironment, encompassing miRNAs, interleukins, macrophages and fibroblasts and modulating iPSC-CMs hence beating rate.

Methods

Multi-omics analysis was conducted to compare human SN vs. right atria (RA). Human iPSC-derived cardiomyocytes (iPSC-CMs) were co-cultured with M2-type macrophages (M2s) and fibroblasts. Mechanistic studies involved the application of IL-34 and the silencing or overexpression of CSF1R and STAT3. mRNA-miRNA interactions were predicted using Ingenuity Pathway Analysis (IPA). miR-363-5p was applied to three cell lines and a co-culture system and, along with IL-34, was tested in human serum samples.

Results

M2s and fibroblasts markers were identified. Co-culturing iPSC-CMs with M2s induced a more nodal-like phenotype. Elevated IL-34 in the co-culture medium suggested that M2s may secrete IL-34, driving these nodal like feature. IL-34 promoted a nodal-like phenotype in iPSC-CMs by upregulating HCN4 expression probably through CSF1R/STAT3 signaling. Three key miRNAs were identified, with miR-363-5p inhibiting the differentiation of macrophages into the M2 phenotype. The nodal-like shift of iPSC-CMs in the co-culture system was reversed by miR-363-5p. While miR-363-5p (together with miR-486-3p) was highly expressed, IL-34 was reduced in aged individuals with SND.

Conclusion

M2 macrophages contribute to the SN microenvironment by secreting IL-34 and thus enhance SN function via upregulating HCN4. Elevated miR-363-5p, seen in age-related SND, may reverse these effects.