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 by which they influence heart rate remain unclear.

Objective: This study aimed to investigate the SN microenvironment, encompassing miRNAs, interleukins, macrophages, and fibroblasts and modulating induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and hence beating rate.

Methods: Multiomics analysis was conducted to compare human SN with the right atria. Human iPSC-CMs were cocultured with M2-type macrophages (M2s) and fibroblasts. Mechanistic studies involved the application of interleukin 34 (IL-34) and the silencing or overexpression of colony-stimulating factor 1 receptor and signal transducer and activator of transcription 3. Messenger RNA-miRNA interactions were predicted using the Ingenuity Pathway Analysis. miR-363-5p was applied to 3 cell lines and a coculture system and, along with IL-34, was tested in human serum samples.

Results: M2 and fibroblast markers were identified. Coculturing iPSC-CMs with M2s induced a more nodal-like phenotype. Elevated IL-34 in the coculture medium suggested that M2s may secrete IL-34, driving these nodal-like features. IL-34 promoted a nodal-like phenotype in iPSC-CMs by upregulating hyperpolarization-activated cyclic nucleotide-gated channel subtype 4 expression probably through colony-stimulating factor 1 receptor/signal transducer and activator of transcription 3 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 coculture system was reversed by miR-363-5p. Although miR-363-5p (together with miR-486-3p) was highly expressed, IL-34 was reduced in aged individuals with SN dysfunction.

Conclusion: M2s contribute to the SN microenvironment by secreting IL-34 and thus enhance SN function by upregulating hyperpolarization-activated cyclic nucleotide-gated channel subtype 4. Elevated miR-363-5p, seen in age-related SN dysfunction, may reverse these effects.