Abstract
Dysregulation of cardiac-enriched microRNAs (CE-miRNAs) contributes to age-related cardiovascular diseases (CVDs). However, the age- and sex-specific expression patterns of CE-miRNAs in the heart remain poorly understood. Given their evolutionary conservation, we conducted a cross-species analysis to investigate changes in CE-miRNA expression associated with cardiac conduction (miR-1), hypertrophy (miR-9, -208), senescence (miR-34a), angiogenesis (miR-126), and fibrosis (miR-133) in male and female Drosophila melanogaster (flies), mice, and humans.
Heart tissues were collected at defined intervals across the lifespan of flies (7–70 days, n= 5/sex/timepoint), mice (18–72 weeks, n= 6/sex/timepoint), and humans (40–90 years, n=7-10/sex/timepoint). CE-miRNA and target gene expression were quantified using RT-qPCR. In flies, cardiac myofibril diameter was measured using Masson’s trichrome staining to assess structural changes associated with miR-9.
miR-9 exhibited consistent age-related downregulation across all species. miR-1 and miR-133 were selectively downregulated in female flies (P <0.0001), mice (P = 0.0013), and humans (P = 0.0006), whereas miR-34 and miR-126 were upregulated in aged male mice (P = 0.01) and humans (P <0.0001). miR-208 increased specifically in female mice (P = 0.0016). Corresponding target gene expression in flies (MRTF, CCN, KCNQ) showed sex-dependent dysregulation. Cardiac myofibril thickness increased with age in flies of both sexes.
To therapeutically restore downregulated miR-1 expression, we conducted a pilot study in aged female mice using lipid nanoparticle (LNP)-mediated delivery of miR-1 mimics. In vitro transfection of miR-1 mimic-LNPs increased (P <0.0001) miR-1 expression in AC-16 cardiomyocytes. However, subcutaneous administration of miR-1-LNPs in mice did not alter miR-1 expression, suggesting the need to optimise in vivo miRNA delivery.
This is the first cross-species study to characterize sex-specific, age-related CE-miRNA dysregulation. Our findings highlight the importance of considering sex differences in miRNA-mediated cardiac aging, and show the potential of miRNAs as diagnostic tools in age-related CVDs.