Abstract
Since the 1950s, the average human life expectancy has increased rapidly owing to advancements in medicine, nutrition, and living standards. This increase in life expectancy is undoubtedly a victory of decades of research and modern medical technology, but it’s also worrisome as an aging population poses a major healthcare and socio-economic challenge. Aging is strongly correlated with an increased risk and prevalence of cardiovascular diseases (CVDs), cancer, neurodegenerative diseases, and diabetes. Globally, CVDs resulted in 19.8 million deaths in 2022 and is one of the leading causes of mortality. Aging is an independent risk factor for CVDs. The risk of CVDs increases drastically when combined with other age-related comorbidities such as diabetes. With age, the cardiovascular system undergoes several age-related pathophysiological and molecular changes including cardiac conduction defects leading to arrhythmia, cardiac hypertrophy, fibrosis, microvascular dysfunction, and cellular apoptosis. These changes occur primarily due to the age-related molecular changes in the cardiac cells. Among the several molecular changes in the cardiac cells, change in the expression of microRNAs (miRNA) have emerged as one of the potential contributors for age-related cardiac dysfunction. miRNAs are non-coding RNAs that inhibit the expression of their target protein by repressing translation. Previous studies have highlighted dysregulated expression of miRNAs to be a causative factor for age-related cardiac dysfunction. However, the specific age at which the dysregulation occurs and the sex-specific effects of miRNA expression changes remain largely unexplored. Given the evolutionary conservation of miRNAs, we conducted a cross-species analysis to investigate changes in the expression of cardiac-enriched miRNAs associated with cardiac conduction (miR-1-3p), hypertrophy (miR-9-5p, miR-208a-3p), senescence (miR-34a-5p), angiogenesis (miR-126-3p), and fibrosis (miR-133-3p) in male and female Drosophila melanogaster (fruit flies), mice, and humans. The six target miRNAs (miR-1, -9, -34a, -126, - 133, -208a) were selected based on existing literature and prior work from our laboratory, highlighting their established roles in major cardiac pathophysiological pathways. For this, heart tissues were collected at defined intervals across the lifespan of flies (7–70 days, n = 5/sex/timepoint), C57BL/6 mice (18–72 weeks, n= 6/sex/timepoint), and humans (40–90 years, n = 7-10/sex/timepoint). The expression of the target cardiac-enriched miRNAs (miR-1, -9, - 34a, -126, -133, and -208aa) were measured by RT-qPCR. Across species, aging was associated with strong sex-specific changes in target miRNA expression and corresponding changes in their target proteins. In Drosophila, miR-1 was selectively downregulated in aged females, whereas miR-9 and miR-34a were downregulated in aged males. In contrast, miR-133 declined in both sexes. Consistent with these changes, expression of the target genes KCNQ (miR-1 target), MRTF (miR-9 target), and CCN (miR133 target) showed age and sex-dependent alteration. KCNQ increased in females, MRTF increased in both sexes, and CCN increased in males but decreased in females. Elevated MRTF expression promotes cardiac hypertrophy, which was confirmed with a significant increase in myofibril diameter in both male and female fly hearts. In mice and humans, target miRNA expression were altered in a sex-dependent pattern. In mice, miR-1 and miR-9 were reduced in both sexes, miR-34a and miR-133 changed only in males (miR-34a increased, miR-133 reduced), and miR-208a increased only in females. Similarly in humans, miR-9 was downregulated and miR-34a was upregulated in both sexes, miR-126 showed opposite patterns between males and females, and miR-133 was reduced only in females. Furthermore, western blot analysis using mouse heart tissue confirmed changes in miRNA expression alters the expression of its downstream target protein. In mice, IRX-5 and myocardin (targets of miR-1 and miR-9) increased in both sexes, SIRT-1 (miR-34a target) decreased in males, and TR-α (miR-208a target) decreased in females. Collectively, these findings reveal distinct species and sex-specific patterns of cardiac miRNA regulation with age. Next, to therapeutically modulate the observed downregulation of miR-1 expression in female mice, we conducted a pilot study in aged female mice (54-72 weeks old) using lipid nanoparticle (LNP)-mediated delivery of miR-1 mimics. LNP-loaded with miR-1 mimics were formulated using microfluidics and characterised for optimal size (98.2 ± 8.06 nm) and charge (+23.51 ± 0.84 mV). In vitro transfection of miR-1 mimic-LNPs resulted in a four-fold increase in (P <0.0001) miR-1 expression in AC-16 cardiomyocytes. However, subcutaneous administration of miR-1-LNPs in mice did not increase cardiac miR-1 expression but caused elevated miR-1 expression in the liver. Subsequent western blot analysis revealed reduced IRX5 expression in the heart indicating a transient increase in expression of miR-1 released by LNPs repressed IRX-5 expression. Repression of IRX-5 is known to confer protection against arrhythmia under baseline conditions. To evaluate this, a pharmacological stress test electrocardiogram was performed on 72 week old mice that had received four doses of miR-1 loaded LNPs. In this study, reduced IRX-5 levels did not protect against pharmacologically induced arrhythmia as assessed by electrocardiography. Overall, these findings suggest that cardiac-specific delivery of miRNA-loaded LNPs requires optimization to achieve functional therapeutic outcomes. In conclusion, this is the first cross-species study to investigate species- and sex-specific, agerelated dysregulation in the expression of cardiac enriched miRNAs. The findings of this study highlights the complex, sex-dependent regulation of cardiac miRNA expression and emphasise the importance of incorporating sex as a biological variable and translation of rodent model findings to human studies.