Non-coding RNAs (ncRNAs) in Cardiovascular Aging

National Institute On Aging

Investigators

Linked publications, trials & patents

Abstract

a) miR-200c role in cardiac diastolic and systolic dysfunction associated with aging and comorbidities frequently associated with cardiovascular aging, i. e. diabetes and obesity Oxidative stress enhances miR-200 family (co-transcribed miR-200c and -141; co-transcribed miR-200a, -200b and -429) expression with miR-200c exhibiting the highest oxidative stress-induced expression level, and miR-200c further increases oxidative stress. Aging, obesity and diabetes are associated with increased oxidative stress. The expression of miR-200 family members was evaluated in young vs old rats and in young diabetic vs normoglycemic rats. We found that miR-200c and, to a lower extent, miR-141 expression increases in myocardial cells of old rats and diabetic rats. In contrast, co-transcribed miR-200a, -200b and -429 expression does not increase. The forced expression of miR-200c in isolated rat myocardial cells induced a significant prolongation of the electrically stimulated action potential and associated cytosolic calcium (Cai) transient. Further, we found a significant prolongation of the Cai transient, in response to a caffeine spritz, i.e. in the absence of electrically stimulated action potential. Importantly, action potential and Cai transient prolongation are key features of diastolic dysfunction in the old heart and in diabetic cardiomyopathy. To establish the role of miR-200c in myocardial function and its potential link to diastolic and systolic dysfunction in aging, obesity and diabetes, we developed a novel miR-200c knockout (KO) mouse in collaboration with Francesco Tommasoni-Ardori, PhD, and Lino Tessarolo, PhD, at NCI/CCR. In order to induce diabetes, mice are fed a high-fat diet (HFD) and are treated with a single high-dose of streptozotocin after 8 weeks on HFD. Then, diabetic mice remain on HFD for additional 8 weeks. In summary, we have 6 animal groups: WT and miR-200c KO fed a normal diet, obese, obese and diabetic. In this experimental protocol we evaluate whole-body metabolism (CLAMS), body composition (mini-NMR), glucose metabolism (oral glucose tolerance test and insulin tolerance test) and cardiac performance (echocardiography). Further, we collaborate with Dr. Kenneth Fishbein and Dr. Richard Spencer of the NIA MRI Core Facility to evaluate cardiac function, morphometry and fibrosis by MRI. Additional experiments are performed with isolated left ventricular myocytes to evaluate cell contraction and the associated Cai transient in response to an electrical stimulus and a caffeine spritz. We analyze the transcriptome (RNA-sequencing) of isolated cardiomyocytes from wild-type and miR-200c KO mice. In collaboration with Dr Adamo, we evaluate cardiac immune cells. b) miR-34a role in vascular aging: Vascular aging is characterized by a “sterile” pro-inflammatory state, largely driven by Renin-Angiotensin Aldosterone System (RAAS) activation and enhanced Ang II signaling via Ang II type 1 receptor (AT1R), leading to structural and functional alterations, including vascular smooth muscle cell (VSMC) proliferation, migration and secretion of extracellular matrix and inflammatory molecules. Further, miR-34a expression is modulated by aging and increases in different tissues, including the aorta, but its role in RAAS activation is unknown. AT1R-associated protein (ATRAP/Agtrap) binds to AT1R, promoting its internalization and inhibiting Ang II signaling. This study aims at establishing if miR-34a interacts with ATRAP/Agtrap and modulates Ang II signaling in arterial VSMC. miR-34a and gene expression was quantified by RT-qPCR. Protein levels were assessed by immunoblotting and immunohistochemistry. The direct binding of miR-34a to target mRNA was assessed by 3’UTR-luciferase assay. Results: miR-34a exhibits an age-associated increase in Rhesus Monkeys common carotid artery and rat aorta. Further, AGTRAP protein expression is lower in old rat VSMC and in old mice aorta. Ang II enhances miR-34a in old rat VSMC and human aortic smooth muscle cells (HASMC), and inhibits AGTRAP and SIRT1 mRNA/protein expression. In miR-34a-overexpressing HASMC, AGTRAP and SIRT1 mRNA/protein decrease, and these effects are rescued by AGTRAP forced expression. We found that miR-34a directly targets AGTRAP and that AGTRAP downmodulation enhances miR-34a expression and decreases SIRT1 in HASMC. Finally, Ang II and miR-34a-induced upregulation of proinflammatory genes, IL-6, COX-2, MCP-1 and MFGE8 in HASMC, is abolished by AGTRAP forced expression. Conclusions: Our results show that Ang II upregulates miR-34a, activating a negative feedback-loop on AGTRAP that reinforces Ang II signaling. Further, we found an age-associated AGTRAP decrease in central arteries and VSMC, underlying a potential miR-34a/AGTRAP role in vascular aging. These results on miR-34a and vascular aging have been presented at the Poster session of the 2024 Basic Cardiovascular Science – American Heart Association meeting and a full manuscript has been submitted for publication.