Non-coding RNAs (ncRNAs) in Cardiovascular Aging

National Institute On Aging

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Abstract

Some projects that require expertise in microRNAs and, specifically, in miR-200 family and miR-34a, have been initiated. It has been shown that miR-200 family members (co-transcribed miR-200c, -141; co-transcribed miR-200b, -200a, -429) and miR-34a increase in response to oxidative stress suggesting that these miRNAs may play a functional role in conditions associated with enhanced production of reactive oxygen species (ROS) such as aging and diabetes. Some miR-200 family members exhibit an age-associated increase in human skin fibroblasts and liver, and in non-human primates skeletal muscle. Further, miR-200c increases within the myocardium of diabetic rodents, in femoral arteries of diabetic rodents and in skin cells of patients with type 2 diabetes and diabetic foot ulcers. Dr Capogrossi directs the conception and implementation of the following ongoing projects on aging, diabetes and obesity: 1. Establish the role of miR-200 family members in myocardial function and cardiac fibrosis, and its potential link to systolic and diastolic dysfunction; 2. Establish the role of miR-200 family members and miR-34a in vascular dysfunction; 3. Establish the expression level of circulating miR-200 family members in human aging, diabetes and obesity; 4. Establish the role of miR-200 family members in skeletal muscle function. REPORTS 2023 1. miR-200 family members in cardiac function. Oxidative stress is defined as a dysregulation between the production of ROS and endogenous antioxidant defense mechanisms. Reactive oxygen species (ROS) upregulate miR-200c via a p53-dependent mechanism and p53 is implicated in the induction of apoptosis and senescence. MiR-200c upregulation induces growth arrest, senescence, and apoptosis through the inhibition of ZEB1 and SIRT1/eNOS/FOXO1 regulatory loop by directly targeting all of them. Diminished SIRT1 expression associates with cell senescence. Moreover, miR-200c increases ROS production by 2 mechanisms: (1) it decreases ROS scavengers by targeting peroxiredoxin 2 (PRDX2), and FOXO1, a transcription factor required for catalase (CAT) and manganese superoxide dismutase (MnSOD) expression; (2) it induces ROS production via p66Shc phosphorylation in Serine 36. Therefore, a positive feedback loop occurs between enhanced oxidative stress increasing miR-200c expression and miR-200c further increasing oxidative stress. Conditions such as aging, diabetes, obesity are associated with increased oxidative stress in the heart and lead to the impairment of cellular differentiation and proliferation, alterations in excitation-contraction coupling, cardiac fibrosis and heart failure. Further, miR-200c targets ACE2, responsible for the conversion of pro-inflammatory and pro-fibrotic Angiotensin II into anti-inflammatory and anti-fibrotic Angiotensin 1,7; the decreased ACE2 expression is expected to enhance proinflammatory AT1 receptor signaling and decrease anti-inflammatory MAS receptor signaling. The expression of miR-200 family members was evaluated in diabetic rats myocardial cells. Diabetes was induced with the intraperitoneal injection of 50 mg/kg streptozotocin (STZ). Co-transcribed miR-141 and miR-200c significantly increased in cardiomyocytes isolated form diabetic rats; in contrast, co-transcribed miR-200a, -200b and -429 expression did 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 transient. Further, we also found a significant prolongation of the caffeine-triggered cytosolic calcium transient, in the absence of electrically stimulated action potential. Moreover, miR-200c and miR-141 are upregulated in vitro, in human umbilical vein endothelial cells grown in high glucose, and in vivo, in mouse endothelial cells, bone marrow-derived endothelial progenitor cells, human skin fibroblasts, and human skin. To establish the role of miR-200 family members in myocardial function and its potential link to systolic and diastolic dysfunction in aging and diabetes, we used a mouse model where the locus with miR-141 and -200c is deleted. Cardiac fractional shortening evaluated by echocardiography increased in KO mice when compared to WT animals. However, it was not possible to induce diabetes in this model since the ablation of miR-141/-200c protects against STZ-induced beta cell death and the consequent diabetes induction. To better evaluate the role of miR-200c, we created, in collaboration with the Mouse Cancer Genetics Program at the NCI/CCR, a mouse model where only miR-200c is deleted. The CRISPR/Cas9 genome editing technology was used to introduce loxP sites flanking the miR-200c genomic sequence in mouse. Specifically, two guide RNAs were designed to target the region upstream and downstream of the miR-200c genomic locus. A single strand DNA oligonucleotide (ssDNA oligo; 907 nt) containing the loxP sites flanking the specific miR-200c sequence and homology regions was used as donor template for the homologous recombination process. In this new animal model it was possible to induce diabetes with the combination of high fat diet and a single high dose of STZ (as reported by Bengt-Frederik Belgardt et al, Nat Med 2015 in miR-141/-200c KO mice). In addition of a variety of metabolic parameters (e.g. glycemia and insulin resistance) we are using echocardiography and MRI to serially assess changes in cardiac morphometry and function and MRI to quantify cardiac fibrosis. The MRI experiments are implemented in collaboration with Dr. Richard Spencer and Dr Ken Fishbein at the NIA. A number of histologic and molecular end-points will be evaluated following animal sacrifice at 20 weeks of age. This approach, will allow us to establish if miR-200c deletion prevents cardiac dysfunction and cardiac fibrosis associated with diabetes, obesity and insulin resistance. Some preliminary results have been presented as a poster presentation at the Cardiac Regulatory mechanisms Gordon Research Seminar and Conference in June 2022. 2. miR-34a in vascular dysfunction in cardiovascular aging. The Renin-angiotensin-aldosterone system (RAAS) plays a key role in cardiovascular aging and disease. Renin secretion is the first step in the activation of the RAAS pathway. Renin cleaves angiotensinogen to form Ang I, which is then transformed into Ang II by ACE and chymase enzyme. Specific receptors, AT1R and AT2R, can then bind Ang II; AT1R has a pro-inflammatory and pro-fibrotic action, increases blood pressure, promotes cardiac remodeling and atherosclerosis; in contrast, AT2R and MAS receptor signaling have opposite effects. ACE2 cleaves Ang I and Ang II to form Ang(19) to Ang(17), respectively; Ang(17) has anti-inflammatory, antifibrotic, and anti-remodeling effects through the MAS receptor. Importantly, ATIR signaling is inhibited by Angiotensin II receptor-associated protein (AGTRAP), a transmembrane protein that enhances AT1R internalization. Presently, is still unknown if AGTRAP expression in central arteries is modulated by aging. We demonstrated that miR-34a increases in monkey common carotid artery (CCA), in rat aortic wall, and CCA in an age dependent manner. BY the luciferase activity assay, we demonstrated that AGTRAP is directly targeted by miR-34a. AGTRAP mRNA and protein expression were lower in rat vascular smooth muscle cells (VSMCs) isolated from old rats and human VSMCs overexpressing miR-34a. Moreover, miR-34a expression negatively correlated with SIRT1 and AGTRAP in human atherosclerotic carotid plaques. The results of our work point to miR-34a-induced age-dependent decrease in AGTRAP as a novel mechanism for the increased AT1R signaling, a key player in cardiovascular aging. A manuscript is currently in preparation.