Role of Human Epicardial Adipose Tissue–Derived miR-92a-3p in Myocardial Redox State

Background Visceral obesity is directly linked to increased cardiovascular risk, including heart failure. Objectives This study explored the ability of human epicardial adipose tissue (EAT)-derived microRNAs (miRNAs) to regulate the myocardial redox state and clinical outcomes. Methods This study screened for miRNAs expressed and released from human EAT and tested for correlations with the redox state in the adjacent myocardium in paired EAT/atrial biopsy specimens from patients undergoing cardiac surgery. Three miRNAs were then tested for causality in an in vitro model of cardiomyocytes. At a clinical level, causality/directionality were tested using genome-wide association screening, and the underlying mechanisms were explored using human biopsy specimens, as well as overexpression of the candidate miRNAs and their targets in vitro and in vivo using a transgenic mouse model. The final prognostic value of the discovered targets was tested in patients undergoing cardiac surgery, followed up for a median of 8 years. Results EAT miR-92a-3p was related to lower oxidative stress in human myocardium, a finding confirmed by using genetic regulators of miR-92a-3p in the human heart and EAT. miR-92a-3p reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase–derived superoxide (O2.–) by targeting myocardial expression of WNT5A, which regulated Rac1-dependent activation of NADPH oxidases. Finally, high miR-92a-3p levels in EAT were independently related with lower risk of adverse cardiovascular events. Conclusions EAT-derived miRNAs exert paracrine effects on the human heart. Indeed miR-92a-3p suppresses the wingless-type MMTV integration site family, member 5a/Rac1/NADPH oxidase axis and improves the myocardial redox state. EAT-derived miR-92a-3p is related to improved clinical outcomes and is a rational therapeutic target for the prevention and treatment of obesity-related heart disease.

can affect cardiac physiology in a paracrine way, due to its close anatomical relationship with the myocardium. [2][3][4] Under physiological conditions, there is a continuous cross-talk between EAT and the myocardium, essential for the maintenance of myocardial health.
Although the EAT secretome in chronic diseases may have cardioprotective properties, metabolic dysregulation leads to a shift of the EAT secretome to a pro-oxidant and proinflammatory profile, with detrimental effects on the human heart. The nature of these communication signals between EAT and the heart is poorly understood.

MicroRNAs
(miRNAs) are highly conserved, small, single-stranded noncoding RNAs that mainly act as negative posttranscriptional regulators and are rational therapeutic targets in CVD. 5 Evidence suggests that adipose tissue secretes miRNAs encapsulated into extracellular vesicles that can travel to other organs, affecting their transcriptomic profile. 6,7 The role of EATderived miRNAs in the regulation of myocardial biology is unclear. The current study investigated the hypothesis that EAT could release miRNAs able to affect the myocardial redox state and hence CVD development.
We identified lead miRNAs secreted by human EAT and explored their role in the modulation of myocardial redox signaling, as well as their implication for clinical outcomes in patients with coronary heart disease. The demographic characteristics of these studies are presented in Table 1. Supplemental Figure 1 depicts the study population, goals, and research methodologies of every study arm.

HUMAN TISSUE HARVESTING AND PROCESSING.
Human EAT (Supplemental Figure 2) and its secretome, as well as myocardial biopsy samples, were harvested and processed as described in the Supplemental Methods.
To support our hypothesis that EAT-derived miR-92a-3p is causally associated with myocardial redox state in humans, we performed genetic screening   16 Therefore, we investigated whether the observed inhibitory effects of miR-92a-3p on NADPH oxidase activity were mediated via the Akt/Rac1 axis.
Seven SNPs were significantly associated with high miR-92a-3p levels in EAT (EAT-miR-92a-3p) and 31 with high levels of miR-92a-3p in the myocardium (MYO-miR-92a-3p). The presence of any SNP from EAT-miR-92a-3p was associated with higher miR-92a-3p levels only in EAT, not in the myocardium (B, n ¼ 149; C, n ¼ 265), whereas the presence of any MYO-miR-92a-3p SNP was associated with higher miR-92a-3p levels only in the myocardium, not in EAT (G, n ¼ 265; F, n ¼ 149). The presence of any EAT-miR-92a-3p SNP led to a statistically significant reduction of myocardial superoxide production (D and H, n ¼ 196). In B to D and F to H, data are presented as median (25th-75th percentile).
To understand the relevance of these findings in humans, we assessed miR-92a-3p levels in EAT and the expression levels of these 2 potential miR-92a-3p targets in myocardial samples from patients of study arm 2. Patients with high miR-92a-3p levels in EAT exhibited lower myocardial levels of WNT5A, whereas there were no significant differences in PHLPP2 expression in the myocardium of patients with low vs high EAT miR-92a-3p levels ( Figures 4D and 4F).
Collectively, these data suggest that Wnt5a could be a target of miR-92a-3p with a role in myocardial Rac1-   cardiomyocytes transfected with either miR-NC or a miR-92a-3p mimic AE Akt inhibitor perifosine (n ¼ 5). Lines on scatterplots represent medians. *P < 0.05, **P < 0.01 vs controls by Wilcoxon signed-rank test. Abbreviations as in Figure 1.
Although there was no significant difference in most of the risk factors between groups, patients with high miR-92a-3p levels were more likely to be taking antiplatelet medication or to have hypercholesterolemia (Supplemental Table 1).

DISCUSSION
Adipose tissue communicates with the cardiovascular system via the secretion of endocrine and paracrine signals, which include adipokines/adipocytokines, 3,24 lipid species, 25 microRNAs, 26  Finally, the paired RAA and the EAT from the atrioventricular groove were in anatomical proximity but not directly adjacent to each other; however, samples from the right atrioventricular groove have similar miR-92a-3p expression compared with samples directly adjacent to the RAA (Supplemental Figure 2).

CONCLUSIONS
The current study shows that high EAT-derived miR-