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Rationale: Previous translational studies implicate plasma extracellular microRNA-30d (miR-30d) as a biomarker in left ventricular remodeling and clinical outcome in heart failure (HF) patients, although precise mechanisms remain obscure. Objective: To investigate the mechanism of miR-30d–mediated cardioprotection in HF. Methods and Results: In rat and mouse models of ischemic HF, we show that miR-30d gain of function (genetic, lentivirus, or agomiR-mediated) improves cardiac function, decreases myocardial fibrosis, and attenuates cardiomyocyte (CM) apoptosis. Genetic or locked nucleic acid–based knock-down of miR-30d expression potentiates pathological left ventricular remodeling, with increased dysfunction, fibrosis, and cardiomyocyte death. RNA sequencing of in vitro miR-30d gain and loss of function, together with bioinformatic prediction and experimental validation in cardiac myocytes and fibroblasts, were used to identify and validate direct targets of miR-30d. miR-30d expression is selectively enriched in cardiomyocytes, induced by hypoxic stress and is acutely protective, targeting MAP4K4 (mitogen-associate protein kinase 4) to ameliorate apoptosis. Moreover, miR-30d is secreted primarily in extracellular vesicles by cardiomyocytes and inhibits fibroblast proliferation and activation by directly targeting integrin α5 in the acute phase via paracrine signaling to cardiac fibroblasts. In the chronic phase of ischemic remodeling, lower expression of miR-30d in the heart and plasma extracellular vesicles is associated with adverse remodeling in rodent models and human subjects and is linked to whole-blood expression of genes implicated in fibrosis and inflammation, consistent with observations in model systems. Conclusions: These findings provide the mechanistic underpinning for the cardioprotective association of miR-30d in human HF. More broadly, our findings support an emerging paradigm involving intercellular communication of extracellular vesicle–contained miRNAs (microRNAs) to transregulate distinct signaling pathways across cell types. Functionally validated RNA biomarkers and their signaling networks may warrant further investigation as novel therapeutic targets in HF.

Rationale:Cardiac CITED4 is induced by exercise and is sufficient to cause physiological hypertrophy and mitigate adverse ventricular remodeling after ischemic injury. However, the role of endogenous CITED4 in response to physiological or pathological stress is unknown.Objective:To investigate the role of CITED4 in murine models of exercise and pressure overload.Methods and Results:We generated cardiomyocyte-specific CITED4 knockout mice (C4KO) and subjected them to an intensive swim exercise protocol as well as transverse aortic constriction (TAC). Echocardiography, western blotting, qPCR, immunohistochemistry, immunofluorescence, and transcriptional profiling for mRNA and miRNA expression were performed. Cellular crosstalk was investigated in vitro. CITED4 deletion in cardiomyocytes did not affect baseline cardiac size or function in young adult mice. C4KO mice developed modest cardiac dysfunction and dilation in response to exercise. After TAC, C4KOs developed severe heart failure with left ventricular dilation, impaired cardiomyocyte growth accompanied by reduced mammalian target of rapamycin (mTOR) activity and maladaptive cardiac remodeling with increased apoptosis, autophagy, and impaired mitochondrial signaling. Interstitial fibrosis was markedly increased in C4KO hearts after TAC. RNAseq revealed induction of a pro-fibrotic miRNA network. miR30d was decreased in C4KO hearts after TAC and mediated crosstalk between cardiomyocytes and fibroblasts to modulate fibrosis. miR30d inhibition was sufficient to increase cardiac dysfunction and fibrosis after TAC.Conclusions:CITED4 protects against pathological cardiac remodeling by regulating mTOR activity and a network of miRNAs mediating cardiomyocyte to fibroblast crosstalk. Our findings highlight the importance of CITED4 in response to both physiological and pathological stimuli.

Long noncoding-RNAs (lncRNAs) are critical regulators of cardiac development as well as pathological hypertrophy and heart failure (HF). However, their roles in exercise-induced physiological hypertrophy are unclear. Here, we used RNAseq to identify a novel class of cardiac lncRNAs that are dynamically regulated by exercise. We call these l ong n on c oding Ex ercise A ssociated C ardiac T ranscripts (lncExACTs). Among them, lncExACT1, a highly conserved lncRNA, is down-regulated in exercised hearts but upregulated in transverse aortic constriction (TAC)-induced pathological hypertrophy and HF. In primary neonatal cardiomyocytes (CMs), transfection of LNA antisense oligonucleotide complementary to lncExACT1 (GapmeR) was sufficient to inhibit lncExACT1 expression and increase CM size with a gene expression pattern consistent with physiological hypertrophy (increased PCG1α and α/βMHC ratio, all p vs . control). In contrast, lentiviral overexpression of lncExACT1 in primary CMs induced a pathological gene expression pattern (decreased PCG1α and β/αMHC ratio, with increased ANP and BNP, all p vs . control). In vivo, GapmeR treatment for two weeks reduced cardiac lncExACT1 expression (0.5-fold at 2 weeks), increased ventricular wall thickness and fractional shortening (FS, p =0.034 vs . control), increased heart weight to tibial length ratio (HW/TL) and α/βMHC. In contrast, injection of AAV9-lncExACT1 increased cardiac lncExACT1 (6-fold at 5 weeks) increased HW/TL as well as βMHC and ANP. Further, lncExACT1 acted as a sponge for microRNA-222 (a microRNA we previously reported is necessary for physiological cardiac growth) thereby increasing expression of microRNA-222 targets, including p27, a cell cycle inhibitor protein. Moreover, GapmeR injection reduced TAC-induced increase of interventricular septal end diastole (IVSd) and left Ventricular Posterior Wall Dimensions (LVPWd) without affecting FS. We conclude that inhibition of lncExACT1 is sufficient to induce physiological hypertrophy and protect against pathological hypertrophy, while induction of lncExACT1 promotes pathological hypertrophy. lncExACT1 appears to mediate these effects, at least in part, by acting as competing endogenous RNA for microRNA-222.

Aerobic exercise confers myriad benefits to human health but the mechanism by which it does so remain incompletely understood. We hypothesize that exercise works in part through circulating protein signaling. Healthy adult men (n, 12) participated in treadmill running sessions at both low (6 mph) and high intensity (maximal effort). We used pre- and post-exercise plasma and a high-throughput aptamer-based assay (SomaScan) to examine the acute impact of exercise on the plasma proteome. Acute aerobic exercise consistently alters the resting plasma proteome in an intensity-dependent fashion. Of 1,305 proteins assayed 184 (14%) and 598 (46%) change at low and high intensity respectively (FDR p < 0.05). 159 protein species (12%) are common to both intensities. Gene ontology analysis revealed enrichment of pathways associated with leukocyte chemotaxis and chylomicron metabolism at low intensity and Wnt signaling, neuronal axonogenesis, and nitric oxide metabolism pathways at high intensity. We used human sequencing data from the GTEX Consortium (Broad, Cambridge MA) to computationally infer the sources of increasing proteins and found major contributions from the cardiovascular, gastrointestinal, and nervous systems. We identified 43 cis-SNPs that approximate the upregulated proteomic response to acute exercise and used Mendelian randomization to infer a causal relationship between the exercise proteome and decreased muscle wasting in a UK Biobank cohort. Although guidelines present low and high intensity exercise as equivalent alternatives for health, these data suggest that distinct exercise intensities might offer common and distinct exercise benefits.

Background: Physiological cardiac hypertrophy occurs commonly in response to exercise and can protect against pathological stress. In contrast, pathological hypertrophy occurs in disease and often precedes heart failure . Although physiological and pathological hypertrophy often involve distinct signaling mechanisms, miR-222 is an exercise-induced microRNA that is required for physiological hypertrophy but is also induced in pathological hypertrophy. Here, we sought to define the role of miR-222 in pathological hypertrophy. Methods and Results: We generated miR-222 gain-of-function (GOF) models through cardiac-specific constitutive transgenic miR-222 expression (TgC-miR-222) or somatic expression with AAV9-miR-222. Loss-of-function (LOF) was achieved using a locked nucleic antimiR (LNA) specific for miR-222. miR-222 GOF and LOF models manifested normal cardiac structure and function at baseline. After transverse aortic constriction (TAC), TgC-miR-222 had less pathological hypertrophy as well as better cardiac function (FS = 37.9% vs 28.7%, p Conclusions: Although miR-222 is upregulated in both physiological and pathological hypertrophy, its roles in these conditions are quite different. In response to pressure overload, miR-222 inhibits pathological hypertrophy, adverse remodeling, and cardiac dysfunction. These findings reinforce the conceptual model that physiological and pathological hypertrophy are qualitatively distinct, rather than differing only in degree. Further, they suggest that miR-222 may hold promise as a potential therapeutic target in pathological cardiac hypertrophy and heart failure.

Despite significant declines in early mortality after acute myocardial infarction (MI), heart failure (HF) remains a significant chronic complication following MI. Small non-coding RNAs are short (Figure 1 ). Principal components of candidate RNAs selected from differential expression n RNA-seq were associated with LV phenotypes post-MI, specifically LV mass and fibrosis at 4-6 weeks post-MI and change in LV mass at 6 months. In animal and cellular models of ischemia, we found that (1) candidate miRNAs found in humans were expressed in a temporally and cell-specific fashion in the myocardium and (2) candidate miRNA expression increased in cardiomyocyte culture after hypoxia/reoxygenation. Collectively, these data add to a burgeoning literature implicating plasma circulating ex-RNAs as functional markers of acute cardiovascular disease involved in cardiac remodeling en route to HF.