Mir-30d Regulates Cardiac Remodeling by Intracellular and Paracrine Signaling.

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.

Y RNAs: Biogenesis, Function and Implications for the Cardiovascular System.

In recent years, progress in the field of high-throughput sequencing technology and its application to a wide variety of biological specimens has greatly advanced the discovery and cataloging of a diverse set of non-coding RNAs (ncRNAs) that have been found to have unexpected biological functions. Y RNAs are an emerging class of highly conserved, small ncRNAs. There is a growing number of reports in the literature demonstrating that Y RNAs and their fragments are not just random degradation products but are themselves bioactive molecules. This review will outline what is currently known about Y RNA including biogenesis, structure and functional roles. In addition, we will provide an overview of studies reporting the presence and functions attributed to Y RNAs in the cardiovascular system.

MicroRNAs are associated with cardiac biomarkers, cardiac structure and function and incident outcomes in heart failure.

AIMS: The association between microRNAs (miRNAs) and established cardiac biomarkers is largely unknown. We aimed to measure the association between plasma miRNAs and N-terminal pro-B-type natriuretic peptide (NT-proBNP), cardiac troponin I, soluble urokinase-type plasminogen activator receptor (suPAR), and galectin-3 with cardiac structure and function and clinical outcomes. METHODS AND RESULTS: We quantified 32 plasma miRNAs using the FirePlex miRNA assay and measured biomarkers in 139 individuals with symptomatic heart failure (HF). We used principal component (PC) analysis and linear regression to evaluate the association between miRNAs and biomarkers with ventricular size and function by echocardiography and Cox modelling for the incidence of a first composite event of HF hospitalization, heart transplant, left ventricular assist device implant, or death. The mean (standard deviation) age at baseline was 64.3 (12.4) years, 33 (24%) were female, and 122 (88%) were White. A total of 45 events occurred over a median follow-up of 368 (interquartile range 234, 494) days. Baseline NT-proBNP (ß = -2.0; P = 0.001) and miRNA PC2 (ß = 2.6; P = 0.002) were associated with baseline left ventricular ejection fraction. NT-proBNP (ß = 20.6; P = 0.0004), suPAR (ß = -39.6; P = 0.005), and PC4 (ß = 21.1; P = 0.02) were associated with baseline left ventricular end-diastolic volumes. NT-proBNP [hazard ratio (HR) 1.67, 95% confidence interval (CI) 1.28-2.18, P = 0.0002], galectin-3 (HR 2.02, 95% CI 1.05-3.91, P = 0.036), PC3 (HR 1.75, 95% CI 1.23-2.49, P = 0.002), and PC4 (HR 1.67, 95% CI 1.1-2.52, P = 0.016) were independently associated with incident events. CONCLUSIONS: Biomarkers and miRNA PCs are associated with cardiac structure and function and incident cardiovascular outcomes. Combining information from miRNAs provides prognostic information beyond biomarkers in HF.

Mitochondrial Ca 2+ uniporter (MCU) variants form plasma-membrane channels.

MCU is widely recognized as a responsible gene for encoding a pore-forming subunit of highly mitochondrial-specific and Ca 2+ -selective channel, mitochondrial Ca 2+ uniporter complex (mtCUC). Here, we report a novel short variant derived from the MCU gene (termed MCU-S) which lacks mitochondria-targeted sequence and forms a Ca 2+ - permeable channel outside of mitochondria. MCU-S was ubiquitously expressed in all cell-types/tissues, with particularly high expression in human platelets. MCU-S formed Ca 2+ channels at the plasma membrane, which exhibited similar channel properties to those observed in mtCUC. MCU-S channels at the plasma membrane served as an additional Ca 2+ influx pathway for platelet activation. Our finding is completely distinct from the originally reported MCU gene function and provides novel insights into the molecular basis of MCU variant-dependent cellular Ca 2+ handling.

A Novel Tissue Atlas and Online Tool for the Interrogation of Small RNA Expression in Human Tissues and Biofluids.

One promising goal for utilizing the molecular information circulating in biofluids is the discovery of clinically useful biomarkers. Extracellular RNAs (exRNAs) are one of the most diverse classes of molecular cargo, easily assayed by sequencing and with expressions that rapidly change in response to subject status. Despite diverse exRNA cargo, most evaluations from biofluids have focused on small RNA sequencing and analysis, specifically on microRNAs (miRNAs). Another goal of characterizing circulating molecular information, is to correlate expression to injuries associated with specific tissues of origin. Biomarker candidates are often described as being specific, enriched in a particular tissue or associated with a disease process. Likewise, miRNA data is often reported to be specific, enriched for a tissue, without rigorous testing to support the claim. Here we provide a tissue atlas of small RNAs from 30 different tissues and three different blood cell types. We analyzed the tissues for enrichment of small RNA sequences and assessed their expression in biofluids: plasma, cerebrospinal fluid, urine, and saliva. We employed published data sets representing physiological (resting vs. acute exercise) and pathologic states (early- vs. late-stage liver fibrosis, and differential subtypes of stroke) to determine differential tissue-enriched small RNAs. We also developed an online tool that provides information about exRNA sequences found in different biofluids and tissues. The data can be used to better understand the various types of small RNA sequences in different tissues as well as their potential release into biofluids, which should help in the validation or design of biomarker studies.

SnRNA sequencing defines signaling by RBC-derived extracellular vesicles in the murine heart.

Extracellular vesicles (EVs) mediate intercellular signaling by transferring their cargo to recipient cells, but the functional consequences of signaling are not fully appreciated. RBC-derived EVs are abundant in circulation and have been implicated in regulating immune responses. Here, we use a transgenic mouse model for fluorescence-based mapping of RBC-EV recipient cells to assess the role of this intercellular signaling mechanism in heart disease. Using fluorescent-based mapping, we detected an increase in RBC-EV-targeted cardiomyocytes in a murine model of ischemic heart failure. Single cell nuclear RNA sequencing of the heart revealed a complex landscape of cardiac cells targeted by RBC-EVs, with enrichment of genes implicated in cell proliferation and stress signaling pathways compared with non-targeted cells. Correspondingly, cardiomyocytes targeted by RBC-EVs more frequently express cellular markers of DNA synthesis, suggesting the functional significance of EV-mediated signaling. In conclusion, our mouse model for mapping of EV-recipient cells reveals a complex cellular network of RBC-EV-mediated intercellular communication in ischemic heart failure and suggests a functional role for this mode of intercellular signaling.

MicroRNA-1-Mediated Inhibition of Cardiac Fibroblast Proliferation Through Targeting Cyclin D2 and CDK6.

MicroRNA-1 (miRNA-1) has been long viewed as a muscle-specific miRNA and plays a critical role in myocardium and cardiomyocytes by controlling myocyte growth and rhythm. We identified that miRNA-1 is expressed in cardiac fibroblasts, which are one of the major non-muscle cell types in myocardium and are responsible for cardiac fibrosis in pathological conditions. In this study, we aimed to investigate the effect and mechanism of action of miRNA-1 on cardiac fibroblast proliferation. Subcutaneous angiotensin II (Ang II) infusion via osmotic minipumps for 4 weeks was used to induce myocardial interstitial fibrosis in male Sprague-Dawley rats. MiRNA-1 expression was significantly down-regulated by 68% in freshly isolated ventricular fibroblasts from Ang II-infused rats than that from control rats. Similar results were obtained in adult rat ventricular fibroblasts that were stimulated in culture by Ang II or TGFß for 48 h. Functionally, overexpression of miRNA-1 inhibited fibroblast proliferation, whereas knockdown of endogenous miRNA-1 increased fibroblast proliferation. We then identified and validated cyclin D2 and cyclin-dependent kinase 6 (CDK6) as direct targets of miRNA-1 in cardiac fibroblasts using biochemical assays. Moreover, we showed that the inhibitory effects of miRNA-1 on cardiac fibroblast proliferation can be blunted by overexpression of its target, cyclin D2. In conclusion, our findings demonstrate miRNA-1 expression and regulation in adult ventricular fibroblasts, where it acts as a novel negative regulator of adult cardiac fibroblast proliferation that is at least partially mediated by direct targeting of two cell cycle regulators. Our results expand the understanding of the regulatory roles of miRNA-1 in cardiac cells (i.e., from myocytes to a major non-muscle cells in the heart).