Background-Recently, elevation of circulating muscle-specific microRNA (miRNA) levels has been reported in patients with acute myocardial infarction. However, it is still unclear from which part of the myocardium or under what conditions miRNAs are released into circulating blood. The purpose of this study was to identify the source of elevated levels of circulating miRNAs and their function in cardiovascular diseases. Methods and Results-Serum levels of miRNA (miR)-1 and miR-133a were increased significantly in patients not only with acute myocardial infarction but also with unstable angina pectoris and Takotsubo cardiomyopathy without elevation of serum creatine phosphokinase or cardiac troponin. MicroRNA microarray analysis of the heart from a mouse model of myocardial infarction indicated that the levels of miR-1, miR-133a, miR-208a, and miR-499 were significantly reduced in the infarcted myocardium. In situ hybridization of miR-133a also showed that miR-133a levels were very low in the infarcted and peri-infarcted myocardium. It has been shown that circulating miRNAs are localized inside exosomes, which are released after Ca 2ϩ stimulation. We stimulated H9c2 cardiomyoblasts with A23187 and measured miR-133a levels in the exosome fraction of the culture medium. A23187 induced a dose-dependent release of miR-133a, and significant elevation was observed only at concentrations where dead cells were detected. We also found that miR-133a-containing exosomes reduced the luciferase activity of 293FT cells transfected with an miR-133a sensor vector. Conclusions-These results suggest that elevated levels of circulating miR-133a in patients with cardiovascular diseases originate mainly from the injured myocardium. Circulating miR-133a can be used as a marker for cardiomyocyte death, and it may have functions in cardiovascular diseases. (Circ Cardiovasc Genet. 2011;4:446-454.)Key Words: circulating microRNA Ⅲ myocardial infarction Ⅲ cell death Ⅲ calcium ionophore M icroRNAs (miRNAs) are endogenous, single-stranded, Ϸ22-nucleotide noncoding RNAs. MicroRNAs are generally regarded as negative regulators of gene expression through inhibition of translation and/or promotion of mRNA degradation by base-pairing to complementary sequences within the 3Ј untranslated region (3ЈUTR) of protein-coding mRNA transcripts. 1 The first miRNA assigned to a specific function was lin-4, which targets lin-14 during temporal pattern formation in Caenorhabditis elegans. 2 Since then, a variety of miRNAs have been discovered. More than 500 miRNAs have been cloned and sequenced in humans, and the estimated number of miRNA genes may be as high as 1000 in the human genome. 3 Each miRNA regulates dozens to hundreds of distinct target genes; thus miRNAs are estimated to regulate the expression of more than one-third of human protein-coding genes. 4 Clinical Perspective on p 454The implications of miRNAs in the pathological process of the cardiovascular system have been recognized recently, and research on miRNAs in relation to cardiovascular dise...

Sterol regulatory element-binding protein 2 (SREBP-2) transcription factor has been identified as a key protein in cholesterol metabolism through the transactivation of the LDL receptor and cholesterol biosynthesis genes. Here, we generated mice lacking microRNA (miR)-33, encoded by an intron of the Srebp2, and showed that miR-33 repressed the expression of ATP-binding cassette transporter A1 (ABCA1) protein, a key regulator of HDL synthesis by mediating cholesterol efflux from cells to apolipoprotein A (apoA)-I. In fact, peritoneal macrophages derived from miR-33-deficient mice showed a marked increase in ABCA1 levels and higher apoA-I-dependent cholesterol efflux than those from WT mice. ABCA1 protein levels in liver were also higher in miR-33-deficient mice than in WT mice. Moreover, miR-33-deficient mice had significantly higher serum HDL cholesterol levels than WT mice. These data establish a critical role for miR-33 in the regulation of ABCA1 expression and HDL biogenesis in vivo.A TP-binding cassette transporter A1 (ABCA1), a 254-kDa cytoplasmic membrane protein, is a pivotal regulator of lipid efflux from cells to apolipoproteins (1). ABCA1 mediates the ratecontrolling step in HDL particle formation and plays an important role in reverse cholesterol transfer (2, 3). Mutations in the ABCA1 gene cause Tangier disease, which is characterized by the near absence of plasma HDL cholesterol associated with storage of cholesterol esters in reticuloendothelial tissues (4-7). Abca1 mRNA and protein are very unstable, with a half life of 1-2 h in murine macrophages (8), which indicates that new transcription and translation are major factors in ensuring constant and inducible ABCA1 expression.Sterol regulatory element-binding proteins (SREBPs), including SREBP-1a, -1c, and -2, modulate the transcription of a number of genes involved in the synthesis and receptor-mediated uptake of cholesterol and fatty acids (9-11). In sterol-depleted cells, SREBPs are cleaved by proteases in the Golgi, releasing the N-termini, which translocate into the nucleus and bind to SREs in the enhancers of multiple genes encoding enzymes and proteins involved in cholesterol biosynthesis and lipid uptake (11-13). Results to date support the notion that SREBP-1 primarily activates the fatty acid triglyceride and phospholipid pathways, whereas SREBP-2 is the prominent isoform for cholesterol synthesis and uptake (9,10,12).MicroRNAs (miRs) are small, non-protein-coding RNAs that base pair with specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports have indicated that miR-33 controls cholesterol homeostasis based on knockdown experiments using antisense technology (14-16). Antisense inhibition of miRNA function has been an important tool for elucidating miRNA biology. However, to determine the potential developmental function of specific miRNAs and to perform longer-term studies, it is necessary to generate mice lacking each miRNA. We generated miR-33-deficient mice, which were born at the expected Mendelian ratio, a...

Objective We previously showed that cholesterol loading in vitro converts mouse aortic vascular smooth muscle cells (VSMC) from a contractile state to one resembling macrophages. In human and mouse atherosclerotic plaques it has become appreciated that ~40% of cells classified as macrophages by histological markers may be of VSMC origin. We therefore sought to gain insight into the molecular regulation of this clinically relevant process. Approach and Results VSMC of mouse (or human) origin were incubated with cyclodextrin-cholesterol complexes for 72 hours, at which time the expression at the protein and mRNA levels of contractile-related proteins were reduced and of macrophage markers increased. Concurrent was down regulation of miR-143/145, which positively regulate the master VSMC-differentiation transcription factor myocardin (MYOCD). Mechanisms were further probed in mouse VSMC. Maintaining the expression of MYOCD or miR-143/145 prevented and reversed phenotypic changes caused by cholesterol loading. Reversal was also seen when cholesterol efflux was stimulated after loading. Notably, despite expression of macrophage markers, bioinformatic analyses showed that cholesterol-loaded cells remained closer to the VSMC state, consistent with impairment in classical macrophage functions of phagocytosis and efferocytosis. In apoE-deficient atherosclerotic plaques, cells positive for VSMC and macrophage markers were found lining the cholesterol-rich necrotic core. Conclusions Cholesterol loading of VSMC converts them to a macrophage–appearing state by downregulating the miR-143/145-myocardin axis. Though these cells would be classified by immunohistochemistry as macrophages in human and mouse plaques, their transcriptome and functional properties imply that their contributions to atherogenesis would not be those of classical macrophages.

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AimsA significant increase in congestive heart failure (CHF) was reported when the anti-ErbB2 antibody trastuzumab was used in combination with the chemotherapy drug doxorubicin (Dox). The aim of the present study was to investigate the role(s) of miRNAs in acute Dox-induced cardiotoxicity.Methods and resultsNeuregulin-1-ErbB signalling is essential for maintaining adult cardiac function. We found a significant reduction in ErbB4 expression in the hearts of mice after Dox treatment. Because the proteasome pathway was only partially involved in the reduction of ErbB4 expression, we examined the involvement of microRNAs (miRs) in the reduction of ErbB4 expression. miR-146a was shown to be up-regulated by Dox in neonatal rat cardiac myocytes. Using a luciferase reporter assay and overexpression of miR-146a, we confirmed that miR-146a targets the ErbB4 3′UTR. After Dox treatment, overexpression of miR-146a, as well as that of siRNA against ErbB4, induced cell death in cardiomyocytes. Re-expression of ErbB4 in miR-146a-overexpressing cardiomyocytes ameliorated Dox-induced cell death. To examine the loss of miR-146a function, we constructed ‘decoy’ genes that had tandem complementary sequences for miR-146a in the 3′UTR of a luciferase gene. When miR-146a ‘decoy’ genes were introduced into cardiomyocytes, ErbB4 expression was up-regulated and Dox-induced cell death was reduced.ConclusionThese findings suggested that the up-regulation of miR-146a after Dox treatment is involved in acute Dox-induced cardiotoxicity by targeting ErbB4. Inhibition of both ErbB2 and ErbB4 signalling may be one of the reasons why those patients who receive concurrent therapy with Dox and trastuzumab suffer from CHF.

GLUT4 shows decreased levels in failing human adult hearts. We speculated that GLUT4 expression in cardiac muscle may be fine-tuned by microRNAs. Forced expression of miR-133 decreased GLUT4 expression and reduced insulin-mediated glucose uptake in cardiomyocytes. A computational miRNA target prediction algorithm showed that KLF15 is one of the targets of miR-133. It was confirmed that over-expression of miR-133 reduced the protein level of KLF15, which reduced the level of the downstream target GLUT4. Cardiac myocytes infected with lenti-decoy, in which the 3'UTR with tandem sequences complementary to miR-133 was linked to the luciferase reporter gene, had decreased miR-133 levels and increased levels of GLUT4. The expression levels of KLF15 and GLUT4 were decreased at the left ventricular hypertrophy and congestive heart failure stage in a rat model. The present results indicated that miR-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiomyocytes.

MicroRNAs (miRNAs or miRs) are small, non-coding RNAs that modulate mRNA stability and post-transcriptional translation. A growing body of evidence indicates that specific miRNAs can affect the cellular function of cardiomyocytes. In the present study, miRNAs that are highly expressed in the heart were overexpressed in neonatal rat ventricular myocytes, and cellular ATP levels were assessed. As a result, miR-15b, -16, -195, and -424, which have the same seed sequence, the most critical determinant of miRNA targeting, decreased cellular ATP levels. These results suggest that these miRNAs could specifically down-regulate the same target genes and consequently decrease cellular ATP levels. Through a bioinformatics approach, ADPribosylation factor-like 2 (Arl2) was identified as a potential target of miR-15b. It has already been shown that Arl2 localizes to adenine nucleotide transporter 1, the exchanger of ADP/ATP in mitochondria. Overexpression of miR-15b, -16, -195, and -424 suppressed the activity of a luciferase reporter construct fused with the 3-untranslated region of Arl2. In addition, miR-15b overexpression decreased Arl2 mRNA and protein expression levels. The effects of Arl2 siRNA on cellular ATP levels were the same as those of miR-15b, and the expression of Arl2 could restore ATP levels reduced by miR-15b. A loss-of-function study of miR-15b resulted in increased Arl2 protein and cellular ATP levels. Electron microscopic analysis revealed that mitochondria became degenerated in cardiomyocytes that had been transduced with miR-15b and Arl2 siRNA. The present results suggest that miR-15b may decrease mitochondrial integrity by targeting Arl2 in the heart. MicroRNAs (miRNAs or miRs)2 are small, non-coding RNAs that modulate mRNA stability and post-transcriptional translation. A growing body of evidence indicates that miRNAs are involved in basic cell functions, including early development and oncogenesis. In the heart, microarray analysis has shown that the expression profile of miRNAs is altered in human heart disease and animal models of cardiac hypertrophy or heart failure (1-3). Numerous studies have revealed that various subcellular organelles, such as the extracellular matrix, myofibrils, sarcoplasmic reticulum, nucleus, and mitochondria, undergo various changes in biochemical composition and structure in cardiac diseases (4 -8, 10, 11). These findings imply an association between miRNAs and subcellular organelles, and the identification of the miRNAs and their target genes that affect subcellular organelles may lead to new understandings in cardiac pathophysiology.The present study focused on the relationship in cardiomyocytes between specific miRNAs, cellular ATP levels, and mitochondria, which are highly abundant and constitute ϳ40% of the total cardiomyocyte volume in the heart (9) and mostly generate cellular ATP. To identify miRNAs that can affect cellular ATP levels and mitochondria, the cellular ATP levels of cardiac myocytes, in which a specific miRNA was overexpressed, were assessed. A seri...

Retrovirus insertion-mediated random mutagenesis was applied in 3T3-L1 preadipocyte cells to better understand the molecular basis of obesity (the expansion of individual adipocytes). We found that tryptophan hydroxylase-1, a rate-limiting enzyme for the synthesis of serotonin (5-HT), is expressed in adipocytes and is required for their differentiation. A 5-HT type 2A receptor (5-HT(2A)R) antagonist, ketanserin, and a 5-HT(2c)R antagonist, SB-242084, inhibited adipocyte differentiation. Because 5-HT(2c)R mRNA levels are up-regulated during adipocyte differentiation and micro-RNA (miR)-448 is located in the fourth intron of Htr2c, we also studied the role of miR-448 in 3T3-L1 cells. Through a bioinformatics approach, Krüppel-like factor 5 (KLF5) was identified as a potential target of miR-448. Using a luciferase reporter assay, we confirmed that miR-448 targets the Klf5 3'-intranslated region. Overexpression of miR-448 reduced the expression of Klf5 and adipocyte differentiation, which was confirmed by the reduced expression of adipogenic genes and triglyceride accumulation. To examine the loss of miR-448 function, we constructed a decoy gene that had tandem complementary sequences for miR-448 in the 3'-untranslated region of a luciferase gene under the control of a cytomegalovirus promoter. When the miR-448 decoy gene was introduced into 3T3-L1 preadipocytes, KLF5 was up-regulated and triglyceride concentration was increased. In this study, we identified the regulation of adipocyte differentiation by 5-HT, 5-HT(2A)R, and 5-HT(2C)R. miR-448-mediated repression of KLF5 was identified as a negative regulator for adipocyte differentiation.