MicroRNAs Are Involved in Homocysteine-Induced Cardiac Remodeling

Elevated level of homocysteine (Hcy) called hyperhomocysteinemia (HHcy) is one of the major risk factors for chronic heart failure. Although the role of Hcy in cardiac remodeling is documented, the regulatory mechanism involved therein is still nebulous. MicroRNAs (miRNAs) and dicer have been implicated in regulation of cardiovascular diseases. Dicer is the only known enzyme involved in miRNA maturation. We investigated the involvement of dicer and miRNA in Hcy-induced cardiac remodeling. HL-1 cardiomyocytes were cultured in different doses of Hcy. Total RNA was isolated and RT-PCR and real-time PCR was performed for dicer, MMP-2,-9, TIMP-1,-3, and NOX-4. MiRNA microarray was used for analyzing the differential expression of miRNAs. Individual miRNA assay was also done. Western blotting was used to assess the MMP-9 expression in HHcy cardiomyocytes. The RT-PCR results suggest that dicer expression is enhanced in HHcy cardiomyocytes suggesting its involvement in cardiac remodeling caused due to high dose of Hcy. On the other hand, high dose of Hcy increased NOX-4 expression, a marker for oxidative stress. Additionally, HHcy cardiomyocytes showed elevated levels of MMP-2,-9 and TIMP-1,-3, and reduced expression of TIMP-4, suggesting cardiac remodeling due to oxidative stress. The miRNA microarray assay revealed differential expression of 11 miRNAs and among them miR-188 show dramatic downregulation. These findings suggest that dicer and miRNAs especially miR-188 are involved in Hcy-induced cardiac remodeling.     

Regulation of zebrafish heart regeneration by miR-133

Zebrafish regenerate cardiac muscle after severe injuries through the activation and proliferation of spared cardiomyocytes. Little is known about factors that control these events. Here we investigated the extent to which miRNAs regulate zebrafish heart regeneration. Microarray analysis identified many miRNAs with increased or reduced levels during regeneration. miR-133, a miRNA with known roles in cardiac development and disease, showed diminished expression during regeneration. Induced transgenic elevation of miR-133 levels after injury inhibited myocardial regeneration, while transgenic miR-133 depletion enhanced cardiomyocyte proliferation. Expression analyses indicated that cell cycle factors mps1, cdc37, and PA2G4, and cell junction components cx43 and cldn5, are miR-133 targets during regeneration. Using pharmacological inhibition and EGFP sensor interaction studies, we found that cx43 is a new miR-133 target and regeneration gene. Our results reveal dynamic regulation of miRNAs during heart regeneration, and indicate that miR-133 restricts injury-induced cardiomyocyte proliferation.     

MicroRNA-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiac myocytes

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.     

Gadd45α: A Novel Diabetes-Associated Gene Potentially Linking Diabetic Cardiomyopathy and Baroreflex Dysfunction

Both diabetic cardiomyopathy (DCM) and baroreflex dysfunction independently contribute to sudden cardiac death (SCD), however the inherent connections between them under diabetic state remains unclear. As microRNAs (miRNAs) have been reported to participate in various physiological and pathological processes, we presume they may also be involved in DCM and DM-induced impairment of baroreflex sensitivity. Two sets of gene expression profiles data from streptozotocin (STZ)-induced diabetic heart and diabetic dorsal root ganglia (DDRG) were retrieved from GEO and ArrayExpress. Co-differentially-expressed genes in diabetic heart and DDRG were identified by t test and intersection analysis. Human Protein Reference Database (HPRD) was applied to find direct interacting proteins of Gadd45α. Differentially-expressed miRNAs in left ventricle from 4-week STZ-induced diabetic rats were screened by miRNA microarray. Expression of miR-499 and its regulating effect on Gadd45α were then verified by quantitative real-time PCR (qRT-PCR), western blot, computational predication, and dual-luciferase reporter analysis. Four co-differentially-expressed genes in DCM and DDRG were identified. Among these genes, Gadd45α has 16 direct interacting proteins and 11 of them are documentedly associated with DM. Accompanied with significantly increased miR-499 expression, Gadd45α expression was increased at mRNA level but decreased at protein level in both diabetic heart and nucleus ambiguous. Furthermore, miR-499 was confirmed negatively regulating Gadd45α by targeting its 3′UTR. Collectively, reduced Gadd45α protein expression by forced miR-499 expression indicated it''s a diabetes-associated gene which might potentially be involved in both DCM and DM-induced baroreflex dysfunction.     

Differential Expression of Dicer, miRNAs, and Inflammatory Markers in Diabetic Ins2+/− Akita Hearts

Diabetic cardiomyopathy is a leading cause of morbidity and mortality, and Insulin2 mutant (Ins2+/?) Akita is a genetic mice model of diabetes relevant to humans. Dicer, miRNAs, and inflammatory cytokines are associated with heart failure. However, the differential expression of miRNAs, dicer, and inflammatory molecules are not clear in diabetic cardiomyopathy of Akita. We measured the levels of miRNAs, dicer, pro-inflammatory tumor necrosis factor alpha (TNFα), and anti-inflammatory interleukin 10 (IL-10) in C57BL/6J (WT) and Akita hearts. The results revealed increased heart to body weight ratio and robust expression of brain natriuretic peptide (BNP: a hypertrophy marker) suggesting cardiac hypertrophy in Akita. The multiplex RT-PCR, qPCR, and immunoblotting showed up regulation of dicer, whereas miRNA array elicited spread down regulation of miRNAs in Akita including dramatic down regulation of let-7a, miR-130, miR-142-3p, miR-148, miR-338, miR-345-3p, miR-384-3p, miR-433, miR-450, miR-451, miR-455, miR-494, miR-499, miR-500, miR-542-3p, miR-744, and miR-872. Conversely, miR-295 is induced in Akita. Cardiac TNFα is upregulated at mRNA (RT-PCR and qPCR), protein (immunoblotting), and cellular (immunohistochemistry and confocal microscopy) levels, and is robust in hypertrophic cardiomyocytes suggesting direct association of TNFα with hypertrophy. Contrary to TNFα, cardiac IL-10 is downregulated in Akita. In conclusion, induction of dicer and TNFα, and attenuation of IL-10 and majority of miRNA are associated with cardiomyopathy in Akita and could be used for putative therapeutic target for heart failure in diabetics.     

Urinary Exosomal MicroRNAs in Incipient Diabetic Nephropathy

MicroRNAs (miRNAs), a class of small non-protein-encoding RNAs, regulate gene expression via suppression of target mRNAs. MiRNAs are present in body fluids in a remarkable stable form as packaged in microvesicles of endocytic origin, named exosomes. In the present study, we have assessed miRNA expression in urinary exosomes from type 1 diabetic patients with and without incipient diabetic nephropathy. Results showed that miR-130a and miR-145 were enriched, while miR-155 and miR-424 reduced in urinary exosomes from patients with microalbuminuria. Similarly, in an animal model of early experimental diabetic nephropathy, urinary exosomal miR-145 levels were increased and this was paralleled by miR-145 overexpression within the glomeruli. Exposure of cultured mesangial cells to high glucose increased miR-145 content in both mesangial cells and mesangial cells-derived exosomes, providing a potential mechanism for diabetes-induced miR-145 overexpression. In conclusion, urinary exosomal miRNA content is altered in type 1 diabetic patients with incipient diabetic nephropathy and miR-145 may represent a novel candidate biomarker/player in the complication.     

MiRNA-1/133a Clusters Regulate Adrenergic Control of Cardiac Repolarization

The electrical properties of the heart are primarily determined by the activity of ion channels and the activity of these molecules is permanently modulated and adjusted to the physiological needs by adrenergic signaling. miRNAs are known to control the expression of many proteins and to fulfill distinct functions in the mammalian heart, though the in vivo effects of miRNAs on the electrical activity of the heart are poorly characterized. The miRNAs miR-1 and miR-133a are the most abundant miRNAs of the heart and are expressed from two miR-1/133a genomic clusters. Genetic modulation of miR-1/133a cluster expression without concomitant severe disturbance of general cardiomyocyte physiology revealed that these miRNA clusters govern cardiac muscle repolarization. Reduction of miR-1/133a dosage induced a longQT phenotype in mice especially at low heart rates. Longer action potentials in cardiomyocytes are caused by modulation of the impact of β-adrenergic signaling on the activity of the depolarizing L-type calcium channel. Pharmacological intervention to attenuate β-adrenergic signaling or L-type calcium channel activity in vivo abrogated the longQT phenotype that is caused by modulation of miR-1/133a activity. Thus, we identify the miR-1/133a miRNA clusters to be important to prevent a longQT-phenotype in the mammalian heart.     

Cardiac hypertrophy and impaired relaxation in transgenic mice overexpressing triadin 1

Triadin 1 is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum (SR), which forms a quaternary complex with the ryanodine receptor (Ca(2+) release channel), junctin, and calsequestrin. To better understand the role of triadin 1 in excitation-contraction coupling in the heart, we generated transgenic mice with targeted overexpression of triadin 1 to mouse atrium and ventricle, employing the alpha-myosin heavy chain promoter to drive protein expression. The protein was overexpressed 5-fold in mouse ventricles, and overexpression was accompanied by cardiac hypertrophy. The levels of two other junctional SR proteins, the ryanodine receptor and junctin, were reduced by 55% and 73%, respectively, in association with triadin 1 overexpression, whereas the levels of calsequestrin, the Ca(2+)-binding protein of junctional SR, and of phospholamban and SERCA2a, Ca(2+)-handling proteins of the free SR, were unchanged. Cardiac myocytes from triadin 1-overexpressing mice exhibited depressed contractility; Ca(2+) transients decayed at a slower rate, and cell shortening and relengthening were diminished. The extent of depression of cell shortening of triadin 1-overexpressing cardiomyocytes was rate-dependent, being more depressed under low stimulation frequencies (0.5 Hz), but reaching comparable levels at higher frequencies of stimulation (5 Hz). Spontaneously beating, isolated work-performing heart preparations overexpressing triadin 1 also relaxed at a slower rate than control hearts, and failed to adapt to increased afterload appropriately. The fast time inactivation constant, tau(1), of the l-type Ca(2+) channel was prolonged in transgenic cardiomyocytes. Our results provide evidence for the coordinated regulation of junctional SR protein expression in heart independent of free SR protein expression, and furthermore suggest an important role for triadin 1 in regulating the contractile properties of the heart during excitation-contraction coupling.     

microRNA在小鼠乳腺不同发育时期差异表达谱及作用

microRNA是一类大小约22个核苷酸的非编码RNA分子,是一种广泛存在的对基因表达进行微调的分子。microRNA可以通过与靶基因mRNA的特定位点结合,抑制该蛋白的合成或诱导该mRNA的降解,从而参与基因的表达调控。一般来源于染色体的非编码区域,由大约70个核苷酸大小的可形成发夹结构的前体经Dicer酶加工而来。这类小RNA在表达上具有组织和时间的特异性,是调节其他功能基因表达的重要调控分子,在生物的生长发育过程中发挥着重要作用。因此,虽然microRNA的研究仅有很短的历史,但已成为基因表达调控研究的热点领域。以中国昆明小鼠不同发育时期的乳腺组织为实验材料,应用芯片技术及荧光定量PCR技术,分析发育不同时期的乳腺组织microRNA差异表达图谱。本文研究发现microRNA在乳腺不同的发育时期表达图谱不同;与青春期、退化期比较,妊娠期、哺乳期有十余种microRNAs表达上调,20余种microRNAs表达下调;microRNAs在乳腺发育和泌乳周期中发挥重要的作用。     

Attenuation of microRNA-22 derepressed PTEN to effectively protect rat cardiomyocytes from hypertrophy

Cardiac hypertrophy, which is characterized by the enlargement of cell size, reactivation of fetal genes, remains one of the most important triggers to heart failure. Increasing evidence shows that microRNA (miRNA) is extensively involved in the pathogenesis of cardiac hypertrophy. But the effects of miRNAs on cardiomyocyte hypertrophy have not been completely solved yet. Here, we showed that a collection of miRNAs was aberrantly expressed in hypertrophic cardiomyocytes induced by phenylephrine (PE) or angiotensin II (Ang II). Among them, miR-22 was the most strikingly up-regulated miRNA. To investigate the role of miR-22 in hypertrophy, both over-expression and knock-down assays were performed on cardiomyocytes. The results showed that up-regulation of miR-22 significantly increased the cell size and markedly influenced the expression of hypertrophic markers, including induction of nppa and reduction of myh6. In contrast, reduction of miR-22 level attenuated either PE- or Ang II-induced hypertrophic reaction. Furthermore, several genes, including PTEN, were identified as potential targets of miR-22 by bioinformatic algorithms. Using luciferase analysis, miR-22 could significantly suppress the luciferase activity of reporter fused with 3' untranslated region of PTEN mRNA. Furthermore, up-regulation of miR-22 could suppress the protein level of PTEN and reduction of miR-22 level markedly increased the protein level of PTEN in cardiomyocytes by Western blot analysis, suggesting that the contribution of miR-22 to cardiomyocyte hypertrophy may be partially through targeting PTEN. Taken together, miRNAs were dynamically regulated in cardiomyocyte hypertrophy and attenuation of miR-22 in rat cardiomyocytes efficiently protected from hypertrophic effects through derepressing PTEN.     

On the role of junctin in cardiac Ca2+ handling, contractility, and heart failure

Junctin is a transmembrane protein located at the cardiac junctional sarcoplasmic reticulum (SR) and forms a quaternary complex with the Ca(2+) release channel, triadin and calsequestrin. Impaired protein interactions within this complex may alter the Ca(2+) sensitivity of the Ca(2+) release channel and may lead to cardiac dysfunction, including hypertrophy, depressed contractility, and abnormal Ca(2+) transients. To study the expression of junctin and, for comparison, triadin, in heart failure, we measured the levels of these proteins in SR from normal and failing human hearts. Junctin was below our level of detection in SR membranes from failing human hearts, and triadin was downregulated by 22%. To better understand the role of junctin in the regulation of Ca(2+) homeostasis and contraction of cardiac myocytes, we used an adenoviral approach to overexpress junctin in isolated rat cardiac myocytes. A recombinant adenovirus encoding the green fluorescent protein served as a control. Infection of myocytes with the junctin-expressing virus resulted in an increased RNA and protein expression of junctin. Ca(2+) transients showed a decreased maximum Ca(2+) amplitude, and contractility of myocytes was depressed. Our results demonstrate that an increased expression of junctin is associated with an impaired Ca(2+) homeostasis. Downregulation of junctin in human heart failure may thus be a compensatory mechanism.     

Altered miRNA Expression is Associated with Differentiation, Invasion, and Metastasis of Esophageal Squamous Cell Carcinoma (ESCC) in Patients from Huaian, China

Esophageal squamous cell carcinoma (ESCC) is the leading malignancy in Huaian, China. Recently, emerging studies have suggested that an aberrant microRNA (miRNA) expression signature exists in ESCC. However, there is discordant information available on specific miRNA expression in patients from different regions. In this study, we identified 12 miRNAs that are differentially expressed in patients with ESCC from Huaian, China. Among these miRNAs that displayed unique miRNA expression signatures, miR-1, miR-29c, miR-100, miR-133a, miR-133b, miR-143, miR-145, and miR-195 were downregulated, and miR-7, miR-21, miR-223, and miR-1246 were upregulated in cancerous tissue compared with the adjacent normal tissue. Bioinformatics analyses identified the major biological processes and signaling pathways that are targeted by these differentially expressed miRNAs. Accordingly, miR-29c, miR-100, miR-133a, and miR-133b were found to be involved in invasion and metastasis of ESCC, and miR-7 and miR-21 were found to be related to the differentiation of ESCC. Thus, our data present new evidence for the important roles of miRNAs in ESCC.     

microRNA在多能干细胞向心肌细胞分化中的作用

microRNAs(miRNAs)是长约22 nt的非编码RNAs,广泛参与细胞的增殖、分化、病变、修复和凋亡等多种生命活动．多能干细胞（pluripotent stem cells）是指体外具有自我更新和多向分化潜能的细胞,在一定条件下可被定向诱导分化为多种细胞类型．miRNAs在多能干细胞中表达丰富,并通过调控基因表达影响其自我更新及分化．由多能干细胞向心肌细胞分化的方法主要有3种,即拟胚体形成法、与内胚层细胞共培养法和特定诱导物添加法．虽然这3种方法均可成功诱导多能干细胞向心肌细胞分化,但重复率很低. 所以,人们把研究的视野逐渐转向miRNAs--这个广泛参与细胞生命活动的小分子物质．大量研究表明,在多能干细胞中,不同的miRNAs可通过打靶不同基因影响其向心肌细胞分化．在间充质干细胞中,miR-1、miR-133 和miR-499可分别打靶Hes-1、SRF和Pdcd4| 而在胚胎干细胞中,miR-1和miR-499分别打靶 Hand2和Pacs2促进其向心肌细胞分化．miRNAs在多能干细胞向心肌分化作用机制的研究必将促进再生医学在心脏疾病治疗上的应用．     

Triadin is a critical determinant of cellular Ca cycling and contractility in the heart

Triadin is involved in the regulation of cardiac excitation-contraction coupling. However, the extent of its contribution to the regulation of sarcoplasmic reticulum (SR) Ca release remains unclear, because overexpression of triadin in single-transgenic mice was associated with the downregulation of its homologous protein, junctin. In the present study, this problem was circumvented by cross-breeding of mice with heart-directed overexpression of triadin and junctin (JxT). This resulted in a stable approximately threefold expression of total triadin but unchanged junctin protein. Transgenic mice exhibited cardiac hypertrophy and structural abnormalities of myofibrils. Measurement of cardiac function by echocardiography and edge detection in myocytes revealed an impaired relaxation in JxT mice. The stimulation of beta-adrenergic receptors resulted in a depressed contractility and an impaired relaxation in catheterized hearts and myocytes of JxT mice. The use of a maximum stimulation frequency (5 Hz) was associated with both a lower shortening and relengthening in isolated myocytes of JxT mice. The contractile effects in JxT myocytes were paralleled by similar changes of the intracellular Ca concentration ([Ca](i)) peak amplitude and Ca transient decay kinetics at basal conditions, under administration of isoproterenol, and with high-frequency stimulation. Finally, we found a higher caffeine-induced [Ca](i) peak amplitude in JxT myocytes. Our data show that the stable expression of triadin, independent of junctin expression, resulted in cardiac hypertrophy, prolonged basal relaxation, a depressed response to beta-adrenergic agonists, and altered Ca transients. Thus the maintenance of triadin expression is essential for normal SR Ca cycling and contractile function.