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Due to the lack of typical clinical symptoms, the average delay time for diagnosis of pulmonary hypertension (PH) is longer than 2 years. It is urgent to find biomarkers for PH diagnosis. In this study we investigated whether plasma microRNAs (miRNAs) can be used as biomarkers for PH diagnosis. We used microarray to identify dynamic miRNAs between PH and non-PH patients. The candidate miRNAs were verified using qRT-PCR in a mouse model of PH, which was induced by monocrotaline (MCT) injection. We observed that miR-21, miR-126, miR-145, miR-191 and miR-150 had no differences between control mice and MCT-treated mice; but plasma miR-451 was significantly decreased in the 2wk-MCT group, with no further decrease in the 4wk-MCT group. Plasma miR-451 was also markedly decreased in PH patients, whereas miR-21, miR-126, miR-150 and miR-320 did not show differences between 53 PH patients and 54 non-PH patients. Receiver operating characteristic curves (ROCs) were constructed from the patient data to assess the clinical diagnostic values of circulating miR-451 and Doppler echocardiography (D-ECHO). The areas under the curve (AUCs) of ROCs for miR-451 and D-ECHO were 0.710 and 0.766, respectively. Combination of miR-451 and D-ECHO with AUC of 0.825 was superior to the use of either miR-451 or D-ECHO alone for PH diagnosis. In conclusion, plasma miR-451 has a moderate diagnostic value in PH comparable to that of D-ECHO, and the combination of miR-451 with D-ECHO has better diagnostic value than either method alone, which may have implications for PH diagnosis.

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. In an intact rat model of myocardial ischemia caused by coronary artery ligation, this study identified 17 miRNAs that changed more than 1.5-fold in the myocardium subjected to 4-h ischemia. Using miRNA microarray analysis, most of these aberrantly expressed miRNAs were confirmed by quantitative RT-PCR. MiR-378, a significantly down-regulated miRNA, was selected for further function study. In serum deprived rat H9c2 cardiomyocytes exposed to hypoxia (1% O(2)), miR-378 expression was down-regulated as well. The overexpression of miR-378 resulting from miR-378 mimic transfection significantly enhanced cell viability, reduced lactate dehydrogenase release, and inhibited apoptosis and necrosis. By contrast, miR-378 deficiency resulting from miR-378 inhibitor transfection aggravated the hypoxia-induced apoptosis and cell injury. In accordance, miR-378 inhibitor caused significant apoptosis and cell injury to cardiomyocytes cultured under normoxia. Using bioinformatic algorithms, caspase-3, a key apoptosis executioner, was predicted as a putative target of miR-378. The quantitative RT-PCR showed no effects of miR-378 mimic or inhibitor on caspase-3 mRNA level. However, the amount of caspase-3 proteins was reduced by miR-378 mimic, whereas increased by miR-378 inhibitor. Furthermore, the luciferase reporter assay confirmed caspase-3 to be a target of miR-378, and the apoptosis and cell injury caused by miR-378 inhibitor in both normoxic and hypoxic cells were abolished by a caspase-3 inhibitor. This study first showed that miR-378 inhibited caspase-3 expression and attenuated ischemic injury in cardiomyocytes. It may represent a potential novel treatment for apoptosis and ischemic heart disease.

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., (Copyright © 2011 Wiley Periodicals, Inc.)

Objective: To investigate the role of miRNA-199a on cardiac hypertrophy., Methods: (1) Male Sprague-Dawley rats were subjected to pressure overload induced by abdominal aortic constriction (AAC, n = 6) and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the change of microRNAs (miRNAs). (2) Neonatal rat ventricular myocytes were isolated from 2-day old Sprague-Dawley rats. The myocytes were divided into two groups: adenovirus miRNA-199a (Ad-miRNA-199a) or adenovirus vector (Ad-vector). They were transfected in cardiomyocytes for 48 h using Lipofectamine 2000. qRT-PCR was used to detect the change of myocardial hypertrophy markers α-myosin heavy chain (αMHC, myh6), β-myosin heavy chain (βMHC, myh7) and atrial natriuretic peptide (ANP, Nppa). Software Axio Vision was used to detect the change of cardiomyocytes surface areas. (3) Neonatal rat ventricular myocytes were divided into two groups: antisense oligonucleotide-miRNA-199a (As-miRNA-199a) and scramble oligonucleotides (As-ctl). They were transfected to cardiomyocytes respectively for 48 h. qRT-PCR was used to detect the change of miRNA-199a. (4) Neonatal rat ventricular myocytes were divided into four groups: A: control (ctl), B: phenylephrine (PE), C: PE + As-ctl, D: PE + As-miRNA-199a. qRT-PCR was used to detect the change of myh6, myh7 and Nppa. Software Axio Vision was used to detect the change of cardiomyocytes surface areas., Results: (1) qRT-PCR results showed that miRNA-1, miRNA-133, miRNA-181a and miRNA-499 were significantly decreased, while the miRNA-199a was significantly increased at 1 week post AAC hearts compared with the sham group. (2) qRT-PCR results showed that miRNA-199a and myh7 were increased and myh6 was decreased significantly in Ad-miRNA-199a group compared with Ad-vector group. The cardiomyocytes surface area was increased in Ad-miRNA-199a group detected by immunofluorescence. (3) qRT-PCR results showed that miRNA-199a was significantly decreased in As-miRNA-199a group compared with Ad-vector group. (4) The Nppa and myh7 were significantly increased and myh6 was decreased in cardiomyocytes stimulated by PE for 48 h. The cardiomyocytes surface area determined by immunofluorescence was increased in PE + As-miRNA-199a groups compared with PE + As-ctl groups., Conclusion: miRNA-199a may play a regulatory role in cardiac hypertrophy.

Cardiac hypertrophy, which is characterized by an increase in cell size and reactivation of fetal genes, occurs as an adaptive response to diverse forms of stress and often results in heart failure and sudden death. Growing evidence indicates that microRNAs (miRNAs) are involved in cardiac hypertrophy, but the function of these miRNAs remains elusive. Here, using real time PCR analysis, we showed that several miRNAs were dynamically regulated in the rat hypertrophic hearts and miR-199a was up-regulated by 10-fold in hypertrophic hearts after abdominal aorta constriction for 12 weeks. With tissue profiling analysis, we showed that miR-199a was predominantly expressed in cardiomyocytes, but was also faintly detected in cardiac fibroblasts. To investigate whether miR-199a was involved in cardiac hypertrophy, both over-expression and knockdown of miR-199a were performed in cultured cardiomyocytes. Over-expression of miR-199a in cardiomyocytes increased the cell size as measured by cell surface area, and also reduced the mRNA expression level of alpha-myosin heavy chain. In accordance, knockdown of endogenous miR-199a in cardiomyocytes reduced the cell size. Down-regulation of miR-199a also attenuated the phenylephrine-induced increase of cell size. Furthermore, bioinformatic algorithms were used to predict the potential targets of miR-199a in cardiac hypertrophy, and hypoxia-inducible factor 1 alpha was confirmed by the luciferase reporter assay to be a potential target of miR-199a. Taken together, our results demonstrated that miR-199a, which was predominantly expressed in cardiomyocytes, was essential for the maintenance of cell size of cardiomyocytes and might play a role in the regulation of cardiac hypertrophy., ((c) 2010 Wiley-Liss, Inc.)

MicroRNAs are involved in several aspects of cardiac hypertrophy, including cardiac growth, conduction, and fibrosis. However, their effects on the regulation of the cardiomyocyte cytoskeleton in this pathological process are not known. Here, with microRNA microarray and small RNA library sequencing, we show that microRNA-1 (miR-1) is the most abundant microRNA in the human heart. By applying bioinformatic target prediction, a cytoskeleton regulatory protein twinfilin-1 was identified as a potential target of miR-1. Overexpression of miR-1 not only reduced the luciferase activity of the reporter containing the 3' untranslated region of twinfilin-1 mRNA, but also suppressed the endogenous protein expression of twinfilin-1, indicating that twinfilin-1 is a direct target of miR-1. miR-1 was substantially downregulated in the rat hypertrophic left ventricle and phenylephrine-induced hypertrophic cardiomyocytes, and accordingly, the protein level of twinfilin-1 was increased. Furthermore, overexpression of miR-1 in hypertrophic cardiomyocytes reduced the cell size and attenuated the expression of hypertrophic markers, whereas silencing of miR-1 in cardiomyocytes resulted in the hypertrophic phenotype. In accordance, twinfilin-1 overexpression promoted cardiomyocyte hypertrophy. Taken together, our results demonstrate that the cytoskeleton regulatory protein twinfilin-1 is a novel target of miR-1, and that reduction of miR-1 by hypertrophic stimuli induces the upregulation of twinfilin-1, which in turn evokes hypertrophy through the regulation of cardiac cytoskeleton.