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It is known that secreted proteins from the anterior lateral endoderm, FGF8 and BMP2, are involved in mesodermal cardiac differentiation, which determines the first cardiac field, defined by the expression of the earliest specific cardiac markers Nkx-2.5 and Gata4. However, the molecular mechanisms responsible for early cardiac development still remain unclear. At present, microRNAs represent a novel layer of complexity in the regulatory networks controlling gene expression during cardiovascular development. This paper aims to study the role of miR130 during early cardiac specification. Our model is focused on developing chick at gastrula stages. In order to identify those regulatory factors which are involved in cardiac specification, we conducted gain- and loss-of-function experiments in precardiac cells by administration of Fgf8, Bmp2 and miR130, through in vitro electroporation technique and soaked beads application. Embryos were subjected to in situ hybridization, immunohistochemistry and qPCR procedures. Our results reveal that Fgf8 suppresses, while Bmp2 induces, the expression of Nkx-2.5 and Gata4. They also show that Fgf8 suppresses Bmp2, and vice versa. Additionally, we observed that Bmp2 regulates miR-130 -a putative microRNA that targets Erk1/2 (Mapk1) 3'UTR, recognizing its expression in precardiac cells which overlap with Erk1/2 pattern. Finally, we evidence that miR-130 is capable to inhibit Erk1/2 and Fgf8, resulting in an increase of Bmp2, Nkx-2.5 and Gata4. Our data present miR-130 as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2, establishing a negative feed-back loop responsible to achieve early cardiac specification., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Exosomes play an important role in intercellular signaling and exert regulatory function by carrying bioactive molecules. This study investigated (1) the cardioprotective capabilities of exosomes derived from mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSC(GATA-4)) and (2) its underlying regulatory mechanisms for expression of target proteins in recipient cells., Methods and Results: Exosomes were isolated and purified from MSC(GATA-4) (Exo(GATA-4)) and control MSCs (Exo(Null)). Cell injury was investigated in primary cultured rat neonatal cardiomyocytes (CM) and in the rat heart. Exosomes contributed to increased CM survival, reduced CM apoptosis, and preserved mitochondrial membrane potential in CM cultured under a hypoxic environment. Direct intramyocardial transplantation of exosomes at the border of an ischemic region following ligation of the left anterior descending coronary artery significantly restored cardiac contractile function and reduced infarct size. Real-time PCR revealed that several anti-apoptotic miRs were highly expressed in Exo(GATA-4). Rapid internalization of Exo(GATA-4) by CM was documented using time-lapse imaging. Subsequent expression of these miRs, particularly miR-19a was higher in CM and in the myocardium treated with Exo(GATA-4) compared to those treated with Exo(Null). The enhanced protective effects observed in CM were diminished by the inhibition of miR-19a. The expression level of PTEN, a predicted target of miR-19a, was reduced in CM treated with Exo(GATA-4), which resulted in the activation of the Akt and ERK signaling pathways., Conclusions: Exo(GATA-4) upon transplantation in the damaged tissue mediate protection by releasing multiple miRs responsible for activation of the cell survival signaling pathway., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background/purpose: MicroRNA-208a (miR208a) and mechanical stress play a key role in cardiac hypertrophy. The relationship between miR208a and mechanical stress in cultured cardiomyocytes has not been investigated. The molecular mechanisms underlying miR208a-induced hypertrophy of cardiomyocytes by mechanical stress is poorly understood. This study investigated whether miR208a is a critical regulator in cardiomyocyte hypertrophy under mechanical stretch., Methods: Neonatal rat cardiomyocytes grown on a flexible membrane base were stretched at 60 cycles/minute. MiR real-time quantitative assays were used to quantify miRs. A quantitative sandwich enzyme immunoassay technique was used to measure transforming growth factor-β1 (TGF-β1). A (3)H-proline incorporation assay was used to measure protein synthesis., Results: Mechanical stretch significantly enhanced miR208a expression. Stretch significantly induced cardiomyocyte hypertrophic protein expression such as β-myosin heavy chain (MHCβ), thyroid hormone receptor-associated protein 100, myostatin, connexin 40, GATA4, and brain natriuretic peptide. MHCα was not induced by stretch. Overexpression of miR208a significantly increased MHCβ protein expression while pretreatment with antagomir208a significantly attenuated MHCβ protein expression induced by stretch and overexpression of miR208a. Mechanical stretch significantly increased the secretion of TGF-β1 from cultured cardiomyocytes. Exogenous addition of TGF-β1 recombinant protein significantly increased miR208a expression and pretreatment with TGF-β1 antibody attenuated miR208a expression induced by stretch. Mechanical stretch and overexpression of miR208a increased protein synthesis while antagomir208a attenuated protein synthesis induced by stretch and overexpression of miR208a., Conclusion: Cyclic stretch enhances miR208a expression in cultured rat cardiomyocytes. MiR208a plays a role in stretch-induced cardiac hypertrophy. The stretch-induced miR208a is mediated by TGF-β1., (Copyright © 2013. Published by Elsevier B.V.)

Somatic mutations and dysregulation by microRNAs (miRNAs) may have a pivotal role in the Congenital Heart Defects (CHDs). The purpose of the study was to assess both somatic and germline mutations in the GATA4 and NKX2.5 genes as well as to identify 3'UTR single nucleotide polymorphisms (SNPs) in the miRNA target sites. We enrolled 30 patients (13 males; 13.4±8.3 years) with non-syndromic CHD. GATA4 and NKX2.5 genes were screened in cardiac tissue of sporadic and in blood samples of familial cases. Computational methods were used to detect putative miRNAs in the 3'UTR region and to assess the Minimum Free Energy of hybridization (MFE, kcal/mol). Difference of MFEs (ΔMFE) ≥4 kcal/mol between alleles was considered biologically relevant on miRNA binding. The sum of all ΔMFEs (|ΔMFEtot|=∑|ΔMFE|) was calculated in order to predict the biological importance of SNPs binding more miRNAs. No evidence of novel GATA4 and NKX2.5 mutations was found both in sporadic and familial patients. Bioinformatic analysis revealed 27 putative miRNAs binding to identified SNPs in the 3'UTR of GATA4. ΔMFE ≥4 kcal/mol between alleles was obtained for the +354A>C (miR-4299), +587A>G (miR-604), +1355G>A (miR-548v, miR-139-5p) and +1521C>G (miR-583, miR-3125, miR-3928) SNPs. The +1521C>G SNP showed the highest ΔMFEtot (21.66 kcal/mol). Luciferase reporter assays indicated that miR-583 was dose-dependently effective in regulating +1521 C allele compared with +1521 G allele. Based on the analysis of 100 CHD cases and 204 healthy newborns, the +1521 G allele was also associated with a lower risk of CHD (OR=0.5, 95% CI 0.3-0.9, p=0.03), likely due to the relatively low binding of the miRNA and high levels of protein. These results suggest that common SNPs in the 3'UTR of GATA4 alter miRNA gene regulation contributing to the pathogenesis of CHDs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

West Nile virus (WNV) belongs to a group of medically important single-stranded, positive-sense RNA viruses causing deadly disease outbreaks around the world. The 3' untranslated region (3'-UTR) of the flavivirus genome, in particular the terminal 3' stem-loop (3'SL) fulfils multiple functions in virus replication and virus-host interactions. Using the Kunjin strain of WNV (WNV(KUN)), we detected a virally encoded small RNA, named KUN-miR-1, derived from 3'SL. Transcription of WNV(KUN) pre-miRNA (3'SL) in mosquito cells either from plasmid or Semliki Forest virus (SFV) RNA replicon resulted in the production of mature KUN-miR-1. Silencing of Dicer-1 but not Dicer-2 led to a reduction in the miRNA levels. Further, when a synthetic inhibitor of KUN-miR-1 was transfected into mosquito cells, replication of viral RNA was significantly reduced. Using cloning and bioinformatics approaches, we identified the cellular GATA4 mRNA as a target for KUN-miR-1. KUN-miR-1 produced in mosquito cells during virus infection or from plasmid DNA, SFV RNA replicon or mature miRNA duplex increased accumulation of GATA4 mRNA. Depletion of GATA4 mRNA by RNA silencing led to a significant reduction in virus RNA replication while a KUN-miR-1 RNA mimic enhanced replication of a mutant WNV(KUN) virus producing reduced amounts of KUN-miR-1, suggesting that GATA4-induction via KUN-miR-1 plays an important role in virus replication.