Epigenetic coordination of embryonic heart transcription by dynamically regulated long noncoding RNAs.

The vast majority of mammalian DNA does not encode for proteins but instead is transcribed into noncoding (nc)RNAs having diverse regulatory functions. The poorly characterized subclass of long ncRNAs (lncRNAs) can epigenetically regulate protein-coding genes by interacting locally in cis or distally in trans. A few reports have implicated specific lncRNAs in cardiac development or failure, but precise details of lncRNAs expressed in hearts and how their expression may be altered during embryonic heart development or by adult heart disease is unknown. Using comprehensive quantitative RNA sequencing data from mouse hearts, livers, and skin cells, we identified 321 lncRNAs present in the heart, 117 of which exhibit a cardiac-enriched pattern of expression. By comparing lncRNA profiles of normal embryonic (∼E14), normal adult, and hypertrophied adult hearts, we defined a distinct fetal lncRNA abundance signature that includes 157 lncRNAs differentially expressed compared with adults (fold-change ≥ 50%, false discovery rate = 0.02) and that was only poorly recapitulated in hypertrophied hearts (17 differentially expressed lncRNAs; 13 of these observed in embryonic hearts). Analysis of protein-coding mRNAs from the same samples identified 22 concordantly and 11 reciprocally regulated mRNAs within 10 kb of dynamically expressed lncRNAs, and reciprocal relationships of lncRNA and mRNA levels were validated for the Mccc1 and Relb genes using in vitro lncRNA knockdown in C2C12 cells. Network analysis suggested a central role for lncRNAs in modulating NFκB- and CREB1-regulated genes during embryonic heart growth and identified multiple mRNAs within these pathways that are also regulated, but independently of lncRNAs.

Cardiac Disease Status Dictates Functional mRNA Targeting Profiles of Individual MicroRNAs.

BACKGROUND: MicroRNAs are key players in cardiac stress responses, but the mRNAs, whose abundance and translational potential are primarily affected by changes in cardiac microRNAs, are not well defined. Stimulus-induced, large-scale alterations in the cardiac transcriptome, together with consideration of the law of mass action, further suggest that the mRNAs most substantively targeted by individual microRNAs will vary between unstressed and stressed conditions. To test the hypothesis that microRNA target profiles differ in health and disease, we traced the fate of empirically determined miR-133a and miR-378 targets in mouse hearts undergoing pressure overload hypertrophy. METHODS AND RESULTS: Ago2 immunoprecipitation with RNA sequencing (RNA-induced silencing complex sequencing) was used for unbiased definition of microRNA-dependent and microRNA-independent alterations occurring among ≈13 000 mRNAs in response to transverse aortic constriction (TAC). Of 37 direct targets of miR-133a defined in unstressed hearts (fold change ≥25%, false discovery rate <0.02), only 4 (11%) continued to be targeted by miR-133a during TAC, whereas for miR-378 direct targets, 3 of 32 targets (9%) were maintained during TAC. Similarly, only 16% (for miR-133a) and 53% (for miR-378) of hundreds of indirectly affected mRNAs underwent comparable regulation, demonstrating that the effect of TAC on microRNA direct target selection resulted in widespread alterations of signaling function. Numerous microRNA-mediated regulatory events occurring exclusively during pressure overload revealed signaling networks that may be responsive to the endogenous decreases in miR-133a during TAC. CONCLUSIONS: Pressure overload-mediated changes in overall cardiac RNA content alter microRNA targeting profiles, reinforcing the need to define microRNA targets in tissue-, cell-, and status-specific contexts.

A novel strategy to increase the proliferative potential of adult human ß-cells while maintaining their differentiated phenotype.

Our previous studies demonstrated that Wnt/GSK-3/ß-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human ß-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases ß-catenin nuclear translocation and ß-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human ß-cell proliferation while maintaining a ß-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human ß-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced ß-catenin-mediated and ß-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/ß-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human ß-cell proliferation while maintaining the ß-cell phenotype.