Abstract-MicroRNAs are naturally existing, small, noncoding RNA molecules that downregulate posttranscriptional gene expression. Their expression pattern and function in the heart remain unknown. Here we report an array of microRNAs that are differentially and temporally regulated during cardiac hypertrophy. Significantly, the muscle-specific microRNA-1 (miR-1) was singularly downregulated as early as day 1 (0.56Ϯ0.036), persisting through day 7 (0.29Ϯ0.14), after aortic constriction-induced hypertrophy in a mouse model. Overexpression experiments showed that miR-1 inhibited its in silico-predicted, growth-related targets, including Ras GTPase-activating protein (RasGAP), cyclin-dependent kinase 9 (Cdk9), fibronectin, and Ras homolog enriched in brain (Rheb), in addition to protein synthesis and cell size. Thus, we propose that microRNAs play an essential regulatory role in the development of cardiac hypertrophy, wherein downregulation of miR-1 is necessary for the relief of growth-related target genes from its repressive influence and induction of hypertrophy. (Circ Res. 2007;100:416-424.)

The posttranscriptional regulator, microRNA-21 (miR-21), is up-regulated in many forms of cancer, as well as during cardiac hypertrophic growth. To understand its role, we overexpressed it in cardiocytes where it revealed a unique type of cell-to-cell "linker" in the form of long slender outgrowths and branches. We subsequently confirmed that miR-21 directly targets and down-regulates the expression of Sprouty2 (SPRY2), an inhibitor of branching morphogenesis and neurite outgrowths. We found that beta-adrenergic receptor (betaAR) stimulation induces up-regulation of miR-21 and down-regulation of SPRY2 and is, likewise, associated with connecting cell branches. Knockdown of SPRY2 reproduced the branching morphology in cardiocytes, and vice versa, knockdown of miR-21 using a specific 'miRNA eraser' or overexpression of SPRY2 inhibited betaAR-induced cellular outgrowths. These structures enclose sarcomeres and connect adjacent cardiocytes through functional gap junctions. To determine how this aspect of miR-21 function translates in cancer cells, we knocked it down in colon cancer SW480 cells. This resulted in disappearance of their microvillus-like protrusions accompanied by SPRY2-dependent inhibition of cell migration. Thus, we propose that an increase in miR-21 enhances the formation of various types of cellular protrusions through directly targeting and down-regulating SPRY2.

In this study we have shown that the histone variant H2A.z is up-regulated during cardiac hypertrophy. Upon its knockdown with RNA interference, hypertrophy and the underlying increase in growth-related genes, protein synthesis, and cell size were down-regulated. During attempts to understand the mode of regulation of H2A.z, we found that overexpression of silent information regulator 2alpha (Sir2␣) specifically induced down-regulation of H2A.z via NAD-dependent activity. This effect was reversed by the proteasome inhibitor epoxomicin, suggesting a Sir2␣-mediated ubiquitin/proteasome-dependent mechanism for degradation of H2A.z. An increase in Sir2␣ also resulted in a dose-dependent reduction of the response to hypertrophic stimuli, whereas its inhibition resulted in enhanced hypertrophy and apoptosis. We have shown that Sir2␣ directly deacetylates H2A.z. Mutagenesis proved that lysines 4, 7, 11, and 13 do not play a role in the stability of H2A.z, whereas Lys-15 was indispensable. Meanwhile, Lys-115 and conserved, ubiquitinatable Lys-121 are critical for Sir2␣-mediated degradation. Fusion of the C terminus of H2A.z (amino acids 115-127) to H2A.x or green fluorescence protein conferred Sir2␣-inducible degradation to the former protein only. Because H2A.x and H2A.z have conserved N-tails, this implied that both the C and N termini are critical for mediating the effect of Sir2␣. In short, the results suggest that H2A.z is required for cardiac hypertrophy, where its stability and the extent of cell growth and apoptosis are moderated by Sir2␣. We also propose that Sir2␣ is involved in deacetylation of H2A.z, which results in ubiquitination of Lys-115 and Lys-121 and its degradation via a ubiquitin/proteasomedependent pathway.H2A.z is an essential histone variant. Disruption of H2A.z in mice is embryonic lethal at a very early stage (E 4.5) (1). Likewise, H2A.z is vital for the development of Drosophila melanogaster (2) and is indispensable in Tetrahymena thermophila (3). In yeast, disruption of H2A.z results in slow growth and formamide sensitivity and is not rescued by the major H2A but is rescued by T. thermophila H2A.z variant (4). These reports provide evidence that H2A.z has a vital and non-redundant role and that it is conserved among species.One of the mechanisms for transcriptional regulation involves the replacement of core histones with more specialized variants. A report by Abbott et al. (5) shows that nucleosomes harboring H2A.z are destabilized, being less compact than nucleosomes harboring the core histone H2A. This is also supported by the crystal structure of a nucleosome core containing H2A.z that exhibits subtle differences compared with the H2A nucleosomes, which account for instability between the H2A.z-H2B dimer and the H3-H4 tetramer (6). This is consistent with a role for H2A.z in nucleosomal remodeling to reduce its structural compactness, thereby facilitating the binding of transcription factors. It should be noted though that this might not be the sole function of H2A.z in the cell. It has...

We have previously reported that Ras GTPase-activating protein (RasGAP) is involved in a pathway that regulates total cellular mRNA and protein synthesis in cardiac myocytes. A yeast two-hybrid screen resulted in identification of filamin C (FLN-C) as one of its targets. Knockdown of RasGAP or FLN-C, or severing their interaction, resulted in down-regulation of the RNA polymerase II kinase, cyclin-dependent kinase 7 (Cdk7). This appeared to be provoked by the release of cdk7 mRNA from RasGAP SH3 domain-binding protein, G3BP, and its subsequent degradation. In parallel, myocyte growth was also inhibited. On the other hand, overexpression of RasGAP induced a Cdk7-and FLN-C-dependent growth. Thus, we propose that the physical interaction between RasGAP and FLN-C facilitates an interaction between G3BP and cdk7 mRNA. This results in stabilization of cdk7 mRNA, an increase in its protein, which is required for cell growth.