The long noncoding RNA CARDINAL attenuates cardiac hypertrophy by modulating protein translation.

One of the features of pathological cardiac hypertrophy is enhanced translation and protein synthesis. Translational inhibition has been shown to be an effective means of treating cardiac hypertrophy, although system-wide side effects are common. Regulators of translation, such as cardiac-specific long noncoding RNAs (lncRNAs), could provide new, more targeted therapeutic approaches to inhibit cardiac hypertrophy. Therefore, we generated mice lacking a previously identified lncRNA named CARDINAL to examine its cardiac function. We demonstrate that CARDINAL is a cardiac-specific, ribosome-associated lncRNA and show that its expression was induced in the heart upon pathological cardiac hypertrophy and that its deletion in mice exacerbated stress-induced cardiac hypertrophy and augmented protein translation. In contrast, overexpression of CARDINAL attenuated cardiac hypertrophy in vivo and in vitro and suppressed hypertrophy-induced protein translation. Mechanistically, CARDINAL interacted with developmentally regulated GTP-binding protein 1 (DRG1) and blocked its interaction with DRG family regulatory protein 1 (DFRP1); as a result, DRG1 was downregulated, thereby modulating the rate of protein translation in the heart in response to stress. This study provides evidence for the therapeutic potential of targeting cardiac-specific lncRNAs to suppress disease-induced translational changes and to treat cardiac hypertrophy and heart failure.

The translational landscape of human vascular smooth muscle cells identifies novel short open reading frame-encoded peptide regulators for phenotype alteration.

AIMS: The plasticity of vascular smooth muscle cells (VSMCs) enables them to alter phenotypes under various physiological and pathological stimuli. The alteration of VSMC phenotype is a key step in vascular diseases, including atherosclerosis. Although the transcriptome shift during VSMC phenotype alteration has been intensively investigated, uncovering multiple key regulatory signalling pathways, the translatome dynamics in this cellular process, remain largely unknown. Here, we explored the genome-wide regulation at the translational level of human VSMCs during phenotype alteration. METHODS AND RESULTS: We generated nucleotide-resolution translatome and transcriptome data from human VSMCs undergoing phenotype alteration. Deep sequencing of ribosome-protected fragments (Ribo-seq) revealed alterations in protein synthesis independent of changes in messenger ribonucleicacid levels. Increased translational efficiency of many translational machinery components, including ribosomal proteins, eukaryotic translation elongation factors and initiation factors were observed during the phenotype alteration of VSMCs. In addition, hundreds of candidates for short open reading frame-encoded polypeptides (SEPs), a class of peptides containing 200 amino acids or less, were identified in a combined analysis of translatome and transcriptome data with a high positive rate in validating their coding capability. Three evolutionarily conserved SEPs were further detected endogenously by customized antibodies and suggested to participate in the pathogenesis of atherosclerosis by analysing the transcriptome and single cell RNA-seq data from patient atherosclerotic artery samples. Gain- and loss-of-function studies in human VSMCs and genetically engineered mice showed that these SEPs modulate the alteration of VSMC phenotype through different signalling pathways, including the mitogen-activated protein kinase pathway and p53 pathway. CONCLUSION: Our study indicates that an increase in the capacity of translation, which is attributable to an increased quantity of translational machinery components, mainly controls alterations of VSMC phenotype at the level of translational regulation. In addition, SEPs could function as important regulators in the phenotype alteration of human VSMCs.

Cardiac ISL1-Interacting Protein, a Cardioprotective Factor, Inhibits the Transition From Cardiac Hypertrophy to Heart Failure.

Heart failure is characterized by the inability of the heart to pump effectively and generate proper blood circulation to meet the body's needs; it is a devastating condition that affects more than 100 million people globally. In spite of this, little is known about the mechanisms regulating the transition from cardiac hypertrophy to heart failure. Previously, we identified a cardiomyocyte-enriched gene, CIP, which regulates cardiac homeostasis under pathological stimulation. Here, we show that the cardiac transcriptional factor GATA4 binds the promotor of CIP gene and regulates its expression. We further determined that both CIP mRNA and protein decrease in diseased human hearts. In a mouse model, induced cardiac-specific overexpression of CIP after the establishment of cardiac hypertrophy protects the heart by inhibiting disease progression toward heart failure. Transcriptome analyses revealed that the IGF, mTORC2 and TGFß signaling pathways mediate the inhibitory function of CIP on pathologic cardiac remodeling. Our study demonstrates GATA4 as an upstream regulator of CIP gene expression in cardiomyocytes, as well as the clinical significance of CIP expression in human heart disease. More importantly, our investigation suggests CIP is a key regulator of the transition from cardiac hypertrophy to heart failure. The ability of CIP to intervene in the onset of heart failure suggests a novel therapeutic avenue of investigation for the prevention of heart disease progression.

The cardiac translational landscape reveals that micropeptides are new players involved in cardiomyocyte hypertrophy.

Hypertrophic growth of cardiomyocytes is one of the major compensatory responses in the heart after physiological or pathological stimulation. Protein synthesis enhancement, which is mediated by the translation of messenger RNAs, is one of the main features of cardiomyocyte hypertrophy. Although the transcriptome shift caused by cardiac hypertrophy induced by different stimuli has been extensively investigated, translatome dynamics in this cellular process has been less studied. Here, we generated a nucleotide-resolution translatome as well as transcriptome data from isolated primary cardiomyocytes undergoing hypertrophy. More than 10,000 open reading frames (ORFs) were detected from the deep sequencing of ribosome-protected fragments (Ribo-seq), which orchestrated the shift of the translatome in hypertrophied cardiomyocytes. Our data suggest that rather than increase the translational rate of ribosomes, the increased efficiency of protein synthesis in cardiomyocyte hypertrophy was attributable to an increased quantity of ribosomes. In addition, more than 100 uncharacterized short ORFs (sORFs) were detected in long noncoding RNA genes from Ribo-seq with potential of micropeptide coding. In a random test of 15 candidates, the coding potential of 11 sORFs was experimentally supported. Three micropeptides were identified to regulate cardiomyocyte hypertrophy by modulating the activities of oxidative phosphorylation, the calcium signaling pathway, and the mitogen-activated protein kinase (MAPK) pathway. Our study provides a genome-wide overview of the translational controls behind cardiomyocyte hypertrophy and demonstrates an unrecognized role of micropeptides in cardiomyocyte biology.

Transcribed Ultraconserved Regions, Uc.323, Ameliorates Cardiac Hypertrophy by Regulating the Transcription of CPT1b (Carnitine Palmitoyl transferase 1b).

Transcribed ultraconserved regions (T-UCRs) are a novel class of long noncoding RNAs transcribed from UCRs, which exhibit 100% DNA sequence conservation among humans, mice, and rats. However, whether T-UCRs regulate cardiac hypertrophy remains unclear. We aimed to explore the effects of T-UCRs on cardiac hypertrophy. First, we performed long noncoding RNA microarray analysis on hearts of mice subjected to sham surgery or aortic banding and found that the T-UCR uc.323 was decreased significantly in mice with aortic banding-induced cardiac hypertrophy. In vitro loss- and gain-of-function experiments demonstrated that uc.323 protected cardiomyocytes against hypertrophy induced by phenylephrine. Additionally, we discovered that mammalian target of rapamycin 1 contributed to phenylephrine-induced uc.323 downregulation and uc.323-mediated cardiomyocyte hypertrophy. We further mapped the possible target genes of uc.323 through global microarray mRNA expression analysis after uc.323 knockdown and found that uc.323 regulated the expression of cardiac hypertrophy-related genes such as CPT1b (Carnitine Palmitoyl transferase 1b). Then, chromatin immunoprecipitation proved that EZH2 (enhancer of zeste homolog 2) bound to the promoter of CPT1b via H3K27me3 (trimethylation of lysine 27 of histone H3) to induce CPT1b downregulation. And overexpression of CPT1b could block uc.323-mediated cardiomyocyte hypertrophy. Finally, we found that uc.323 deficiency induced cardiac hypertrophy. Our results reveal that uc.323 is a conserved T-UCR that inhibits cardiac hypertrophy, potentially by regulating the transcription of CPT1b via interaction with EZH2.

Maf1 ameliorates cardiac hypertrophy by inhibiting RNA polymerase III through ERK1/2.

Rationale: An imbalance between protein synthesis and degradation is one of the mechanisms of cardiac hypertrophy. Increased transcription in cardiomyocytes can lead to excessive protein synthesis and cardiac hypertrophy. Maf1 is an RNA polymerase III (RNA pol III) inhibitor that plays a pivotal role in regulating transcription. However, whether Maf1 regulates of cardiac hypertrophy remains unclear. Methods: Cardiac hypertrophy was induced in vivo by thoracic aortic banding (AB) surgery. Both the in vivo and in vitro gain- and loss-of-function experiments by Maf1 knockout (KO) mice and adenoviral transfection were used to verify the role of Maf1 in cardiac hypertrophy. RNA pol III and ERK1/2 inhibitor were utilized to identify the effects of RNA pol III and ERK1/2. The possible interaction between Maf1 and ERK1/2 was clarified by immunoprecipitation (IP) analysis. Results: Four weeks after surgery, Maf1 KO mice exhibited significantly exacerbated AB-induced cardiac hypertrophy characterized by increased heart size, cardiomyocyte surface area, and atrial natriuretic peptide (ANP) expression and by exacerbated pulmonary edema. Also, the deficiency of Maf1 causes more severe cardiac dilation and dysfunction than wild type (WT) mice after pressure overload. In contrast, compared with adenoviral-GFP injected mice, mice injected with adenoviral-Maf1 showed significantly ameliorated AB-induced cardiac hypertrophy. In vitro study has demonstrated that Maf1 could significantly block phenylephrine (PE)-induced cardiomyocyte hypertrophy by inhibiting RNA pol III transcription. However, application of an RNA pol III inhibitor markedly improved Maf1 knockdown-promoted cardiac hypertrophy. Moreover, ERK1/2 was identified as a regulator of RNA pol III, and ERK1/2 inhibition by U0126 significantly repressed Maf1 knockdown-promoted cardiac hypertrophy accompanied by suppressed RNA pol III transcription. Additionally, IP analysis demonstrated that Maf1 could directly bind ERK1/2, suggesting Maf1 could interact with ERK1/2 and then inhibit RNA pol III transcription so as to attenuate the development of cardiac hypertrophy. Conclusions: Maf1 ameliorates PE- and AB-induced cardiac hypertrophy by inhibiting RNA pol III transcription via ERK1/2 signaling suppression.

Direct induction of functional neuronal cells from fibroblast-like cells derived from adult human retina.

Obtaining and manipulating neuronal cells are critical for neural biology basic mechanism studies and translational applications. Recent advances in protocol development and mechanism dissections have made direct induction of neuronal cells from other somatic cells (iN) a promising strategy for such purposes. In this study, we established a protocol to expand a population of fibroblast-like cells from adult human retinal tissues, which can be reprogrammed into iNs by forced expression of neurogenic transcription factors. Interestingly, the combination of Ascl1, Brn2, Myt1l, and NeuroD1 transcription factors, which has been demonstrated to be sufficient to reprogram human embryonic and dermal fibroblasts into iNs, failed to reprogram the fibroblast-like cells from human retinas into iNs. Instead, supplementing Ascl1 with Pax6 sufficed to convert the cells into iNs, which exhibited a typical neuronal morphology, expressed neural marker genes, displayed active and passive neuronal membrane activities, and made synaptic communications with other neurons. Moreover, iNs converted from retina-derived fibroblast-like cells contained high ratios of γ-Aminobutyric acid- (GABA-) and tyrosine hydroxylase- (TH-) positive neurons. Thus, the present study proposes a protocol that makes use of discarded retinal tissues from eye banks for iN generation, and suggests that different sources of somatic cells require different iN induction recipes and may also affect the iN subtype outputs. Our study may also facilitate the future development of methods to convert resident cells in situ into retinal neurons for treating retinal degeneration disease purpose.

Overexpression of Arachis hypogaea NAC3 in tobacco enhances dehydration and drought tolerance by increasing superoxide scavenging.

Drought stress can severely affect plant growth and substantially diminish crop yields. We previously isolated Arachis hypogaea NAC3 (AhNAC3), a dehydration-induced NAM/ATAF/CUC (NAC) gene from peanut. In this study, to examine the role of AhNAC3 in stress tolerance, we constructed transgenic tobacco lines overexpressing AhNAC3. The transgenic plants showed hyper-resistance to dehydration and drought stresses and accumulated more proline and less superoxide anion (O2(-)) than wild type under dehydration and drought conditions. Moreover, the transgenic plants showed upregulation of four functional genes, superoxide dismutase (SOD), pyrroline-5-carboxylate synthetase (P5SC), late embryogenic abundant proteins (LEA), and early response to drought 10 (ERD10C). Protein localization and transactivation analysis suggested that AhNAC3 activates its specific targets in the nucleus. These results suggest that AhNAC3 is a dehydration-induced transcription factor that improves water stress tolerance by increasing superoxide scavenging and promoting the accumulation of various protective molecules.

Involvement of G1-to-S transition and AhAUX-dependent auxin transport in abscisic acid-induced inhibition of lateral root primodia initiation in Arachis hypogaea L.

Previous study indicated that increasing endogenous abscisic acid (ABA) level could inhibit the lateral root (LR) formation of peanuts. In this study, we investigated the mechanisms by which ABA regulated lateral root primordia (LRP) initiation in peanuts (Arachis hypogaea L). Results suggested that ABA inhibited LRP initiation through blocking G1-to-S transition in seedlings and mature roots: e.g. 5.8% increase in the proportion of G1 phase and 18% decrease in the proportion of S phase after ABA treatment for 6 days. Further study of the expression of the cell cycle marker gene for G2-to-M transition in peanut roots suggested that AhCYCB1 expression was regulated by ABA. We also investigated the cooperative regulation of LRP initiation by ABA and indole-3-acetic acid (IAA). ABA treatment greatly reduced the effects of endogenous IAA on mature roots. The expression of the IAA polar transport gene AhAUX1 appeared to be regulated by ABA since ABA inhibited auxin-mediated LRP initiation by suppressing AhAUX-dependent auxin transport in peanut roots. We further examined the effect of ABA on the expression of DR5::GUS and AtAUX1 in the model plant Arabidopsis. The results of Arabidopsis were consistent with that of the peanut.