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.
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.
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