No abstract

RNA G-quadruplexes (G4s) play important roles in RNA biology. However, the function and regulation of mRNA G-quadruplexes in embryonic development remain elusive. Previously, we identified RHAU (DHX36, G4R1) as an RNA helicase that resolves mRNA G-quadruplexes. Here, we find that cardiac deletion of Rhau leads to heart defects and embryonic lethality in mice. Gene expression profiling identified Nkx2-5 mRNA as a target of RHAU that associates with its 5' and 3' UTRs and modulates its stability and translation. The 5' UTR of Nkx2-5 mRNA contains a G-quadruplex that requires RHAU for protein translation, while the 3' UTR of Nkx2-5 mRNA possesses an AU-rich element (ARE) that facilitates RHAU-mediated mRNA decay. Thus, we uncovered the mechanisms underlying Nkx2-5 post-transcriptional regulation during heart development. Meanwhile, this study demonstrates the function of mRNA 5' UTR G-quadruplex-mediated protein translation in organogenesis.

A previous study indicated that Rheb1 is required for mammalian target of TOR complex 1 (mTORC1) signaling in the brain. However, the function of Rheb1 in the heart is still elusive. In the present study, we deleted Rheb1 specifically in cardiomyocytes and found that reduced Rheb1 levels conferred cardioprotection against pathologic remodeling in myocardial infarction (MI) and pressure overload (transverse aortic constriction) mouse models. Cardiomyocyte apoptosis was reduced and mTORC1 activity was suppressed in cardiomyocyte Rheb1-deletion mice, suggesting that Rheb1 regulates mTORC1 activation in myocardium. Furthermore, we demonstrated that astragaloside IV (As-IV) could inhibit mTORC1, and As-IV treatment displayed similar protection against MI and transverse aortic constriction as Rheb1 genetic inhibition. This study indicates that Rheb1 is essential for mTORC1 activation in cardiomyocytes and suggests that targeting Rheb1-mTORC1 signaling, such as by As-IV treatment, may be an effective therapeutic method for treating patients with adverse cardiac remodeling after MI and hypertrophy.

G-quadruplex (G4) DNA and G4 DNA resolvase are involved in a variety of biological processes. To understand the biological function of G4 DNA structures and their resolvases in spermatogenesis, we investigated the distribution of G4 structures in mouse testis and identified their alterations during spermatogenesis. Meanwhile, we studied the function of RNA helicase associated with AU-rich element (RHAU), a G4 DNA resolvase, in spermatogenesis with a germ-cell-specific knockout mouse model. The results showed that the ablation of RHAU in germ cells caused the increase of G4 structures and thus resulted in the decrease of spermatogonial differentiation. c-kit, a spermatogonia differentiation-related gene, contains two G4 DNA motifs on its promoter. We found its expression was significantly downregulated in RHAU conditional knockout testis. A further analysis demonstrated that RHAU directly bound to the G4 structures to activate c-kit expression. We concluded that RHAU regulates spermatogonia differentiation by promoting c-kit expression via directly binding to the G4 DNA motifs c-kit promoter.

Actin dynamics are critical for muscle development and function, and mutations leading to deregulation of actin dynamics cause various forms of heritable muscle diseases. AIP1 is a major cofactor of the actin depolymerizing factor/cofilin in eukaryotes, promoting actin depolymerizing factor/cofilin-mediated actin disassembly. Its function in vertebrate muscle has been unknown. To investigate functional roles of AIP1 in myocardium, we generated conditional knockout (cKO) mice with cardiomyocyte-specific deletion of Wdr1, the mammalian homolog of yeast AIP1. Wdr1 cKO mice began to die at postnatal day 13 (P13), and none survived past P24. At P12, cKO mice exhibited cardiac hypertrophy and impaired contraction of the left ventricle. Electrocardiography revealed reduced heart rate, abnormal P wave, and abnormal T wave at P10 and prolonged QT interval at P12. Actin filament (F-actin) accumulations began at P10 and became prominent at P12 in the myocardium of cKO mice. Within regions of F-actin accumulation in myofibrils, the sarcomeric components α-actinin and tropomodulin-1 exhibited disrupted patterns, indicating that F-actin accumulations caused by Wdr1 deletion result in disruption of sarcomeric structure. Ectopic cofilin colocalized with F-actin aggregates. In adult mice, Wdr1 deletion resulted in similar but much milder phenotypes of heart hypertrophy, F-actin accumulations within myofibrils, and lethality. Taken together, these results demonstrate that AIP1-regulated actin dynamics play essential roles in heart function in mice.

eThe protein kinase Akt plays a critical role in heart function and is activated by phosphorylation of threonine 308 (T308) and serine 473 (S473). While phosphoinositide-dependent kinase 1 (PDK1) is responsible for Akt T308 phosphorylation, the identities of the kinases for Akt S473 phosphorylation in the heart remain controversial. Here, we disrupted mTOR complex 2 (mTORC2) through deletion of Rictor in the heart and found normal heart growth and function. Rictor deletion caused significant reduction of Akt S473 phosphorylation but enhanced Akt T308 phosphorylation, suggesting that a high level of Akt T308 phosphorylation maintains Akt activity and heart function. Deletion of Pdk1 in the heart caused significantly enhanced Akt S473 phosphorylation that was suppressed by removal of Rictor, leading to worsened dilated cardiomyopathy (DCM) and accelerated heart failure in Pdk1-deficient mice. In addition, we found that increasing Akt S473 phosphorylation through deletion of Pten or chemical inhibition of PTEN reversed DCM and heart failure in Pdk1-deficient mice. Investigation of heart samples from human DCM patients revealed changes similar to those in the mouse models. These results demonstrated that PDK1 and mTORC2 synergistically promote postnatal heart growth and maintain heart function in postnatal mice.

BackgroundThe Twist1-family basic helix-loop-helix (bHLH) transcription factors including Twist1, Hand1 and Hand2, play an essential role in heart development and are implicated in pathological heart remodeling. Previously, it was reported that these bHLH transcription factors can be regulated by phosphorylation within the basic-helix I domain, which is involved in developmental processes such as limb formation and trophoblast differentiation. However, how phosphorylation of Twist1 family functions in post-natal heart is elusive.Principal FindingsHere, we generated transgenic mice with over-expression of Hand1 and Twist1 mutants (to mimic or to abolish phosphorylation) in cardiomyocytes and found pathological cardiac remodeling leading to heart failure and sudden death. Gene expression profile analysis revealed up-regulation of growth-promoting genes and down-regulation of metabolic genes. It is well known that aberrant activation of Akt signaling causes pathological cardiac remodeling and results in heart failure. The basic-helix I domain of Twist1 family members contain Akt substrate consensus motif and may be downstream targets of Akt signaling. Using biochemical analysis, we demonstrated that Hand1 and Twist1 were phosphorylated by Akt in the basic-helix I domain. Phosphorylation of Hand1 regulated its transcriptional activation of luciferase reporter genes and DNA binding ability.ConclusionsThis study provides novel insights into the regulation of Twist1 family in cardiac remodeling and suggests that the Twist1 family can be regulated by Akt signaling.

Background/Aims: Previous studies have indicated that long non-coding RNAs (lncRNA) are related to the occurrence and development of many human diseases, such as cancer and the HELLP and the brachydactyly syndromes. However, studies of LncRNA in heart failure have not yet been reported. Here, we investigated cardiac lncRNA expression profiles in the myocardial-specific knockout pdk1 gene (KO) mouse model of heart failure. Methods: Cardiac samples were obtained from PDK1 KO and WT mice on postnatal (P) day 8 (P8) and day 40 (P40), and lncRNA expression profiles were analyzed by sequencing and screening using the Arraystar mouse lncRNA microarray. Quantitative real-time PCR analysis of these lncRNAs confirmed the identity of some genes. Results: Comparisons of the KO and control groups showed fold changes of >1.5 in the expression levels of 2,024 lncRNAs at P8, while fold changes of >2 in the expression levels of 4,095 lncRNAs were detected at P40. Nineteen lncRNAs were validated by RT-PCR. Bioinformatic and pathway analyses indicated that mkk7, a sense overlap lncRNA, may be involved in the pathological processes of heart failure through the MAPK signaling pathway. Conclusion: These data reveal differentially expressed lncRNA in mice with a myocardial-specific deletion of the pdk1 gene, which may provide new insights into the mechanism of heart failure in PDK1 knockout mice.