Novel roles of an intragenic G-quadruplex in controlling microRNA expression and cardiac function.

Simultaneous dysregulation of multiple microRNAs (miRs) affects various pathological pathways related to cardiac failure. In addition to being potential cardiac disease-specific markers, miR-23b/27b/24-1 were reported to be responsible for conferring cardiac pathophysiological processes. In this study, we identified a conserved guanine-rich RNA motif within the miR-23b/27b/24-1 cluster that can form an RNA G-quadruplex (rG4) in vitro and in cells. Disruption of this intragenic rG4 significantly increased the production of all three miRs. Conversely, a G4-binding ligand tetrandrine (TET) stabilized the rG4 and suppressed miRs production in human and rodent cardiomyocytes. Our further study showed that the rG4 prevented Drosha-DGCR8 binding and processing of the pri-miR, suppressing the biogenesis of all three miRs. Moreover, CRISPR/Cas9-mediated G4 deletion in the rat genome aberrantly elevated all three miRs in the heart in vivo, leading to cardiac contractile dysfunction. Importantly, loss of the G4 resulted in reduced targets for the aforementioned miRs critical for normal heart function and defects in the L-type Ca2+ channel-ryanodine receptor (LCC-RyR) coupling in cardiomyocytes. Our results reveal a novel mechanism for G4-dependent regulation of miR biogenesis, which is essential for maintaining normal heart function.

DCA-TGR5 signaling activation alleviates inflammatory response and improves cardiac function in myocardial infarction.

AIMS: The progression of myocardial infarction (MI) involves multiple metabolic disorders. Bile acid metabolites have been increasingly recognized as pleiotropic signaling molecules that regulate multiple cardiovascular functions. G protein-coupled bile acid receptor (TGR5) is one of the receptors sensing bile acids to mediate their biological functions. In this study, we aimed to elucidate the effects of bile acids-TGR5 signaling pathways in myocardial infarction (MI). METHODS AND RESULTS: Blood samples of AMI patients or control subjects were collected and plasma was used for bile acid metabolism analysis. We discovered that bile acid levels were altered and deoxycholic acid (DCA) was substantially reduced in the plasma of AMI patients. Mice underwent either the LAD ligation model of MI or sham operation. Both MI and sham mice were gavaged with 10 mg/kg/d DCA or vehicle control since 3-day before the operation. Cardiac function was assessed by ultrasound echocardiography, infarct area was evaluated by TTC staining and Masson trichrome staining. Administration of DCA improved cardiac function and reduced ischemic injury at the 7th-day post-MI. The effects of DCA were dependent on binding to its receptor TGR5. Tgr5-/- mice underwent the same MI model. Cardiac function deteriorated and infarct size was increased at the 7th-day post-MI, which were not savaged by DCA administration. Moreover, DCA inhibited interleukin (IL)-1ß expression in the infarcted hearts, and ameliorated IL-1ß activation at 1-day post-MI. DCA inhibited NF-κB signaling and further IL-1ß expression in cultured neonatal mouse cardiomyocytes under hypoxia as well as cardio-fibroblasts with the treatment of LPS. CONCLUSIONS: DCA-TGR5 signaling pathway activation decreases inflammation and ameliorates heart function post-infarction. Strategies that control bile acid metabolism and TGR5 signaling to ameliorate the inflammatory responses may provide beneficial effects in patients with myocardial infarction.

Cystathionine γ lyase-hydrogen sulfide increases peroxisome proliferator-activated receptor γ activity by sulfhydration at C139 site thereby promoting glucose uptake and lipid storage in adipocytes.

Adipocytes express the cystathionine γ lyase (CSE)-hydrogen sulfide (H2S) system. CSE-H2S promotes adipogenesis but ameliorates adipocyte insulin resistance. We investigated the mechanism of how CSE-H2S induces these paradoxical effects. First, we confirmed that an H2S donor or CSE overexpression promoted adipocyte differentiation. Second, we found that H2S donor inhibited but CSE inhibition increased phosphodiesterase (PDE) activity. H2S replacing isobutylmethylxanthine in the differentiation program induced adipocyte differentiation in part. Inhibiting PDE activity by H2S induced peroxisome proliferator activated receptor γ (PPARγ) protein and mRNA expression. Of note, H2S directly sulfhydrated PPARγ protein. Sulfhydrated PPARγ increased its nuclear accumulation, DNA binding activity and adipogenesis gene expression, thereby increasing glucose uptake and lipid storage, which were blocked by the desulfhydration reagent DTT. H2S induced PPARγ sulfhydration, which was blocked by mutation of the C139 site of PPARγ. In mice fed a high-fat diet (HFD) for 4 weeks, the CSE inhibitor decreased but H2S donor increased adipocyte numbers. In obese mice fed an HFD for 13 weeks, H2S treatment increased PPARγ sulfhydration in adipose tissues and attenuated insulin resistance but did not increase obesity. In conclusion, CSE-H2S increased PPARγ activity by direct sulfhydration at the C139 site, thereby changing glucose into triglyceride storage in adipocytes. CSE-H2S-mediated PPARγ activation might be a new therapeutic target for diabetes associated with obesity.

The Role of METTL3-Mediated N6-Methyladenosine (m6A) of JPH2 mRNA in Cyclophosphamide-Induced Cardiotoxicity.

Cyclophosphamide (CYP)-induced cardiotoxicity is a common side effect of cancer treatment. Although it has received significant attention, the related mechanisms of CYP-induced cardiotoxicity remain largely unknown. In this study, we used cell and animal models to investigate the effect of CYP on cardiomyocytes. Our data demonstrated that CYP-induced a prolonged cardiac QT interval and electromechanical coupling time courses accompanied by JPH2 downregulation. Moreover, N6-methyladenosine (m6A) methylation sequencing and RNA sequencing suggested that CYP induced cardiotoxicity by dysregulating calcium signaling. Importantly, our results demonstrated that CYP induced an increase in the m6A level of JPH2 mRNA by upregulating methyltransferases METTL3, leading to the reduction of JPH2 expression levels, as well as increased field potential duration and action potential duration in cardiomyocytes. Our results revealed a novel mechanism for m6A methylation-dependent regulation of JPH2, which provides new strategies for the treatment and prevention of CYP-induced cardiotoxicity.

Sulfhydrated Sirtuin-1 Increasing Its Deacetylation Activity Is an Essential Epigenetics Mechanism of Anti-Atherogenesis by Hydrogen Sulfide.

Aims: Hydrogen sulfide (H2S) has a protective role in the pathogenesis of atherosclerosis by multiple pathways. Sirtuin-1 (SIRT1) is a histone deacetylase, as an essential mediated longevity gene, and has an anti-atherogenic effect by regulating the acetylation of some functional proteins. Whether SIRT1 is involved in protecting H2S in atherosclerosis and its mechanism remains unclear. Results: In ApoE-knockout atherosclerosis mice, treatment with an H2S donor (NaHS or GYY4137) reduced atherosclerotic plaque area, macrophage infiltration, aortic inflammation, and plasma lipid level. H2S treatment increased aorta and liver SIRT1 mRNA expression. Overexpression or slicing cystathionine gamma lyase (CSE) also changed intracellular SIRT1 expression. CSE/H2S treatment increased SIRT1 deacetylation in endothelium and hepatocytes and macrophages, then induced deacetylation of its target proteins (P53, P65, and sterol response element binding protein), thereby reducing endothelial and macrophage inflammation and inhibiting macrophage cholesterol uptake and cholesterol de novo synthesis of liver. Also, CSE/H2S induced SIRT1 sulfhydration at its two zinc finger domains, increased its zinc ion binding activity to stabilize the alpha-helix structure, lowered its ubiquitination, and reduced its degradation. Innovation: H2S is a novel SIRT1 activator by direct sulfhydration. Because SIRT1 has a role in longevity, H2S may be a protector for aging-related diseases. Conclusion: Endogenous CSE/H2S directly sulfhydrated SIRT1, enhanced SIRT1 binding to zinc ion, then promoted its deacetylation activity, and increased SIRT1 stability, thus reducing atherosclerotic plaque formation.

AK098656, a Novel Vascular Smooth Muscle Cell-Dominant Long Noncoding RNA, Promotes Hypertension.

Recent studies reported some long noncoding RNAs (lncRNAs)-mediated vascular smooth muscle cells (VSMCs) phenotypic switch, which was a common pathophysiological process of vascular diseases. However, whether human-specific expressed lncRNAs would modulate VSMCs phenotype and participate into the pathogenesis of essential hypertension remains unclear. By comparing the circulating lncRNAs expression profiles between hypertensive patients and healthy controls, we identified a lncRNA-AK098656, strongly upregulated in the plasma of hypertensive patients, and predominantly expressed in VSMCs. AK098656 promoted VSMCs synthetic phenotype evidenced by increasing VSMC proliferation and migration, elevating extracellular matrix proteins, whereas lowering contractile proteins. Furthermore, AK098656 was demonstrated to directly bind with the VSMCs-specific contractile protein, myosin heavy chain-11, and an essential component of extracellular matrix, fibronectin-1, and finally lowered these protein levels through protein degradation. AK098656 was also shown to bind with 26S proteasome non-ATPase regulatory subunit 11 and facilitated myosin heavy chain-11 to interact with this protein. In vivo, AK098656 transgenic rats showed spontaneous development of hypertension, with elevated VSMCs synthetic phenotype and narrowed resistant arteries. Transgenic rats also showed slight cardiac hypertrophy without other complications, which was similar with early pathophysiological changes of hypertension. All these data indicated AK098656 as a new human VSMC-dominant lncRNA, which could promote hypertension through accelerating contractile protein degradation, increasing VSMC synthetic phenotype, and finally narrowed resistance arteries.

Cystathionine γ-Lyase-Hydrogen Sulfide Induces Runt-Related Transcription Factor 2 Sulfhydration, Thereby Increasing Osteoblast Activity to Promote Bone Fracture Healing.

AIMS: Hydrogen sulfide (H2S) plays an essential role in bone formation, in part, by inhibiting osteoclast differentiation, maintaining mesenchymal stem cell osteogenesis ability, or reducing osteoblast injury. We aimed to investigate the role of H2S in osteoblast function and its possible molecular target. RESULTS: In this study, we found that cystathionine γ-lyase (CSE) majorly contributed to endogenous H2S production in the primary osteoblast. Overexpressed CSE increased osteoblast differentiation and maturation with higher bone morphogenetic protein 2 and osteopontin expression, alkaline phosphatase activity, and calcium nodule formation; in contrast, knockdown of CSE had opposite effects. Runt-related transcript factor 2 (RUNX2) is required for osteoblast biologic function. CSE-H2S increased nuclear RUNX2 accumulation, DNA binding activity, and target gene transcription. Protein sulfhydration is a common signal by H2S. We confirmed that RUNX2 was also sulfhydrated by H2S. This chemical modification enhanced RUNX2 transactivation, which was blocked by dithiothreitol (DTT, sulfhydration remover). Mutation of two cysteine sites in the runt domain of RUNX2 abolished H2S-induced RUNX2 sulfhydration and transactivation. In a bone -fracture rat model, overexpressed CSE promoted bone healing, which confirmed the effect of CSE-H2S on osteoblasts. INNOVATION: CSE-H2S is a dominant H2S generation system in osteoblasts and promotes osteoblast activity by the RUNX2 pathway, with RUNX2 sulfhydration as a novel transactivation regulation. CONCLUSION: CSE-H2S sulfhydrated RUNX2 enhanced its transactivation and increased osteoblast differentiation and maturation, thereby promoting bone healing. Antioxid. Redox Signal. 27, 742-753.