Suppression of RBFox2 by Multiple MiRNAs in Pressure Overload-Induced Heart Failure.

Heart failure is the final stage of various cardiovascular diseases and seriously threatens human health. Increasing mediators have been found to be involved in the pathogenesis of heart failure, including the RNA binding protein RBFox2. It participates in multiple aspects of the regulation of cardiac function and plays a critical role in the process of heart failure. However, how RBFox2 itself is regulated remains unclear. Here, we dissected transcriptomic signatures, including mRNAs and miRNAs, in a mouse model of heart failure after TAC surgery. A global analysis showed that an asymmetric alternation in gene expression and a large-scale upregulation of miRNAs occurred in heart failure. An association analysis revealed that the latter not only contributed to the degradation of numerous mRNA transcripts, but also suppressed the translation of key proteins such as RBFox2. With the aid of Ago2 CLIP-seq data, luciferase assays verified that RBFox2 was targeted by multiple miRNAs, including Let-7, miR-16, and miR-200b, which were significantly upregulated in heart failure. The overexpression of these miRNAs suppressed the RBFox2 protein and its downstream effects in cardiomyocytes, which was evidenced by the suppressed alternative splicing of the Enah gene and impaired E-C coupling via the repression of the Jph2 protein. The inhibition of Let-7, the most abundant miRNA family targeting RBFox2, could restore the RBFox2 protein as well as its downstream effects in dysfunctional cardiomyocytes induced by ISO treatment. In all, these findings revealed the molecular mechanism leading to RBFox2 depression in heart failure, and provided an approach to rescue RBFox2 through miRNA inhibition for the treatment of heart failure.

Kv4.2 phosphorylation by PKA drives Kv4.2-KChIP2 dissociation, leading to Kv4.2 out of lipid rafts and internalization.

Sympathetic regulation of the Kv4.2 transient outward potassium current (Ito) is critical for the acute electrical and contractile response of the myocardium under physiological and pathological conditions. Previous studies have suggested that KChIP2, the key auxiliary subunit of Kv4 channels, is required for the sympathetic regulation of Kv4.2 current densities. Of interest, Kv4.2 and KChIP2, and key components mediating acute sympathetic signaling transduction are present in lipid rafts, which are profoundly involved in regulation of Ito densities in rat ventricular myocytes. However, little is known about the mechanisms of Kv4.2-raft association and its connection with acute sympathetic regulation. With the aid of high-resolution fluorescent microscope, we demonstrated that KChIP2 assisted Kv4.2 localization in lipid rafts in HEK293 cells. Moreover, PKA-mediated Kv4.2 phosphorylation, the downstream signaling event of acute sympathetic stimulation, induced dissociation between Kv4.2 and KChIP2, resulting in Kv4.2 shifting out of lipid rafts in KChIP2-expressed HEK293. The mutation that mimics Kv4.2 phosphorylation by PKA (K4.2-S552D) similarly disrupted Kv4.2 interaction with KChIP2 and also decreased the surface stability of Kv4.2. The attenuated Kv4.2-KChIP2 interaction was also observed in native neonatal rat ventricular myocytes (NRVMs) upon acute adrenergic stimulation with phenylephrine (PE). Furthermore, PE stimulation decreased Kv4.2 location at lipid rafts and induced internalization of Kv4.2 as well as the effect of lipid rafts disruption. In conclusion, KChIP2 contributes to targeting Kv4.2 to lipid rafts. Acute adrenergic stimulation induces Kv4.2-KChIP2 dissociation, leading to Kv4.2 out of lipid rafts and internalization, reinforcing the critical role of Kv4.2-lipid raft association in the essential physiological response of Ito to acute sympathetic regulation.

TFEB insufficiency promotes cardiac hypertrophy by blocking autophagic degradation of GATA4.

Autophagosome-lysosome pathway (ALP) insufficiency has been suggested to play a critical role in the pathogenesis of cardiac hypertrophy. However, the mechanisms underlying ALP insufficiency remain largely unknown, and strategies to specifically manipulate ALP insufficiency for treating cardiac hypertrophy are lacking. Transcription factor EB (TFEB), as a master regulator of ALP, regulates the generation and function of autophagosomes and lysosomes. We found that TFEB was significantly decreased, whereas autophagosome markers were increased in phenylephrine (PE)-induced and transverse aortic constriction-induced cardiomyocyte hypertrophy and failing hearts from patients with dilated cardiomyopathy. Knocking down TFEB induced ALP insufficiency, as indicated by increased autophagosome markers, decreased light chain 3II flux, and cardiomyocyte hypertrophy manifested through increased levels of atrial natriuretic peptide and ß-myosin heavy chain and enlarged cell size. The effects of TFEB knockdown were abolished by promoting autophagy. TFEB overexpression improved autophagic flux and attenuated PE-stimulated cardiomyocyte hypertrophy and transverse aortic constriction-induced hypertrophic remodeling, fibrosis, and cardiac dysfunction. Curcumin analog compound C1, a specific TFEB activator, similarly attenuated PE-induced ALP insufficiency and cardiomyocyte hypertrophy. TFEB knockdown increased the accumulation of GATA4, a transcription factor for several genes causing cardiac hypertrophy by blocking autophagic degradation of GATA4, whereas knocking down GATA4 attenuated TFEB downregulation-induced cardiomyocyte hypertrophy. Both TFEB overexpression and C1 promoted GATA4 autophagic degradation and alleviated PE-induced cardiomyocyte hypertrophy. In conclusion, TFEB downregulation plays a vital role in the development of pressure overload-induced cardiac hypertrophy by causing ALP insufficiency and blocking autophagic degradation. Activation of TFEB represents a potential therapeutic strategy for treating cardiac hypertrophy.

HIMF deletion ameliorates acute myocardial ischemic injury by promoting macrophage transformation to reparative subtype.

Appropriately manipulating macrophage M1/M2 phenotypic transition is a promising therapeutic strategy for tissue repair after myocardial infarction (MI). Here we showed that gene ablation of hypoxia-induced mitogenic factor (HIMF) in mice (Himf-/- and HIMFflox/flox;Lyz2-Cre) attenuated M1 macrophage-dominated inflammatory response and promoted M2 macrophage accumulation in infarcted hearts. This in turn reduced myocardial infarct size and improved cardiac function after MI. Correspondingly, expression of HIMF in macrophages induced expression of pro-inflammatory cytokines; the culturing medium of HIMF-overexpressing macrophages impaired the cardiac fibroblast viability and function. Furthermore, macrophage HIMF was found to up-regulate C/EBP-homologous protein (CHOP) expression, which exaggerated the release of pro-inflammatory cytokines via activating signal transducer of activator of transcription 1 (STAT1) and 3 (STAT3) signaling. Together these data suggested that HIMF promotes M1-type and prohibits M2-type macrophage polarization by activating the CHOP-STAT1/STAT3 signaling pathway to negatively regulate myocardial repair. HIMF might thus constitute a novel target to treat MI.

HIMF (Hypoxia-Induced Mitogenic Factor)-IL (Interleukin)-6 Signaling Mediates Cardiomyocyte-Fibroblast Crosstalk to Promote Cardiac Hypertrophy and Fibrosis.

HIMF (hypoxia-induced mitogenic factor) is a secreted proinflammatory cytokine with a critical role in cardiac hypertrophy development. Loss of HIMF attenuates transverse aortic constriction-induced cardiac hypertrophy and fibrosis, but the underlying mechanisms are unknown. We show that IL (interleukin)-6 production increases following transverse aortic constriction in wild-type mice; this effect is inhibited in HIMF gene knockout ( Himf-/-) mice. IL-6 production also increases in cultured cardiac myocytes overexpressing HIMF and neutralizing IL-6 with an anti-IL-6 antibody prohibits HIMF-induced cardiomyocyte hypertrophy. HIMF expression in cardiac fibroblasts cannot be stimulated by transverse aortic constriction or exposure to prohypertrophic factors, including phenylephrine, Ang II (angiotensin II), TGF (transform growth factor)-ß, and hypoxia. However, conditioned medium from cardiomyocytes overexpressing HIMF can increase IL-6 production, and cardiac fibroblast proliferation, migration, and myofibroblast differentiation to a similar level as exposure to exogenous rHIMF (recombinant HIMF). Again, neutralizing IL-6 prevented cardiac fibroblasts activation. Finally, the MAPK (mitogen-activated protein kinase) and CaMKII (Ca2+/calmodulin-dependent protein kinase II)-STAT3 (signal transducers and activators of transcription 3) pathways are activated in HIMF-overexpressing cardiomyocytes and rHIMF-stimulated cardiac fibroblasts; this effect can be inhibited on neutralizing IL-6. These data support that HIMF induces cardiac fibrosis via a cardiomyocyte-to-fibroblast paracrine effect. IL-6 is a downstream signal of HIMF and has a central role in cardiomyocyte hypertrophy and myocardial fibrosis that is mediated by activating the MAPK and CaMKII-STAT3 pathways.

MG53, A Novel Regulator of KChIP2 and Ito,f, Plays a Critical Role in Electrophysiological Remodeling in Cardiac Hypertrophy.

BACKGROUND: KChIP2 (K+ channel interacting protein) is the auxiliary subunit of the fast transient outward K+ current ( Ito,f) in the heart, and insufficient KChIP2 expression induces Ito,f downregulation and arrhythmogenesis in cardiac hypertrophy. Studies have shown muscle-specific mitsugumin 53 (MG53) has promiscuity of function in the context of normal and diseased heart. This study investigates the possible roles of cardiac MG53 in regulation of KChIP2 expression and Ito,f, and the arrhythmogenic potential in hypertrophy. METHODS: MG53 expression is manipulated by genetic ablation of MG53 in mice and adenoviral overexpression or knockdown of MG53 by RNA interference in cultured neonatal rat ventricular myocytes. Cardiomyocyte hypertrophy is produced by phenylephrine stimulation in neonatal rat ventricular myocytes, and pressure overload-induced mouse cardiac hypertrophy is produced by transverse aortic constriction. RESULTS: KChIP2 expression and Ito,f density are downregulated in hearts from MG53-knockout mice and MG53-knockdown neonatal rat ventricular myocytes, but upregulated in MG53-overexpressing cells. In phenylephrine-induced cardiomyocyte hypertrophy, MG53 expression is reduced with concomitant downregulation of KChIP2 and Ito,f, which can be reversed by MG53 overexpression, but exaggerated by MG53 knockdown. MG53 knockout enhances Ito,f remodeling and action potential duration prolongation and increases susceptibility to ventricular arrhythmia in mouse cardiac hypertrophy. Mechanistically, MG53 regulates NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity and subsequently controls KChIP2 transcription. Chromatin immunoprecipitation demonstrates NF-κB protein has interaction with KChIP2 gene. MG53 overexpression decreases, whereas MG53 knockdown increases NF-κB enrichment at the 5' regulatory region of KChIP2 gene. Normalizing NF-κB activity reverses the alterations in KChIP2 in MG53-overexpressing or knockdown cells. Coimmunoprecipitation and Western blotting assays demonstrate MG53 has physical interaction with TAK1 (transforming growth factor-b [TGFb]-activated kinase 1) and IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), critical components of the NF-κB pathway. CONCLUSIONS: These findings establish MG53 as a novel regulator of KChIP2 and Ito,f by modulating NF-κB activity and reveal its critical role in electrophysiological remodeling in cardiac hypertrophy.

A Novel, Multi-Target Natural Drug Candidate, Matrine, Improves Cognitive Deficits in Alzheimer's Disease Transgenic Mice by Inhibiting Aß Aggregation and Blocking the RAGE/Aß Axis.

The treatment of AD is a topic that has puzzled researchers for many years. Current mainstream theories still consider Aß to be the most important target for the cure of AD. In this study, we attempted to explore multiple targets for AD treatments with the aim of identifying a qualified compound that could both inhibit the aggregation of Aß and block the RAGE/Aß axis. We believed that a compound that targets both Aß and RAGE may be a feasible strategy for AD treatment. A novel and small natural compound, Matrine (Mat), was identified by high-throughput screening of the main components of traditional Chinese herbs used to treat dementia. Various experimental techniques were used to evaluate the effect of Mat on these two targets both in vitro and in AD mouse model. Mat could inhibit Aß42-induced cytotoxicity and suppress the Aß/RAGE signaling pathway in vitro. Additionally, the results of in vivo evaluations of the effects of Mat on the two targets were consistent with the results of our in vitro studies. Furthermore, Mat reduced proinflammatory cytokines and Aß deposition and attenuated the memory deficits of AD transgenic mice. We believe that this novel, multi-target strategy to inhibit both Aß and RAGE, is worthy of further exploration. Therefore, our future studies will focus on identifying even more effective multi-target compounds for the treatment of AD based on the molecular structure of Mat.

A Highly Selective Inhibitor of Glycine Transporter-1 Elevates the Threshold for Maximal Electroshock-Induced Tonic Seizure in Mice.

Many anti-epileptic drugs (AEDs) that mainly target ion channels or post-synaptic receptors are in clinical use, but a proportion of patients are resistant to these traditional AEDs and experience repeated severe break-out seizures. Given its involvement in the etiology of epilepsy, the neurotransmitter glycine may serve as a novel target for epilepsy treatment. Increasing evidence suggests that inhibitors of glycine transporter 1 (GlyT1) exhibit anti-seizure properties in mouse models and show potential as anti-convulsions drugs. In the present study, we investigated the effect of a highly selective GlyT1 inhibitor (named M22) on glycine transport kinetics using a radioactive substrate uptake assay and investigated the anti-seizure effects of M22 on the maximal electroshock seizure threshold (MEST) test and the timed intravenous (i.v.) pentylenetetrazole (PTZ) intravenous test. Our results demonstrate that M22 was capable of elevating the seizure threshold in the MEST test but did not alter the seizure threshold in the PTZ i.v. test. Strychnine, an inhibitor of glycine receptor activity, reversed the threshold elevation at a subconvulsive dosage (0.1 mg/kg subcutaneously) in the MEST test and did not affect M22 plasma levels in mice, suggesting that the anti-seizure effect in this model may be mediated by increased glycine receptor activity. Moreover, M22 administration did not influence motor function and coordination in mice. In combination with the previously reported excellent pharmacokinetic features of M22, our present results suggested that M22 has the potential to serve as a new anti-convulsive drug or as a lead compound for the development of AEDs.

An ADAM10 promoter polymorphism is a functional variant in severe sepsis patients and confers susceptibility to the development of sepsis.

INTRODUCTION: Although genetic variants of the A disintegrin and metalloproteinase 10 (ADAM10) gene have been shown to be associated with susceptibility to several inflammatory-related diseases, to date little is known about the clinical relationship in the development of sepsis. METHODS: Two genetic variants in the promoter of ADAM10 were selected to analyze the potential association with the risk of sepsis. A total of 440 sepsis patients and 450 matched healthy individuals in two independent Chinese Han population were enrolled. Pyrosequencing and polymerase chain reaction-length polymorphism was used to determine the genotypes of the rs514049 and rs653765. A real-time qPCR method was used to detect the mRNA level of ADAM10. Enzyme-linked immunosorbent assay was used to measure the expression levels of substrates CX3CL1, interleukin (IL)-6R, tumor necrosis factor alpha (TNF-α), and the pro-inflammatory cytokines IL-1ß and IL-6. Luciferase assay was used to analyze the activities of the promoter haplotypes of ADAM10. RESULTS: No statistically significant differences between sepsis cases and controls in the genotype or allele frequencies were observed, suggesting that ADAM10 single nucleotide polymorphisms (SNPs) may not be risk factors for the occurrence of sepsis. A significant difference in the genotype and allele frequencies of the rs653765 SNP between patients with sepsis subtype and severe sepsis (P = 0.0014) or severe sepsis/sepsis shock (P = 0.0037) were observed. Moreover, the rs653765 CC genotype in severe sepsis showed a higher ADAM10 level compared to healthy groups, and the rs653765 CC polymorphism had a strong impact on the production of the ADAM10 substrates CX3CL1, IL-6R and TNF-α. Furthermore, the functional assay showed that ADAM10 C-A haplotype carriers exhibited significantly higher reporter activity compared with the T-A carriers and T-C carriers in human acute monocytic leukemia cell line. CONCLUSIONS: Our data initially indicated the ADAM10 rs653765 polymorphism was associated with the development of severe sepsis; the risk CC genotype could functionally affect the expression level of ADAM10 mRNA and was accompanied by the up-regulation of its substrates. Thus, ADAM10 might be clinically important and play a critical role in the pathogenesis of the development of sepsis, with potentially important therapeutic implications.

A functional polymorphism of the microRNA-146a gene is associated with susceptibility to drug-resistant epilepsy and seizures frequency.

PURPOSE: Epilepsy is the third most common chronic brain disorder and is characterized by an enduring predisposition for seizures. Recently, a growing body of evidence has suggested that microRNA-146a (miR-146a) is upregulated in the brains of epilepsy patients and of mouse models; furthermore, miR-146a may be involved in the development and progression of seizures through the regulation of inflammation and immune responses. In this report, we performed a case-control study to analyze the relationship between the two potentially functional single nucleotide polymorphisms (SNPs) of the miR-146a gene (rs2910464 and rs57095329) and the risk of epilepsy in a Chinese population comprising 249 cases and 249 healthy controls. METHOD: Our study comprised 249 epilepsy patients and 249 healthy controls in two regions of China. The DNA was genotyped using the ABI PRISM SNapShot method. The statistical analysis was estimated using the chi-square test or Fisher's exact test. RESULTS: Our results indicated a significant association between the rs57095329 SNP of the miR-146a gene and the risk of drug resistant epilepsy (DRE) (genotypes, p = 0.0258 and alleles, p = 0.0108). Moreover, the rs57095329 A allele was found to be associated with a reduced risk of seizures frequency in DRE patients (all p < 0.001). However, the rs2910164 variant was not associated with epilepsy. CONCLUSION: Our data indicate that the rs57095329 polymorphism in the promoter region of miR-146a is involved in the genetic susceptibility to DRE and the seizures frequency.