Deficiency of Transcription Factor Sp1 Contributes to Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disorder. However, the pathogenesis of HCM, especially its nongenetic mechanisms, remains largely unclear. Transcription factors are known to be involved in various biological processes including cell growth. We hypothesized that SP1 (specificity protein 1), the first purified TF in mammals, plays a role in the cardiomyocyte growth and cardiac hypertrophy of HCM. METHODS: Cardiac-specific conditional knockout of Sp1 mice were constructed to investigate the role of SP1 in the heart. The echocardiography, histochemical experiment, and transmission electron microscope were performed to analyze the cardiac phenotypes of cardiac-specific conditional knockout of Sp1 mice. RNA sequencing, chromatin immunoprecipitation sequencing, and adeno-associated virus experiments in vivo were performed to explore the downstream molecules of SP1. To examine the therapeutic effect of SP1 on HCM, an SP1 overexpression vector was constructed and injected into the mutant allele of Myh6 R404Q/+ (Myh6 c. 1211C>T) HCM mice. The human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with HCM were used to detect the potential therapeutic effects of SP1 in human HCM. RESULTS: The cardiac-specific conditional knockout of Sp1 mice developed a typical HCM phenotype, displaying overt myocardial hypertrophy, interstitial fibrosis, and disordered myofilament. In addition, Sp1 knockdown dramatically increased the cell area of hiPSC-CMs and caused intracellular myofibrillar disorganization, which was similar to the hypertrophic cardiomyocytes of HCM. Mechanistically, Tuft1 was identified as the key target gene of SP1. The hypertrophic phenotypes induced by Sp1 knockdown in both hiPSC-CMs and mice could be rescued by TUFT1 (tuftelin 1) overexpression. Furthermore, SP1 overexpression suppressed the development of HCM in the mutant allele of Myh6 R404Q/+ mice and also reversed the hypertrophic phenotype of HCM hiPSC-CMs. CONCLUSIONS: Our study demonstrates that SP1 deficiency leads to HCM. SP1 overexpression exhibits significant therapeutic effects on both HCM mice and HCM hiPSC-CMs, suggesting that SP1 could be a potential intervention target for HCM.

Cadherin-11-Interleukin-6 Signaling between Cardiac Fibroblast and Cardiomyocyte Promotes Ventricular Remodeling in a Mouse Pressure Overload-Induced Heart Failure Model.

Heart failure is a serious and life-threatening disease worldwide. Cadherin-11 (Cad-11) is highly expressed in the heart and closely associated with inflammation. There is currently limited understanding on how Cad-11 contributes to cardiac remodeling and its underline molecular mechanism. We found an increased expression of Cad-11 in biopsy heart samples from heart failure patients, suggesting a link between Cad-11 and heart failure. To determine the role of Cad-11 in cardiac remodeling, Cad-11-deficient mice were used in a well-established mouse transverse aortic constriction (TAC) model. Loss of Cad11 greatly improved pressure overload-induced LV structural and electrical remodeling. IL (interleukin)-6 production was increased following TAC in WT mice and this increase was inhibited in cadherin-11-/- mice. We further tested the effect of IL-6 on myocyte hypertrophy and fibrosis in a primary culture system. The addition of hCad-11-Fc to cultured cardiac fibroblasts increased IL-6 production and fibroblast cell activation, whereas neutralizing IL-6 with an IL-6 antibody resulted in alleviating the fibroblast activation induced by hCad-11-Fc. On the other hand, cardiomyocytes were promoted to cardiomyocyte hypertrophy when cultured in condition media collected from cardiac fibroblasts stimulated by hCad-11-Fc.Similarly, neutralizing IL-6 prevented cardiomyocyte hypertrophy. Finally, we found that MAPKs and CaMKII-STAT3 pathways were activated in both hCad-11-Fc stimulated fibroblasts and cardiomyocytes treated with hCad-11-Fc stimulated fibroblast condition medium. IL-6 neutralization inhibited such MAPK and CaMKII-STAT3 signaling activation. These data demonstrate that Cad-11 functions in pressure overload-induced ventricular remodeling through inducing IL-6 secretion from cardiac fibroblasts to modulate the pathophysiology of neighboring cardiomyocytes.

Cadherin-11 deficiency mitigates high-fat diet-induced inflammatory atrial remodeling and vulnerability to atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia nowadays. The occurrence of AF is closely associated with obesity. Cadherin-11 (Cad-11), as a member of the cadherin family, can make a contribution to diet-induced obesity and it will be informative to know whether Cad-11 exerts its effects on atrial remodeling and AF vulnerability in a diet-induced obesity model. In this study, we demonstrated that the expression of Cad-11 was significantly upregulated in the left atrium of AF patients with obesity and mice following 16 weeks of high-fat diet (HFD) feeding. Further confirmed that Cad-11 could regulate the activity of atrial fibroblasts by participating in inducing proinflammatory cytokines production. At animal levels, we found that although there was a lack of statistical difference in body weight, Cad-11-/- mice could markedly improve impaired glucose tolerance and hyperlipidemia. Adverse atrial structural remodeling, including atrial enlargement, inflammation, and fibrosis provoked by HFD feeding were mitigated in Cad-11-/- mice. Mechanistically, Cad-11 activated mitogen-activated protein kinases and nuclear factor-κB for interleukin-6 production in atrial fibroblasts that may contribute to the atrial fibrosis process in obesity-related AF, suggesting Cad-11 might be a new therapeutic target for obesity-related AF.

EZH2 as an Epigenetic Regulator of Cardiovascular Development and Diseases.

ABSTRACT: Enhancer of zeste homolog 2(EZH2) is an enzymatic subunit of polycomb repressive complex 2 (PRC2) and is responsible for catalyzing mono-, di-, and trimethylation of histone H3 at lysine-27(H3K27me1/2/3). Many noncoding RNAs or signaling pathways are involved in EZH2 functional alterations. This new epigenetic regulation of target genes is able to silence downstream gene expression and modify physiological and pathological processes in heart development, cardiomyocyte regeneration, and cardiovascular diseases, such as hypertrophy, ischemic heart diseases, atherosclerosis, and cardiac fibrosis. Targeting the function of EZH2 could be a potential therapeutic approach for cardiovascular diseases.

EZH2 as a novel therapeutic target for atrial fibrosis and atrial fibrillation.

Angiotensin II (Ang-II)-induced fibroblast differentiation plays an important role in the development of atrial fibrosis and atrial fibrillation (AF). Here, we show that the expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is increased in atrial muscle and atrial fibroblasts in patients with AF, accompanied by significant atrial fibrosis and atrial fibroblast differentiation. In addition, EZH2 is induced in murine models of atrial fibrosis. Furthermore, either pharmacological GSK126 inhibition or molecular silencing of EZH2 can inhibit the differentiation of atrial fibroblasts and the ability to produce ECM induced by Ang-II. Simultaneously, inhibition of EZH2 can block the Ang-II-induced migration of atrial fibroblasts. We found that EZH2 promotes fibroblast differentiation mainly through the Smad signaling pathway and can form a transcription complex with Smad2 to bind to the promoter region of the ACTA2 gene. Finally, our in vivo experiments demonstrated that the EZH2 inhibitor GSK126 significantly inhibited Ang-II-induced atrial enlargement and fibrosis and reduced AF vulnerability. Our results demonstrate that targeting EZH2 or EZH2-regulated genes might present therapeutic potential in AF.

Inhibition of BRD4 attenuates transverse aortic constriction- and TGF-ß-induced endothelial-mesenchymal transition and cardiac fibrosis.

Cardiac fibrosis (CF), a process characterized by potentiated proliferation of cardiac fibroblasts and excessive secretion and deposition of extracellular matrix (ECM) from the cells, contributes strongly to the pathogenesis of a series of cardiovascular (CV) diseases, including AMI, heart failure and atrial fibrillation. Endothelial-mesenchymal transition (EndMT), one of the sources of transformed cardiac fibroblasts, has been reported as a key factor involved in CF. However, the molecular basis of EndMT has not been thoroughly elucidated to date. At the posttranscriptional level, of the three epigenetic regulators, writer and eraser are reported to be involved in EndMT, but the role of reader in the process is still unknown. In this study, we aimed to explore the role of Bromodomain-containing protein 4 (BRD4), an acetyl-lysine reader protein, in EndMT-induced CF and related mechanisms. We found that BRD4 was upregulated in endothelial cells (ECs) in the pressure-overload mouse heart and that its functional inhibitor JQ1 potently attenuated the TAC-induced CF and preserved cardiac function. In umbilical vein endothelial cells (HUVECs) and mouse aortic endothelial cells (MAECs), bothJQ1 and shRNA-mediated silencing of BRD4 blocked TGF-ß-induced EC migration, EndMT and ECM synthesis and preserved the EC sprouting behavior, possibly through the downregulation of a group of transcription factors specific for EndMT (Snail, Twist and Slug), the Smads pathway and TGF-ß receptor I. In the absence of TGF-ß stimulation, ectopic expression of BRD4 alone could facilitate EndMT, accelerate migration and increase the synthesis of ECM. In vivo, JQ1 also attenuated TAC-induced EndMT and CF, which was consistent with JQ1's intracellular mechanisms of action. Our results showed that BRD4 plays a critical role in EndMT-induced CF and that targeting BRD4 might be a novel therapeutic option for CF.

Foxm1 is a critical driver of TGF-ß-induced EndMT in endothelial cells through Smad2/3 and binds to the Snail promoter.

Endothelial-to-mesenchymal transition (EndMT) was first reported in heart development. Recent studies have shown that EndMT also occurs in the progression of cardiac fibrosis. Herein, we demonstrated a critical role of the Forkhead Box M1 (Foxm1) transcription factor in transforming growth factor beta (TGF-ß)-induced EndMT in endothelial cells (ECs) and a possible underlying molecular mechanism. Foxm1 was induced in ECs following TGF-ß stimulation. Using both pharmacological and molecular approaches to inhibit Foxm1 function can attenuate the TGF-ß-induced EndMT and cell migration. In contrast, lentivirus-mediated overexpression of Foxm1 allowed EndMT to proceed despite the absence of TGF-ß in ECs. Moreover, we found that the activation of the Smad2/3 signaling pathway and EndMT-related transcription factors played important roles in the pathogenesis of Foxm1-mediated EndMT. Further analysis revealed that Foxm1 bound to and increased the promoter activity of the Snail gene encoding a critical transcriptional regulator of EndMT. In conclusion, our results identify FOXM1 as a driver of TGF-ß-induced EndMT and underscore the therapeutic potential of targeting FOXM1 for cardiac fibrosis.

Weft-knitted silk-poly(lactide-co-glycolide) mesh scaffold combined with collagen matrix and seeded with mesenchymal stem cells for rabbit Achilles tendon repair.

Natural silk fibroin fiber scaffolds have excellent mechanical properties, but degrade slowly. In this study, we used poly(lactide-co-glycolide) (PLGA, 10:90) fibers to adjust the overall degradation rate of the scaffolds and filled them with collagen to reserve space for cell growth. Silk fibroin-PLGA (36:64) mesh scaffolds were prepared using weft-knitting, filled with type I collagen, and incubated with rabbit autologous bone marrow-derived mesenchymal stem cells (MSCs). These scaffold-cells composites were implanted into rabbit Achilles tendon defects. At 16 weeks after implantation, morphological and histological observations showed formation of tendon-like tissues that expressed type I collagen mRNA and a uniformly dense distribution of collagen fibers. The maximum load of the regenerated Achilles tendon was 58.32% of normal Achilles tendon, which was significantly higher than control group without MSCs. These findings suggest that it is feasible to construct tissue engineered tendon using weft-knitted silk fibroin-PLGA fiber mesh/collagen matrix seeded with MSCs for rabbit Achilles tendon defect repair.

BRD4 as a therapeutic target for atrial fibrosis and atrial fibrillation.

OBJECTIVE: This study aimed to elucidate the molecular mechanisms by which BRD4 play a role in atrial fibrillation (AF). METHODS AND RESULTS: We used a discovery-driven approach to detect BRD4 expression in the atria of patients with AF and in various murine models of atrial fibrosis. We used a BRD4 inhibitor (JQ1) and atrial fibroblast (aFB)-specific BRD4-knockout mice to elucidate the role of BRD4 in AF. We further examined the underlying mechanisms using RNA-seq and ChIP-seq analyses in vitro, to identify key downstream targets of BRD4. We found that BRD4 expression is significantly increased in patients with AF, with accompanying atrial fibrosis and aFB differentiation. We showed that JQ1 treatment and shRNA-based molecular silencing of BRD4 blocked ANG-II-induced extracellular matrix production and cell-cycle progression in aFBs. BRD4-related RNA-seq and ChIP-seq analyses in aFBs demonstrated enrichment of a subset of promoters related to the expression of profibrotic and proliferation-related genes. The pharmacological inhibition of BRD4 in vivo or in aFB-specific BRD4-knockout in mice limited ANG-II-induced atrial fibrosis, atrial enlargement, and AF susceptibility. CONCLUSION: Our findings suggest that BRD4 plays a key role in pathological AF, at least partially by activating aFB proliferation and ECM synthesis. This study provides mechanistic insights into the development of BRD4 inhibitors as targeted antiarrhythmic therapies.

A GABAergic system in atrioventricular node pacemaker cells controls electrical conduction between the atria and ventricles.

Physiologically, the atria contract first, followed by the ventricles, which is the prerequisite for normal blood circulation. The above phenomenon of atrioventricular sequential contraction results from the characteristically slow conduction of electrical excitation of the atrioventricular node (AVN) between the atria and the ventricles. However, it is not clear what controls the conduction of electrical excitation within AVNs. Here, we find that AVN pacemaker cells (AVNPCs) possess an intact intrinsic GABAergic system, which plays a key role in electrical conduction from the atria to the ventricles. First, along with the discovery of abundant GABA-containing vesicles under the surface membranes of AVNPCs, key elements of the GABAergic system, including GABA metabolic enzymes, GABA receptors, and GABA transporters, were identified in AVNPCs. Second, GABA synchronously elicited GABA-gated currents in AVNPCs, which significantly weakened the excitability of AVNPCs. Third, the key molecular elements of the GABAergic system markedly modulated the conductivity of electrical excitation in the AVN. Fourth, GABAA receptor deficiency in AVNPCs accelerated atrioventricular conduction, which impaired the AVN's protective potential against rapid ventricular frequency responses, increased susceptibility to lethal ventricular arrhythmias, and decreased the cardiac contractile function. Finally, interventions targeting the GABAergic system effectively prevented the occurrence and development of atrioventricular block. In summary, the endogenous GABAergic system in AVNPCs determines the slow conduction of electrical excitation within AVNs, thereby ensuring sequential atrioventricular contraction. The endogenous GABAergic system shows promise as a novel intervention target for cardiac arrhythmias.

I2-mediated Csp2-P bond formation via tandem cyclization of o-alkynylphenyl isothiocyanates with organophosphorus esters.

A novel, I2-mediated tandem cyclization of o-alkynylphenyl isothiocyanates with organophosphorus esters has been developed under mild conditions. Different kinds of 4H-benzo[d][1,3]thiazin-2-ylphosphonate could be synthesized in moderate to excellent yields. This method has the advantages of easy access to raw materials, free-metal catalyst, simple operation, high yield and high functional group tolerance.

Ezh2 Inhibits Replicative Senescence of Atrial Fibroblasts Through Promotion of H3K27me3 in the Promoter Regions of CDKN2a and Timp4 Genes.

Background: In most cell types, replicative senescence (RS) is supposed to be a principle causative factor for aging. Atrial fibrosis, pathologically characterized by proliferation of atrial fibroblasts (AFs) and excessive accumulation of extracellular matrix proteins, is the most common substrate of atrial fibrillation (Afib) in the elderly. However, whether AFs' RS develops in the aged and fibrotic left atrium (LA) and, if yes, what is the key regulator for the pathogenesis of AFs' RS remain largely unknown. Methods: We obtained the left atrial tissues from young (6-8 weeks old) and aged (24 months old) C57BL/6 male mice. Screening and validation of differential genes were performed using comparative analysis of RNA-seq results. Replicative senescence was examined in primary AFs after cell passage. Further gain-of-function and loss-of-function experiments were performed to explore the regulation of the AFs' RS progression. Results: In the present study, we demonstrated that there was a considerable extent of AFs' RS in the aged and fibrotic LA. Transcriptome screening showed that Ezh2 (Enhancer of zeste homolog 2) was significantly downregulated in the LA tissue of aged mice. Ezh2 is a histone methyltransferase that catalyzes H3K27me3 and mediates transcriptional silencing. We confirmed that Ezh2 was downregulated in the isolated pure senescent AFs. Knockdown of Ezh2 by siRNA or inhibition of Ezh2's methyltransferase activities by GSK-126 and GSK-343 accelerated RS in the early passage of AFs, while its overexpression deaccelerated RS in the late passage of AFs. Mechanistically, Ezh2 suppressed CDKN2a (p16, p19) and Timp4 gene transcription by forming canonical H3K27me3 modifications in their promoter regions. Furthermore, the functional balance between Timp4 and MMP8 in AFs could be collapsed by changes in Ezh2 expression. Conclusion: These results thus indicate that Ezh2 is a key regulator of AFs' RS and this work may provide a basis for future treatments for atrial fibrosis in the elderly.

Inhibition of Wdr5 Attenuates Ang-II-Induced Fibroblast-to-Myofibroblast Transition in Cardiac Fibrosis by Regulating Mdm2/P53/P21 Pathway.

Cardiac fibrosis is an important pathological process in many diseases. Wdr5 catalyzes the trimethylation of lysine K4 on histone H3. The effects of Wdr5 on the cardiac fibrosis phenotype and the activation or transformation of cardiac fibroblasts were investigated by Ang-II-infused mice by osmotic mini-pump and isolated primary neonatal rat cardiac fibroblasts. We found that the Wdr5 expression and histone H3K4me3 modification were significantly increased in Ang-II-infused mice. By stimulating primary neonatal rat cardiac fibroblasts with Ang II, we detected that the expression of Wdr5 and H3K4me3 modification were also significantly increased. Two Wdr5-specific inhibitors, and the lentivirus that transfected Sh-Wdr5, were used to treat primary mouse cardiac fibroblasts, which not only inhibited the histone methylation by Wdr5 but also significantly reduced the activation and migration ability of Ang-II-treated fibroblasts. To explore its mechanism, we found that the inhibition of Wdr5 increased the expression of P53, P21. Cut&Tag-qPCR showed that the inhibition of Wdr5 significantly reduced the enrichment of H3K4me3 in the Mdm2 promoter region. For in vivo experiments, we finally proved that the Wdr5 inhibitor OICR9429 significantly reduced Ang-II-induced cardiac fibrosis and increased the expression of P21 in cardiac fibroblasts. Inhibition of Wdr5 may mediate cardiac fibroblast cycle arrest through the Mdm2/P53/P21 pathway and alleviate cardiac fibrosis.

Cadherin-11 Deficiency Attenuates Ang-II-Induced Atrial Fibrosis and Susceptibility to Atrial Fibrillation.

BACKGROUND: Atrial fibrosis serves as a disease initiating mechanism in the development of atrial fibrillation. Angiotensin II (Ang-II), a key mediator for atrial fibrosis, aberrantly activates atrial fibroblasts (AFs) into myofibroblasts, resulting in subsequent excessive synthesis and deposition of extracellular matrix (ECM). Cadherin-11 (CDH11) is essential in the development of non-cardiac fibrotic diseases. In this study, we investigated its role in the pathogenesis and underlying mechanism of atrial fibrillation. METHODS: We obtained left atrial tissues from either patients with atrial fibrillation or Ang-II-induced atrial fibrosis mice. We utilized a global CDH11 knockout mouse (CDH11-/-) model to determine the effect of CDH11 on AF cell proliferation, migration, ECM synthesis/deposition. RNA-Seq of isolated AFs from CDH11-/- or normal mice was performed and differential expressed genes were analyzed. The mouse susceptibility to atrial fibrillation was examined by cardiac electrophysiology. RESULTS: We found that cadherin-11 was significantly up-regulated in fibrotic atrial tissue from patients with atrial fibrillation and Ang-II-induced mice. Both normal and CDH11-/- mice did not develop atrial fibrosis at resting state. However, after Ang-II infusion, unlike severe atrial fibrosis occurred in normal mice, CDH11-/- mice displayed a reduced atrial fibrosis. Atrial fibroblasts with CDH11 deletion from CDH11-/- mice showed reduction in Ang-II-induced cell proliferation, migration and ECM synthesis/deposition, indicating the involvement of CDH11 in atrial fibrosis. Consistently, RNA-Seq of CDH11-null AFs uncovered significant decrease in pro-fibrotic gene expression. In addition, we identified reduction of transcripts associated with Smad2/3, ERK1/2 and JNK pathways. Further, CDH11-/- mice showed a significantly attenuated Ang-II-induced susceptibility to atrial fibrillation. CONCLUSION: Our results indicate that CDH11 potentiates Ang-II-induced activation of AFs. The pathogenesis of atrial fibrosis is through CDH11 mediated stimulation of Smad2/3, ERK1/2 and JNK pathways. Thus, CDH11 might serve as a novel therapeutic target for ameliorating the development of atrial fibrillation.

Human membrane protein Tim-3 facilitates hepatitis A virus entry into target cells.

In this study, a cellular surface membrane protein of immunoglobulin (Ig) superfamily (IgSF) was identified from a human dendritic cell (DC) cDNA library by large-scale random sequencing, which is identical to previously reported Tim-3 (T-cell Ig- and mucin-domain-containing molecule 3). Recent data have suggested the association of the 281-residue mouse Tim-3 molecule with Th1-related T cell responses and disease in mice. Human Tim-3 is a 301-residue type I membrane protein whose extracellular region contains a Cys-rich Ig-like domain and a mucin domain, the characteristics of Tim proteins. It shows significant homology to human hepatitis A virus (HAV) cellular receptor-1 (HuHAVcr-1)/Tim-1. Human Tim-3 mRNA was highly expressed in monocytes or monocyte-derived cells, and the expression level decreased when DC underwent maturation and activation. There is no previous report on the biological functions of human Tim-3, especially the involvement in virus infection. We demonstrated that HeLa cells, which are refractory to HAV infection, acquired a limited susceptibility to HAV infection after stably overexpressing human Tim-3 as confirmed by Western blot analysis using anti-Tim-3 antibody, but Tim-3-Fc fusion protein had no direct HAV-binding activity. The results indicated that human Tim-3 can promote HAV entry into target cells but itself may not function as a cellular receptor of HAV.

Scalded skin of rat treated by using fibrin glue combined with allogeneic bone marrow mesenchymal stem cells.

BACKGROUND: It is difficult to achieve satisfactory results with the traditional treatment of large-area skin defects and deep burns. OBJECTIVE: To test the treatment effect of an active dressing film made of a mixture of fibrin glue and bone marrow mesenchymal stem cells (BMSCs) for repairing burn wounds on the skin of rats. METHODS: Two scald wounds were made on the back of each rat. A total of 30 scald wounds were randomly divided into 3 groups, with 10 wounds in each group. In the experimental treatment group, the scald wounds were covered with the fibrin glue and BMSC mixture. The wounds of the experimental control group were covered with fibrin glue only. No intervention was administered to the blank control group. Thirty days after treatment, pathological sections were cut from the scalded local tissues of all rats from the 3 groups and observed with a microscope. RESULTS: The speed of scald wound healing in the experimental treatment group was faster than the other 2 groups. In the experimental treatment group, histopathological analysis revealed that the sebaceous glands showed obviously proliferous at the edge of the new tissue and gradually extended to the deep dermal layer of the new tissue. CONCLUSION: BMSCs may have an active role in promoting skin tissue repair and generating skin appendages. Allogeneic BMSCs mixed with fibrin glue can contribute to the quick formation of a film-like gel over the scald wounds, which might be of significance for emergency treatment and skin-grafting operations.