Dynamic changes in P300 enhancers and enhancer-promoter contacts control mouse cardiomyocyte maturation.

Cardiomyocyte differentiation continues throughout murine gestation and into the postnatal period, driven by temporally regulated expression changes in the transcriptome. The mechanisms that regulate these developmental changes remain incompletely defined. Here, we used cardiomyocyte-specific ChIP-seq of the activate enhancer marker P300 to identify 54,920 cardiomyocyte enhancers at seven stages of murine heart development. These data were matched to cardiomyocyte gene expression profiles at the same stages and to Hi-C and H3K27ac HiChIP chromatin conformation data at fetal, neonatal, and adult stages. Regions with dynamic P300 occupancy exhibited developmentally regulated enhancer activity, as measured by massively parallel reporter assays in cardiomyocytes in vivo, and identified key transcription factor-binding motifs. These dynamic enhancers interacted with temporal changes of the 3D genome architecture to specify developmentally regulated cardiomyocyte gene expressions. Our work provides a 3D genome-mediated enhancer activity landscape of murine cardiomyocyte development.

Development of Yin-Yang ligand for cannabinoid receptors.

Cannabinoid receptors (CBs), including CB1 and CB2, are the key components of a lipid signaling endocannabinoid system (ECS). Development of synthetic cannabinoids has been attractive to modulate ECS functions. CB1 and CB2 are structurally closely related subtypes but with distinct functions. While most efforts focus on the development of selective ligands for single subtype to circumvent the undesired off-target effect, Yin-Yang ligands with opposite pharmacological activities simultaneously on two subtypes, offer unique therapeutic potential. Herein we report the development of a new Yin-Yang ligand which functions as an antagonist for CB1 and concurrently an agonist for CB2. We found that in the pyrazole-cored scaffold, the arm of N1-phenyl group could be a switch, modification of which yielded various ligands with distinct activities. As such, the ortho-morpholine substitution exerted the desired Yin-Yang bifunctionality which, based on the docking study and molecular dynamic simulation, was proposed to be resulted from the hydrogen bonding with S173 and S285 in CB1 and CB2, respectively. Our results demonstrated the feasibility of structure guided ligand evolution for challenging Yin-Yang ligand.

Bioactive components of Banxia Xiexin Decoction for the treatment of gastrointestinal diseases based on flavor-oriented analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Banxia Xiexin Decoction (BXD) was first recorded in a Chinese medical classic, Treatise on Febrile Diseases and Miscellaneous Diseases, which was written in the Eastern Han dynasty of China. This ancient prescription consists of seven kinds of Chinese herbal medicine, namely, Pinellia ternata, Rhizoma Coptidis, Radix scutellariae, Rhizoma Zingiberis, Ginseng, Jujube, and Radix Glycyrrhizaepreparata. In clinic practice, its original application in China mainly has focused on the treatment of chronic gastritis for several hundred years. BXD is also effective in treating other gastrointestinal diseases (GIDs) in modern medical application. Despite available literature support and clinical experience, the treatment mechanisms or their relationships with the bioactive compounds in BXD responsible for its pharmacological actions, still need further explorations in more diversified channels. According to the analysis based on the five-flavor theory of TCM, BXD is traditionally viewed as the most representative prescription for pungent-dispersion, bitter-purgation and sweet-tonification. Consequently, based on the flavor-oriented analysis, the compositive herbs in BXD can be divided into three flavor groups, namely, the pungent, bitter, and sweet groups, each of which has specific active ingredients that are possibly relevant to GID treatment. AIM OF THE REVIEW: This paper summarized recent literatures on BXD and its bioactive components used in GID treatment, and provided the pharmacological or chemical basis for the further exploration of the ancient prescription and the relative components. METHOD: ology: Relevant literature was collected from various electronic databases such as Pubmed, Web of Science, and China National Knowledge Infrastructure (CNKI). Citations were based on peer-reviewed articles published in English or Chinese during the last decade. RESULTS: Multiple components were found in the pungent, bitter, and sweet groups in BXD. The corresponding bioactive components include gingerol, shogaol, stigmasterol, and ß-sitosterol in the pungent group; berberine, palmatine, coptisine, baicalein, and baicalin in the bitter group; and ginsenosides, polysaccharides, liquiritin, and glycyrrhetinic acid in the sweet group. These components have been found directly or indirectly responsible for the remarkable effects of BXD on GID. CONCLUSION: This review provided some valuable reference to further clarify BXD treatment for GID and their possible material basis, based on the perspective of the flavor-oriented analysis.

Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation.

The forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

CASAAV: A CRISPR-Based Platform for Rapid Dissection of Gene Function In Vivo.

In vivo loss-of-function studies are currently limited by the need for appropriate conditional knockout alleles. CRISPR/Cas9 is a powerful tool commonly used to induce loss-of-function mutations in vitro. However, CRISPR components have been difficult to deploy in vivo. To address this problem, we developed the CASAAV (CRISPR/Cas9/AAV-based somatic mutagenesis) platform, in which recombinant adeno-associated virus (AAV) is used to deliver tandem guide RNAs and Cre recombinase to Cre-dependent Cas9-P2A-GFP mice. Because Cre is under the control of a tissue-specific promoter, this system allows temporally controlled, cell type-selective knockout of virtually any gene to be obtained within a month using only one mouse line. Here, we focus on gene disruption in cardiomyocytes, but the system could easily be adapted to inactivate genes in other cell types transduced by AAV. © 2017 by John Wiley & Sons, Inc.

Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo.

RATIONALE: Loss-of-function studies in cardiac myocytes (CMs) are currently limited by the need for appropriate conditional knockout alleles. The factors that regulate CM maturation are poorly understood. Previous studies on CM maturation have been confounded by heart dysfunction caused by whole organ gene inactivation. OBJECTIVE: To develop a new technical platform to rapidly characterize cell-autonomous gene function in postnatal murine CMs and apply it to identify genes that regulate transverse tubules (T-tubules), a hallmark of mature CMs. METHODS AND RESULTS: We developed CRISPR/Cas9/AAV9-based somatic mutagenesis, a platform in which AAV9 delivers tandem guide RNAs targeting a gene of interest and cardiac troponin-T promoter-driven Cre to RosaCas9GFP/Cas9GFP neonatal mice. When directed against junctophilin-2 (Jph2), a gene previously implicated in T-tubule maturation, we achieved efficient, rapid, and CM-specific JPH2 depletion. High-dose AAV9 ablated JPH2 in 64% CMs and caused lethal heart failure, whereas low-dose AAV9 ablated JPH2 in 22% CMs and preserved normal heart function. In the context of preserved heart function, CMs lacking JPH2 developed T-tubules that were nearly morphologically normal, indicating that JPH2 does not have a major, cell-autonomous role in T-tubule maturation. However, in hearts with severe dysfunction, both adeno-associated virus-transduced and nontransduced CMs exhibited T-tubule disruption, which was more severe in the transduced subset. These data indicate that cardiac dysfunction disrupts T-tubule structure and that JPH2 protects T-tubules in this context. We then used CRISPR/Cas9/AAV9-based somatic mutagenesis to screen 8 additional genes for required, cell-autonomous roles in T-tubule formation. We identified RYR2 (Ryanodine Receptor-2) as a novel, cell-autonomously required T-tubule maturation factor. CONCLUSIONS: CRISPR/Cas9/AAV9-based somatic mutagenesis is a powerful tool to study cell-autonomous gene functions. Genetic mosaics are invaluable to accurately define cell-autonomous gene function. JPH2 has a minor role in normal T-tubule maturation but is required to stabilize T-tubules in the failing heart. RYR2 is a novel T-tubule maturation factor.

Protective and Therapeutic Effects of Chinese Medicine Formula Jiajian Yunvjian on Experimental Cardiac Remodeling after Myocardial Infarction Induced by Coronary Artery Ligation.

Introduction. This study was designed to explore the effect and mechanism of a classic Chinese medicine formula Jiajian Yunvjian (JJYNJ) on cardiac remodeling. Cardiac remodeling after myocardial infarction (MI) model was achieved by coronary artery ligation (CAL). Methodology. When dosed orally once daily, the effects of JJYNJ on hemodynamics, left ventricular weight index (LVWI), heart weight index (HWI), concentration, and gene expression of neuroendocrine factors as well as the histomorphological observation were determined. Results. After 4 weeks, mild cardiac remodeling in CAL group was characterized compared with sham group, but after 4 weeks of treatment of JJYNJ, hemodynamics improved, HWI reduced, and circulating angiotensin II (Ang II), endothelin-1 (ET-1), tumor necrosis factor-α (TNF-α), and hydroxyproline (Hyp) concentrations as well as Ang II receptor type 1 (AT1R) mRNA, transforming growth factor ß 1 (TGF-ß 1) mRNA, and TNF-α mRNA levels in myocardium were lower than in CAL group. Decreased plasma aldosterone (ALD) concentration, cross-sectional area of cardiomyocyte, collagen volume fraction (CVF), collagen types I and III, perivascular collagen area (PVCA), and upregulated nitric oxide (NO) levels were observed at the same time. Conclusions. These findings suggest that JJYNJ may have a protective and therapeutic function on cardiac remodeling related to MI.

Effect of gentiopicroside on experimental acute pancreatitis induced by retrograde injection of sodium taurocholate into the biliopancreatic duct in rats.

Gentiopicroside (otherwise known as Gentiopicrin), one of the main active ingredients from the traditional Chinese herb medicine Gentiana manshurica Kitag, presents the effect of attenuating acute pancreatitis in rats. The experimental acute pancreatitis was made by retrograde injection of sodium taurocholate into the biliopancreatic duct in rats. Gentiopicroside was given orally and it markedly reduced the pancreatitis-evoked increase of serum amylase and lipase activity, decreased the pancreas mass/body mass index, tissue water content, TNF-α and IL-1ß concentrations, and attenuated the histopathological changes and NF-κB p65 protein expression in pancreatic tissue. The results indicate that the function of gentiopicroside on acute pancreatitis may be related to inhibiting the release of inflammatory mediators and NF-κB p65 protein expression.

[Effect of Chrysanthemum indicum on ventricular remodeling in rats].

OBJECTIVE: To explore the effects and mechanism of Chrysanthemum indicum on experimental ventricular remodeling induced by isoprenaline (ISO) and L-thyroxine (L-Thy). METHODS: The ventricular remodeling of mice were induced by subcutaneous injection of ISO with the dosage of 2 mg/kg daily for 7 d and the rats with L-Thy intraperitoneally with the dosage of 0.25 mg/kg daily for 9 d. After 7 days' treatment, the cardiac index and the Ang II content in myocardium of mice were measured. After 9 days' treatment, the ratios of LVW/BW, HW/BW of rats were calculated, the Ang II content in heart tissue and the ALD, TNF-alpha concentration in serun were determined by radioimmunoassay, the Hydroxy proline (Hyp) content in heart tissue were measured by hydrolysis method. RESULTS: After 7 - 9 days of treatment, Chrysanthemum indicum significantly reduced the left ventricular weight index and heart weight index in mice and rats with myocardial hypertrophy, decreased the content of Ang II in ventricular tissue in mice and rats, and reduced the ALD, TNF-alpha concentration in serum and the Hyp content in ventricular tissue in rats (P < 0.05). CONCLUSION: Chrysanthemum indicum can significantly attenuate the experimental ventricular remodeling; the mechanism may be related with restricting the activity of the sympathetic nervous system and decreasing the levels of Ang II, ALD and TNF-alpha.

Effects of Chinese herb medicine Radix Scrophulariae on ventricular remodeling.

The effects and mechanism of the extract of Radix Scrophulariae (ERS), a traditional Chinese herb, on experimental ventricular remodeling in rats was studied. Rats were separated randomly into 5 groups: sham, model, captopril (40 mg x kg(-1)) and ERS (8, 16 g x kg(-1)). The experimental ventricular remodeling was induced with ligating the left anterior descending branch of the coronary artery of the rats. The sham group was conducted the same procedure without ligation. After 4 weeks treatment with intragastric administration of the corresponding drugs, the left ventricular weight index (LVWI) and heart weight index (HWI) were determined. The concentrations of angiotensin II (Ang II) and hydroxyproline (Hyp) in myocardium were detected. Myocardium tissue was stained with HE and picric acid/Sirius red for cardiocyte cross-section area and collagen content measurements. Real-time RT-PCR was used to detect the gene expressions of AT1R, TNF-alpha and TGF-beta1 mRNA. ERS could significantly reduce the LVWI, HWI, decrease the content of Ang II, Hyp, diminish cardiocyte cross-section area and ameliorate collagen deposition. In addition, ERS could down regulate the gene expressions of AT1R, TNF-alpha and TGF-beta1 mRNA in myocardium. ERS has beneficial effect against ventricular remodeling. The mechanism may be related to decreasing the level of Ang II and cardiac fibrosis, modulating some gene expressions associated with cardiac hypertrophy.

[Influence of Chrysanthemum indium on collagen accumulation and signaling transduction pathways in left ventricular tissue of cardiac hypertrophy in rats].

OBJECTIVE: To evaluate the influence of Chrysanthemum indium on collagen accumulation and signaling transduction pathways in left ventricle tissue of cardiac hypertrophy induced by abdominal aortic banding in rats. METHOD: Ventricular remodeling was induced by abdominal aortic banding (AAB) in rats. After 35 day treatment, the blood pressure was measured, then the ratios of LVW/BW and HW/BW were calculated. The histological assay was performed by HE staining for determining the myocardium cell cross section and picric acid/sirius red staining for determining collagen content. Immunohistochemistry was used to detect the protein expressions of PKC, bFGF and P38. RESULT: The experimental data demonstrated that C. indium could decrease blood pressure and the cardiac indexes of LVW/BW and HW/BW, significantly diminish cross sectional area of cardiomyocyte, ameliorate collagen accumulation such as collagen volume fraction, perivascular collagen area and collagen distributions of type I and II and significantly down regulate the protein expressions of PKC, bFGF and P38 (P<0.05). CONCLUSION: C. indium can significantly attenuate the experimental ventricular remodeling. The mechanism may be related to reducing the blood pressure, decreasing the total collagen content of left ventricle tissue and modulating signaling transduction pathway.

[Progress in treatment of chronic heart failure in Western medicine and treatment strategies in traditional Chinese medicine].

The concept of modern medicine in treating chronic heart failure (CHF) has changed markedly in recent years. To improve the quality of life and prolong life, the treatment goal is no longer just temporary improvement of symptoms, more importantly, is to prevent and delay the occurrence and development of ventricular remodeling. Long-term chronic over-activation of sympathetic system, renin-angiotensin-aldosterone system and other neuroendocrine factors promotes myocardial remodeling, increases myocardial injury and deteriorates cardiac function. Despite short-term use can significantly improve the blood flow dynamics, long-term use of beta-adrenergic receptor stimulators and phosphodiesterase inhibitors does not prolong life, but increases the rate of sudden death caused by cardiac arrhythmia. Angiotensin converting enzyme inhibitors and beta-blockers have become the preferred drugs in treating chronic heart failure. In fact, after long-term use, beta-blockers can significantly improve ventricular remodeling, enhance ventricular function and reduce the incidence of sudden death of patients with CHF. In traditional Chinese medicine practice, short-term use of drugs for warming yang and reinforcing qi can improve symptoms of CHF, but long-term use may have adverse effects, for these medicines can stimulate sympathetic system. Early treatment with medicines of cold and cool property may be more favorable to patients with CHF, except the advanced patients who need special intervention. Eliminating heat and nourishing yin may play more active role in controlling the occurrence and development of CHF. Drugs with good efficacy and value in treating CHF may be developed from the Chinese herbal medicines with eliminating heat and nourishing yin property.

[The study of Xuanshen on ventricular remodeling and cardiac endothelin-1 expression in rats treated with pressure-overload].

OBJECTIVE: To investigate the influence of Xuanshen on cardiac endothelin-1 expression, ventricular remodeling and its mechanism in rats treated with pressure-overload. METHODS: The ventricular remodeling model was induced by abdominal aortic stenosis in rats. Meanwhile, sham-operated rats were established as the control group. 8 weeks after drug interference, heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), left ventricular weight and heart weight index (LVWI and HWI), the activity of superoxide dismutase (SOD), cardiac endothelin-1 concentration and its gene expression were determined. RESULTS: Compared with those of sham-operated rats, the HR, SBP, DBP, LVWI and HWI of the model rats were increased significantly. The activity of SOD decreased, the concentration of cardiac endothelin-1 and its gene expression increased. In groups treated with Xuanshen, the HR, SBP, DBP, LVWI and HWI declined and the activity of SOD was improved. Moreover, the concentration of cardiac endothelin-1 and its gene expression decreased. CONCLUSION: Pressure-overload may induce oxidative stress and over-expression of cardiac endothelin-1. Xuanshen can inhibit ventricular remodeling. The mechanism may be related to the inhibition of oxidative stress and down regulation of endothelin-1 expression.

[Influence of Tinglizi on collagen volume fraction and perivascular collagen area in left ventricle tissue of cardiac hypertrophy induced by abdominal aortic banding in rats].

OBJECTIVE: To evaluate the influence of Tinglizi on collagen volume fraction (CVF) and perivascular collagen volume area (PVCA ) in left ventricle tissue of cardiac hypertrophy induced by abdominal aortic banding in rats. METHOD: Ventricular remodeling was induced by abdominal aortic banding (AAB) in rats. After 30 day treatment, the systolic blood pressure (SBP), diastolic blood pressure (DBP); heart rate (HR) were measured. The histological assay consisted of the HE stain for determining the myo-cardium cell cross section and collagen stain (Van Gieson' method) for determining collagen content, including collagen volume fracton (CVF) and perivascular collagen volume area (PVCA). RESULT: The experimental data demonstrated that Tinglizi decreased SBP, DBP, HR and could significantly reduce the total collagen content (CVF, PVCA) and lessen the myocardium cell cross section (P < 0.05). CONCLUSION: Tinglizi may decrease the total collagen content of ventricle and attenuate the ventricular remodeling induced by abdominal aortic banding.

[Comparative study of Scrophulariae and Aconite in inhibiting myocardial hypertrophy in rats and mice].

OBJECTIVE: To explore the effects of Scrophulariae of cold nature and Aconite of hot nature on myocardial hypertrophy and neuroendocrine factors in rats and mice. METHODS: A mouse model of myocardial hypertrophy was established by hypodermic injection of isoproterenol. Sixty myocardial hypertrophy mice were randomly divided into five groups: normal control group, untreated group, metoprolol-treated group, Scrophulariae-treated group and Aconite-treated group. A rat model of myocardial hypertrophy was established by peritoneal injection of L-thyroxin. Fifty rats were randomly divided into five groups: normal control group, untreated group, captopril-treated group, Scrophulariae-treated group and Aconite-treated group. After 7-9 days of treatment with intragastric administration of the corresponding drugs, the effects of Scrophulariae and Aconite on left ventricular weight index (LVWI) and heart weight index (HWI) were determined. The concentrations of cyclic adenosine monophosphate (cAMP) in plasma and angiotensin II (Ang II) in myocardium were detected through radio-immunity method. Cardiocyte cross-section area was determined by using HE staining. RESULTS: Scrophulariae of cold nature could significantly reduce the LVWI, HWI and cardiocyte cross-section area, and could decrease the content of cAMP and Ang II. However, Aconite had no such effects. CONCLUSION: Scrophulariae of cold nature can inhibit myocardial hypertrophy through restraining the activity of sympathetic nervous system and decreasing the level of Ang II. The inhibition of Aconite of hot nature on cardiac hypertrophy is not significant.

[Semen descurainiae inhibits CYP11B1, CYP11B2 and TGF-beta1 mRNA expression in left ventricular].

OBJECTIVE: To investigate the effects of Semen descurainiae and Captopril on CYP11B1, CYP11B2 and TGF-beta1 mRNA expression of heart tissue in rats treated with Abdominal Aortic Banding. METHODS: Ventricular remodeling was induced by abdominal aortic banding (AAB) in rats. After 30 days' treatment, the ratios of LVW/BW (left ventricle weight/body weight), HW/BW (heart weight/body weight) were calculated; Then the CYP11B, CYP11B2 and TGF-beta1 mRNA expression of left ventricle were detected by Real-time PCR, respectively. RESULTS: The experimental data demonstrated that Semen descurainiae decreased the indexes of LVW/BW and HW/BW, down-regulated CYP11B, CYP11B2 and TGF-beta1 mRNA expression in left ventricle (P<0.05). CONCLUSION: Semen desceurainiae can significantly inhibit the experimental ventricular remodeling; the mechanism is related to its ability to attenuate the mRNA expression of CYP11B1, CYP11B2 and TGF-beta1 in left ventricle. The inhibition of aldosterone key gene expression by Semen descurainiae may contribute to its effect on restraint cardiac remodeling.