RESUMO
Gnaphalium affine is traditionally used to treat hyperuricemia and gout in China. Recently, the hypouricemic and renal protective effects of G. affine extract (GAD) have been deeply evaluated. However, little is known about the pharmacokinetics (PKs) of bioactive constituents in GAD. This study is aimed at investigating the individual and holistic pharmacokinetics of 10 bioactive components (including caffeic acid, caffeoylquinic acids, and flavonoids) in rats after single and multiple administrations of GAD. GAD is orally dosed to normal male rats at doses of 225, 450, or 900 mg/kg/day for 10 consecutive days and also orally administrated to uric acid nephropathy (UAN) rats at a dose of 900 mg/kg/day for 28 consecutive days. Integrated PKs of multiple components are calculated by area under the curve (AUC)-based weighting approach. All the components show a double-peak phenomenon in terms of their plasma concentration-time curves, suggesting that the components undergo enterohepatic circulation. The integrated AUC increases in a good dose-proportional manner with GAD dose. Compared with that in normal rats, the plasma exposure of caffeic acid and caffeoylquinic acids increases by 2.3- to 4.3-fold after 10-day chronic treatment of 900 mg/kg GAD in UAN rats. Modest drug accumulation is observed after 28-day chronic treatment.
Assuntos
Gnaphalium , Hiperuricemia , Ratos , Animais , Rim/metabolismo , Hiperuricemia/tratamento farmacológico , Área Sob a Curva , Administração Oral , Extratos Vegetais/farmacologiaRESUMO
BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) manifests with sudden death, arrhythmias, heart failure, apoptosis, and myocardial fibro-adipogenesis. The phenotype typically starts at the epicardium and advances transmurally. Mutations in genes encoding desmosome proteins, including DSP (desmoplakin), are major causes of ACM. METHODS: To delineate contributions of the epicardium to the pathogenesis of ACM, the Dsp allele was conditionally deleted in the epicardial cells in mice upon expression of tamoxifen-inducible Cre from the Wt1 locus. Wild type (WT) and Wt1-CreERT2:DspW/F were crossed to Rosa26mT/mG (R26mT/mG) dual reporter mice to tag the epicardial-derived cells with the EGFP (enhanced green fluorescent protein) reporter protein. Tagged epicardial-derived cells from adult Wt1-CreERT2:R26mT/mG and Wt1-CreERT2: R26mT/mG:DspW/F mouse hearts were isolated by fluorescence-activated cell staining and sequenced by single-cell RNA sequencing. RESULTS: WT1 (Wilms tumor 1) expression was progressively restricted postnatally and was exclusive to the epicardium by postnatal day 21. Expression of Dsp was reduced in the epicardial cells but not in cardiac myocytes in the Wt1-CreERT2:DspW/F mice. The Wt1-CreERT2:DspW/F mice exhibited premature death, cardiac dysfunction, arrhythmias, myocardial fibro-adipogenesis, and apoptosis. Single-cell RNA sequencing of ≈18 000 EGFP-tagged epicardial-derived cells identified genotype-independent clusters of endothelial cells, fibroblasts, epithelial cells, and a very small cluster of cardiac myocytes, which were confirmed on coimmunofluorescence staining of the myocardial sections. Differentially expressed genes between the paired clusters in the 2 genotypes predicted activation of the inflammatory and mitotic pathways-including the TGFß1 (transforming growth factor ß1) and fibroblast growth factors-in the epicardial-derived fibroblast and epithelial clusters, but predicted their suppression in the endothelial cell cluster. The findings were corroborated by analysis of gene expression in the pooled RNA-sequencing data, which identified predominant dysregulation of genes involved in epithelial-mesenchymal transition, and dysregulation of 146 genes encoding the secreted proteins (secretome), including genes in the TGFß1 pathway. Activation of the TGFß1 and its colocalization with fibrosis in the Wt1-CreERT2:R26mT/mG:DspW/F mouse heart was validated by complementary methods. CONCLUSIONS: Epicardial-derived cardiac fibroblasts and epithelial cells express paracrine factors, including TGFß1 and fibroblast growth factors, which mediate epithelial-mesenchymal transition, and contribute to the pathogenesis of myocardial fibrosis, apoptosis, arrhythmias, and cardiac dysfunction in a mouse model of ACM. The findings uncover contributions of the epicardial-derived cells to the pathogenesis of ACM.
Assuntos
Cardiomiopatias/fisiopatologia , Comunicação Parácrina/imunologia , Pericárdio/fisiopatologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Cardiomiopatias/mortalidade , Modelos Animais de Doenças , Humanos , Camundongos , Análise de SobrevidaRESUMO
A new sesquiterpene, chlorantholide G (1: ), a new sesquiterpene dimer, elatiolactone (2: ), and 2 new diterpenes, elatiorlabdane B (3: ) and elatiorlabdane C (4: ), together with 51 known compounds, were isolated from the aerial parts of Chloranthus elatior. The new structures including their absolute configurations were mainly established by mass spectrometric, NMR, and electronic circular dichroism experiments. All isolated compounds were tested for their anti-hDHODH activity. (4S,6R)-4-hydroxy-6-isopropyl-3-methylcyclohex-2-enone (5: ) and (4S,5R,9S,10R)-8(17),12,14-labdatrien-18-oic acid (29: ) were the most active compounds with IC50 values of 18.7 and 30.7 µM, respectively.
Assuntos
Di-Hidro-Orotato Desidrogenase , Sesquiterpenos , Dicroísmo Circular , Humanos , Estrutura Molecular , Sesquiterpenos/química , Sesquiterpenos/farmacologia , EstereoisomerismoRESUMO
AIMS: The study aims to describe the long-term outcome of radiofrequency catheter ablation for ventricular tachycardia (VT) in a large cohort arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. METHODS AND RESULTS: Radiofrequency catheter ablation was performed in 284 ARVC patients due to VT between July 2000 and January 2019. An endocardial approach was used initially, with epicardial ablation procedures reserved for those patients who failed an endocardial ablation. Activation, entrainment, pace and substrate mapping strategies were used with regional ablation applied. A total of 393 ablation procedures were performed including endocardial approach only (n = 377) and endo and epicardial combined (n = 16). Right ventricular basal free wall was accounted as the primary substrate of VT in 258 (65.6%) patients. There were 81 patients underwent redo ablation procedure (second time = 81; ≥3 times = 28). New targets were observed in 68.8% of redo procedures. There were 171 VT recurrences and 19 deaths occurred during the follow-up. Ventricular tachycardia-free survival rate of the first, second, and last ablation procedure was 56.7%, 73.2%, and 78.1%, respectively. Multivariate analysis showed ≥3 induced VTs in the procedure was correlated with rehospitalized VT recurrence [hazard ratio (HR) 1.467, 95% confidence interval (CI) 1.052-2.046; P = 0.024]. For all-cause mortality, rehospitalized VT and ≥3 induced VTs were the independent risk factors (HR 2.954, 95% CI 1.8068.038; P = 0.034; HR 3.189, 95% CI 1.073-9.482; P = 0.037). CONCLUSION: Endocardial ablation is effective to ARVC VT though it may require repeated procedures. Induced multiple VTs was correlated with worse outcomes.
Assuntos
Displasia Arritmogênica Ventricular Direita , Ablação por Cateter , Taquicardia Ventricular , Displasia Arritmogênica Ventricular Direita/diagnóstico , Displasia Arritmogênica Ventricular Direita/cirurgia , Endocárdio/cirurgia , Humanos , Recidiva , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/cirurgia , Resultado do TratamentoRESUMO
BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a heritable myocardium disorder that predominantly affects the ventricle. Little is known about atrial involvement. This study aimed to assess atrial involvement, especially the role of genotype on atrium in ARVC. METHODS: The incidence, characterization and predictors of atrial involvement were investigated. Nine known ARVC-causing genes were screened and the correlation between genotype and atrial involvement was assessed. RESULTS: Right atrium (RA) dilation, left atrium (LA) dilation, and sustained atrial tachyarrhythmias (ATa) were found in 45, 16 and 3 patients, respectively. Gene mutations were identified in 64 (64.0%) patients. Mutation carriers showed more RA dilation than noncarriers (54.7% vs. 27.8%, P = 0.009), and no difference in LA dilation and ATa. Multivariate analysis showed tricuspid regurgitation (OR: 18.867; 95% CI: 1.466-250.000; P = 0.024) increased the risk of RA dilation and decreased left ventricular ejection fraction (LVEF) (OR: 1.134; 95% CI: 1.002-1.272; P = 0.031) correlated with LA dilation, whereas genotype showed no significant effect. At a median follow-up time of 91 months, 7 patients died and 1 patient accepted heart transplantation. New-onset RA dilation, LA dilation, and sustained ATa were found in 8, 7, and 6 patients, respectively. Atrial involvement was not associated with the long-term survival. Despite mutation carriers showing more RA dilation, Kaplan-Meier analysis showed genotype was not associated with atrial involvement. CONCLUSION: Atrial involvement was common in ARVC. Tricuspid regurgitation and decreased LVEF increased the risk for atrial dilation. Genotype was not associated with atrial involvement.
Assuntos
Displasia Arritmogênica Ventricular Direita/genética , Fibrilação Atrial/etiologia , Flutter Atrial/etiologia , Função do Átrio Esquerdo , Função do Átrio Direito , Ablação por Cateter , Átrios do Coração/fisiopatologia , Mutação , Taquicardia Supraventricular/etiologia , Potenciais de Ação , Adulto , Antiarrítmicos/uso terapêutico , Displasia Arritmogênica Ventricular Direita/complicações , Displasia Arritmogênica Ventricular Direita/diagnóstico , Displasia Arritmogênica Ventricular Direita/fisiopatologia , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/fisiopatologia , Fibrilação Atrial/terapia , Flutter Atrial/diagnóstico , Flutter Atrial/fisiopatologia , Flutter Atrial/terapia , Remodelamento Atrial , Feminino , Predisposição Genética para Doença , Átrios do Coração/cirurgia , Frequência Cardíaca , Transplante de Coração , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Encaminhamento e Consulta , Fatores de Risco , Taquicardia Supraventricular/diagnóstico , Taquicardia Supraventricular/fisiopatologia , Taquicardia Supraventricular/terapia , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Tissue engineering combines cell biology and material science to construct tissues or organs for disease modeling, drug testing, and regenerative medicine. The cell sheet is a newly developed tissue engineering technology that has brought about scaffold-free tissue and shows great application potential. In this review, we summarized recent progress and future possibilities in preclinical research into and clinical applications of cell sheets fabricated by differing cell types from various sources for cardiac tissue repair, and the manufacturing strategies and promising application potential of 3D cell-dense tissue constructed from cell sheets. Special attention was paid to the mechanisms of mesenchymal stem cell (MSC) sheets in the prevention of myocardial ischemia and left ventricle remodeling. Comparing MSCs sheets with other types of cell sheets and 3D cardiac tissues, engineering tissues' potential safety and effectiveness concerns were also discussed.
Assuntos
Isquemia Miocárdica , Remodelação Ventricular , Humanos , Engenharia Tecidual , Isquemia Miocárdica/terapia , Medicina Regenerativa , CoraçãoRESUMO
Introduction: Arrhythmogenic cardiomyopathy (ACM) is hereditary cardiomyopathy caused by pathogenic variants (mutations) in genes encoding the intercalated disc (ID), particularly desmosome proteins. ACM caused by mutations in the DSP gene encoding desmoplakin (DSP) is characterized by the prominence of cell death, myocardial fibrosis, and inflammation, and is referred to as desmoplakin cardiomyopathy. Aim: The aim of this article was to gain insight into the pathogenesis of DSP cardiomyopathy. Methods and Results: The Dsp gene was exclusively deleted in cardiac myocytes using tamoxifen-inducible MerCreMer (Myh6-Mcm Tam) and floxed Dsp (Dsp F/F) mice (Myh6-Mcm Tam:Dsp F/F). Recombination was induced upon subcutaneous injection of tamoxifen (30 mg/kg/d) for 5 days starting post-natal day 14. Survival was analyzed by Kaplan-Meier plots, cardiac function by echocardiography, arrhythmias by rhythm monitoring, and gene expression by RNA-Seq, immunoblotting, and immunofluorescence techniques. Cell death was analyzed by the TUNEL assay and the expression levels of specific markers were by RT-PCR and immunoblotting. Myocardial fibrosis was assessed by picrosirius red staining of the myocardial sections, RT-PCR, and immunoblotting. The Myh6-Mcm Tam: Dsp F/F mice showed extensive molecular remodeling of the IDs and the differential expression of ~10,000 genes, which predicted activation of KDM5A, IRFs, and NFκB and suppression of PPARGC1A and RB1, among others in the DSP-deficient myocytes. Gene set enrichment analysis predicted activation of the TNFα/NFκB pathway, inflammation, cell death programs, and fibrosis. Analysis of cell death markers indicated PANoptosis, comprised of apoptosis (increased CASP3, CASP8, BAD and reduced BCL2), necroptosis (increased RIPK1, RIPK3, and MLKL), and pyroptosis (increased GSDMD and ASC or PYCARD) in the DSP-deficient myocytes. Transcript levels of the pro-inflammatory and pro-fibrotic genes were increased and myocardial fibrosis comprised ~25% of the myocardium in the DSP-deficient hearts. The Myh6-Mcm Tam:Dsp F/F mice showed severe cardiac systolic dysfunction and ventricular arrhythmias, and died prematurely with a median survival rate of ~2 months. Conclusion: The findings identify PANoptosis as a prominent phenotypic feature of DSP cardiomyopathy and set the stage for delineating the specific molecular mechanisms involved in its pathogenesis. The model also provides the opportunity to test the effects of pharmacological and genetic interventions on myocardial fibrosis and cell death.
RESUMO
AIMS: Mutations in the DSP gene encoding desmoplakin, a constituent of the desmosomes at the intercalated discs (IDs), cause a phenotype that spans arrhythmogenic cardiomyopathy (ACM) and dilated cardiomyopathy. It is typically characterized by biventricular enlargement and dysfunction, myocardial fibrosis, cell death, and arrhythmias. The canonical wingless-related integration (cWNT)/ß-catenin pathway is implicated in the pathogenesis of ACM. The ß-catenin is an indispensable co-transcriptional regulator of the cWNT pathway and a member of the IDs. We genetically inactivated or activated ß-catenin to determine its role in the pathogenesis of desmoplakin cardiomyopathy. METHODS AND RESULTS: The Dsp gene was conditionally deleted in the 2-week-old post-natal cardiac myocytes using tamoxifen-inducible MerCreMer mice (Myh6-McmTam:DspF/F). The cWNT/ß-catenin pathway was markedly dysregulated in the Myh6-McmTam:DspF/F cardiac myocytes, as indicated by a concomitant increase in the expression of cWNT/ß-catenin target genes, isoforms of its key co-effectors, and the inhibitors of the pathway. The ß-catenin was inactivated or activated upon inducible deletion of its transcriptional or degron domain, respectively, in the Myh6-McmTam:DspF/F cardiac myocytes. Genetic inactivation of ß-catenin in the Myh6-McmTam:DspF/F mice prolonged survival, improved cardiac function, reduced cardiac arrhythmias, and attenuated myocardial fibrosis, and cell death caused by apoptosis, necroptosis, and pyroptosis, i.e. PANoptosis. In contrast, activation of ß-catenin had the opposite effects. The deleterious and the salubrious effects were independent of changes in the expression levels of the cWNT target genes and were associated with changes in several molecular and biological pathways, including cell death programmes. CONCLUSION: The cWNT/ß-catenin was markedly dysregulated in the cardiac myocytes in a mouse model of desmoplakin cardiomyopathy. Inactivation of ß-catenin attenuated, whereas its activation aggravated the phenotype, through multiple molecular pathways, independent of the cWNT transcriptional activity. Thus, suppression but not activation of ß-catenin might be beneficial in desmoplakin cardiomyopathy.
Assuntos
Displasia Arritmogênica Ventricular Direita , Cardiomiopatias , Camundongos , Animais , Displasia Arritmogênica Ventricular Direita/genética , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Cardiomiopatias/genética , Arritmias Cardíacas/metabolismo , FibroseRESUMO
The presence of multiple pathogenic variants in desmosomal genes (DSC2, DSG2, DSP, JUP, and PKP2) in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to a severe phenotype. However, the pathogenicity of variants is reclassified frequently, which may result in a changed clinical risk prediction. Here, we present the collection, reclassification, and clinical outcome correlation for the largest series of ARVC patients carrying multiple desmosomal pathogenic variants to date (n = 331). After reclassification, only 29% of patients remained carriers of two (likely) pathogenic variants. They reached the composite endpoint (ventricular arrhythmias, heart failure, and death) significantly earlier than patients with one or no remaining reclassified variant (hazard ratios of 1.9 and 1.8, respectively). Periodic reclassification of variants contributes to more accurate risk stratification and subsequent clinical management strategy. Graphical Abstract.
Assuntos
Displasia Arritmogênica Ventricular Direita , Humanos , Displasia Arritmogênica Ventricular Direita/diagnóstico , Displasia Arritmogênica Ventricular Direita/genética , Placofilinas/genética , Fenótipo , Arritmias Cardíacas , MutaçãoRESUMO
BACKGROUND: Gnaphalium affine D. Don extract (GAD) enhanced efficacy and reduced toxicity of benzbromarone (BBR) in combination use. However, little is known about effects of GAD on the pharmacokinetics (PKs) and metabolic enzymes of BBR. PURPOSE: To investigate the pharmacokinetic (PK) and pharmacodynamic (PD) mechanism of the herb-drug interactions (HDIs) between GAD and BBR. STUDY DESIGN AND METHODS: Intragastric single BBR (4.5 or 50 mg/kg), single BBR (4.5 or 50 mg/kg) + single GAD (450 mg/kg, 2 h after BBR-administration), or single BBR (4.5 or 50 mg/kg) + multiple GAD (450 mg/kg/day, once daily for 7 days) were administered to both sexes for BBR PK studies in normal rats. Intragastric multiple BBR (4.5 mg/kg/day), or multiple BBR (4.5 mg/kg/day) + multiple GAD (450 mg/kg/day, 2 h after BBR-administration) were administered for BBR PK and PD studies in male rats with hyperuricemic nephropathy (HN). The cumulative anti-hyperuricemic effects of BBR and BBR+GAD were determined by plasma uric acid (UA) concentration-time curve and area under curve (AUCUA). An in vivo cocktail approach was employed to determine the effects of GAD on cytochrome P450 (CYP) 2C11(9) and 1A2 - mediated drug metabolism. RESULTS: In normal rats, the repeated dose administration of GAD induced a significant increase of BBR AUC and prolonged the mean residence time (MRT) (p < 0.05). systemic exposure to BBR and metabolically derived hydroxybenzbromarones was significantly greater in female compared with male rats (p < 0.05). In HN rats, post-administration of GAD resulted in significantly higher bioavailability and enterohepatic recycling (ER) of BBR relative to the BBR alone administrated group from the prolongation of terminal elimination half-life (T1/2) and MRT of BBR (p < 0.05). Significantly higher urate-lowering effect of BBR+GAD compared with BBR alone was generally observed at post-dosing most time points with a maximal effect of 84.3% (acute treatment), 71.4% (7-day subchronic treatment) and 82.5% (14-day subchronic treatment) reduction in UA levels. Additionally, GAD showed a significant inhibitory effect on CYP2C11(9)-mediated tolbutamide (probe substrate) metabolism with ≥ 1.25 but < 2-fold increase in AUCtolbutamide. CONCLUSIONS: PD synergism demonstrated with the BBR+GAD combination could be explained by the PK interaction observed partially from CYP2C11(9)-mediation and enterohepatic recycling.
Assuntos
Gnaphalium , Interações Ervas-Drogas , Animais , Benzobromarona/farmacologia , Feminino , Masculino , Extratos Vegetais/farmacologia , Ratos , Tolbutamida/farmacocinéticaRESUMO
BACKGROUND: Cardioneuroablation is an emerging therapy for refractory vasovagal syncope (VVS), but the standard enrollment criterion is undetermined. Mainstream studies empirically enroll patients with cardioinhibitory and mixed types of VVS on the basis of the head-up tilt (HUT). However, a variety of studies have shown that the results of HUT exhibit unpleasant reproducibility. OBJECTIVE: We aimed to investigate the prognostic value of baseline deceleration capacity in patients with refractory VVS after cardioneuroablation. METHODS: This study enrolled 123 patients (mean age 42.2±17.7 years; 54 males [43.9%]) with the diagnosis of VVS, of whom 16 patients had a negative result of HUT (13.0%), 8 (6.5%) had vasodepressive VVS, 32 (26.0%) cardioinhibitory VVS, and 67 (54.5%) mixed VVS. All patients underwent cardioneuroablation that was performed in the left atrium. RESULTS: After a mean follow-up of 4.0±1.1 years, 33 patients experienced syncope/presyncope events (26.8%). Patients with recurrent syncope/presyncope have a lower baseline deceleration capacity level than do those without (8.9±3.2 ms vs 11.3±3.7 ms; P < .001). Each 1-ms increase in deceleration capacity had a 34% (95% confidence interval [CI] 12%-50%) reduced risk of syncope/presyncope recurrence after cardioneuroablation. Nighttime deceleration capacity had the highest discrimination value (area under the curve 0.757; 95% confidence interval 0.657-0.858). At a high-risk threshold of 51% (nighttime deceleration capacity at a cutoff of 9.9 ms), the nighttime deceleration capacity enrollment strategy additionally benefited 18 per 100 patients after cardioneuroablation without syncope/presyncope recurrence as compared with the HUT strategy in decision curve analyses. CONCLUSION: Baseline nighttime deceleration capacity ≥ 10 ms may act as an indication for cardioneuroablation in patients with refractory VVS.
Assuntos
Síncope Vasovagal , Adulto , Desaceleração , Átrios do Coração , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Síncope Vasovagal/diagnóstico , Síncope Vasovagal/cirurgia , Teste da Mesa Inclinada/métodos , Adulto JovemRESUMO
INTRODUCTION: Aging is associated with cardiac myocyte loss, sarcopenia, and cardiac dysfunction. Adult cardiac myocytes are postmitotic cells with an insufficient proliferative capacity to compensate for myocyte loss. The canonical WNT (cWNT) pathway is involved in the regulation of cell cycle reentry in various cell types. The effects of the cWNT pathway on the expression of genes involved in cell cycle reentry in the postmitotic cardiac myocytes are unknown. AIM: The aim of the study was to identify genes whose expression is regulated by the ß-catenin, the indispensable component to the cWNT signaling, in the postmitotic myocytes. METHODS AND RESULTS: Cardiac myocyte-specific tamoxifen-inducible MerCreMer (Myh6-Mcm) mice were used to delete the floxed exon 3 or exons 8 to 13 of the Ctnnb1 gene to induce gain-of-function (GoF) or loss-of-function (LoF) the ß-catenin, respectively. Deletion of exon 3 leads to the expression of a stable ß-catenin. In contrast, deletion of exons 8-13 leads to the expression of transcriptionally inactive truncated ß-catenin, which is typically degraded. GoF or LoF of the ß-catenin was verified by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting, and immunofluorescence. Myocyte transcripts were analyzed by RNA-Sequencing (RNA-Seq) at 4 weeks of age. The GoF of ß-catenin was associated with differential expression of ~1700 genes, whereas its LoF altered expression of ~400 genes. The differentially expressed genes in the GoF myocytes were enriched in pathways regulating the cell cycle, including karyokinesis and cytokinesis, whereas the LoF was associated with increased expression of genes involved in mitochondrial oxidative phosphorylation. These findings were validated by RT-PCR in independent samples. Short-term GoF nor LoF of ß-catenin did not affect the number of cardiac myocytes, cardiac function, myocardial fibrosis, myocardial apoptosis, or adipogenesis at 4 weeks of age. CONCLUSION: Activation of the ß-catenin of the cWNT pathway in postmitotic myocytes leads to cell cycle reentry and expression of genes involved in cytokinesis without leading to an increase in the number of myocytes. In contrast, suppression of the ß-catenin modestly increases the expression of genes involved in oxidative phosphorylation. The findings provide insights into the role of ß-catenin of the cWNT pathway in the regulation of cell cycle reentry and oxidative phosphorylation in the postmitotic cardiac myocytes.
RESUMO
The Cre-LoxP technology, including the tamoxifen (TAM) inducible MerCreMer (MCM), is increasingly used to delineate gene function, understand the disease mechanisms, and test therapeutic interventions. We set to determine the effects of TAM-MCM on cardiac myocyte transcriptome. Expression of the MCM was induced specifically in cardiac myocytes upon injection of TAM to myosin heavy chain 6-MCM (Myh6-Mcm) mice for 5 consecutive days. Cardiac function, myocardial histology, and gene expression (RNA-sequencing) were analyzed 2 weeks after TAM injection. A total of 346 protein coding genes (168 up- and 178 down-regulated) were differentially expressed. Transcript levels of 85 genes, analyzed by a reverse transcription-polymerase chain reaction in independent samples, correlated with changes in the RNA-sequencing data. The differentially expressed genes were modestly enriched for genes involved in the interferon response and the tumor protein 53 (TP53) pathways. The changes in gene expression were relatively small and mostly transient and had no discernible effects on cardiac function, myocardial fibrosis, and apoptosis or induction of double-stranded DNA breaks. Thus, TAM-inducible activation of MCM alters cardiac myocytes gene expression, provoking modest and transient interferon and DNA damage responses without exerting other discernible phenotypic effects. Thus, the effects of TAM-MCM on gene expression should be considered in discerning the bona fide changes that result from the targeting of the gene of interest.
RESUMO
AIMS: Arrhythmogenic cardiomyopathy (ACM) is a primary myocardial disease that typically manifests with cardiac arrhythmias, progressive heart failure, and sudden cardiac death (SCD). ACM is mainly caused by mutations in genes encoding desmosome proteins. Desmosomes are cell-cell adhesion structures and hubs for mechanosensing and mechanotransduction. The objective was to identify the dysregulated molecular and biological pathways in human ACM in the absence of overt heart failure. METHODS AND RESULTS: Transcriptomes in the right ventricular endomyocardial biopsy samples from three independent individuals carrying truncating mutations in the DSP gene and five control samples were analysed by RNA-Seq (discovery group). These cases presented with cardiac arrhythmias and had a normal right ventricular function. The RNA-Seq analysis identified â¼5000 differentially expressed genes (DEGs), which predicted suppression of the Hippo and canonical WNT pathways, among others. Dysregulated genes and pathways, identified by RNA-Seq, were tested for validation in the right and left ventricular tissues from five independent autopsy-confirmed ACM cases with defined mutations (validation group), who were victims of SCD and had no history of heart failure. Protein levels and nuclear localization of the cWNT and Hippo pathway transcriptional regulators were reduced in the right and left ventricular validation samples. In contrast, levels of acetyltransferase EP300, known to suppress the Hippo and canonical WNT pathways, were increased and its bona fide target TP53 was acetylated. RNA-Seq data identified apical junction, reflective of cell-cell attachment, as the most disrupted biological pathway, which were corroborated by disrupted desmosomes and intermediate filament structures. Moreover, the DEGs also predicted dysregulation of over a dozen canonical signal transduction pathways, including the Tec kinase and integrin signalling pathways. The changes were associated with increased apoptosis and fibro-adipogenesis in the ACM hearts. CONCLUSION: Altered apical junction structures are associated with activation of the EP300-TP53 and suppression of the Hippo/cWNT pathways in human ACM caused by defined mutations in the absence of an overt heart failure. The findings implicate altered mechanotransduction in the pathogenesis of ACM.
Assuntos
Displasia Arritmogênica Ventricular Direita , Cardiomiopatias , Insuficiência Cardíaca , Arritmias Cardíacas/metabolismo , Cardiomiopatias/metabolismo , Morte Súbita Cardíaca/etiologia , Proteína p300 Associada a E1A/metabolismo , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/genética , Humanos , Mecanotransdução Celular , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Via de Sinalização WntRESUMO
Four new compounds: an abietane (1), two isopimarane (2 and 3) diterpenes, and a dihydrobenzofuran neolignan (5), together with a sesquiterpene glycoside (6) firstly isolated from a natural source, and a known phenol glycoside (4) were isolated from leaves of Platycladus orientalis. The structures and relative configurations of these compounds were assigned on the basis of MS, IR, 1D and 2D NMR data. The absolute configuration of compound 1 was determined via density functional theory calculations of its electronic circular dichroism. In addition, the results of bioassays indicated that compounds 4 and 6 showed a potent inhibitory effect on NO production with IC50 values of 24.4 and 11.9 µM, respectively. Meanwhile, compounds 1, 2, and 3 moderately inhibited TNF- α release.
Assuntos
Cupressaceae/química , Diterpenos/farmacologia , Medicamentos de Ervas Chinesas/química , Lignanas/farmacologia , Óxido Nítrico/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular Tumoral , Diterpenos/química , Diterpenos/isolamento & purificação , Glicosídeos/química , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Lignanas/química , Lignanas/isolamento & purificação , Lipopolissacarídeos/efeitos adversos , Medicina Tradicional Chinesa , Camundongos , Conformação Molecular , Óxido Nítrico/antagonistas & inibidores , Folhas de Planta/química , Plantas Medicinais/química , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
INTRODUCTION: Arrhythmogenic cardiomyopathy (ACM) is a genetic disease of the myocardium, characterized by cardiac arrhythmias, dysfunction, and sudden cardiac death. The pathological hallmark of ACM is fibro-adipocytes replacing cardiac myocytes. The canonical WNT pathway is implicated in the pathogenesis of ACM. AIM: The study aimed to determine the effects of the suppression of the WNT pathway on cardiac phenotype in a mouse model of ACM. METHODS AND RESULTS: One copy of the Dsp gene, a known cause of ACM in humans, was deleted specifically in cardiac myocytes (Myh6-Cre-Dsp W/F). Three-month-old wild type and Myh6-Cre-Dsp W/F mice, without a discernible phenotype, were randomized to either untreated or daily administration of a vehicle (placebo), or WNT974, the latter an established inhibitor of the WNT pathway, for three months. The Myh6-Cre-Dsp W/F mice in the untreated or placebo-treated groups exhibited cardiac dilatation and dysfunction, increased myocardial fibrosis, and apoptosis upon completion of the study, which was verified by complementary methods. Daily administration of WNT974 prevented and/or attenuated evolving cardiac dilatation and dysfunction, normalized myocardial fibrosis, and reduced apoptosis, compared to the untreated or placebo-treated groups. However, administration of WNT974 increased the number of adipocytes only in the Myh6-Cre-Dsp W/F hearts. There were no differences in the incidence of cardiac arrhythmias and survival rates. CONCLUSION: Suppression of the WNT pathway imparts salutary phenotypic effects by preventing or attenuating age-dependent expression of cardiac dilatation and dysfunction, myocardial fibrosis, and apoptosis in a mouse model of ACM. The findings set the stage for large-scale studies and studies in larger animal models to test the beneficial effects of the suppression of the WNT pathway in ACM. ONE SENTENCE SUMMARY: Suppression of the WNT signaling pathway has beneficial effects on cardiac dysfunction, myocardial apoptosis, and fibrosis in a mouse model of arrhythmogenic cardiomyopathy.
RESUMO
Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are promising candidates for regenerative medicine; however, the lack of scalable methods for high quantity EV production limits their application. In addition, signature EV-derived proteins shared in 3D environments and 2D surfaces, remain mostly unknown. Herein, we present a platform combining MSC microfiber culture with ultracentrifugation purification for high EV yield. Within this platform, a high quantity MSC solution (â¼3 × 108total cells) is encapsulated in a meter-long hollow hydrogel-microfiber via coaxial bioprinting technology. In this 3D core-shell microfiber environment, MSCs express higher levels of stemness markers (Oct4, Nanog, Sox2) than in 2D culture, and maintain their differentiation capacity. Moreover, this platform enriches particles by â¼1009-fold compared to conventional 2D culture, while preserving their pro-angiogenic properties. Liquid chromatography-mass spectrometry characterization results demonstrate that EVs derived from our platform and conventional 2D culturing have unique protein profiles with 3D-EVs having a greater variety of proteins (1023 vs 605), however, they also share certain proteins (536) and signature MSC-EV proteins (10). This platform, therefore, provides a new tool for EV production using microfibers in one culture dish, thereby reducing space, labor, time, and cost.
Assuntos
Bioimpressão , Vesículas Extracelulares , Células-Tronco Mesenquimais , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Medicina Regenerativa , TecnologiaRESUMO
AIMS: Arrhythmogenic cardiomyopathy (ACM) encompasses a genetically heterogeneous group of myocardial diseases whose manifestations are sudden cardiac death, cardiac arrhythmias, heart failure, and in a subset fibro-adipogenic infiltration of the myocardium. Mutations in the TMEM43 gene, encoding transmembrane protein 43 (TMEM43) are known to cause ACM. The purpose of the study was to gain insights into the molecular pathogenesis of ACM caused by TMEM43 haploinsufficiency. METHODS AND RESULTS: The Tmem43 gene was specifically deleted in cardiac myocytes by crossing the Myh6-Cre and floxed Tmem43 mice. Myh6-Cre:Tmem43W/F mice showed an age-dependent phenotype characterized by an increased mortality, cardiac dilatation and dysfunction, myocardial fibrosis, adipogenesis, and apoptosis. Sequencing of cardiac myocyte transcripts prior to and after the onset of cardiac phenotype predicted early activation of the TP53 pathway. Increased TP53 activity was associated with increased levels of markers of DNA damage response (DDR), and a subset of senescence-associated secretary phenotype (SASP). Activation of DDR, TP53, SASP, and their selected downstream effectors, including phospho-SMAD2 and phospho-SMAD3 were validated by alternative methods, including immunoblotting. Expression of SASP was associated with epithelial-mesenchymal transition and age-dependent expression of myocardial fibrosis and apoptosis in the Myh6-Cre:Tmem43W/F mice. CONCLUSION: TMEM43 haploinsufficiency is associated with activation of the DDR and the TP53 pathways, which lead to increased expression of SASP and an age-dependent expression of a pro-fibrotic cardiomyopathy. Given that TMEM43 is a nuclear envelope protein and our previous data showing deficiency of another nuclear envelope protein, namely lamin A/C, activates the DDR/TP53 pathway, we surmise that DNA damage is a shared mechanism in the pathogenesis of cardiomyopathies caused by mutations involving nuclear envelope proteins.
Assuntos
Cardiomiopatias/metabolismo , Senescência Celular , Dano ao DNA , Reparo do DNA , Haploinsuficiência , Proteínas de Membrana/deficiência , Miócitos Cardíacos/metabolismo , Animais , Apoptose , Cardiomiopatias/genética , Cardiomiopatias/patologia , Modelos Animais de Doenças , Fibrose , Predisposição Genética para Doença , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas de Membrana/genética , Camundongos da Linhagem 129 , Camundongos Knockout , Miócitos Cardíacos/patologia , Fenótipo , Fosforilação , Fenótipo Secretor Associado à Senescência , Transdução de Sinais , Proteína Smad2 , Proteína Smad3/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Less is known about pregnancy in women with arrhythmogenic right ventricular cardiomyopathy (ARVC). From April 1995 to May 2018, 157 women with ARVC were retrospectively enrolled. Data on pregnancy and cardiac outcomes were analyzed. There were 224 pregnancies in 120 patients including 30 (13.4%) spontaneous and 2 (0.9%) medical abortions, 12 cardiac adverse events were recorded including new onset frequent premature ventricular contractions (PVC) in 3 (2.5%) patients, previous PVC numbers increased more than 100% in 5 (4.2%), syncope in 2 (1.7%), sustained ventricular tachycardia and heart failure required hospitalization each in one patient (0.8%). Women with cardiac events showed lower left ventricular ejection fraction (LVEF) (50.3 ± 2.7 vs 60.0 ± 7.3; pâ¯=â¯0.004). No significant change in cardiac structure and function was found at 1 year follow-up postpartum. At a median follow-up of 8 (1 to 32) years, 36 (22.9%) women died. Earlier symptom onset age (hazard ratio 1.046; 95% confidence interval 1.017 to 1.075; pâ¯=â¯0.002) and decreased LVEF (hazard ratio 1.127; 95% confidence interval 1.001 to 1.154; pâ¯=â¯0.041) increased the risk of all-cause mortality, pregnancy had no negative influence on survival. In all the 192 offsprings (mean age 26.3 ± 13.5 years), 2 died of sudden death, no definite ARVC was found. Pregnancy seemed to be acceptable in ARVC, decreased LVEF increased the risk of pregnancy and was associated with poorer long-term survival.
Assuntos
Displasia Arritmogênica Ventricular Direita/complicações , Adulto , Ecocardiografia , Eletrocardiografia , Feminino , Humanos , Imageamento por Ressonância Magnética , Gravidez , Resultado da GravidezRESUMO
AIMS: Arrhythmogenic cardiomyopathy (ACM) is a myocardial disease caused mainly by mutations in genes encoding desmosome proteins ACM patients present with ventricular arrhythmias, cardiac dysfunction, sudden cardiac death, and a subset with fibro-fatty infiltration of the right ventricle predominantly. Endurance exercise is thought to exacerbate cardiac dysfunction and arrhythmias in ACM. The objective was to determine the effects of treadmill exercise on cardiac phenotype, including myocyte gene expression in myocyte-specific desmoplakin (Dsp) haplo-insufficient (Myh6-Cre:DspW/F) mice. METHODS AND RESULTS: Three months old sex-matched wild-type (WT) and Myh6-Cre:DspW/F mice with normal cardiac function, as assessed by echocardiography, were randomized to regular activity or 60 min of daily treadmill exercise (5.5 kJ work per run). Cardiac myocyte gene expression, cardiac function, arrhythmias, and myocardial histology, including apoptosis, were analysed prior to and after 3 months of routine activity or treadmill exercise. Fifty-seven and 781 genes were differentially expressed in 3- and 6-month-old Myh6-Cre:DspW/F cardiac myocytes, compared to the corresponding WT myocytes, respectively. Genes encoding secreted proteins (secretome), including inhibitors of the canonical WNT pathway, were among the most up-regulated genes. The differentially expressed genes (DEGs) predicted activation of epithelial-mesenchymal transition (EMT) and inflammation, and suppression of oxidative phosphorylation pathways in the Myh6-Cre:DspW/F myocytes. Treadmill exercise restored transcript levels of two-third (492/781) of the DEGs and the corresponding dysregulated transcriptional and biological pathways, including EMT, inflammation, and secreted inhibitors of the canonical WNT. The changes were associated with reduced myocardial apoptosis and eccentric cardiac hypertrophy without changes in cardiac function. CONCLUSION: Treadmill exercise restored transcript levels of the majority of dysregulated genes in cardiac myocytes, reduced myocardial apoptosis, and induced eccentric cardiac hypertrophy without affecting cardiac dysfunction in a mouse model of ACM. The findings suggest that treadmill exercise has potential beneficial effects in a subset of cardiac phenotypes in ACM.