RESUMO
The coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic as declared by World Health Organization (WHO). In the absence of an effective treatment, different drugs with unknown effectiveness, including antimalarial hydroxychloroquine (HCQ), with or without concurrent administration with azithromycin (AZM), have been tested for treating COVID-19 patients with developed pneumonia. However, the efficacy and safety of HCQ and/or AZM have been questioned by recent clinical reports. Direct effects of these drugs on the human heart remain very poorly defined. To better understand the mechanisms of action of HCQ +/- AZM, we employed bioengineered human ventricular cardiac tissue strip (hvCTS) and anisotropic sheet (hvCAS) assays, made with human pluripotent stem cell (hPSC)-derived ventricular cardiomyocytes (hvCMs), which have been designed for measuring cardiac contractility and electrophysiology, respectively. Our hvCTS experiments showed that AZM induced a dose-dependent negative inotropic effect which could be aggravated by HCQ; electrophysiologically, as revealed by the hvCAS platform, AZM prolonged action potentials and induced spiral wave formations. Collectively, our data were consistent with reported clinical risks of HCQ and AZM on QTc prolongation/ventricular arrhythmias and development of heart failure. In conclusion, our study exposed the risks of HCQ/AZM administration while providing mechanistic insights for their toxicity. Our bioengineered human cardiac tissue constructs therefore provide a useful platform for screening cardiac safety and efficacy when developing therapeutics against COVID-19.
Assuntos
Arritmias Cardíacas/patologia , Azitromicina/efeitos adversos , Cloroquina/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Contração Miocárdica , Miócitos Cardíacos/patologia , Função Ventricular/efeitos dos fármacos , Antibacterianos/efeitos adversos , Antimaláricos/efeitos adversos , Arritmias Cardíacas/induzido quimicamente , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/patologia , Engenharia Tecidual/métodos , Tratamento Farmacológico da COVID-19RESUMO
Autophagy is a process essential for cell survival under stress condition. The patients with autosomal dominant polycystic kidney disease, which is caused by polycystin-1 or polycystin-2 (PKD2) mutation, display cardiovascular abnormalities and dysregulation in autophagy. However, it is unclear whether PKD2 plays a role in autophagy. In the present study, we explored the functional role of PKD2 in autophagy and apoptosis in human embryonic stem cell-derived cardiomyocytes. HES2 hESC line-derived cardiomyocytes (HES2-CMs) were transduced with adenoviral-based PKD2-shRNAs (Ad-PKD2-shRNAs), and then cultured with normal or glucose-free medium for 3 hours. Autophagy was upregulated in HES2-CMs under glucose starvation, as indicated by increased microtubule-associated protein 1 light chain 3-II level in immunoblots and increased autophagosome and autolysosome formation. Knockdown of PKD2 reduced the autophagic flux and increased apoptosis under glucose starvation. In Ca2+ measurement, Ad-PKD2-shRNAs reduced caffeine-induced cytosolic Ca2+ rise. Co-immunoprecipitation and in situ proximity ligation assay demonstrated an increased physical interaction of PKD2 with ryanodine receptor 2 (RyR2) under glucose starvation condition. Furthermore, Ad-PKD2-shRNAs substantially attenuated the starvation-induced activation of AMP-activated protein kinase (AMPK) and inactivation of mammalian target of rapamycin (mTOR). The present study for the first time demonstrates that PKD2 functions to promote autophagy under glucose starvation, thereby protects cardiomyocytes from apoptotic cell death. The mechanism may involve PKD2 interaction with RyR2 to alter Ca2+ release from sarcoplasmic reticulum, consequently modulating the activity of AMPK and mTOR, resulting in alteration of autophagy and apoptosis. Stem Cells 2018;36:501-513.
Assuntos
Autofagia , Glucose/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Cátion TRPP/biossíntese , Apoptose , Linhagem Celular , Glucose/genética , Células-Tronco Embrionárias Humanas/citologia , Humanos , Miócitos Cardíacos/citologia , Canais de Cátion TRPP/genéticaRESUMO
Diagnosing heart failure with preserved ejection fraction (HFpEF) remains challenging. Intraventricular four-dimensional flow (4D flow) phase-contrast cardiovascular magnetic resonance (CMR) can assess different components of left ventricular (LV) flow including direct flow, delayed ejection, retained inflow and residual volume. This could be utilised to identify HFpEF. This study investigated if intraventricular 4D flow CMR could differentiate HFpEF patients from non-HFpEF and asymptomatic controls. Suspected HFpEF patients and asymptomatic controls were recruited prospectively. HFpEF patients were confirmed using European Society of Cardiology (ESC) 2021 expert recommendations. Non-HFpEF patients were diagnosed if suspected HFpEF patients did not fulfil ESC 2021 criteria. LV direct flow, delayed ejection, retained inflow and residual volume were obtained from 4D flow CMR images. Receiver operating characteristic (ROC) curves were plotted. 63 subjects (25 HFpEF patients, 22 non-HFpEF patients and 16 asymptomatic controls) were included in this study. 46% were male, mean age 69.8 ± 9.1 years. CMR 4D flow derived LV direct flow and residual volume could differentiate HFpEF vs combined group of non-HFpEF and asymptomatic controls (p < 0.001 for both) as well as HFpEF vs non-HFpEF patients (p = 0.021 and p = 0.005, respectively). Among the 4 parameters, direct flow had the largest area under curve (AUC) of 0.781 when comparing HFpEF vs combined group of non-HFpEF and asymptomatic controls, while residual volume had the largest AUC of 0.740 when comparing HFpEF and non-HFpEF patients. CMR 4D flow derived LV direct flow and residual volume show promise in differentiating HFpEF patients from non-HFpEF patients.
RESUMO
Aims: Heart failure with preserved ejection fraction (HFpEF) continues to be a diagnostic challenge. Cardiac magnetic resonance atrial measurement, feature tracking (CMR-FT), tagging has long been suggested to diagnose HFpEF and potentially complement echocardiography especially when echocardiography is indeterminate. Data supporting the use of CMR atrial measurements, CMR-FT or tagging, are absent. Our aim is to conduct a prospective case-control study assessing the diagnostic accuracy of CMR atrial volume/area, CMR-FT, and tagging to diagnose HFpEF amongst patients suspected of having HFpEF. Methods and results: One hundred and twenty-one suspected HFpEF patients were prospectively recruited from four centres. Patients underwent echocardiography, CMR, and N-terminal pro-B-type natriuretic peptide (NT-proBNP) measurements within 24â h to diagnose HFpEF. Patients without HFpEF diagnosis underwent catheter pressure measurements or stress echocardiography to confirm HFpEF or non-HFpEF. Area under the curve (AUC) was determined by comparing HFpEF with non-HFpEF patients. Fifty-three HFpEF (median age 78 years, interquartile range 74-82 years) and thirty-eight non-HFpEF (median age 70 years, interquartile range 64-76 years) were recruited. Cardiac magnetic resonance left atrial (LA) reservoir strain (ResS), LA area index (LAAi), and LA volume index (LAVi) had the highest diagnostic accuracy (AUCs 0.803, 0.815, and 0.776, respectively). Left atrial ResS, LAAi, and LAVi had significantly better diagnostic accuracy than CMR-FT left ventricle (LV)/right ventricle (RV) parameters and tagging (P < 0.01). Tagging circumferential and radial strain had poor diagnostic accuracy (AUC 0.644 and 0.541, respectively). Conclusion: Cardiac magnetic resonance LA ResS, LAAi, and LAVi have the highest diagnostic accuracy to identify HFpEF patients from non-HFpEF patients amongst clinically suspected HFpEF patients. Cardiac magnetic resonance feature tracking LV/RV parameters and tagging had low diagnostic accuracy to diagnose HFpEF.
RESUMO
BACKGROUND: Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by a non-coding mutation in the first intron of the frataxin (FXN) gene that suppresses its expression. Compensatory hypertrophic cardiomyopathy, dilated cardiomyopathy, and conduction system abnormalities in FRDA lead to cardiomyocyte (CM) death and fibrosis, consequently resulting in heart failure and arrhythmias. Murine models have been developed to study disease pathology in the past two decades; however, differences between human and mouse physiology and metabolism have limited the relevance of animal studies in cardiac disease conditions. To bridge this gap, we aimed to generate species-specific, functional in vitro experimental models of FRDA using 2-dimensional (2D) and 3-dimensional (3D) engineered cardiac tissues from FXN-deficient human pluripotent stem cell-derived ventricular cardiomyocytes (hPSC-hvCMs) and to compare their contractile and electrophysiological properties with healthy tissue constructs. METHODS: Healthy control and FRDA patient-specific hPSC-hvCMs were derived by directed differentiation using a small molecule-based protocol reported previously. We engineered the hvCMs into our established human ventricular cardiac tissue strip (hvCTS) and human ventricular cardiac anisotropic sheet (hvCAS) models, and functional assays were performed on days 7-17 post-tissue fabrication to assess the electrophysiology and contractility of FRDA patient-derived and FXN-knockdown engineered tissues, in comparison with healthy controls. To further validate the disease model, forced expression of FXN was induced in FXN-deficient tissues to test if disease phenotypes could be rescued. RESULTS: Here, we report for the first time the generation of human engineered tissue models of FRDA cardiomyopathy from hPSCs: FXN-deficient hvCTS displayed attenuated developed forces (by 70-80%) compared to healthy controls. High-resolution optical mapping of hvCAS with reduced FXN expression also revealed electrophysiological defects consistent with clinical observations, including action potential duration prolongation and maximum capture frequency reduction. Interestingly, a clear positive correlation between FXN expression and contractility was observed (ρ > 0.9), and restoration of FXN protein levels by lentiviral transduction rescued contractility defects in FXN-deficient hvCTS. CONCLUSIONS: We conclude that human-based in vitro cardiac tissue models of FRDA provide a translational, disease-relevant biomimetic platform for the evaluation of novel therapeutics and to provide insight into FRDA disease progression.
Assuntos
Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Potenciais de Ação/fisiologia , Cardiomiopatias/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Insuficiência Cardíaca/metabolismo , Humanos , FrataxinaAssuntos
Insuficiência Cardíaca , Volume Sistólico , Humanos , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/fisiopatologia , Volume Sistólico/fisiologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Imageamento por Ressonância Magnética/métodos , Meios de Contraste , Imagem Cinética por Ressonância Magnética/métodosRESUMO
Ectopic expression of viral oncoproteins disrupts cellular functions and limits the value of many existing immortalization models as models for carcinogenesis, especially for cancers without definitive viral etiology. Our newly established telomerase-immortalized human esophageal epithelial cell line, NE2-hTERT, retained nearly-diploid and non-tumorigenic characteristics, but exhibited genetic and genomic alterations commonly found in esophageal cancer, including progressive loss of the p16(INK4a) alleles, upregulation of anti-apoptotic proteins, epithelial-mesenchymal transition, whole-chromosome 7 gain and duplicated 5q arm. Our data also revealed a novel positive regulation of p16(INK4a) on cyclin D1. These findings probably represent early crucial events and mechanisms in esophageal carcinogenesis.