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BACKGROUND: International guidelines recommend natriuretic peptide biomarker-based screening for patients at high heart failure (HF) risk to allow early detection. There have been few reports about the incorporation of screening procedure to existing clinical practice. OBJECTIVE: To implement screening of left ventricular dysfunction in patients with type 2 diabetes mellitus (DM). METHOD: A prospective screening study at the DM complication screening centre was performed. RESULTS: Between 2018 and 2019, 1043 patients (age: 63.7±12.4 years; male: 56.3%) with mean glycated haemoglobin of 7.25%±1.34% were recruited. 81.8% patients had concomitant hypertension, 31.1% had coronary artery disease, 8.0% had previous stroke, 5.5% had peripheral artery disease and 30.7% had chronic kidney disease (CKD) stages 3-5. 43 patients (4.1%) had an elevated N-terminal prohormone of brain natriuretic peptide (NT-proBNP) concentration above the age-specific diagnostic thresholds for HF, and 43 patients (4.1%) had newly detected atrial fibrillation (AF). The prevalence of elevated NT-proBNP increased with age from 0.85% in patients aged <50 years to 7.14% in those aged 70-79 years and worsening kidney function from 0.43% in patients with CKD stage 1 to 42.86% in CKD stage 5. In multivariate logistic regression, male gender (OR: 3.67 (1.47-9.16), p = 0.005*), prior stroke (OR: 3.26 (1.38-7.69), p = 0.007*), CKD (p<0.001*) and newly detected AF (OR: 7.02 (2.65-18.57), p<0.001*) were significantly associated with elevated NT-proBNP. Among patients with elevated NT-proBNP, their mean left ventricular ejection fraction (LVEF) was 51.4%±14.7%, and 45% patients had an LVEF <50%. CONCLUSION: NT-proBNP and ECG screening could be implemented with relative ease to facilitate early detection of cardiovascular complication and improve long-term outcomes.
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Fibrilación Atrial , Diabetes Mellitus Tipo 2 , Insuficiencia Cardíaca , Insuficiencia Renal Crónica , Accidente Cerebrovascular , Disfunción Ventricular Izquierda , Humanos , Masculino , Persona de Mediana Edad , Anciano , Volumen Sistólico , Función Ventricular Izquierda , Estudios Prospectivos , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/diagnóstico , Disfunción Ventricular Izquierda/diagnóstico , Disfunción Ventricular Izquierda/etiología , Biomarcadores , Accidente Cerebrovascular/etiología , Péptido Natriurético Encefálico , Fragmentos de Péptidos , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/diagnósticoRESUMEN
Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by MeCP2 mutations. Nonetheless, the pathophysiological roles of MeCP2 mutations in the etiology of intrinsic cardiac abnormality and sudden death remain unclear. In this study, we performed a detailed functional studies (calcium and electrophysiological analysis) and RNA-sequencing-based transcriptome analysis of a pair of isogenic RTT female patient-specific induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) that expressed either MeCP2wildtype or MeCP2mutant allele and iPSC-CMs from a non-affected female control. The observations were further confirmed by additional experiments, including Wnt signaling inhibitor treatment, siRNA-based gene silencing, and ion channel blockade. Compared with MeCP2wildtype and control iPSC-CMs, MeCP2mutant iPSC-CMs exhibited prolonged action potential and increased frequency of spontaneous early after polarization. RNA sequencing analysis revealed up-regulation of various Wnt family genes in MeCP2mutant iPSC-CMs. Treatment of MeCP2mutant iPSC-CMs with a Wnt inhibitor XAV939 significantly decreased the ß-catenin protein level and CACN1AC expression and ameliorated their abnormal electrophysiological properties. In summary, our data provide novel insight into the contribution of activation of the Wnt/ß-catenin signaling cascade to the cardiac abnormalities associated with MeCP2 mutations in RTT.
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Células Madre Pluripotentes Inducidas , Síndrome de Rett , Humanos , Femenino , Síndrome de Rett/metabolismo , Vía de Señalización Wnt , Miocitos Cardíacos/metabolismo , Línea Celular , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , MutaciónRESUMEN
BACKGROUND: SARS-CoV-2 infection is associated with myocardial injury, but there is a paucity of experimental platforms for the condition.MethodsâandâResults:Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) infected by SARS-CoV-2 for 3 days ceased beating and exhibited cytopathogenic changes with reduced viability. Active viral replication was evidenced by an increase in supernatant SARS-CoV-2 and the presence of SARS-CoV-2 nucleocaspid protein within hiPSC-CMs. Expressions of BNP, CXCL1, CXCL2, IL-6, IL-8 and TNF-α were upregulated, while ACE2 was downregulated. CONCLUSIONS: Our hiPSC-CM-based in-vitro SARS-CoV-2 myocarditis model recapitulated the cytopathogenic effects and cytokine/chemokine response. It could be exploited as a drug screening platform.
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Betacoronavirus/metabolismo , Infecciones por Coronavirus/complicaciones , Células Madre Pluripotentes Inducidas/virología , Miocarditis/complicaciones , Miocitos Cardíacos/virología , Neumonía Viral/complicaciones , Enzima Convertidora de Angiotensina 2 , Betacoronavirus/genética , COVID-19 , Supervivencia Celular , Células Cultivadas , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Proteínas de la Nucleocápside de Coronavirus , Citocinas/metabolismo , Efecto Citopatogénico Viral , Evaluación Preclínica de Medicamentos/métodos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocarditis/metabolismo , Miocarditis/virología , Miocitos Cardíacos/metabolismo , Proteínas de la Nucleocápside/metabolismo , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Fosfoproteínas , Neumonía Viral/metabolismo , Neumonía Viral/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Replicación ViralRESUMEN
BACKGROUND: Danon disease is an X-linked disorder that leads to fatal cardiomyopathy caused by a deficiency in lysosome-associated membrane protein-2 (LAMP2). In female patients, a later onset and less severe clinical phenotype have been attributed to the random inactivation of the X chromosome carrying the mutant diseased allele. We generated a patient-specific induced pluripotent stem cell (iPSCs)-based model of Danon disease to evaluate the therapeutic potential of Xi-chromosome reactivation using a DNA methylation inhibitor. METHODS: Using whole-exome sequencing, we identified a nonsense mutation (c.520C>T, exon 4) of the LAMP2 gene in a family with Danon disease. We generated iPSC lines from somatic cells derived from the affected mother and her 2 sons, and we then differentiated them into cardiomyocytes (iPSC-CMs) for modeling the histological and functional signatures, including autophagy failure of Danon disease. RESULTS: Our iPSC-CM platform provides evidence that random inactivation of the wild-type and mutant LAMP2 alleles on the X chromosome is responsible for the unusual phenotype in female patients with Danon disease. In vitro, iPSC-CMs from these patients reproduced the histological features and autophagy failure of Danon disease. Administration of the DNA demethylating agent 5-aza-2'-deoxycytidine reactivated the silent LAMP2 allele in iPSCs and iPSC-CMs in female patients with Danon disease and ameliorated their autophagy failure, supporting the application of a patient-specific iPSC platform for disease modeling and drug screening. CONCLUSIONS: Our iPSC-CM platform provides novel mechanistic and therapeutic insights into the contribution of random X chromosome inactivation to disease phenotype in X-linked Danon disease.
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Autofagia , Azacitidina/farmacología , Cromosomas Humanos X/genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas , Adulto , Alelos , Autofagia/efectos de los fármacos , Autofagia/genética , Línea Celular , Femenino , Enfermedad por Depósito de Glucógeno de Tipo IIb/genética , Enfermedad por Depósito de Glucógeno de Tipo IIb/metabolismo , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas/biosíntesis , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , MasculinoRESUMEN
Friedreich ataxia (FRDA), a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy, is due to GAA repeat expansions within the first intron of the frataxin (FXN) gene encoding the mitochondrial protein involved in iron-sulfur cluster biosynthesis. The triplet codon repeats lead to heterochromatin-mediated gene silencing and loss of frataxin. Nevertheless, inadequacy of existing FRDA-cardiac cellular models limited cardiomyopathy studies. We tested the hypothesis that iron homeostasis deregulation accelerates reduction in energy synthesis dynamics which contributes to impaired cardiac calcium homeostasis and contractile force. Silencing of FXN expressions occurred both in somatic FRDA-skin fibroblasts and two of the induced pluripotent stem cells (iPSC) clones; a sign of stress condition was shown in FRDA-iPSC cardiomyocytes with disorganized mitochondrial network and mitochondrial DNA (mtDNA) depletion; hypertrophic cardiac stress responses were observed by an increase in α-actinin-positive cell sizes revealed by FACS analysis as well as elevation in brain natriuretic peptide (BNP) gene expression; the intracellular iron accumulated in FRDA cardiomyocytes might be due to attenuated negative feedback response of transferring receptor (TSFR) expression and positive feedback response of ferritin (FTH1); energy synthesis dynamics, in terms of ATP production rate, was impaired in FRDA-iPSC cardiomyocytes, which were prone to iron overload condition. Energetic insufficiency determined slower Ca(2+) transients by retarding calcium reuptake to sarcoplasmic reticulum (SR) and impaired the positive inotropic and chronotropic responses to adrenergic stimulation. Our data showed for the first time that FRDA-iPSCs cardiac derivatives represent promising models to study cardiac stress response due to impaired iron homeostasis condition and mitochondrial damages. The cardiomyopathy phenotype was accelerated in an iron-overloaded condition early in calcium homeostasis aspect.
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Cardiomiopatías , Ataxia de Friedreich/complicaciones , Técnicas In Vitro , Células Madre Pluripotentes , Adulto , Cardiomiopatías/etiología , Femenino , Ataxia de Friedreich/genética , Humanos , Sobrecarga de Hierro/complicaciones , Proteínas de Unión a Hierro/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , FrataxinaRESUMEN
AIMS: Recent studies suggested that the development of neuropathic pain associated with neural injury may be partly due to up-regulation of cyclooxygenase (COX) in the central nervous system. However, the cellular sources of COX-1 and COX-2 up-regulation following nerve injury are unclear. METHODS: We investigated the spinal cellular sources of COX-1 and COX-2 in association with allodynia following L5 spinal nerve ligation (SNL). RESULTS: Post-SNL pain-related behaviour was shown by increased sensitivity to mechanical stimulation. There was a significant increase in both COX-1 and COX-2 immunoreactivity (P < 0.01) on the ipsilateral side of spinal dorsal horn. Double immunofluorescence labelling demonstrated that COX-1 immunoreactive cells colocalized chiefly with dorsal horn neuronal nuclei and microglia, whereas COX-2 was expressed in neuronal cytoplasm. CONCLUSION: These findings demonstrate that while spinal dorsal horn neurones are important source of COX-1 and COX-2 after nerve injury, microglia also contribute to the pathogenesis of neuropathic pain, partly by producing additional COX-1.
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Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Neuralgia/enzimología , Neuroglía/enzimología , Neuronas/enzimología , Asta Dorsal de la Médula Espinal/enzimología , Animales , Hiperalgesia/enzimología , Ligadura , Masculino , Ratas , Ratas Sprague-Dawley , Nervios Espinales/lesiones , Regulación hacia ArribaRESUMEN
BACKGROUND: Researchers have developed machine learning-based ECG diagnostic algorithms that match or even surpass cardiologist level of performance. However, most of them cannot be used in real-world, as older generation ECG machines do not permit installation of new algorithms. OBJECTIVE: To develop a smartphone application that automatically extract ECG waveforms from photos and to convert them to voltage-time series for downstream analysis by a variety of diagnostic algorithms built by researchers. METHODS: A novel approach of using objective detection and image segmentation models to automatically extract ECG waveforms from photos taken by clinicians was devised. Modular machine learning models were developed to sequentially perform waveform identification, gridline removal, and scale calibration. The extracted data were then analysed using a machine learning-based cardiac rhythm classifier. RESULTS: Waveforms from 40 516 scanned and 444 photographed ECGs were automatically extracted. 12 828 of 13 258 (96.8%) scanned and 5399 of 5743 (94.0%) photographed waveforms were correctly cropped and labelled. 11 604 of 12 735 (91.1%) scanned and 5062 of 5752 (88.0%) photographed waveforms achieved successful voltage-time signal extraction after automatic gridline and background noise removal. In a proof-of-concept demonstration, an atrial fibrillation diagnostic algorithm achieved 91.3% sensitivity, 94.2% specificity, 95.6% positive predictive value, 88.6% negative predictive value and 93.4% F1 score, using photos of ECGs as input. CONCLUSION: Object detection and image segmentation models allow automatic extraction of ECG signals from photos for downstream diagnostics. This novel pipeline circumvents the need for costly ECG hardware upgrades, thereby paving the way for large-scale implementation of machine learning-based diagnostic algorithms.
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Electrocardiografía , Aprendizaje Automático , Teléfono Inteligente , Humanos , Electrocardiografía/métodos , Electrocardiografía/instrumentación , Algoritmos , Fotograbar/instrumentación , Fotograbar/métodos , Procesamiento de Señales Asistido por Computador , Aplicaciones Móviles , Frecuencia Cardíaca/fisiología , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/fisiopatologíaRESUMEN
Despite the extraordinary success of immune checkpoint inhibitors (ICIs) in cancer treatment, their use is associated with a high incidence of immune-related adverse events (IRAEs), resulting from therapy-related autoimmunity against various target organs. ICI-induced myocarditis is one of the most severe forms of IRAE, which is associated with risk of hemodynamic compromise and mortality. Despite increasing recognition and prompt treatment by clinicians, there remain significant gaps in knowledge regarding the pathophysiology, diagnosis and treatment of ICI-induced myocarditis. As the newly emerged disease entity is relatively rare, it is challenging for researchers to perform studies involving patients at scale. Alternatively, mouse models have been developed to facilitate research understanding of the pathogenesis of ICI-induced myocarditis and drug discovery. Transgenic mice with immune checkpoint genes knocked out allow induction of myocarditis in a highly reproducible manner. On the other hand, it has not been possible to induce ICI-induced myocarditis in wild type mice by injecting ICIs monotherapy alone. Additional interventions such as combinational ICI, tumor inoculation, cardiac sarcomere immunization, or cardiac irradiation are necessary to mimic the underlying pathophysiology in human cancer patients and to induce ICI-induced myocarditis successfully. This review focuses on the immunopathogenesis of ICI-induced myocarditis, drawing insights from human studies and animal models, and discusses the potential implications for treatment.
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Patients infected with SARS-CoV-2 may deteriorate rapidly and therefore continuous monitoring is necessary. We conducted an observational study involving patients with mild COVID-19 to explore the potentials of wearable biosensors and machine learning-based analysis of physiology parameters to detect clinical deterioration. Thirty-four patients (median age: 32 years; male: 52.9%) with mild COVID-19 from Queen Mary Hospital were recruited. The mean National Early Warning Score 2 (NEWS2) were 0.59 ± 0.7. 1231 manual measurement of physiology parameters were performed during hospital stay (median 15 days). Physiology parameters obtained from wearable biosensors correlated well with manual measurement including pulse rate (r = 0.96, p < 0.0001) and oxygen saturation (r = 0.87, p < 0.0001). A machine learning-derived index reflecting overall health status, Biovitals Index (BI), was generated by autonomous analysis of physiology parameters, symptoms, and other medical data. Daily BI was linearly associated with respiratory tract viral load (p < 0.0001) and NEWS2 (r = 0.75, p < 0.001). BI was superior to NEWS2 in predicting clinical worsening events (sensitivity 94.1% and specificity 88.9%) and prolonged hospitalization (sensitivity 66.7% and specificity 72.7%). Wearable biosensors coupled with machine learning-derived health index allowed automated detection of clinical deterioration.
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Técnicas Biosensibles/métodos , COVID-19 , Aprendizaje Automático , Dispositivos Electrónicos Vestibles , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Observacionales como Asunto , Adulto JovenRESUMEN
BACKGROUND: A high proportion of COVID-19 patients were reported to have cardiac involvements. Data pertaining to cardiac sequalae is of urgent importance to define subsequent cardiac surveillance. METHODS: We performed a systematic cardiac screening for 97 consecutive COVID-19 survivors including electrocardiogram (ECG), echocardiography, serum troponin and NT-proBNP assay 1-4 weeks after hospital discharge. Treadmill exercise test and cardiac magnetic resonance imaging (CMR) were performed according to initial screening results. RESULTS: The mean age was 46.5 ± 18.6 years; 53.6% were men. All were classified with non-severe disease without overt cardiac manifestations and did not require intensive care. Median hospitalization stay was 17 days and median duration from discharge to screening was 11 days. Cardiac abnormalities were detected in 42.3% including sinus bradycardia (29.9%), newly detected T-wave abnormality (8.2%), elevated troponin level (6.2%), newly detected atrial fibrillation (1.0%), and newly detected left ventricular systolic dysfunction with elevated NT-proBNP level (1.0%). Significant sinus bradycardia with heart rate below 50 bpm was detected in 7.2% COVID-19 survivors, which appeared to be self-limiting and recovered over time. For COVID-19 survivors with persistent elevation of troponin level after discharge or newly detected T wave abnormality, echocardiography and CMR did not reveal any evidence of infarct, myocarditis, or left ventricular systolic dysfunction. CONCLUSION: Cardiac abnormality is common amongst COVID-survivors with mild disease, which is mostly self-limiting. Nonetheless, cardiac surveillance in form of ECG and/or serum biomarkers may be advisable to detect more severe cardiac involvement including atrial fibrillation and left ventricular dysfunction.
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COVID-19/fisiopatología , Cardiopatías/fisiopatología , Adulto , Anciano , Arritmias Cardíacas/sangre , Arritmias Cardíacas/epidemiología , Arritmias Cardíacas/fisiopatología , Biomarcadores/sangre , COVID-19/sangre , COVID-19/complicaciones , Electrocardiografía , Femenino , Cardiopatías/sangre , Cardiopatías/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Estudios Prospectivos , SARS-CoV-2/aislamiento & purificación , Análisis de Supervivencia , Sobrevivientes , Disfunción Ventricular Izquierda/sangre , Disfunción Ventricular Izquierda/epidemiología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
AIMS: To recapitulate progressive human dilated cardiomyopathy (DCM) and heart block in the Lmna R225X mutant mice model and investigate the molecular basis of LMNA mutation induced cardiac conduction disorders (CD); To investigate the potential interventional impact of exercise endurance. METHODS AND RESULTS: A Lmna R225X knock-in mice model in either heterozygous or homozygous genotype was generated. Electrical remodeling was observed with higher occurrence of AV block from neonatal and aged mutant mice as measured by surface electrocardiogram and atrio-ventricular Wenckebach point detection. Histological and molecular profiles revealed an increase in apoptotic cells and activation of caspase-3 activities in heart tissue. Upon aging, extracellular cellular matrix (ECM) remodeling appeared with accumulation of collagen in Lmna R225X mutant hearts as visualized by Masson's trichrome stain. This could be explained by the upregulated ECM gene expression, such as Fibronectin: Fn1, collagen: Col12a1, intergrin: Itgb2 and 3, as detected by microarray gene chip. Also, endurance exercise for 3 month improved the ventricular ejection fraction, attenuated fibrosis and cardiomyocytes apoptosis in the aged mutant mice. CONCLUSIONS: The mechanism of LMNA nonsense mutation induced cardiac conduction defects through AV node fibrosis is due to upregulated ECM gene expression upon activation of cardiac apoptosis. Lmna R225X mutant mice hold the potential for serving as in vivo models to explore the mechanism and therapeutic methods for AV block or myopathy associated with the aging process.
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Trastorno del Sistema de Conducción Cardíaco/genética , Cardiomiopatía Dilatada/genética , Codón sin Sentido/genética , Lamina Tipo A/genética , Condicionamiento Físico Animal/fisiología , Animales , Animales Recién Nacidos , Trastorno del Sistema de Conducción Cardíaco/metabolismo , Trastorno del Sistema de Conducción Cardíaco/terapia , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/terapia , Expresión Génica , Técnicas de Sustitución del Gen/métodos , Frecuencia Cardíaca/fisiología , Lamina Tipo A/biosíntesis , Ratones , Condicionamiento Físico Animal/métodosRESUMEN
INTRODUCTION: Normal heart rhythms originate in the sinoatrial node. HCN-encoded funny current (I(f)) and the Kir2-encoded inward rectifier (I(K1)) counteract each other by respectively oscillating and stabilizing the negative resting membrane potential, and controlling action potential firing. Therefore, I(K1) suppression and I(f) overexpression have been independently exploited to convert cardiomyocytes (CMs) into AP-firing bioartificial pacemakers. Although the 2 strategies have been largely assumed synergistic, their complementarity has not been investigated. METHODS AND RESULTS: We explored the interrelationships of automaticity, I(f) and I(K1) by transducing single left ventricular (LV) CMs isolated from guinea pig hearts with the recombinant adenoviruses Ad-CMV-GFP-IRES-HCN1-AAA and/or Ad-CGI-Kir2.1 to mediate their current densities via a whole-cell patch clamp technique at 37 degrees C. Results showed that Ad-CGI-HCN1-AAA but not Ad-CGI-Kir2.1 transduction induced automaticity (181.1 +/- 13.1 bpm). Interestingly, Ad-CGI-HCN1-AAA/Ad-CGI-Kir2.1 cotransduction significantly promoted the induced firing frequency (320.0 +/- 15.8 bpm; P < 0.05). Correlation analysis revealed that the firing frequency, phase-4 slope and APD(90) of AP-firing LV CMs were correlated with I(f) (R(2) > 0.7) only when -2 >I(K1) >-4 pA/pF but not with I(K1) over the entire I(f) ranges examined (0.02 < R(2) < 0.4). Unlike I(f), I(K1) displayed correlation with neither the phase-4 slope (R(2)= 0.02) nor phase-4 length (R(2)= 0.04) when -2 > I(f) > -4 pA/pF. As anticipated, however, APD(90) was correlated with I(K1) (R(2)= 0.4). CONCLUSION: We conclude that an optimal level of I(K1) maintains a voltage range for I(f) to operate most effectively during a dynamic cardiac cycle.
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Potenciales de Acción/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Ingeniería Genética/métodos , Sistema de Conducción Cardíaco/fisiología , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Canales de Potasio/genética , Potasio/metabolismo , Animales , Femenino , Cobayas , Canales Regulados por Nucleótidos Cíclicos Activados por HiperpolarizaciónRESUMEN
Empagliflozin, a sodium-glucose co-transporter (SGLT) inhibitor, reduces heart failure and sudden cardiac death but the underlying mechanisms remain elusive. In cardiomyocytes, SGLT1 and SGLT2 expression is upregulated in diabetes mellitus, heart failure, and myocardial infarction. We hypothesise that empagliflozin exerts direct effects on cardiomyocytes that attenuate diabetic cardiomyopathy. To test this hypothesis, cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) were used to test the potential effects of empagliflozin on neutralization of cardiac dysfunction induced by diabetic-like cultures. Our results indicated that insulin-free high glucose culture significantly increased the size of and NPPB, SGLT1 and SGLT2 expression of hiPSC-derived cardiomyocytes. In addition, high glucose-treated hiPSC-derived cardiomyocytes exhibited reduced contractility regardless of the increased calcium transient capacity. Interestingly, application of empagliflozin before or after high glucose treatment effectively reduced the high glucose-induced cardiac abnormalities. Since application of empagliflozin did not significantly alter viability or glycolytic capacity of the hiPSC-derived cardiomyocytes, it is plausible that empagliflozin exerts its effects via the down-regulation of SGLT1, SGLT2 and GLUT1 expression. These observations provide supportive evidence that may help explain its unexpected benefit observed in the EMPA-REG trial.
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Compuestos de Bencidrilo/farmacología , Glucosa/metabolismo , Glucósidos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Línea Celular , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Cardiomiopatías Diabéticas/tratamiento farmacológico , Cardiomiopatías Diabéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Miocitos Cardíacos/metabolismoRESUMEN
BACKGROUND: Precision medicine is an emerging approach to disease treatment and prevention that takes into account individual variability in the environment, lifestyle, and genetic makeup of patients. Patient-specific human induced pluripotent stem cells hold promise to transform precision medicine into real-life clinical practice. Lamin A/C (LMNA)-related cardiomyopathy is the most common inherited cardiomyopathy in which a substantial proportion of mutations in the LMNA gene are of nonsense mutation. PTC124 induces translational read-through over the premature stop codon and restores production of the full-length proteins from the affected genes. In this study we generated human induced pluripotent stem cells-derived cardiomyocytes from patients who harbored different LMNA mutations (nonsense and frameshift) to evaluate the potential therapeutic effects of PTC124 in LMNA-related cardiomyopathy. METHODS AND RESULTS: We generated human induced pluripotent stem cells lines from 3 patients who carried distinctive mutations (R225X, Q354X, and T518fs) in the LMNA gene. The cardiomyocytes derived from these human induced pluripotent stem cells lines reproduced the pathophysiological hallmarks of LMNA-related cardiomyopathy. Interestingly, PTC124 treatment increased the production of full-length LMNA proteins in only the R225X mutant, not in other mutations. Functional evaluation experiments on the R225X mutant further demonstrated that PTC124 treatment not only reduced nuclear blebbing and electrical stress-induced apoptosis but also improved the excitation-contraction coupling of the affected cardiomyocytes. CONCLUSIONS: Using cardiomyocytes derived from human induced pluripotent stem cells carrying different LMNA mutations, we demonstrated that the effect of PTC124 is codon selective. A premature stop codon UGA appeared to be most responsive to PTC124 treatment.
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Cardiomiopatía Dilatada/tratamiento farmacológico , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Lamina Tipo A/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Oxadiazoles/farmacología , Adulto , Apoptosis/efectos de los fármacos , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/fisiopatología , Codón sin Sentido , Estimulación Eléctrica , Acoplamiento Excitación-Contracción/efectos de los fármacos , Mutación del Sistema de Lectura , Regulación de la Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Lamina Tipo A/genética , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , FenotipoRESUMEN
In this study, we compared the endothelium-dependent and -independent relaxation of the isolated thoracic aorta of control (+db/+m) and diabetic (+db/+db) (C57BL/KsJ) mice. The gene expression (mRNA and protein) level of the muscarinic M(3) receptors, endothelial nitric oxide synthase (eNOS) and caveolin-1 of the aorta was also evaluated. Acetylcholine caused a concentration-dependent, N(G)-nitro-L-arginine methyl-ester (20 microM)-sensitive relaxation, with approximately 100% relaxation at 10 microM, in +db/+m mice. In +db/+db mice, the acetylcholine-induced relaxation was significantly smaller (maximum relaxation: approximately 80%). The sodium nitroprusside-mediated relaxation was slightly diminished in +db/+db mice, compared to +db/+m mice. However, there was no significant difference in the isoprenaline- and cromakalim-induced relaxation observed in both species. The mRNA and protein expression levels of caveolin-1 were significantly higher in the aorta of +db/+db mice. In contrast, there was no difference in the mRNA and protein expression levels of eNOS and muscarinic M(3) receptors between these mice. Our results demonstrate that the impairment of the acetylcholine-induced, endothelium-dependent aortic relaxation observed in +db/+db mice was probably associated with an enhanced expression of caveolin-1 mRNA and protein.
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Aorta Torácica/fisiopatología , Caveolina 1/análisis , Diabetes Mellitus Tipo 2/fisiopatología , Vasodilatación , Acetilcolina/farmacología , Animales , Aorta Torácica/química , Aorta Torácica/efectos de los fármacos , Glucemia/metabolismo , Western Blotting , Cromakalim/farmacología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/fisiopatología , Inhibidores Enzimáticos/farmacología , Femenino , Insulina/sangre , Isoproterenol/farmacología , Ratones , Ratones Endogámicos C57BL , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo II/análisis , Óxido Nítrico Sintasa de Tipo III , Nitroprusiato/farmacología , ARN Mensajero/análisis , Receptor Muscarínico M3/análisis , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
BACKGROUND: Friedreich's ataxia (FRDA), a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy, is caused by silencing of the frataxin (FXN) gene encoding the mitochondrial protein involved in iron-sulfur cluster biosynthesis. METHODS: Application of our previously established FRDA human induced pluripotent stem cell (hiPSC) derived cardiomyocytes model as a platform to assess the efficacy of treatment with either the antioxidant coenzyme Q10 analog, idebenone (IDE) or the iron chelator, deferiprone (DFP), which are both under clinical trial. RESULTS: DFP was able to more significantly suppress synthesis of reactive oxygen species (ROS) than IDE at the dosages of 25 µM and 10nM respectively which agreed with the reduced rate of intracellular accumulation of iron by DFP treatment from 25 to 50 µM. With regard to cardiac electrical-contraction (EC) coupling function, decay velocity of calcium handling kinetics in FRDA-hiPSC-cardiomyocytes was significantly improved by DFP treatment but not by IDE. Further mechanistic studies revealed that DFP also modulated iron induced mitochondrial stress as reflected by mitochondria network disorganization and decline level of respiratory chain protein, succinate dehydrogenase (CxII) and cytochrome c oxidase (COXIV). In addition, iron-response protein (IRP-1) regulatory loop was overridden by DFP as reflected by resumed level of ferritin (FTH) back to basal level and the attenuated transferrin receptor (TSFR) mRNA level suppression thereby reducing further iron uptake. CONCLUSIONS: DFP modulated iron homeostasis in FRDA-hiPSC-cardiomyocytes and effectively relieved stress-stimulation related to cardiomyopathy. The resuming of redox condition led to the significantly improved cardiac prime events, cardiac electrical-coupling during contraction.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Ataxia de Friedreich/terapia , Células Madre Pluripotentes Inducidas , Hierro/metabolismo , Miocitos Cardíacos/metabolismo , Piridonas/farmacología , Ubiquinona/análogos & derivados , Antioxidantes/farmacología , Deferiprona , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Regulación de la Expresión Génica , Homeostasis , Humanos , Quelantes del Hierro/farmacología , Proteínas de Unión a Hierro/biosíntesis , Proteínas de Unión a Hierro/genética , Miocitos Cardíacos/patología , Estrés Oxidativo , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquinona/farmacología , FrataxinaRESUMEN
Laminopathy is a disease closely related to deficiency of the nuclear matrix protein lamin A/C or failure in prelamin A processing, and leads to accumulation of the misfold protein causing progeria. The resultant disrupted lamin function is highly associated with abnormal nuclear architecture, cell senescence, apoptosis, and unstable genome integrity. To date, the effects of loss in nuclear integrity on the susceptible organ, striated muscle, have been commonly associated with muscular dystrophy, dilated cardiac myopathy (DCM), and conduction defeats, but have not been studied intensively. In this review, we aim to summarize recent breakthroughs in an in vivo laminopathy model and in vitro study using patient-specific human induced pluripotent stem cells (iPSCs) that reproduce the pathophysiological phenotype for further drug screening. We describe several in-vivo transgenic mouse models to elucidate the effects of Lmna H222P, N195K mutations, and LMNA knockout on cardiac function, in terms of hemodynamic and electrical signal propagation; certain strategies targeted on stress-related MAPK are mentioned. We will also discuss human iPSC cardiomyocytes serving as a platform to reveal the underlying mechanisms, such as the altered mechanical sensation in electrical coupling of the heart conduction system and ion channel alternation in relation to altered nuclear architecture, and furthermore to enable screening of drugs that can attenuate this cardiac premature aging phenotype by inhibition of prelamin misfolding and oxidative stress, and also enhancement of autophagy protein clearance and cardiac-protective microRNA.
Asunto(s)
Cardiomiopatía Dilatada/genética , Lamina Tipo A/genética , Modelos Cardiovasculares , Mutación , Progeria/genética , Deficiencias en la Proteostasis/genética , Animales , Cardiomiopatía Dilatada/tratamiento farmacológico , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Cardiotónicos/farmacología , Expresión Génica , Inestabilidad Genómica , Sistema de Conducción Cardíaco/efectos de los fármacos , Sistema de Conducción Cardíaco/metabolismo , Sistema de Conducción Cardíaco/fisiopatología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Lamina Tipo A/deficiencia , Ratones , Ratones Transgénicos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Progeria/tratamiento farmacológico , Progeria/metabolismo , Progeria/patología , Pliegue de Proteína , Deficiencias en la Proteostasis/tratamiento farmacológico , Deficiencias en la Proteostasis/metabolismo , Deficiencias en la Proteostasis/patologíaRESUMEN
Patients with Danon disease may suffer from severe cardiomyopathy, skeletal muscle dysfunction as well as varying degrees of mental retardation, in which the primary deficiency of lysosomal membrane-associated protein-2 (LAMP2) is considerably associated. Owing to the scarcity of human neurons, the pathological role of LAMP2 deficiency in neural injury of humans remains largely elusive. However, the application of induced pluripotent stem cells (iPSCs) may shed light on overcoming such scarcity. In this study, we obtained iPSCs derived from a patient carrying a mutated LAMP2 gene that is associated with Danon disease. By differentiating such LAMP2-deficient iPSCs into cerebral cortical neurons and with the aid of various biochemical assays, we demonstrated that the LAMP2-deficient neurons are more susceptible to mild oxidative stress-induced injury. The data from MTT assay and apoptotic analysis demonstrated that there was no notable difference in cellular viability between the normal and LAMP2-deficient neurons under non-stressed condition. When exposed to mild oxidative stress (10 µM H2O2), the LAMP2-deficient neurons exhibited a significant increase in apoptosis. Surprisingly, we did not observe any aberrant accumulation of autophagic materials in the LAMP2-deficient neurons under such stress condition. Our results from cellular fractionation and inhibitor blockade experiments further revealed that oxidative stress-induced apoptosis in the LAMP2-deficient cortical neurons was caused by increased abundance of cytosolic cathepsin L. These results suggest the involvement of lysosomal membrane permeabilization in the LAMP2 deficiency associated neural injury.
RESUMEN
AIMS: We identified an autosomal dominant nonsense mutation (R225X) in exon 4 of the lamin A/C (LMNA) gene in a Chinese family spanning 3 generations with familial dilated cardiomyopathy (DCM). In present study, we aim to generate induced pluripotent stem cells derived cardiomyocytes (iPSCCMs) from an affected patient with R225X and another patient bearing LMNA frameshift mutation for drug screening. METHODS AND RESULTS: Higher prevalence of nuclear bleb formation and micronucleation was present in LMNA(R225X/WT) and LMNA(Framshift/WT) iPSCCMs. Under field electrical stimulation, percentage of LMNAmutated iPSCCMs exhibiting nuclear senescence and cellular apoptosis markedly increased. shRNA knockdown of LMNA replicated those phenotypes of the mutated LMNA field electrical stress. Pharmacological blockade of ERK1/2 pathway with MEK1/2 inhibitors, U0126 and selumetinib (AZD6244) significantly attenuated the proapoptotic effects of field electric stimulation on the mutated LMNA iPSCCMs. CONCLUSION: LMNArelated DCM was modeled invitro using patientspecific iPSCCMs. Our results demonstrated that haploinsufficiency due to R225X LMNA nonsense mutation was associated with accelerated nuclear senescence and apoptosis of iPSC CMs under electrical stimulation, which can be significantly attenuated by therapeutic blockade of stressrelated ERK1/2 pathway.