RESUMEN
Hospitalized patients who die from Covid-19 often have pre-existing heart disease. The SARS-CoV-2 virus is dependent on the ACE2 receptor to be able to infect cells. It is possible that the strong link between cardiovascular comorbidities and a poor outcome following a SARS-CoV-2 infection is sometimes due to viral myocarditis. The aim was to examine the expression of ACE2 in normal hearts and hearts from patients with terminal heart failure. The ACE2 expression was measured by global quantitative proteomics and RT-qPCR in left ventricular (LV) tissue from explanted hearts. Immunohistochemistry was used to examine ACE2 expression in cardiomyocytes, fibroblasts and endothelial cells. In total, tissue from 14 organ donors and 11 patients with terminal heart failure were included. ACE2 expression was 2.6 times higher in 4 hearts from patients with terminal heart failure compared with 6 healthy donor hearts. The results were confirmed by immunohistochemistry where more than half of cardiomyocytes or fibroblasts showed expression of ACE2 in hearts from patients with terminal heart failure. In healthy donor hearts ACE2 was not expressed or found in few fibroblasts. A small subpopulation of endothelial cells expressed ACE2 in both groups. Upregulated ACE2 expression in cardiomyocytes may increase the risk of SARS-CoV-2 myocarditis in patients with heart failure.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Células Endoteliales/patología , Fibroblastos/patología , Insuficiencia Cardíaca/patología , Miocitos Cardíacos/patología , Donantes de Tejidos/provisión & distribución , Adulto , Anciano , Enzima Convertidora de Angiotensina 2/genética , Estudios de Casos y Controles , Células Endoteliales/metabolismo , Femenino , Fibroblastos/metabolismo , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/terapia , Trasplante de Corazón/métodos , Humanos , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/metabolismo , Adulto JovenRESUMEN
The recently reported cell division assay (CDA) was optimized to measure the relative sensitivity of cells to cytotoxic drugs in vitro. Here, we investigated the in vitro hypersensitivity of lymphocytes from Fanconi anemia (FA) patients, to cytotoxic drugs using CDA. Peripheral blood mononuclear cells (PBMC) as well as cell lines derived from FA patients were treated with two DNA interstrand crosslinking (ICL) agents, mitomycin C and cyclophosphamide. Our data indicate that the CDA detects hypersensitivity of cells from FA patients to mitomycin C. Further, cell lines derived from FA-patients were also hypersensitive to mitomycin C as well as cyclophosphamide, when assayed by the CDA. This study suggests that the CDA is a useful alternative for the diagnosis of FA patients' hypersensitivity to ICL agents.
Asunto(s)
División Celular/efectos de los fármacos , Anemia de Fanconi/tratamiento farmacológico , Mitomicina/farmacología , Antineoplásicos/farmacología , Línea Celular , Ciclofosfamida/farmacología , Citometría de Flujo/métodos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Linfocitos/efectos de los fármacosRESUMEN
Blood levels of cardiac troponins (cTn) and myoglobin are analysed when myocardial infarction (MI) is suspected. Here we describe a novel clearance mechanism for muscle proteins by muscle cells. The complete plasma clearance profile of cTn and myoglobin was followed in rats after intravenous or intermuscular injections and analysed by PET and fluorescence microscopy of muscle biopsies and muscle cells. Compared with intravenous injections, only 5 % of cTnT, 0.6 % of cTnI and 8 % of myoglobin were recovered in the circulation following intramuscular injection. In contrast, 47 % of the renal filtration marker FITC-sinistrin and 81 % of cTn fragments from MI-patients were recovered after intramuscular injection. In addition, PET and biopsy analysis revealed that cTn was taken up by the quadriceps muscle and both cTn and myoglobin were endocytosed by cultured muscle cells. This local clearance mechanism could possibly be the dominant clearance mechanism for cTn, myoglobin and other muscle damage biomarkers released by muscle cells.
Asunto(s)
Células Musculares/metabolismo , Proteínas Musculares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Endocitosis , Humanos , Masculino , Ratones , Ratas , Ratas Sprague-DawleyRESUMEN
Cardiac-specific troponins (cTn), troponin T (cTnT) and troponin I (cTnI) are diagnostic biomarkers when myocardial infarction is suspected. Despite its clinical importance it is still not known how cTn is cleared once it is released from damaged cardiac cells. The aim of this study was to examine the clearance of cTn in the rat. A cTn preparation from pig heart was labeled with fluorescent dye or fluorine 18 (18 F). The accumulation of the fluorescence signal using organ extracts, or the 18 F signal using positron emission tomography (PET) was examined after a tail vein injection. The endocytosis of fluorescently labeled cTn was studied using a mouse hepatoma cell line. Close to 99% of the cTnT and cTnI measured with clinical immunoassays were cleared from the circulation two hours after a tail vein injection. The fluorescence signal from the fluorescently labeled cTn preparation and the radioactivity from the 18F-labeled cTn preparation mainly accumulated in the liver and kidneys. The fluorescently labeled cTn preparation was efficiently endocytosed by mouse hepatoma cells. In conclusion, we find that the liver and the kidneys are responsible for the clearance of cTn from plasma in the rat.
Asunto(s)
Riñón/metabolismo , Hígado/metabolismo , Miocardio/metabolismo , Troponina T/farmacocinética , Animales , Colorantes Fluorescentes/química , Radioisótopos de Flúor/química , Masculino , Tasa de Depuración Metabólica , Tomografía de Emisión de Positrones/métodos , Ratas Endogámicas WKY , Porcinos , Troponina T/sangre , Troponina T/químicaRESUMEN
BACKGROUND: Although cardiac troponin I (cTnI) and troponin T (cTnT) form a complex in the human myocardium and bind to thin filaments in the sarcomere, cTnI often reaches higher concentrations and returns to normal concentrations faster than cTnT in patients with acute myocardial infarction (MI). METHODS: We compared the overall clearance of cTnT and cTnI in rats and in patients with heart failure and examined the release of cTnT and cTnI from damaged human cardiac tissue in vitro. RESULTS: Ground rat heart tissue was injected into the quadriceps muscle in rats to simulate myocardial damage with a defined onset. cTnT and cTnI peaked at the same time after injection. cTnI returned to baseline concentrations after 54 h, compared with 168 h for cTnT. There was no difference in the rate of clearance of solubilized cTnT or cTnI after intravenous or intramuscular injection. Renal clearance of cTnT and cTnI was similar in 7 heart failure patients. cTnI was degraded and released faster and reached higher concentrations than cTnT when human cardiac tissue was incubated in 37°C plasma. CONCLUSION: Once cTnI and cTnT are released to the circulation, there seems to be no difference in clearance. However, cTnI is degraded and released faster than cTnT from necrotic cardiac tissue. Faster degradation and release may be the main reason why cTnI reaches higher peak concentrations and returns to normal concentrations faster in patients with MI.
Asunto(s)
Infarto del Miocardio/metabolismo , Troponina I/metabolismo , Troponina T/metabolismo , Animales , Biomarcadores/sangre , Insuficiencia Cardíaca/sangre , Humanos , Cinética , Masculino , Persona de Mediana Edad , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Ratas , Ratas Endogámicas WKY , Sarcómeros/metabolismo , Troponina I/sangre , Troponina T/sangreRESUMEN
OBJECTIVE: The extent of kidney-dependent clearance of the cardiac damage biomarker cardiac troponin T (cTnT) is not known. METHODS AND RESULTS: We examined clearance of cTnT after injection of heart extracts in rats with or without clamped kidney vessels. The extent of degradation of cTnT to fragments able to pass the glomerular membrane and the kidney extraction index of cTnT was examined in human subjects. After a bolus injection of rat cardiac extract, simulating a large myocardial infarction, there was no significant difference in clearance of cTnT with or without kidney function. However, a slower clearance was observed late in the clearance process, when cTnT levels were low. When low levels of rat cardiac extract were infused at a constant rate to steady state, clamping of the renal vessels resulted in significant 2-fold reduction in clearance of cTnT. Over 60% of the measured cTnT in human subjects had a molecular weight below 17kDa, expected to have a relatively free passage over the glomerular membrane. The extraction index of cTnT in three heart failure patients undergoing renal vein catheterization was 8-19%. Kidney function adjusted cTnT levels increased the area under the ROC curve for diagnosis of myocardial infarction of the cTnT analysis in an emergency room cohort. CONCLUSIONS: At high concentrations, often found after a large myocardial infarction, extrarenal clearance of cTnT dominates. At low levels of cTnT, often found in patients with stable cTnT elevations, renal clearance also contribute to the clearance of cTnT. This potentially explains why stable cTnT levels tend to be higher in patients with low kidney function.
Asunto(s)
Membrana Basal Glomerular/metabolismo , Tasa de Filtración Glomerular , Infarto del Miocardio , Miocardio/metabolismo , Proteolisis , Troponina T/metabolismo , Animales , Humanos , Masculino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Ratas , Ratas Sprague-DawleyRESUMEN
Immunofluorescence quantification of γH2AX foci is a powerful approach to quantify DNA double-strand breaks induced by cancer therapy or accidental exposure to ionizing radiation. Here we report a modification to the γH2AX immunofluorescence labeling method, whereby cells are stained in-solution before being spotted and fixed onto microscope slides. Our modified method allows arraying of 16 patient samples/slide ready for foci counting in 2 h and demonstrated reliably detection of γH2AX foci in mononuclear cells prepared from patients who had undergone radiation therapy.
Asunto(s)
Roturas del ADN de Doble Cadena , Técnica del Anticuerpo Fluorescente/métodos , Histonas/análisis , Leucocitos Mononucleares/química , Neoplasias/radioterapia , Análisis de Matrices Tisulares/métodos , Aminoglicósidos/farmacología , Enediinos/farmacología , Fibroblastos/metabolismo , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/efectos de la radiación , Coloración y Etiquetado/métodosRESUMEN
BACKGROUND: Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs. METHODOLOGY/PRINCIPAL FINDINGS: To examine etoposide-induced DNA damage in more detail we compared the relative amount of SSBs and DSBs, survival and H2AX phosphorylation in cells treated with etoposide or calicheamicin, a drug that produces free DSBs and SSBs. With this combination of methods we found that only 3% of the DNA strand breaks induced by etoposide were DSBs. By comparing the level of DSBs, H2AX phosphorylation and toxicity induced by etoposide and calicheamicin, we found that only 10% of etoposide-induced DSBs resulted in histone H2AX phosphorylation and toxicity. There was a close match between toxicity and histone H2AX phosphorylation for calicheamicin and etoposide suggesting that the few etoposide-induced DSBs that activated H2AX phosphorylation were responsible for toxicity. CONCLUSIONS/SIGNIFICANCE: These results show that only 0.3% of all strand breaks produced by etoposide activate H2AX phosphorylation and suggests that over 99% of the etoposide induced DNA damage does not contribute to its toxicity.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Etopósido/farmacología , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Separación Celular , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Histonas/metabolismo , Humanos , Modelos Teóricos , Fosforilación , Factores de TiempoRESUMEN
Phosphorylation of histone protein H2AX on serine 139 (gamma-H2AX) occurs at sites flanking DNA double-stranded breaks (DSBs) and can provide a measure of the number of DSBs within a cell. We describe a flow cytometry-based method optimized to measure gamma-H2AX in nonfixed mononuclear blood cells as well as in cultured cells, which is more sensitive and involves less steps compared with protocols involving fixed cells. This method can be used to monitor induction of gamma-H2AX in mononuclear cells from cancer patients undergoing radiotherapy and for detection of gamma-H2AX throughout the cell cycle in cultured cells. The method is based on the fact that H2AX like other histone proteins are retained in the nucleus when cells are lysed at physiological salt concentrations. Cells are therefore added without fixation to a solution containing detergent to lyse the cells along with a fluorescein isothiocyanate-labeled monoclonal gamma-H2AX antibody, DNA staining dye and blocking agents. The stained nuclei can be analyzed by flow cytometry to monitor the level of gamma-H2AX to determine the level of DSBs and DNA content and to determine the cell cycle stage. The omission of fixation simplifies staining and enhances the sensitivity. This protocol can be completed within 4-6 h.