RESUMEN
The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.
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Autofagosomas/virología , COVID-19/virología , Autofagia , COVID-19/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Endosomas/fisiología , Endosomas/virología , Aparato de Golgi/fisiología , Células HEK293 , Células HeLa , Humanos , Fusión de Membrana , Microscopía Confocal , Fagosomas/metabolismo , Fagosomas/virología , Proteínas Qa-SNARE/biosíntesis , Receptores sigma/biosíntesis , SARS-CoV-2 , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Sinaptotagminas/biosíntesis , Receptor Sigma-1RESUMEN
Interleukin-17 (IL-17), also called IL-17A, is an important regulator of cardiac diseases, but its role in calcium-related cardiac dysfunction remains to be explored. Thus, we investigated the influence of IL-17 on calcium handling process and its contribution to the development of heart failure. Mice were subjected to transaortic constriction (TAC) to induce heart failure. In these mice, the levels of IL-17 in the plasma and cardiac tissue were significantly increased compared with the sham group. In 77 heart failure patients, the plasma level of IL-17 was significantly higher than 49 non-failing subjects, and was negatively correlated with cardiac ejection fraction and fractional shortening. In IL-17 knockout mice, the shortening of isolated ventricular myocytes was increased compared with that in wild-type mice, which was accompanied by significantly increased amplitude of calcium transient and the upregulation of SERCA2a and Cav1.2. In cultured neonatal cardiac myocytes, treatment of with IL-17 (0.1, 1 ng/mL) concentration-dependently suppressed the amplitude of calcium transient and reduced the expression of SERCA2a and Cav1.2. Furthermore, IL-17 treatment increased the expression of the NF-κB subunits p50 and p65, whereas knockdown of p50 reversed the inhibitory effects of IL-17 on SERCA2a and Cav1.2 expression. In mice with TAC-induced mouse heart, IL-17 knockout restored the expression of SERCA2a and Cav1.2, increased the amplitude of calcium transient and cell shortening, and in turn improved cardiac function. In addition, IL-17 knockout attenuated cardiac hypertrophy with inhibition of calcium-related signaling pathway. In conclusion, upregulation of IL-17 impairs cardiac function through NF-κB-mediated disturbance of calcium handling and cardiac remodeling. Inhibition of IL-17 represents a potential therapeutic strategy for the treatment of heart failure.
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Canales de Calcio Tipo L/biosíntesis , Insuficiencia Cardíaca/metabolismo , Interleucina-17/biosíntesis , FN-kappa B/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Regulación hacia Arriba/fisiología , Animales , Animales Recién Nacidos , Canales de Calcio Tipo L/genética , Línea Celular , Células Cultivadas , Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Humanos , Interleucina-17/deficiencia , Interleucina-17/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genéticaRESUMEN
Resveratrol (RSV) is a phytoestrogen which has been related to chemoprevention of several types of cancer. In this work, we show up to a 6-fold increased expression of ATP2A3 gene induced by RSV that triggers apoptosis and changes of intracellular Ca2+ management in MCF-7 and MDA-MB-231 breast cancer cell lines. We explored epigenetic mechanisms for that RSV-induced ATP2A3 up-regulation. The results indicate that RSV-induced ATP2A3 up-regulation correlates with about 50% of reduced HDAC activity and reduced nuclear HDAC2 expression and occupancy on ATP2A3 promoter, increasing the global acetylation of histone H3 and the enrichment of histone mark H3K27Ac on the proximal promoter of the ATP2A3 gene in MDA-MB-231 cells. We also quantified HAT activity, finding that it can be boosted with RSV treatment; however, pharmacological inhibition of p300, one of the main HATs, did not have significant effects in RSV-mediated ATP2A3 gene expression. Additionally, DNMT activity was also reduced in cells treated with RSV, as well as the expression of Methyl-DNA binding proteins MeCP2 and MBD2. However, analysis of the methylation pattern of ATP2A3 gene promoter showed un-methylated promoter in both cell lines. Taken together, the results of this work help to explain, at the molecular level, how ATP2A3 gene is regulated in breast cancer cells, and the benefits of RSV intake observed in epidemiological data, studies with animals, and in vitro models.
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Antioxidantes/farmacología , Neoplasias de la Mama/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Calcio/metabolismo , Línea Celular Tumoral , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Metilación de ADN , Epigénesis Genética , Femenino , Histona Desacetilasa 2/antagonistas & inhibidores , Histona Desacetilasa 2/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Humanos , Células MCF-7 , Regiones Promotoras Genéticas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Sarco(endo)plasmic reticulum Ca2+-ATPases (SERCA) expression is reduced or absent in several types of cancer and cancer cell lines; however, their expression and regulation in hepatocellular carcinoma (HCC) are unknown. Histone deacetylase inhibitors (HDACi) increase SERCA3 mRNA expression in gastric and breast cancer cell lines by increasing H3K9ac and binding of Sp1 and Sp3 transcription factors to the promoter; however, the molecular mechanism is not fully understood. Our results show that ATP2A3 (SERCA3) gene expression is decreased in human HCC samples and rat HCC AS-30D cells compared to normal liver, and HCC patients with high expression of ATP2A3 had longer overall survival than those with low expression. Sodium butyrate (NaB) and trichostatin A (TSA) increase SERCA3 mRNA expression in AS-30D cells, whereas SERCA2b mRNA expression did not change. NaB and TSA increase H3K9ac and H3K27ac in two ATP2A3 promoter regions. Besides, NaB treated cells increased Sp1 and Sp3 occupancy at ATP2A3 promoter; whereas TSA treated cells showed increased p300 levels at ATP2A3 promoter. Inhibition of p300 by C646, a specific inhibitor, mitigates SERCA3 mRNA induction by TSA, and reduces more than 70% of basal SERCA3 mRNA expression, suggesting that p300 is important for ATP2A3 gene transcription in AS-30D cells. Moreover, inhibition of p300 decreases H3K9ac in TSA treated cells. Our results provide evidence of decreased SERCA3 expression in human HCC samples and rat AS-30D cells and a correlation of SERCA3 expression with overall survival in HCC patients. Also, reveal new insights in SERCA3 transcriptional regulation mediated by HDACi.
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Carcinoma Hepatocelular/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias Hepáticas/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Animales , Ácido Butírico/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Expresión Génica/efectos de los fármacos , Histonas/genética , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Regiones Promotoras Genéticas , Ratas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Células Tumorales CultivadasRESUMEN
Abstract Introduction: Hepatic ischemia-reperfusion injury is a common pathophysiological process in liver surgery. Whether Propofol can reduce myocardial ischemia-reperfusion injury induced by hepatic ischemia-reperfusion injury in rats, together with related mechanisms, still needs further studies. Objective: To investigate if propofol would protect the myocardial cells from apoptosis with hepatic ischemia-reperfusion injury. Methods: Male Sprague-Dawley rats (n = 18) were randomly allocated into three groups: Sham Group (Group S, n = 6), Hepatic Ischemia-reperfusion Injury Group (Group IR, n = 6) and Propofol Group (Group P, n = 6). Group S was only subjected to laparotomy. Group IR was attained by ischemia for 30 min and reperfusion for 4 h. Group P was subjected identical insult as in Group IR with the administration of propofol started 10 min before ischemia with 120 mg.kg−1, following by continuous infusion at 20 mg.kg−1.h−1. Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Endoplasmic reticulum Ca2+-ATPase2 (SERCA2) and cysteine-containing aspartic acid cleaved-caspase3 (cleaved-caspase3) were assayed by western blot and Altimeter polymerase chain reaction. Results: Apoptosis rate was increased, with mRNA and protein of SERCA2 down-regulated and cleaved-caspase3 up-regulated in Group IR compared with Group S (p < 0.01). Apoptosis rate was decreased, with mRNA and protein of SERCA2 up-regulated and cleaved-caspase3 down-regulated in Group P compared with Group IR (p < 0.01). Conclusions: Propofol can reduce hepatic ischemia-reperfusion injury-induced myocardial cell apoptosis, meanwhile, can up-regulate mRNA and protein of SERCA2 in rats.
Resumo Introdução: A lesão hepática por isquemia-reperfusão é um processo fisiopatológico comum em cirurgias hepáticas. Mais estudos ainda são necessários para avaliar se o propofol pode reduzir a lesão de isquemia-reperfusão miocárdica induzida pela lesão de isquemia-reperfusão hepática em ratos, juntamente com os mecanismos que estão relacionados. Objetivo: Investigar se propofol protege as células do miocárdio da apoptose com a lesão hepática por isquemia-reperfusão. Métodos: Ratos machos da raça Sprague-Dawley (n = 18) foram alocados aleatoriamente em três grupos: Grupo Sham (Grupo S, n = 6), Grupo Lesão Hepática por Isquemia-reperfusão (Grupo IR, n = 6) e Grupo Propofol (Grupo P, n = 6). O Grupo S foi submetido apenas à laparotomia. O grupo IR foi submetido à isquemia por 30 min e reperfusão por 4 h. O grupo P foi submetido à mesma isquemia do grupo IR, com a administração de 120 mg.kg-1 de propofol iniciada 10min antes da isquemia, seguida de infusão contínua a 20 mg.kg-1.h-1. A apoptose celular foi examinada por meio do ensaio de marcação de terminações dUTP pela deoxinucleotidil transferase. Retículo endoplasmático Ca2+-ATPase2 (SERCA2) e caspase-3 do ácido aspártico contendo cisteína (caspase-3 clivada) foram avaliados com o ensaio western blot e reação em cadeia da polimerase. Resultados: A taxa de apoptose foi maior com mRNA e proteína de SERCA2 regulados para baixo e caspase-3 clivada suprarregulada no Grupo IR, em comparação com o Grupo S (p < 0,01). A taxa de apoptose foi menor com mRNA e proteína de SERCA2 suprarregulada e caspase-3 clivada sub-regulada no Grupo P, em comparação com o Grupo IR (p < 0,01). Conclusões: O propofol pode reduzir a apoptose de células miocárdicas induzida por lesão hepática por isquemia-reperfusão. Entretanto, pode suprarregular o mRNA e a proteína de SERCA2 em ratos.
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Animales , Masculino , Ratas , Daño por Reperfusión/prevención & control , Propofol/administración & dosificación , Apoptosis/efectos de los fármacos , Anestésicos Intravenosos/administración & dosificación , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/efectos de los fármacos , Hígado/irrigación sanguínea , Distribución Aleatoria , Propofol/farmacología , Ratas Sprague-Dawley , Anestésicos Intravenosos/farmacologíaRESUMEN
INTRODUCTION: Hepatic ischemia-reperfusion injury is a common pathophysiological process in liver surgery. Whether Propofol can reduce myocardial ischemia-reperfusion injury induced by hepatic ischemia-reperfusion injury in rats, together with related mechanisms, still needs further studies. OBJECTIVE: To investigate if propofol would protect the myocardial cells from apoptosis with hepatic ischemia-reperfusion injury. METHODS: Male Sprague-Dawley rats (n=18) were randomly allocated into three groups: Sham Group (Group S, n=6), Hepatic Ischemia-reperfusion Injury Group (Group IR, n=6) and Propofol Group (Group P, n=6). Group S was only subjected to laparotomy. Group IR was attained by ischemia for 30min and reperfusion for 4h. Group P was subjected identical insult as in Group IR with the administration of propofol started 10min before ischemia with 120mg.kg-1, following by continuous infusion at 20mg.kg-1.h-1. Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Endoplasmic reticulum Ca2+-ATPase2 (SERCA2) and cysteine-containing aspartic acid cleaved-caspase3 (cleaved-caspase3) were assayed by western blot and Altimeter polymerase chain reaction. RESULTS: Apoptosis rate was increased, with mRNA and protein of SERCA2 down-regulated and cleaved-caspase3 up-regulated in Group IR compared with Group S (p<0.01). Apoptosis rate was decreased, with mRNA and protein of SERCA2 up-regulated and cleaved-caspase3 down-regulated in Group P compared with Group IR (p<0.01). CONCLUSIONS: Propofol can reduce hepatic ischemia-reperfusion injury-induced myocardial cell apoptosis, meanwhile, can up-regulate mRNA and protein of SERCA2 in rats.
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Anestésicos Intravenosos/administración & dosificación , Apoptosis/efectos de los fármacos , Hígado/irrigación sanguínea , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Propofol/administración & dosificación , Daño por Reperfusión/prevención & control , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/efectos de los fármacos , Anestésicos Intravenosos/farmacología , Animales , Masculino , Propofol/farmacología , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Sorting nexin 17 (SNX17) is a critical cytoplasmic adaptor protein that regulates endosomal trafficking of membrane proteins to determine their recycling and/or degradation. The potential role of SNX17 in cardiovascular pathophysiology has not been reported. METHODS AND RESULTS: Cardiac arrhythmias were monitored using standard limb lead II electrocardiograph, and cardiac performances were determined by echocardiography in a rat model of myocardial infarction (MI) created by left anterior descending coronary artery ligation. We found that SNX17 was substantially downregulated in ischemic myocardium. The downregulation contributed to the cardiac electrical disturbances and contractile dysfunction as SNX17 replacement mitigated the detrimental alterations of MI hearts. Specifically, silence of SNX17 expression using RNA interference caused intracellular Ca2+ overload as revealed by the abnormal rise of resting [Ca2+]i and deceleration of Ca2+ decay, whereas SNX17 overexpression using vectors elicited the opposite effects. Moreover, the protein level of SERCA2a was significantly decreased by silencing SNX17. Immunohistochemistry indicated that SNX17 and SERCA2a were co-localized, and co-immunoprecipitation revealed the binding between the phox-homology domain of SNX17 and SERCA2a protein. Furthermore, lysosome inhibitor chloroquine prevented SNX17 silencing-induced reduction of SERCA2a protein level. CONCLUSION: Abnormal downregulation of SNX17 contributes to ischemic damages of cardiac electrophysiology and contractile function. SNX17 is an endogenous anti-arrhythmic factor acting by preserving functional SERCA2a protein in MI thereby offering a new strategy for the management of MI to alleviate ischemic myocardial injuries.
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Infarto del Miocardio/metabolismo , Infarto del Miocardio/prevención & control , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Nexinas de Clasificación/biosíntesis , Animales , Células Cultivadas , Masculino , Infarto del Miocardio/genética , Ratas , Ratas Wistar , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Nexinas de Clasificación/genéticaRESUMEN
BACKGROUND Recent research suggests that abnormal Ca2+ handling plays a role in the occurrence and maintenance of atrial fibrillation (AF). Therefore, Ca2+ release and ingestion depend on properties of the ryanodine receptor (RyR) and sarcoplasmic reticulum Ca2+ATPase2a (SERCA2a). This study aimed to detect whether SERCA2a gene overexpression has a preventive effect on atrial fibrillation caused by rapid pacing right atrium. MATERIAL AND METHODS Forty-eight New Zealand white rabbits were randomly divided into a control group, AF group, AAV9/GFP group, and AAV9/SERCA2a group. The right atrium was rapidly paced at 600 beats/min for 30 days after an intraperitoneal injection of an adeno-associated virus expressing the SERCA2a gene and GFP. The AF induction rate and the effective refraction period (ERP) were measured after 0, 4, 8, 12, and 24 h of pacing. Western blot analysis was used to test for the expression of SERCA2a. Changes in atrial tissue structure were observed by H&E staining and electron microscopy. RESULTS The AF induction rate was higher in the AF groups than in the AAV9/SERCA2a group at different time points of pacing. After 12 h of pacing, ERP was significantly prolonged in the AAV9/SERCA2a group compared to the AF and AAV9/GFP groups (p<0.05). SERCA2a protein expression was significantly lower in the AF and AAV9/GFP groups compared to the control group (p<0.05), while expression was significantly higher in the AAV9/SERCA2a group than in the AF and AAV9/GFP groups (p<0.05). The myocardial structure of the AAV9/SERCA2a group was significantly improved compared with the AF group, indicating that SERCA2a overexpression relieved the structural remodeling of atrial fibrillation. CONCLUSIONS SERCA2a overexpression is capable of suppressing ERP shortening and AF induced by rapid pacing atrium. SERCA2a gene therapy is expected to be a new anti-atrial fibrillation strategy.
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Fibrilación Atrial/prevención & control , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Fibrilación Atrial/enzimología , Fibrilación Atrial/metabolismo , Estimulación Cardíaca Artificial/efectos adversos , Modelos Animales de Enfermedad , Expresión Génica , Terapia Genética/métodos , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Conejos , Distribución Aleatoria , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/enzimología , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesisRESUMEN
Reduced cardiomyocyte excitation-contraction coupling and downregulation of the SERCA2a (sarcoendoplasmic reticulum calcium ATPase 2a) is associated with heart failure. This has led to viral transgene upregulation of SERCA2a in cardiomyocytes as a treatment. We hypothesized that SERCA2a gene therapy expressed under a similar promiscuous cytomegalovirus promoter could also affect the cardiac sympathetic neural axis and promote sympathoexcitation. Stellate neurons were isolated from 90 to 120 g male, Sprague-Dawley, Wistar Kyoto, and spontaneously hypertensive rats. Neurons were infected with Ad-mCherry or Ad-mCherry-hATP2Aa (SERCA2a). Intracellular Ca2+ changes were measured using fura-2AM in response to KCl, caffeine, thapsigargin, and carbonylcyanide-p-trifluoromethoxyphenylhydrazine to mobilize intracellular Ca2+ stores. The effect of SERCA2a on neurotransmitter release was measured using [3H]-norepinephrine overflow from 340 to 360 g Sprague-Dawley rat atria in response to right stellate ganglia stimulation. Upregulation of SERCA2a resulted in greater neurotransmitter release in response to stellate stimulation compared with control (empty: 98.7±20.5 cpm, n=7; SERCA: 186.5±28.41 cpm, n=8; P<0.05). In isolated Sprague-Dawley rat stellate neurons, SERCA2a overexpression facilitated greater depolarization-induced Ca2+ transients (empty: 0.64±0.03 au, n=57; SERCA: 0.75±0.03 au, n=68; P<0.05), along with increased endoplasmic reticulum and mitochondria Ca2+ load. Similar results were observed in Wistar Kyoto and age-matched spontaneously hypertensive rats, despite no further increase in endoplasmic reticulum load being observed in the spontaneously hypertensive rat (spontaneously hypertensive rats: empty, 0.16±0.04 au, n=18; SERCA: 0.17±0.02 au, n=25). In conclusion, SERCA2a upregulation in cardiac sympathetic neurons resulted in increased neurotransmission and increased Ca2+ loading into intracellular stores. Whether the increased Ca2+ transient and neurotransmission after SERCA2A overexpression contributes to enhanced sympathoexcitation in heart failure patients remains to be determined.
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Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Mitocondrias/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Sistema Nervioso Simpático/metabolismo , Transmisión Sináptica/genética , Animales , Modelos Animales de Enfermedad , Retículo Endoplásmico/ultraestructura , Corazón/inervación , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Masculino , Microscopía Electrónica de Transmisión , Mitocondrias/ultraestructura , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Sprague-Dawley , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Sistema Nervioso Simpático/ultraestructuraRESUMEN
Novel strategies are needed to treat the growing population of heart failure patients. While new drug and device based therapies have improved outcomes over the past several decades, heart failure patients continue to experience amongst the lowest quality of life of any chronic disease, high likelihood of being hospitalized and marked reduction in survival. Better understanding of many of the basic mechanisms involved in the development of heart failure has helped identify abnormalities that could potentially be targeted by gene transfer. Despite success in experimental animal models, translating gene transfer strategies from the laboratory to the clinic remains at an early stage. This review provides an introduction to gene transfer as a therapy for treating heart failure, describes some of the many factors that need to be addressed in order for it to be successful and discusses some of the recent studies that have been carried out in heart failure patients. Insights from these studies highlight both the enormous promise of gene transfer and the obstacles that still need to be overcome for this treatment approach to be successful.
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Terapia Genética/métodos , Insuficiencia Cardíaca/terapia , Adenoviridae/genética , Adenilil Ciclasas/biosíntesis , Adenilil Ciclasas/genética , Animales , Quimiocina CXCL12/biosíntesis , Quimiocina CXCL12/genética , Dependovirus/genética , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Humanos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Resultado del TratamientoRESUMEN
An impairment of cardiac function is a key feature of the cardiovascular failure associated with sepsis. Although there is some evidence that suppression of sarcoplasmic reticulum Ca2+-ATP-ase (SERCA2) contributes to septic cardiomyopathy, it is not known whether prevention of the down-regulation of SERCA2 improves outcome in sepsis. Thus, we investigated whether the administration of the synthetic antimicrobial peptide Pep2.5 may attenuate the cardiac dysfunction in murine polymicrobial sepsis through regulating SERCA2 expression. We show here for the first time that the infusion of Pep2.5 reduces the impaired systolic and diastolic contractility and improves the survival time in polymicrobial sepsis. Preservation of cardiac function in sepsis by Pep2.5 is associated with prevention of the activation of NF-κB and activation of the Akt/eNOS survival pathways. Most notably, Pep2.5 prevented the down-regulation of SERCA2 expression in a) murine heart samples obtained from mice with sepsis and b) in cardiomyocytes exposed to serum from septic shock patients. Thus, we speculate that Pep2.5 may be able to prevent down-regulation of cardiac SERCA2 expression in patients with sepsis, which, in turn, may improve cardiac function and outcome in these patients.
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Péptidos Catiónicos Antimicrobianos/farmacología , Cardiomiopatías/tratamiento farmacológico , Regulación hacia Abajo/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Sepsis/tratamiento farmacológico , Animales , Péptidos Catiónicos Antimicrobianos/química , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/fisiopatología , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sepsis/metabolismo , Sepsis/patología , Sepsis/fisiopatologíaRESUMEN
BACKGROUND: Aberrant calcium signaling may contribute to arrhythmias and adverse remodeling in hypertrophic cardiomyopathy (HCM). Mutations in sarcomere genes may distinctly alter calcium handling pathways. METHODS: We analyzed gene expression, protein levels, and functional assays for calcium regulatory pathways in human HCM surgical samples with (n=25) and without (n=10) sarcomere mutations compared with control hearts (n=8). RESULTS: Gene expression and protein levels for calsequestrin, L-type calcium channel, sodium-calcium exchanger, phospholamban, calcineurin, and calcium/calmodulin-dependent protein kinase type II (CaMKII) were similar in HCM samples compared with controls. CaMKII protein abundance was increased only in sarcomere-mutation HCM (P<0.001). The CaMKII target pT17-phospholamban was 5.5-fold increased only in sarcomere-mutation HCM (P=0.01), as was autophosphorylated CaMKII (P<0.01), suggestive of constitutive activation. Calcineurin (PPP3CB) mRNA was not increased, nor was RCAN1 mRNA level, indicating a lack of calcineurin activation. Furthermore, myocyte enhancer factor 2 and nuclear factor of activated T cell transcription factor activity was not increased in HCM, suggesting that calcineurin pathway activation is not an upstream cause of increased CAMKII protein abundance or activation. SERCA2A mRNA transcript levels were reduced in HCM regardless of genotype, as was sarcoplasmic endoplasmic reticular calcium ATPase 2/phospholamban protein ratio (45% reduced; P=0.03). 45Ca sarcoplasmic endoplasmic reticular calcium ATPaseuptake assay showed reduced uptake velocity in HCM regardless of genotype (P=0.01). The cardiac ryanodine receptor was not altered in transcript, protein, or phosphorylated (pS2808, pS2814) protein abundance, and [3H]ryanodine binding was not different in HCM, consistent with no major modification of the ryanodine receptor. CONCLUSIONS: Human HCM demonstrates calcium mishandling through both genotype-specific and common pathways. Posttranslational activation of the CaMKII pathway is specific to sarcomere mutation-positive HCM, whereas sarcoplasmic endoplasmic reticular calcium ATPase 2 abundance and sarcoplasmic reticulum Ca uptake are depressed in both sarcomere mutation-positive and -negative HCM.
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Señalización del Calcio/genética , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Estudios de Casos y Controles , Regulación hacia Abajo , Expresión Génica , Genotipo , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sarcómeros/genética , Sarcómeros/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
BACKGROUND Calcium overload, inflammation, and apoptosis play important roles in myocardial ischemia-reperfusion injury (MIRI). Gastrodin pretreatment can alleviate MIRI. This study observed sarcoplasmic reticulum calcium transport ATPase (Ca2+-ATPase, SERCA) and calcium phosphate (PLB) protein expression in the ventricular remodeling process after myocardial infarction to explore the effect of gastrodin pretreatment on MIRI. MATERIAL AND METHODS Healthy 7-week-old male SD rats were randomly divided into a sham group (A), a model group (B), and gastrodin pretreatment groups C, D, and E (100, 200, and 400 mg/kg, respectively) with 20 in each group. Anterior descending coronary artery ligation method was used to establish a rat MIRI model with 30-min ischemia and 120-min reperfusion. Cardiac electrophysiological activity was recorded. Serum IL-6 and IL10 levels were determined by ELISA. SERCA activity was tested by colorimetric phosphorus method. SERCA, PLB, and pSer-PLB protein expression were detected by Western blot. RESULTS Compared with the sham group, IL-6 and IL-10 levels were elevated, SERCA2a expression was downregulated, and PLB protein was elevated in the model group (P<0.05). pSer16-PLB showed no significant difference among groups, and the ratio of pSer16-PLB/PLB obviously decreased (P<0.05). IL-6 level gradually declined and IL-10 increased in the gastrodin group following concentration elevation. SERCA 2a expression rose in the gastrodin group in a dose-dependent manner (P<0.05). Elevated PLB protein expression showed no significant difference, while pSer16-PLB protein increased (P<0.05), leading to elevated pSer16 PLB/PLB ratio (P<0.05). CONCLUSIONS Gastrodin pretreatment alleviates MIRI and inflammation injury by regulating SERCA and PLB expression to decrease calcium overload.
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Alcoholes Bencílicos/farmacología , Fosfatos de Calcio/metabolismo , Glucósidos/farmacología , Isquemia Miocárdica/terapia , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/metabolismo , Reperfusión Miocárdica/métodos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Enfermedad de la Arteria Coronaria/metabolismo , Masculino , Isquemia Miocárdica/metabolismo , Ratas , Ratas Sprague-Dawley , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismoRESUMEN
The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca(2+)-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes.
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Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Animales , Proteínas de Transporte de Anión/biosíntesis , Proteínas de Transporte de Anión/genética , Anoctaminas/biosíntesis , Anoctaminas/genética , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Conejos , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genéticaRESUMEN
BACKGROUND: Psammomys obesus gerbils are particularly prone to develop diabetes and obesity after brief period of abundant food intake. A hypercaloric high fat diet has been shown to affect cardiac function. Here, we sought to determine whether a short period of high fat feeding might alter myocardial structure and expression of calcium handling proteins in this particular strain of gerbils. METHODS: Twenty Psammomys obesus gerbils were randomly assigned to receive a normal plant diet (controls) or a high fat diet. At baseline and 16-week later, body weight, plasma biochemical parameters (including lipid and carbohydrate levels) were evaluated. Myocardial samples were collected for pathobiological evaluation. RESULTS: Sixteen-week high fat dieting resulted in body weight gain and hyperlipidemia, while levels of carbohydrates remained unchanged. At myocardial level, high fat diet induced structural disorganization, including cardiomyocyte hypertrophy, lipid accumulation, interstitial and perivascular fibrosis and increased number of infiltrating neutrophils. Myocardial expressions of pro-apoptotic Bax-to-Bcl-2 ratio, pro-inflammatory cytokines [interleukin (IL)-1ß and tumor necrosis factor (TNF)-α], intercellular (ICAM1) and vascular adhesion molecules (VCAM1) increased, while gene encoding cardiac muscle protein, the alpha myosin heavy polypeptide (MYH6), was downregulated. Myocardial expressions of sarco(endo)plasmic calcium-ATPase (SERCA2) and voltage-dependent calcium channel (Cacna1c) decreased, while protein kinase A (PKA) and calcium-calmodulin-dependent protein kinase (CaMK2D) expressions increased. Myocardial expressions of ryanodine receptor, phospholamban and sodium/calcium exchanger (Slc8a1) did not change. CONCLUSIONS: We conclude that a relative short period of high fat diet in Psammomys obesus results in severe alterations of cardiac structure, activation of inflammatory and apoptotic processes, and altered expression of calcium-cycling determinants.
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Grasas de la Dieta/efectos adversos , Hiperlipidemias/metabolismo , Hiperlipidemias/mortalidad , Miocardio/metabolismo , Miocardio/patología , Obesidad/metabolismo , Animales , Canales de Calcio/biosíntesis , Grasas de la Dieta/farmacología , Ingestión de Alimentos , Gerbillinae , Hiperlipidemias/inducido químicamente , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-1beta/biosíntesis , Cadenas Pesadas de Miosina/biosíntesis , Obesidad/inducido químicamente , Obesidad/patología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
OBJECTIVE: Heart failure is accompanied by up-regulation of transforming growth factor beta signaling, accumulation of collagen and dysregulation of sarcoplasmic reticulum calcium adenosine triphosphatase cardiac isoform 2a (SERCA2a). We examined the fibrotic response in small and large myocardial infarct, and the effect of overexpression of the SERCA2a gene. METHODS: Ischemic cardiomyopathy was induced via creation of large or small infarct in 26 sheep. Animals were divided into 4 groups: small infarct; large infarct with heart failure; gene-treated (large infarct with heart failure followed by adeno-associated viral vector, serotype 1.SERCA2a gene construct transfer by molecular cardiac surgery with recirculating delivery); and control. RESULTS: Heart failure was significantly less pronounced in the gene-treated and small-infarct groups than in the large-infarct group. Expression of transforming growth factor beta signaling components was significantly higher in the large-infarct group, compared with the small-infarct and gene-treated groups. Both the angiotensin II type 1 receptor and angiotensin II were significantly elevated in the small- and large-infarct groups, whereas gene treatment diminished this effect. Active fibrosis with de novo collagen synthesis was evident in the large-infarct group; the small-infarct and gene-treated groups showed less fibrosis, with a lower ratio of de novo to mature collagen. CONCLUSIONS: The data presented provide evidence that progression of fibrosis is mediated through increased transforming growth factor beta and angiotensin II signaling, which is mitigated by increased SERCA2a gene expression.
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Cardiomiopatías/terapia , Terapia Genética/métodos , Insuficiencia Cardíaca/prevención & control , Infarto del Miocardio/terapia , Miocardio/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Angiotensina II/metabolismo , Animales , Calcio/metabolismo , Cardiomiopatías/enzimología , Cardiomiopatías/genética , Cardiomiopatías/patología , Cardiomiopatías/fisiopatología , Colágeno/metabolismo , Modelos Animales de Enfermedad , Inducción Enzimática , Fibronectinas/metabolismo , Fibrosis , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Hemodinámica , Masculino , Infarto del Miocardio/enzimología , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Miofibroblastos/enzimología , Miofibroblastos/patología , Receptor de Angiotensina Tipo 1/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Ovinos , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismoAsunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Antimaláricos/metabolismo , Artemisininas/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/genética , Animales , ATPasas Transportadoras de Calcio/biosíntesis , ATPasas Transportadoras de Calcio/genética , Oocitos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Xenopus laevisRESUMEN
Cylindrical spirals (CSs) are rare but distinct subsarcolemmal accumulations in skeletal muscle fibers. To date, CSs have been reported in only 16 patients with a variety of neuromuscular conditions. The origin and composition of CSs are unknown, although there are some morphologic similarities between CSs and tubular aggregates (TAs). To clarify the nature of CSs, we characterized the sarcoplasmic reticulum (SR) and other intracellular membrane system proteins in CSs of muscle biopsies from 2 adult Chinese siblings. Immunohistochemical studies revealed subsarcolemmal immunoreactivity for sarco/endoplasmic reticulum Ca2þ-ATPase 1 (SERCA 1) in the longitudinal SR, but no immunoreactivity for calsequestrin in the terminal cisternae or type 1 ryanodine receptor (RYR1) in the junctional SR. Muscles biopsied from 2 patients with TAs showed immunoreactivity not only for SERCA1 but also for other SR proteins, including calsequestrin and RYR1. CSs exhibited no immunoreactivity for the Golgi apparatus marker GM130, the nuclear membrane emerin, desmin, the autophagosome marker LC3, the lysosomal membrane marker LAMP2, dystrophin, or myosin. Our results suggest CSs may originate only from the longitudinal SR, whereas TAs are composed of both the junctional and longitudinal SR. Immunochemical staining with antibodies against calsequestrin and RYR1 help to distinguish these 2 pathological alterations.
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Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/ultraestructura , Sarcolema/ultraestructura , Retículo Sarcoplasmático/ultraestructura , Adulto , Pueblo Asiatico , Canales de Calcio/biosíntesis , Canales de Calcio/genética , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Femenino , Humanos , Inmunohistoquímica , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Persona de Mediana Edad , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Enfermedades Neuromusculares/genética , Enfermedades Neuromusculares/patología , Proteína ORAI1 , Sarcolema/genética , Sarcolema/patología , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/patología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2RESUMEN
The experiments of modeling hypogravity using fluorescent microscopy have shown a decrease of expression of b1 of Na +/K+-ATPase and Ca2+-ATPase subunits and the increase in the insensitivity of synthesis of a1S subunit of the L-type Ca2+-channel of the plasmatic membrane, whereas the synthesis of a2 subunit of Na+/K+-ATPase does not change. In «fast¼ muscle only observed similar for «slow¼ muscle decrease in the expression of b1 subunit without changing other parameters were studied. However, the decrease in fluorescence b1 subunit due to spread of data was not statistically significant. Thus hypogravity adversely affects the functioning primarily skeletal muscles, providing static load.