Alteraciones en la producción y secreción de los péptidos natriuréticos y sus moléculas relacionadas en pacientes con insuficiencia cardiaca. Relación con la función del ventrículo izquierdo

La insuficiencia cardiaca es un síndrome multifactorial de gran impacto en la asistencia sanitaria debido al aumento de la morbilidad y la mortalidad y la ausencia de un tratamiento curativo. Es el resultado, no solo de una sobrecarga o daño cardiaco, sino también de una compleja interacción de cambios genéticos, neurohormonales, inflamatorios y bioquímicos que actúan sobre el miocito y/o el intersticio. Como consecuencia de estas alteraciones, el paciente con insuficiencia cardiaca tiene niveles anómalos de diversas moléculas (enzimas, hormonas, citocinas…) que son utilizadas como biomarcadores de gran importancia clínica. En este sentido, el papel de los péptidos natriuréticos en la clínica de la insuficiencia cardiaca está bien establecido, utilizando sus concentraciones en plasma para ayudar en el diagnóstico, pronóstico y en la determinación de la gravedad de este síndrome. A pesar de ello, la comprensión de su compleja naturaleza aún está lejos de ser completa y exhaustiva, siendo esta información de vital importancia como objetivo para nuevos tratamientos que disminuyan la morbilidad y mortalidad de este síndrome. Además, estudios previos en modelos animales, han sugerido que la secreción de los péptidos natriuréticos durante la insuficiencia cardiaca está alterada y, a su vez, el aparato de Golgi presenta cambios morfológicos en este síndrome. Por ello, nos planteamos estudiar, en un grupo de pacientes con insuficiencia cardiaca, la producción tisular de los péptidos natriuréticos y las moléculas relacionadas implicadas en su biología, así como su secreción a través de las vesículas del aparato de Golgi. Nuestros resultados muestran que en los corazones explantados de pacientes con insuficiencia cardiaca, los niveles génicos de ANP, BNP y el receptor NPR3 se encuentran elevados y los niveles de CORINA disminuidos, sin que se detecte variación en la expresión proteica de ANP y CNP. En los pacientes con miocardiopatía isquémica la expresión génica de CNP y la expresión proteica de corina reflejan el deterioro de la función ventricular izquierda. Además, muestran elevación de la expresión de BNP, corina y del ARNm de CNP y disminución del ARNm de FURINA, indetectable en los pacientes con insuficiencia cardiaca de etiología dilatada. Estos hallazgos podrían indicar la existencia de mecanismos moleculares diferentes en función de la etiología de este síndrome proporcionando nuevas opciones diagnósticas y terapéuticas específicas. Por otro lado, hemos aislado por primera vez las vesículas del aparato de Golgi de tejido cardiaco humano en las cuales detectamos un importante incremento de la concentración de péptidos natriuréticos ANP, BNP y CNP, a diferencia de lo que ocurre cuando estudiamos el conjunto del tejido miocárdico. También encontramos cambios en la expresión génica de los componentes del aparato de Golgi implicados en la secreción, así como de sus vesículas, que reflejan en su número y morfología el deterioro funcional y el aumento del tráfico de péptidos natriuréticos hacia el plasma. Estos hallazgos proporcionan a este orgánulo un papel importante en la biología de los péptidos natriuréticos, regulando el régimen de secreción al que están sometidos estos pacientes, y en el proceso de adaptación cardiovascular morfológica y funcional que experimentan.Heart failure is a multifactorial syndrome with a major impact on health care due to increased morbidity and mortality and the absence of curative treatment. It is the result, not only of an overload or heart damage, but also of a complex interaction of genetic, neurohormonal, inflammatory and biochemical changes acting on the myocyte and / or the interstice. As a consequence of these alterations, the heart failure patient has abnormal levels of various molecules (enzymes, hormones, cytokines...) that are used as biomarkers of great clinical importance. In this regard, the role of natriuretic peptides in clinical heart failure is well established, using their concentrations in plasma to aid in the diagnosis, prognosis and in determining the severity of this syndrome. However, understanding their complex nature is still far from complete and exhaustive, and this information is of vital importance as a target for new treatments that reduce the morbidity. Furthermore, previous studies in animal models have suggested that secretion of natriuretic peptides in heart failure is impaired and, in turn, the Golgi apparatus presents morphological changes in this syndrome. Therefore, we propose to study, in a group of patients with heart failure, the tissue production of natriuretic peptides and related molecules involved in their biology, and their secretion through the Golgi apparatus vesicles. Our results show that explanted hearts from heart failure patients have elevated mRNA levels of ANP, BNP and NPR3 receptor and have reduced levels of CORIN without variation in protein expression of ANP and CNP. In patients with ischaemic cardiomyopathy, CNP gene expression and corin protein expression reflect the impaired left ventricular function. Also, they show elevated expression of BNP, corin and mRNA of CNP and decreased mRNA levels of furin, undetectable in patients with dilated cardiomyopathy. These findings may indicate the existence of different molecular mechanisms depending on the aetiology of this syndrome by providing specific new diagnostic and therapeutic options. On the other hand, we have isolated for the first time vesicles from the Golgi apparatus of human heart tissue in which we detect a significant increase in the ANP, BNP and CNP natriuretic peptides concentration, unlike what happens when studying the whole myocardial tissue. We also found changes in gene expression of the Golgi apparatus components involved in secretion, and their vesicles, which reflect in their number and morphology a functional impairment and increased traffic of natriuretic peptides to the plasma. These findings provide to this organelle an important role in the biology of natriuretic peptides regulating the secretion system which are subject these patients, and in the process of morphological and functional cardiovascular adaptation

El complejo del poro nuclear en la insuficiencia cardiaca

[ES] Las manifestaciones clínicas de la insuficiencia cardiaca (IC) son resultado de cambios en los componentes celulares y moleculares del corazón para mantener un control homeostático. La IC se ha asociado con alteraciones mitocondriales y del citoesqueleto, así como cambios en la expresión y actividad de enzimas glicolíticos y del metabolismo del calcio. Pero, a pesar del papel crítico que ejerce el núcleo en el funcionamiento celular y su implicación en numerosas enfermedades, existen pocos estudios que examinen su importancia en la IC. Por ello, se pretende evaluar si existe alguna alteración en la estructura del complejo del poro nuclear (CPN) que conecta nucleoplasma y citoplasma en la IC. En este proyecto analizamos diferentes nucleoporinas que representan las estructuras del CPN: anillo transmembrana (NDC1), anillo interno (Nup155), anillo externo (Nup160), proteínas ¿linker¿ (Nup93), ¿FG¿ (Nup153) y periféricas (TPR); todo ello en muestras de tejido cardiaco humano. Además, algunas de estas proteínas se han asociado al origen de ciertas enfermedades del sistema nervioso, inmune, o algunos tipos de cáncer. El objetivo del proyecto es determinar los niveles de estas proteínas del CPN en ventrículo izquierdo en pacientes con cardiopatía isquémica y dilatada mediante Western-blot y su localización subcelular mediante microscopía de fluorescencia y electrónica de transmisión.[EN] The clinical manifestations of heart failure (HF) are the result from changes in cellular and molecular components of the heart to maintain a homeostatic control. The HF has been associated with mitochondrial and cytoskeletal alterations and changes in the expression and activity of glycolytic enzymes and calcium metabolism. But, despite critical role exerted by the nucleus in cell function and its involvement in numerous diseases, few studies examine their importance in HF. Thus, we wanted to assess whether there is any alteration in the structure of the nuclear pore complex (NPC) connecting nucleoplasm and cytoplasm in HF. In this project, we analyze nucleoporins that represent different structures of NPC: transmembrane ring (NDC1), inner ring (Nup155), outer ring (Nup160) proteins "linker" (Nup93), "FG" (Nup153) and peripheral (TPR), all from human cardiac tissue samples. In addition, some of these proteins are associated in the origin of certain diseases of the nervous or immune system, or some cancers. The project aim is determine the levels of these proteins in NPC of left ventricle heart in patients with ischemic and dilated cardiomyopathy by Western-blot and subcellular localization by fluorescence and transmission electron microscopy.Tarazón Melguizo, E. (2012). El complejo del poro nuclear en la insuficiencia cardiaca. http://hdl.handle.net/10251/17862Archivo delegad

MMP-2 and sTNF-R1 variability in patients with essential hypertension: 1-year follow-up study

The aim of this study is to analyze MMP-2 and sTNF-R1 variability, potent predictors of cardiovascular events, in stable hypertensive patients during a 12-month followup. 234 asymptomatic patients (age 6 0 ± 1 3 , 136 male) out of 252 patients with essential hypertension were followed up. MMP-2 and sTNF-R1 were measured at baseline and after 12 months (stage I). To compare MMP-2 and sTNF-R1 levels over time interval, we used the statistical method of Bland-Altman. MMP-2 and sTNF-R1 reproducibility was good in our patients for the two intervals with a coefficient of reproducibility of 8.2% and 11.3%, respectively. The percentages of patients within 1.96 × standard deviation of the mean were 93.6% and 92.7%. An elevated coefficient of correlation was obtained for MMP-2, basal versus stage I

The Treatment With the SGLT2 Inhibitor Empagliflozin Modifies the Hepatic Metabolome of Male Zucker Diabetic Fatty Rats Towards a Protective Profile

[Abstract] The EMPA-REG OUTCOME (Empagliflozin, Cardiovascular Outcome Event Trial in patients with Type 2 Diabetes Mellitus (T2DM)) trial evidenced the potential of sodium-glucose cotransporter 2 (SGLT2) inhibitors for the treatment of patients with diabetes and cardiovascular disease. Recent evidences have shown the benefits of the SGLT2 inhibitor empagliflozin on improving liver steatosis and fibrosis in patients with T2DM. Metabolomic studies have been shown to be very useful to improve the understanding of liver pathophysiology during the development and progression of metabolic hepatic diseases, and because the effects of empagliflozin and of other SGLT2 inhibitors on the complete metabolic profile of the liver has never been analysed before, we decided to study the impact on the liver of male Zucker diabetic fatty (ZDF) rats of a treatment for 6 weeks with empagliflozin using an untargeted metabolomics approach, with the purpose to help to clarify the benefits of the use of empagliflozin at hepatic level. We found that empagliflozin is able to change the hepatic lipidome towards a protective profile, through an increase of monounsaturated and polyunsaturated glycerides, phosphatidylcholines, phosphatidylethanolamines, lysophosphatidylinositols and lysophosphatidylcholines. Empagliflozin also induces a decrease in the levels of the markers of inflammation IL-6, chemerin and chemerin receptor in the liver. Our results provide new evidences regarding the molecular pathways through which empagliflozin could exert hepatoprotector beneficial effects in T2DM.This work was supported by Boehringer Ingelheim Pharma GmbH and Co., by the National Institute of Health “Fondo de Investigaciones Sanitarias del Instituto de Salud Carlos III” Madrid, Spain (PI15/00681, PI17/00409, PI18/00821, PI20/00902, RETICS Programme RD16/0012/0014 and CIBER de Enfermedades Cardiovasculares (CIBERCV)); European Regional Development Fund (FEDER) and European Union framework MSCA-RISE-H2020 Programme (Project number 734899). AH-A was funded by predoctoral research grants from Xunta de Galicia and FPU Program of the Spanish Ministry of Science, Innovation and Universities (Spain); MF-S was funded by the predoctoral research grants “Programa Científico do Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS) (Spain) and Xunta de Galicia; and AV-L was funded by the predoctoral research grant from the PFIS Program of the Spanish Ministry of Science and Instituto de Salud Carlos III (Spain

Electron Microscopy Reveals Evidence of Perinuclear Clustering of Mitochondria in Cardiac Biopsy-Proven Allograft Rejection

Acute cellular rejection is a major complication in heart transplantation. We focus on the analysis of new ultrastructural findings in cardiac biopsy rejection based on mitochondrial intracellular organization. This study includes heart transplanted patients from a single center who were referred for endomyocardial biopsies as a scheduled routine screening. Participants were divided into two groups: patients transplanted without allograft rejection (Grade 0R), and patients with biopsy-proven allograft rejection (Grade ≥ 2R). Using electronic microscopy, we detected a significant increase in the volume density of mitochondria (p < 0.0001) and dense bodies (p < 0.01) in the rejection group. The most relevant finding was the presence of local accumulations of mitochondria close to the nuclear envelope, pressing and molding the morphology of this membrane in all rejection samples (100%). We identified this perinuclear clustering of mitochondria phenomenon in a 68 ± 27% of the total cardiac nucleus observed from rejection samples. We did not observe this phenomenon in any non-rejection samples, reflecting excellent sensitivity and specificity. We have identified a specific phenomenon affecting the architecture of the nuclear membrane—perinuclear clustering of mitochondria—in endomyocardial biopsies from patients with cardiac rejection. This ultrastructural approach might complement and improve the diagnosis of rejection

Electron Microscopy Reveals Evidence of Perinuclear Clustering of Mitochondria in Cardiac Biopsy-Proven Allograft Rejection

Acute cellular rejection is a major complication in heart transplantation. We focus on the analysis of new ultrastructural findings in cardiac biopsy rejection based on mitochondrial intracellular organization. This study includes heart transplanted patients from a single center who were referred for endomyocardial biopsies as a scheduled routine screening. Participants were divided into two groups: patients transplanted without allograft rejection (Grade 0R), and patients with biopsy-proven allograft rejection (Grade ≥ 2R). Using electronic microscopy, we detected a significant increase in the volume density of mitochondria (p p < 0.01) in the rejection group. The most relevant finding was the presence of local accumulations of mitochondria close to the nuclear envelope, pressing and molding the morphology of this membrane in all rejection samples (100%). We identified this perinuclear clustering of mitochondria phenomenon in a 68 ± 27% of the total cardiac nucleus observed from rejection samples. We did not observe this phenomenon in any non-rejection samples, reflecting excellent sensitivity and specificity. We have identified a specific phenomenon affecting the architecture of the nuclear membrane—perinuclear clustering of mitochondria—in endomyocardial biopsies from patients with cardiac rejection. This ultrastructural approach might complement and improve the diagnosis of rejection

Implication of Sphingolipid Metabolism Gene Dysregulation and Cardiac Sphingosine-1-Phosphate Accumulation in Heart Failure

Disturbances in sphingolipid metabolism lead to biological function dysregulation in many diseases, but it has not been described in heart failure (HF). Sphingosine-1-phosphate (S1P) levels have not ever been measured in the myocardium. Therefore, we analyze the gene dysregulation of human cardiac tissue by mRNA-seq (n = 36) and ncRNA-seq (n = 50). We observed most major changes in the expression of genes belonging to de novo and salvage pathways, and the tight gene regulation by their miRNAs is largely dysregulated in HF. We verified using ELISA (n = 41) that ceramide and S1P accumulate in HF cardiac tissue, with an increase in the ceramide/S1P ratio of 57% in HF. Additionally, changes in left ventricular mass and diameters are directly related to CERS1 expression and inversely related to S1P levels. Altogether, we define changes in the main components of the sphingolipid metabolism pathways in HF, mainly de novo and salvage, which lead to an increase in ceramide and S1P in cardiac tissue, as well as an increase in the ceramide/S1P ratio in HF patients. Therapeutic gene modulation focused on restoring ceramide levels or reversing the ceramide/S1P ratio could be a potential therapy to be explored for HF patients

Intercalated disc in failing hearts from patients with dilated cardiomyopathy : Its role in the depressed left ventricular function

Alterations in myocardial structure and reduced cardiomyocyte adhesions have been previously described in dilated cardiomyopathy (DCM). We studied the transcriptome of cell adhesion molecules in these patients and their relationships with left ventricular (LV) function decay. We also visualized the intercalated disc (ID) structure and organization. The transcriptomic profile of 23 explanted LV samples was analyzed using RNA-sequencing (13 DCM, 10 control [CNT]), focusing on cell adhesion genes. Electron microscopy analysis to visualize ID structural differences and immunohistochemistry experiments of ID proteins was also performed. RT-qPCR and western blot experiments were carried out on ID components. We found 29 differentially expressed genes, most of all, constituents of the ID structure. We found that the expression of GJA3, DSP and CTNNA3 was directly associated with LV ejection fraction (r = 0.741, P = 0.004; r = 0.674, P = 0.011 and r = 0.565, P = 0.044, respectively), LV systolic (P = 0.003, P = 0.003, P = 0.028, respectively) and diastolic dimensions (P = 0.006, P = 0.001, P = 0.025, respectively). Electron microscopy micrographs showed a reduced ID convolution index and immunogold labeling of connexin 46 (GJA gene), desmoplakin (DSP gene) and catenin α-3 (CTNNA3 gene) proteins in DCM patients. Moreover, we observed that protein and mRNA levels analyzed by RT-qPCR of these ID components were diminished in DCM group. In conclusion, we report significant gene and protein expression changes and found that the ID components GJA3, DSP and CTNNA3 were highly related to LV function. Microscopic observations indicated that ID is structurally compromised in these patients. These findings give new data for understanding the ventricular depression that characterizes DCM, opening new therapeutic perspectives for these critically diseased patients

MiR-138-5p Suppresses Cell Growth and Migration in Melanoma by Targeting Telomerase Reverse Transcriptase

Recent evidence suggests the existence of a miRNA regulatory network involving human telomerase reverse transcriptase gene (hTERT), with miR-138-5p playing a central role in many types of cancers. However, little is known about the regulation of hTERT expression by microRNA (miRNAs) in melanocytic tumors. Here, we investigated the effects of miR-138-5p in hTERT regulation in melanoma cells lines. In vitro studies demonstrated higher miR-138-5p and lower hTERT messenger RNA (mRNA) expression in human epidermal melanocytes, compared with melanoma cell lines (A2058, A375, SK-MEL-28) by quantitative polymerase chain reaction (qPCR) observing a negative correlation between them. A2058 melanoma cells were selected to be transfected with miR-138-5p mimic or inhibitor. Using luciferase assay, hTERT was identified as a direct target of this miRNA. Overexpression of miR-138-5p detected by Western blot revealed a decrease in hTERT protein expression (p = 0.012), and qPCR showed a reduction in telomerase activity (p p = 0.035) and migration abilities (p = 0.015) were observed in A2058-transfected cells using thiazolyl blue tetrazolium bromide and flow cytometry, respectively. This study identifies miR-138-5p as a crucial tumor suppressor miRNA involved in telomerase regulation. Targeting it as a combination therapy with immunotherapy or targeted therapies could be used in advanced melanoma treatment; however, more preclinical studies are necessary