Modulation du transport du Ca²+ et du Na+ cytosolique et nucléaire par la taurine dans les cellules ventriculaires cardiaques

Abstract: Taurine has been reported to produce (i) a positive inotropic effect in heart muscle, (ii) beneficial effects in treatment of congestive heart failure and (iii) protective effects against Ca2+ overload. The objective of this work was to determine the short and long-term effect of normal physiological concentration of taurine on [Ca2+]i and [Na+]i of ventricular heart cells, and to determine whether long-term exposure to normal concentration of taurine ( 1mM) block and/or prevent Ca2+ overload in heart cells induced by sustained depolarization of the cell membrane. Using the fura-2 Ca2+ fluorescence microfluorometry and imaging techniques as well as fluo-3 and Na+-Green 3-D confocal microscopy measurement of free Ca2+ and Na+ respectively, short-term exposure to normal physiological concentration of taurine (1 mM) had no effect on the steady-state basal total [Ca2+]i and [Na+]i in isolated ventricular heart cells. However, exposure to relatively high concentrations of taurine (10-80 mM) induced a significant concentration-dependent sustained increase of the resting steady-state basal total [Ca2+]i and [Na+]i of ventricular heart cells. Pretreatment with β-alanine, a blocker of taurine- Na+ cotransporter, was found to block taurine-induced dose-dependent increase in [Ca2+]i in heart cells. Also a low concentration of the Na+/ Ca2+-exchanger blocker, CBDBMB (an amiloride derivative), was found to attenuate the increase in total [Ca2+]i induced by short-term treatment with taurine. Thus, the increase of basal total [Na+]i by short-term treatment with taurine could be due to Na+ entry through the taurine- Na+ cotransporter and the increase in the basal steady-state [Ca2+]i could then be due to an increase in [Na+]i which in tum favors sarcolemrnal Ca2+ influx through the Na+/ Ca2+-exchanger. Thus, taurine at relatively high concentration may exert a positive inotropic effect by such mechanism. Using the same techniques, it was found that the increase of basal total intracellular Ca2+·and Na+ levels by short-term treatment with high concentration of taurine ( ≥ 5mM) is mainly nuclear. The increase of basal nuclear sustained Na+ by taurine could be due to possible presence of a taurine- Na+ cotransporter on the nuclear membrane whereas the increase of nuclear sustained Ca2+ could be due to cytosolic Ca2+ buffering by the nucleus. Long-term exposure of heart cells to normal physiological concentration of taurine ( 1 mM) was found to decrease both cytosolic and nuclear sustained Ca2+ as well as nuclear Na+ without affecting cytosolic Na+. Moreover, long-term exposure to taurine was found to prevent nuclear increase of Ca2+ induced by permanent depolarization of heart cells with high [K+]o . This preventive effect of taurine on nuclear Ca2+ overload was associated with an increase of both cytosolic and nuclear Na+. Thus, the effect of long-term exposure to taurine on intranuclear Ca2+ overload in heart cells seems to be mediated via stimulation ofsarcolemma and nuclear Ca2+ outflow through the Na+/ Ca2+-exchanger. The fact that taurine failed to block the observed sustained elevation of nuclear Ca2+ pre-induced by sustained long-term depolarization of the membrane suggests that this amino acid possesses only a preventive effect on nuclear Ca2+ overload. [Symboles non conformes]Il est rapporté que Ia taurine produit (i) un effet inotrope positif dans le muscle cardiaque, (ii) des effets bénéfiques dans le traitement de l'insuffisance cardiaque et (iii) des effets protecteurs contre la surcharge du Ca2+. L'objectif de cette étude est de déterminer l'effet à long-terme et à court-terme de la taurine a une concentration physiologique normale sur [Ca2+]i et [Na+]i sur des cellules ventriculaires cardiaques et de déterminer si le traitement à long-terme avec une concentration normale de taurine (1mM) bloque et/ou empêche Ia surcharge calcique induite par une dépolarisation soutenue de la membrane cellulaire. En utilisant les techniques de microfluometrie et d'imagerie avec la sonde calcique fura-2 et la méthode de mesure en 3-D a l'aide de Ia microscopie confocale et les sondes fluo-3 et Na+-Green pour le Ca2+ et le Na+ respectivement, la présence de taurine à une concentration physiologique normale a court-terme, n'a pas d'effet sur le niveau basal total du [Ca2+]i et [Na+]i chez les cellules cardiaques ventriculaires isolées. Cependant, I'ajout de Ia taurine à des concentrations relativement élevées ( 10-80 mM) a induit une augmentation soutenue significative concentration-dépendente du niveau basal total du [Ca2+]i et [Na+]i des les cellules cardiaques. Le pré-traitement avec de la β-alanine, un bloqueur du co-transporteur taurine- Na+, a bloqué l'augmentation du [Ca2+]i induite par Jes concentrations croissantes de taurine dans les cellules cardiaques. De même, une faible concentration du bloqueur de l'échangeur Na+/ Ca2+ , CBDBMB (dérivé de l'amiloride), a atténué I' augmentation totale du [Ca2+]i induite par le traitement avec la taurine a court-terme. L'augmentation du niveau basal total du [Na+]i par le traitement à court-terme avec la taurine peut être due à l'entrée du Na+ à travers le co-transporteur taurine- Na+ et l'augmentation du niveau basal du [Ca2+]i peut être due alors à l'augmentation du [Na+]i qui favorise l'influx calcique à travers le sarcolemme via l'échangeur Na+/ Ca2+ . La taurine exerce alors un effet inotrope positif par un mécanisme semblable. En utilisant les mêmes techniques, il a été démontré que I'élévation du niveau basal total intracellulaire du Ca2+ et Na+ par le traitement à court-terme avec des concentrations élevées de taurine ( ≥ 5mM) est principalement nucléaire. L'augmentation soutenue du niveau basal nucléaire de Na+ par la taurine peut être due à Ia présence possible du co-transporteur taurine- Na+ sur la membrane nucléaire, tandis que l'augmentation soutenue du Ca2+ nucléaire peut être due à l'effet tampon calcique du noyau. Le traitement des cellules cardiaques a long-terme avec une concentration physiologique normale de taurine a diminué le Ca2+ cytosolique et nucléaire ainsi que le Na+ nucléaire sans affecter le Na+ cytosolique. De plus, le traitement à long-terme avec la taurine a empêché l'augmentation nucléaire du Ca2+ induite par la dépolarisation soutenue des cellules cardiaques avec une concentration élevée de [K+]o. Cet effet préventif de la taurine sur la surcharge calcique nucléaire était associé à l'augmentation du Na+ cytosolique et nucléaire. L' effet du traitement à long-terme avec la taurine sur la surcharge du Ca2+ intranucleaire dans les cellules cardiaques se fait suite à la sortie du Ca2+ à travers la membrane nucléaire et le sarcolemme via la stimulation de I' échangeur Na+/ Ca2+ Le fait que la taurine n'a pas bloqué l'élévation soutenue du Ca2+ nucléaire observée pré-induite par la dépolarisation soutenue de la membrane à long-terme suggère que cet acide aminé possède uniquement un effet préventif sur la surcharge du Ca2+ nucléaire. [Symboles non conformes

A self-inactivating retrovector incorporating the IL-2 promoter for activation-induced transgene expression in genetically engineered T-cells

BACKGROUND: T-cell activation leads to signaling pathways that ultimately result in induction of gene transcription from the interleukin-2 (IL-2) promoter. We hypothesized that the IL-2 promoter or its synthetic derivatives can lead to T-cell specific, activation-induced transgene expression. Our objective was to develop a retroviral vector for stable and activation-induced transgene expression in T-lymphocytes. RESULTS: First, we compared the transcriptional potency of the full-length IL-2 promoter with that of a synthetic promoter composed of 3 repeats of the Nuclear Factor of Activated T-Cells (NFAT) element following activation of transfected Jurkat T-cells expressing the large SV40 T antigen (Jurkat TAg). Although the NFAT3 promoter resulted in a stronger induction of luciferase reporter expression post stimulation, the basal levels of the IL-2 promoter-driven reporter expression were much lower indicating that the IL-2 promoter can serve as a more stringent activation-dependent promoter in T-cells. Based on this data, we generated a self-inactivating retroviral vector with the full-length human IL-2 promoter, namely SINIL-2pr that incorporated the enhanced green fluorescent protein (EGFP) fused to herpes simplex virus thymidine kinase as a reporter/suicide "bifunctional" gene. Subsequently, Vesicular Stomatitis Virus-G Protein pseudotyped retroparticles were generated for SINIL-2pr and used to transduce the Jurkat T-cell line and the ZAP-70-deficient P116 cell line. Flow cytometry analysis showed that EGFP expression was markedly enhanced post co-stimulation of the gene-modified cells with 1 μM ionomycin and 10 ng/ml phorbol 12-myristate 13-acetate (PMA). This activation-induced expression was abrogated when the cells were pretreated with 300 nM cyclosporin A. CONCLUSION: These results demonstrate that the SINIL-2pr retrovector leads to activation-inducible transgene expression in Jurkat T-cell lines. We propose that this design can be potentially exploited in several cellular immunotherapy applications

Inhibition of histone deacetylation in 293GPG packaging cell line improves the production of self-inactivating MLV-derived retroviral vectors

BACKGROUND: Self-inactivating retroviral vectors (SIN) are often associated with very low titers. Promoter elements embedded within SIN designs may suppress transcription of packageable retroviral RNA which in turn results in titer reduction. We tested whether this dominant-negative effect involves histone acetylation state. We designed an MLV-derived SIN vector using the cytomegalovirus immediate early enhancer-promoter (CMVIE) as an embedded internal promoter (SINCMV) and transfected the pantropic 293GPG packaging cell line. RESULTS: The SINCMV retroviral producer had uniformly very low titers (~10,000 infectious retroparticles per ml). Northern blot showed low levels of expression of retroviral mRNA in producer cells in particular that of packageable RNA transcript. Treatment of the producers with the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A reversed transcriptional suppression and resulted in an average 106.3 ± 4.6 – fold (P = 0.002) and 15.5 ± 1.3 – fold increase in titer (P = 0.008), respectively. A histone gel assay confirmed increased histone acetylation in treated producer cells. CONCLUSION: These results show that SIN retrovectors incorporating strong internal promoters such as CMVIE, are susceptible to transcriptional silencing and that treatment of the producer cells with HDAC inhibitors can overcome this blockade suggesting that histone deacetylation is implicated in the mechanism of transcriptional suppression

Lamin A/C and emerin regulate MKL1/SRF activity by modulating actin dynamics

Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss Muscular Dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome (HGPS).1 The majority of LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and disturbed interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes.1 We report here that lamin A/C-deficient (Lmna−/−) and Lmna N195K mutant cells have impaired nuclear translocation and downstream signaling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function.2 Disturbed nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna−/− and N195K mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease etiology for the cardiac phenotype in many laminopathies, whereby lamins A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization

Nuclear Envelope Composition Determines The Ability Of Neutrophil-Type Cells To Passage Through Micron-Scale Constrictions

Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huët anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential

Exploring the Crosstalk Between LMNA and Splicing Machinery Gene Mutations in Dilated Cardiomyopathy

Mutations in the LMNA gene, which encodes for the nuclear lamina proteins lamins A and C, are responsible for a diverse group of diseases known as laminopathies. One type of laminopathy is Dilated Cardiomyopathy (DCM), a heart muscle disease characterized by dilation of the left ventricle and impaired systolic function, often leading to heart failure and sudden cardiac death. LMNA is the second most commonly mutated gene in DCM. In addition to LMNA, mutations in more than 60 genes have been associated with DCM. The DCM-associated genes encode a variety of proteins including transcription factors, cytoskeletal, Ca2+-regulating, ion-channel, desmosomal, sarcomeric, and nuclear-membrane proteins. Another important category among DCM-causing genes emerged upon the identification of DCM-causing mutations in RNA binding motif protein 20 (RBM20), an alternative splicing factor that is chiefly expressed in the heart. In addition to RBM20, several essential splicing factors were validated, by employing mouse knock out models, to be embryonically lethal due to aberrant cardiogenesis. Furthermore, heart-specific deletion of some of these splicing factors was found to result in aberrant splicing of their targets and DCM development. In addition to splicing alterations, advances in next generation sequencing highlighted the association between splice-site mutations in several genes and DCM. This review summarizes LMNA mutations and splicing alterations in DCM and discusses how the interaction between LMNA and splicing regulators could possibly explain DCM disease mechanisms

Myopathic lamin mutations impair nuclear stability in cells and tissue and disrupt nucleo-cytoskeletal coupling

Lamins are intermediate filament proteins that assemble into a meshwork underneath the inner nuclear membrane, the nuclear lamina. Mutations in the LMNA gene, encoding lamins A and C, cause a variety of diseases collectively called laminopathies. The disease mechanism for these diverse conditions is not well understood. Since lamins A and C are fundamental determinants of nuclear structure and stability, we tested whether defects in nuclear mechanics could contribute to the disease development, especially in laminopathies affecting mechanically stressed tissue such as muscle. Using skin fibroblasts from laminopathy patients and lamin A/C-deficient mouse embryonic fibroblasts stably expressing a broad panel of laminopathic lamin A mutations, we found that several mutations associated with muscular dystrophy and dilated cardiomyopathy resulted in more deformable nuclei; in contrast, lamin mutants responsible for diseases without muscular phenotypes did not alter nuclear deformability. We confirmed our results in intact muscle tissue, demonstrating that nuclei of transgenic Drosophila melanogaster muscle expressing myopathic lamin mutations deformed more under applied strain than controls. In vivo and in vitro studies indicated that the loss of nuclear stiffness resulted from impaired assembly of mutant lamins into the nuclear lamina. Although only a subset of lamin mutations associated with muscular diseases caused increased nuclear deformability, almost all mutations tested had defects in force transmission between the nucleus and cytoskeleton. In conclusion, our results indicate that although defective nuclear stability may play a role in the development of muscle diseases, other factors, such as impaired nucleo-cytoskeletal coupling, likely contribute to the muscle phenotyp

Precision oncology revolution: CRISPR-Cas9 and PROTAC technologies unleashed

Cancer continues to present a substantial global health challenge, with its incidence and mortality rates persistently reflecting its significant impact. The emergence of precision oncology has provided a breakthrough in targeting oncogenic drivers previously deemed “undruggable” by conventional therapeutics and by limiting off-target cytotoxicity. Two groundbreaking technologies that have revolutionized the field of precision oncology are primarily CRISPR-Cas9 gene editing and more recently PROTAC (PROteolysis TArgeting Chimeras) targeted protein degradation technology. CRISPR-Cas9, in particular, has gained widespread recognition and acclaim due to its remarkable ability to modify DNA sequences precisely. Rather than editing the genetic code, PROTACs harness the ubiquitin proteasome degradation machinery to degrade proteins of interest selectively. Even though CRISPR-Cas9 and PROTAC technologies operate on different principles, they share a common goal of advancing precision oncology whereby both approaches have demonstrated remarkable potential in preclinical and promising data in clinical trials. CRISPR-Cas9 has demonstrated its clinical potential in this field due to its ability to modify genes directly and indirectly in a precise, efficient, reversible, adaptable, and tissue-specific manner, and its potential as a diagnostic tool. On the other hand, the ability to administer in low doses orally, broad targeting, tissue specificity, and controllability have reinforced the clinical potential of PROTAC. Thus, in the field of precision oncology, gene editing using CRISPR technology has revolutionized targeted interventions, while the emergence of PROTACs has further expanded the therapeutic landscape by enabling selective protein degradation. Rather than viewing them as mutually exclusive or competing methods in the field of precision oncology, their use is context-dependent (i.e., based on the molecular mechanisms of the disease) and they potentially could be used synergistically complementing the strengths of CRISPR and vice versa. Herein, we review the current status of CRISPR and PROTAC designs and their implications in the field of precision oncology in terms of clinical potential, clinical trial data, limitations, and compare their implications in precision clinical oncology

La matriz extracelular: morfología, función y biotensegridad (parte I)

La matriz extracelular (MEC) representa una red tridimensional que engloba todos los órganos, tejidos y células del organismo. Constituye un filtro biofísico de protección, nutrición e inervación celular y el terreno para la respuesta inmune, angiogénesis, fibrosis y regeneración tisular. Y representa el medio de transmisión de fuerzas mecánicas a la membrana basal, que a través de las integrinas soporta el sistema de tensegridad y activa los mecanismos epigenéticos celulares. La alteración de la MEC supone la pérdida de su función de filtro eficaz, nutrición, eliminación, denervación celular, pérdida de la capacidad de regeneración y cicatrización y alteración de la transmisión mecánica o mecanotransducción. También la pérdida del sustrato para una correcta respuesta inmune ante agentes infecciosos, tumorales y tóxicos. Los tumores son tejidos funcionales conectados y dependientes del microambiente. El microambiente tumoral, constituido por la MEC, células del estroma y la propia respuesta inmune, son determinantes de la morfología y clasificación tumoral, agresividad clínica, pronóstico y respuesta al tratamiento del tumor. Tanto en condiciones fisiológicas como patológicas, la comunicación recíproca entre células del estroma y el parénquima dirige la expresión génica. La capacidad oncogénica del estroma procede tanto de los fibroblastos asociados al tumor como de la celularidad de la respuesta inmune y la alteración de la tensegridad por la MEC. La transición epitelio-mesenquimal es el cambio que transforma una célula normal o «benigna» en «maligna». El citoesqueleto pseudomesenquimal otorga las propiedades de migración, invasión y diseminación. Y viceversa, el fenotipo maligno es reversible a través de la corrección de las claves que facilita el microambiente tumoral.Extracellular matrix (ECM) is a three-dimensional network that envelopes all the organs, tissues and cells of the body. A biophysical filter that provides protection, nutrition and cell innervation, it is the site for immune response, angiogenesis, fibrosis and tissue regeneration. It is also the transport medium for mechanical forces to the basal membrane through integrins that support the tensegrity system, activating cellular epigenetic mechanisms. The disruption of the ECM leads to a functional loss of nutrition, elimination, cell innervation, regenerative capacity and wound healing as well as alterations in mechanical transduction. This loss also disrupts the immune response to pathogens, tumour cells and toxins. Tumours are functionally connected tissues which depend on the microenvironment. This tumour microenvironment, made up of ECM, stromal cells and the immune response, determines the morphology and tumour histopathological classification, clinical behaviour, prognosis and immune response to the tumour. Both in physiological and pathological conditions, reciprocity in the communication between stromal and parenchymal cells determine gene expression. The oncogenic capacity of the stroma depends on tumour associated fibroblasts, immune system cellularity and disruption of tensegrity by ECM. Epithelial-mesenchymal transition is the change that transforms a normal or benign cell into a malignant cell. The «pseudo-mensenchymal» cytoskeleton is responsible for migration, invasion and dissemination, and vice-versa, the malignant phenotype is reversible through the correction of the microenvironmental factors that favour tumour growth.Noguera Salva, Rosa, [email protected]