Molecular Template for a Voltage Sensor in a Novel K+ Channel. I. Identification and Functional Characterization of KvLm, a Voltage-gated K+ Channel from Listeria monocytogenes

The fundamental principles underlying voltage sensing, a hallmark feature of electrically excitable cells, are still enigmatic and the subject of intense scrutiny and controversy. Here we show that a novel prokaryotic voltage-gated K+ (Kv) channel from Listeria monocytogenes (KvLm) embodies a rudimentary, yet robust, sensor sufficient to endow it with voltage-dependent features comparable to those of eukaryotic Kv channels. The most conspicuous feature of the KvLm sequence is the nature of the sensor components: the motif is recognizable; it appears, however, to contain only three out of eight charged residues known to be conserved in eukaryotic Kv channels and accepted to be deterministic for folding and sensing. Despite the atypical sensor sequence, flux assays of KvLm reconstituted in liposomes disclosed a channel pore that is highly selective for K+ and is blocked by conventional Kv channel blockers. Single-channel currents recorded in symmetric K+ solutions from patches of enlarged Escherichia coli (spheroplasts) expressing KvLm showed that channel open probability sharply increases with depolarization, a hallmark feature of Kv channels. The identification of a voltage sensor module in KvLm with a voltage dependence comparable to that of other eukaryotic Kv channels yet encoded by a sequence that departs significantly from the consensus sequence of a eukaryotic voltage sensor establishes a molecular blueprint of a minimal sequence for a voltage sensor

Molecular Template for a Voltage Sensor in a Novel K+ Channel. II. Conservation of a Eukaryotic Sensor Fold in a Prokaryotic K+ Channel

KvLm, a novel bacterial depolarization-activated K+ (Kv) channel isolated from the genome of Listeria monocytogenes, contains a voltage sensor module whose sequence deviates considerably from the consensus sequence of a Kv channel sensor in that only three out of eight conserved charged positions are present. Surprisingly, KvLm exhibits the steep dependence of the open channel probability on membrane potential that is characteristic of eukaryotic Kv channels whose sensor sequence approximates the consensus. Here we asked if the KvLm sensor shared a similar fold to that of Shaker, the archetypal eukaryotic Kv channel, by examining if interactions between conserved residues in Shaker known to mediate sensor biogenesis and function were conserved in KvLm. To this end, each of the five non-conserved residues in the KvLm sensor were mutated to their Shaker-like charged residues, and the impact of these mutations on the voltage dependence of activation was assayed by current recordings from excised membrane patches of Escherichia coli spheroplasts expressing the KvLm mutants. Conservation of pairwise interactions was investigated by comparison of the effect of single mutations to the impact of double mutations presumed to restore wild-type fold and voltage sensitivity. We observed significant functional coupling between sites known to interact in Shaker Kv channels, supporting the notion that the KvLm sensor largely retains the fold of its eukaryotic homologue

Myocardial creatine levels do not influence response to acute oxidative stress in isolated perfused heart

Background: Multiple studies suggest creatine mediates anti-oxidant activity in addition to its established role in cellular energy metabolism. The functional significance for the heart has yet to be established, but antioxidant activity could contribute to the cardioprotective effect of creatine in ischaemia/reperfusion injury. Objectives: To determine whether intracellular creatine levels influence responses to acute reactive oxygen species (ROS) exposure in the intact beating heart. We hypothesised that mice with elevated creatine due to over-expression of the creatine transporter (CrT-OE) would be relatively protected, while mice with creatine-deficiency (GAMT KO) would fare worse. Methods and Results: CrT-OE mice were pre-selected for creatine levels 20–100% above wild-type using in vivo 1 H– MRS. Hearts were perfused in isovolumic Langendorff mode and cardiac function monitored throughout. After 20 min equilibration, hearts were perfused with either H2O2 0.5 mM (30 min), or the anti-neoplastic drug doxorubicin 15 mM (100 min). Protein carbonylation, creatine kinase isoenzyme activities and phospho-PKCd expression were quantified in perfused hearts as markers of oxidative damage and apoptotic signalling. Wild-type hearts responded to ROS challenge with a profound decline in contractile function that was ameliorated by co-administration of catalase or dexrazoxane as positive controls. In contrast, the functional deterioration in CrT-OE and GAMT KO hearts was indistinguishable from wildtype controls, as was the extent of oxidative damage and apoptosis. Exogenous creatine supplementation also failed to protect hearts from doxorubicin-induced dysfunction. Conclusions: Intracellular creatine levels do not influence the response to acute ROS challenge in the intact beating heart, arguing against creatine exerting (patho-)physiologically relevant anti-oxidant activity

Aplicación de la minería de datos para determinar propuestas para actividades deportivas en los alumnos

La educación integral contempla la impartición de Actividades Extraescolares en los Institutos Tecnológicos del País. Muchas de estas actividades son deportivas, en donde pueden inscribirse los alumnos. Sin embargo, no se toma en cuenta el historial de salud o alergias que los alumnos puedan tener y que la actividad elegida, puede contribuir a mejorar o empeorar los estados de salud de los alumnos. La presente investigación se llevó a cabo para conocer las en las enfermedades comunes entre los alumnos del Instituto Tecnológico de Roque, y aquellas actividades extraescolares que establecen los expertos para que puedan realizar de acuerdo a su condición e inclusive que les permitan mejorar su estado de salud. Posteriormente, se muestra a aplicación de la minería de datos a la información obtenida, para entregar una propuesta de actividades extraescolares adecuadas para las sintomatologías de los alumnos.Palabra(s) Clave(s): Actividades deportivas, enfermedades, minería de datos

Cardiac responses to β‐adrenoceptor stimulation is partly dependent on mitochondrial calcium uniporter activity

Background and Purpose: Despite the importance of mitochondrial Ca2+ to metabolic regulation and cell physiology, little is known about the mechanisms that regulate Ca2+ entry into the mitochondria. Accordingly, we established a system to determine the role of the mitochondrial Ca2+ uniporter in an isolated heart model, at baseline and during increased workload following β-adrenoceptor stimulation. Experimental Approach: Cardiac contractility, oxygen consumption and intracellular Ca2+ transients were measured in ex vivo perfused murine hearts. Ru360 and spermine were used to modify mitochondrial Ca2+ uniporter activity. Changes in mitochondrial Ca2+ content and energetic phosphate metabolite levels were determined. Key Results: The addition of Ru360, a selective inhibitor of the mitochondrial Ca2+ uniporter, induced progressively and sustained negative inotropic effects that were dose-dependent with an EC50 of 7 μM. Treatment with spermine, a uniporter agonist, showed a positive inotropic effect that was blocked by Ru360. Inotropic stimulation with isoprenaline elevated oxygen consumption (2.7-fold), Ca2+-dependent activation of pyruvate dehydrogenase (5-fold) and mitochondrial Ca2+ content (2.5-fold). However, in Ru360-treated hearts, this parameter was attenuated. In addition, β-adrenoceptor stimulation in the presence of Ru360 did not affect intracellular Ca2+ handling, PKA or Ca2+/calmodulin-dependent PK signalling. Conclusions and Implications: Inhibition of the mitochondrial Ca2+ uniporter decreases β-adrenoceptor response, uncoupling between workload and production of energetic metabolites. Our results support the hypothesis that the coupling of workload and energy supply is partly dependent on mitochondrial Ca2+ uniporter activity.Centro de Investigaciones Cardiovasculare

Detección de apoptosis en enfermedades cardiovasculares mediante las imágenes SPECT de cardiología nuclear

La apoptosis es un proceso biológico de muerte o suicidio celular presente en todas las células de los metazoarios. Mantiene un equilibrio entre la regeneración de las células pluripotenciales o células madre y la eliminación de células que ya han servido su propósito, que se han reproducido en exceso, o en las que existe daño genético irreparable. La activación de la apoptosis en cardiomiocitos es un problema común en una gran variedad de cardiopatías y se ha sugerido que contribuye de manera importante a la dilatación ventricular y al aumento del tamaño del infarto en pacientes con insuficiencia cardíaca y con enfermedad cardiovascular. El diagnóstico clínico de la apoptosis es una realidad en la ciencia médica, cuya aplicación en diferentes facetas de la Cardiología, incluye desde la cardiopatía coronaria hasta los trastornos del ritmo. En este sentido, el uso de la imagenología no-invasiva, puede ser de gran utilidad para la detección in vivo de este tipo de muerte celular, en pacientes con necrosis miocárdica, con isquemia miocárdica aguda, con rechazo agudo del trasplante cardíaco, con miocarditis, con tumores malignos intracardíacos, así como en casos de cardiotoxicidad y de otras cardiomiopatías. Particularmente la unión de la Anexina V, marcada con Tc99m produce imágenes gammagráficas que permiten la identificación de células apoptóticas in vivo, en sistemas con el SPECT y SestaMiBi. En resumen, la utilización de estas técnicas será invaluable en un futuro próximo para la terapia e intervención anti-apoptosis en la rutina de la Cardiología diaria

Genetic Variations on Redox Control in Cardiometabolic Diseases: The Role of Nrf2

The transcription factor Nrf2 is a master regulator of multiple cytoprotective genes that maintain redox homeostasis and exert anti-inflammatory functions. The Nrf2-Keap1 signaling pathway is a paramount target of many cardioprotective strategies, because redox homeostasis is essential in cardiovascular health. Nrf2 gene variations, including single nucleotide polymorphisms (SNPs), are correlated with cardiometabolic diseases and drug responses. SNPs of Nrf2, KEAP1, and other related genes can impair the transcriptional activation or the activity of the resulting protein, exerting differential susceptibility to cardiometabolic disease progression and prevalence. Further understanding of the implications of Nrf2 polymorphisms on basic cellular processes involved in cardiometabolic diseases progression and prevalence will be helpful to establish more accurate protective strategies. This review provides insight into the association between the polymorphisms of Nrf2-related genes with cardiometabolic diseases. We also briefly describe that SNPs of Nrf2-related genes are potential modifiers of the pharmacokinetics that contribute to the inter-individual variability