Calpain activation impairs neuromuscular transmission in a mouse model of the slow-channel myasthenic syndrome.

The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca(2+) overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.

Distribution and status of living colonies of Acropora spp. in the reef crests of a protected marine area of the Caribbean (Jardines de la Reina National Park, Cuba).

The reef crests of the Jardines de la Reina National Park (JRNP) are largely formed by Acropora palmata, but colonies of A. cervicornis and the hybrid A. prolifera are also present. This study shows spatial distribution of colonies, thickets and live fragments of these species in the fore reefs. Snorkeling was used to perform the direct observations. The maximum diameter of 4,399 colonies of A. palmata was measured and the health of 3,546 colonies was evaluated. The same was done to 168 colonies of A. cervicornis and 104 colonies of A. prolifera. The influence of the location and marine currents on a number of living colonies of A. palmata was analyzed. For such purpose, reef crests were divided into segments of 500 m. The marine park was divided into two sectors: East and West. The Caballones Channel was used as the reference dividing line. The park was also divided into five reserve zones. We counted 7,276 live colonies of Acropora spp. 1.4% was A. prolifera, 3.5% A. cervicornis and 95.1% A. palmata. There were 104 thickets of A. palmata, ranging from eight to 12 colonies, and 3,495 fragments; 0.6% was A. cervicornis and the rest A. palmata (99.4%). In the East sector, 263 colonies (3.8% of the total), six thickets (5.8%) and 32 fragments (1%) of A. palmate were recorded. In the same sector, there were 11 fragments (50%) of A.cervicornis and two (2%) colonies of A. prolifera. Health of A. palmata was evaluated as good and not so good in the study area. Health of A. cervicornis was critical and health of A. prolifera was good in all five reserve zones. There was a significant increase in the number of colonies from east to west (Χ2 = 11.5, gl = 3.0, p = 0.009). This corroborates the existence of an important abundance differences between the eastern and the western region of the JRNP. A negative relationship was observed between the number of colonies and the distance from the channel (Χ2 = 65.0, df = 3.0, p < 0.001). The influence of the channel, for the live colonies of A. palmata is greater within the first 2,000 m. It then decreases until approximately 6,000 m, and no significant increase beyond. The orientation of the reef crests significantly influenced the abundance of the colonies (Χ2 = 15.5, df = 2.9, p = 0.001). The results presented here provide a baseline for future research on the status of the populations of Acropora spp., considering that there has been a certain recovery of the species A. palmata during the last 10-16 years. Given the current status of the populations of Acropora spp., conservation actions focusing A. cervicornis should be prioritized.

Exploring Biogeochemistry and Microbial Diversity of Extant Microbialites in Mexico and Cuba.

Microbialites are modern analogs of ancient microbial consortia that date as far back as the Archaean Eon. Microbialites have contributed to the geochemical history of our planet through their diverse metabolic capacities that mediate mineral precipitation. These mineral-forming microbial assemblages accumulate major ions, trace elements and biomass from their ambient aquatic environments; their role in the resulting chemical structure of these lithifications needs clarification. We studied the biogeochemistry and microbial structure of microbialites collected from diverse locations in Mexico and in a previously undescribed microbialite in Cuba. We examined their structure, chemistry and mineralogy at different scales using an array of nested methods including 16S rRNA gene high-throughput sequencing, elemental analysis, X-Ray fluorescence (XRF), X-Ray diffraction (XRD), Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM-EDS), Fourier Transformed Infrared (FTIR) spectroscopy and Synchrotron Radiation-based Fourier Transformed Infrared (SR-FTIR) spectromicroscopy. The resulting data revealed high biological and chemical diversity among microbialites and specific microbe to chemical correlations. Regardless of the sampling site, Proteobacteria had the most significant correlations with biogeochemical parameters such as organic carbon (Corg), nitrogen and Corg:Ca ratio. Biogeochemically relevant bacterial groups (dominant phototrophs and heterotrophs) showed significant correlations with major ion composition, mineral type and transition element content, such as cadmium, cobalt, chromium, copper and nickel. Microbial-chemical relationships were discussed in reference to microbialite formation, microbial metabolic capacities and the role of transition elements as enzyme cofactors. This paper provides an analytical baseline to drive our understanding of the links between microbial diversity with the chemistry of their lithified precipitations.

Inositol-1,4,5-triphosphate receptors mediate activity-induced synaptic Ca2+ signals in muscle fibers and Ca2+ overload in slow-channel syndrome.

Strict control of calcium entry through excitatory synaptic receptors is important for shaping synaptic responses, gene expression, and cell survival. Disruption of this control may lead to pathological accumulation of Ca2+. The slow-channel congenital myasthenic syndrome (SCS), due to mutations in muscle acetylcholine receptor (AChR), perturbs the kinetics of synaptic currents, leading to post-synaptic Ca2+ accumulation. To understand the regulation of calcium signaling at the neuromuscular junction (NMJ) and the etiology of Ca2+ overload in SCS we studied the role of sarcoplasmic Ca2+ stores in SCS. Using fura-2 loaded dissociated fibers activated with acetylcholine puffs, we confirmed that Ca2+ accumulates around wild type NMJ and discovered that Ca2+ accumulates significantly faster around the NMJ of SCS transgenic dissociated muscle fibers. Additionally, we determined that this process is dependant on the activation, altered kinetics, and movement of Ca2+ ions through the AChR, although, surprisingly, depletion of intracellular stores also prevents the accumulation of this cation around the NMJ. Finally, we concluded that the sarcoplasmic reticulum is the main source of Ca2+ and that inositol-1,4,5-triphosphate receptors (IP3R), and to a lesser degree L-type voltage gated Ca2+ channels, are responsible for the efflux of this cation from intracellular stores. These results suggest that a signaling system mediated by the activation of AChR, Ca2+, and IP3R is responsible for localized Ca2+ signals observed in muscle fibers and the Ca2+ overload observed in SCS.

Active calcium accumulation underlies severe weakness in a panel of mice with slow-channel syndrome.

Mutations affecting the gating and channel properties of ionotropic neurotransmitter receptors in some hereditary epilepsies, in familial hyperekplexia, and the slow-channel congenital myasthenic syndrome (SCCMS) may perturb the kinetics of synaptic currents, leading to significant clinical consequences. Although at least 12 acetylcholine receptor (AChR) mutations have been identified in the SCCMS, the altered channel properties critical for disease pathogenesis in the SCCMS have not been identified. To approach this question, we investigated the effect of different AChR subunit mutations on muscle weakness and the function and viability of neuromuscular synapses in transgenic mice. Targeted expression of distinct mutant AChR subunits in skeletal muscle prolonged the decay phases of the miniature endplate currents (MEPCs) over a broad range. In addition, both muscle strength and the amplitude of MEPCs were lower in transgenic lines with greater MEPC duration. SCCMS is associated with calcium overload of the neuromuscular junctional sarcoplasm. We found that the extent of calcium overload of motor endplates in the panel of transgenic mice was influenced by the relative permeability of the mutant AChRs to calcium, on the duration of MEPCs, and on neuromuscular activity. Finally, severe degenerative changes at the motor endplate (endplate myopathy) were apparent by electron microscopy in transgenic lines that displayed the greatest activity-dependent calcium overload. These studies demonstrate the importance of control of the kinetics of AChR channel gating for the function and viability of the neuromuscular junction.

Macroscopic properties of spontaneous mutations in slow-channel syndrome: correlation by domain and disease severity.

The slow-channel syndrome (SCS) is a neuromuscular disorder characterized by fatigability, progressive weakness, and degeneration of the neuromuscular junction. The SCS is caused by missense mutations in the four subunits of the skeletal muscle acetylcholine receptor (AChR), which leads to altered channel gating, prolonged neuromuscular postsynaptic currents, and impaired neuromuscular transmission. Although a diverse set of mutations in different functional domains of the AChR appear to be associated with symptoms of widely ranging severity, there is as yet no mutant channel property or combination that explains the variations in disease severity. By observing the recovery time of AChR from desensitization, the authors determined that this process is significantly enhanced in SCS channels. In addition, as expected, the authors found that SCS macroscopic decay currents in transfected HEK293 cells are slower than wild type currents. While slight differences in relative Ca(2+) permeability between some SCS mutations were identified, they did not correlate with apparent disease severity. These results suggest that of the different AChR kinetic features studied, only recovery from desensitization and slow postsynaptic currents correlate with the severity observed in the different mutations of this syndrome.

Activation of apoptotic pathways at muscle fiber synapses is circumscribed and reversible in a slow-channel syndrome model.

In the slow-channel syndrome (SCS) mutant acetylcholine receptors elicit calcium overload and myonuclear degeneration at the neuromuscular junction (NMJ), without muscle fiber death. Activated caspases are present at SCS motor endplates. We hypothesized that SCS represents a limited form of apoptosis. We found condensed chromatin and occasional single-strand DNA nicks in degenerating synaptic nuclei. Cleaved forms of caspases-3 and -9 were present in mouse SCS muscle homogenates and were specifically localized to NMJs. Finally, interruption of cholinergic activity by axotomy markedly reduced NMJ caspase activity and improved the morphological features of apoptosis at NMJs. These results demonstrate that in SCS processes leading to apoptosis may remain compartmentalized and reversible. Use of cysteine protease inhibitors may aid in treatment of this and other dystrophic muscle and excitotoxic disorders. Identification of extrasynaptic factors that prevent the spread of apoptosis in SCS muscle fibers may aid in developing treatments for neurological disorders characterized by excitotoxicity or apoptosis.

Novel beta subunit mutation causes a slow-channel syndrome by enhancing activation and decreasing the rate of agonist dissociation.

We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V --> F substitution in the M1 domain of the beta subunit (betaV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation betaV229F expressed normal amounts of AChRs and decreased the ACh EC50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the betaV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the betaV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the betaV229F mutation on the miniature endplate currents and thus are direct evidence that the betaV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.

Estudios electrofisiológicos en las taquicardias paroxísticas supraventriculares / Electrophysiologic studies in paroxysmal supraventricular tachycardia

Se destaca la importancia de la electrofisiología clínica para el diagnóstico y tratamiento de las taquicardias paroxísticas supraventriculares. Se señalan los propósitos de este tipo de estudios en los pacientes con taquicardia supraventricular y sus objetivos específicos en el caso de las taquicardias que involucren vías accesorias. Finalmente, se indica que la experiencia resultante en estos estudios ha permitido correlacionar numerosos signos electrocardiográficos con cada tipo de taquicardia, lo cual se resume en el presente trabajo con vistas a permitir una orientación adecuada al cardiólogo clínico que no disponga del estudio electrofisiológico

Marcapasos subpectoral en la prevención de la necrosis del bolsillo / Subpectoral pacemakers for the prevention of necrosis of the pocket

Se estudian 38 pacientes de bloqueo auriculoventricular completo, a quienes se les implantó un marcapasos subpectoral en 10 de los casos, por presentar necrosis del bolsillo en forma repetitiva con la colocación del generador en el tejido celular subcutáneo y en el resto, por tener una constitución física delgada. Se observa que en ninguno de los enfermos apareció necrosis de la piel, ni se detectó otro tipo de complicación posterior a la ubicación subpectoral del generador. Se concluye que la implantación del marcapasos debajo del músculo pectoral mayor es un método eficaz para prevenir las necrosis del bolsillo. Se sugiere realizar la extracción del generador en los casos necesarios por vía axilar

Hipertension arterial sistemica después del cierre quirurgico del conducto arterioso persistente: nuestra experiencia / Systemic hypertension after surgical closure of patent ductus arterious: our experience

Se estudian todos los pacientes operados de PCA aislados en un período consecutivo de 2 años, para un total de 56 niños. Se destaca que el 62,5% presenta algún grado de hipertensión arterial sistémica (35 pacientes). Se expresa que en el 74% de éstos, se encuentran signos auscultatorios de PCA de ciertoa magnitud, y sucede lo mismo en el 80% de los examinados radiográfica y electrocardiográficamente. Se señala que las manifestaciones hipertensivas aparecen el primer día y en la mayoría (57%) desaparecen en las primeras 24 horas de evolución. Se indica que el 55% de ellos recibe tratamiento con furosemida, reserpina o ambas. No se comprobó mortalidad por esta causa. Se concluye que este cuadro es una complicación bastante frecuente en la cirugía de PCA, escasa mortalidad y que su aparición guarda relación con la importancia hemodinámica del PCA por lo que se hace necesario su tratamiento en la mitad de los casos, con hipotensores

Experiencia del uso del desfibrilador cardioverter automático implantable / The automatic implantable cardioverter desfibrillator. cuban experience

Por primera vez en Cuba a 10 pacientes con arritmias ventricualares malignas, muerte súbita y/o taquicardias ventriculares sincopales les ha sido implatntado un desfibrilador automático implantable, con marcapasos incorporado, después de estimulación eléctrica programada seriada. Esto permitió caracterizar sus arritmias y evaluar la respuesta a diferentes fármacos. Los pacintes fueron 9 hombres con edad media de 48 (23-70) añosy FE 32% (18-62): 7 portadores de infarto antiguo y 3 con miocardiopatía dilatada. En el seguimiento (entre 2 y 25 mese), 4 pacientes han recibido choques apropiados y efectivos precedidos de palpitaciones rápidas y pre-sincopales. No se han detectado choques erróneos. Dos pacientes fallecieron, uno por TV incesante y otro por causa no aatribuíble a fallo del equipo. Se concluye que el Guardian 4201 y 4202 ofrece un alto grado de efectividad en la prevención de muerte súbita para pacientes de alto riesgo de tal complicación

Cirugía en el síndrome de Wolff-Parkinson-White, experiencia cubana / Surgery for Wolff-Parkinson-White syndrome, cuban experience

El tratamiento quirúrgico de las arritmias constituye una técnica novedosa en el campo de la cardiocirugía. El síndrome de Wolff-Parkinson-White fue la primera patología abordada con éxito por la cirugía. Fueron oprados 25 pacientes con 28 vías accesorias, empleando las dos técnicas clásicas de abordaje: la epicárdica y la endocárdica. Tres pacientes permanecieron con preexitación pero están asintomáticos, en un caso hubo bloqueo AV completo y unpaciente falleción. En todos los casos, se pudieron realizar ambas técnicas de manera satisfactoria. El análisis de los resultados constituye el objeto de este trabajo.