Electrophysiological characterization of the hERG R56Q LQTS variant and targeted rescue by the activator RPR260243

Human Ether-à-go-go (hERG) channels contribute to cardiac repolarization, and inherited variants or drug block are associated with long QT syndrome type 2 (LQTS2) and arrhythmia. Therefore, hERG activator compounds present a therapeutic opportunity for targeted treatment of LQTS. However, a limiting concern is over-activation of hERG resurgent current during the action potential and abbreviated repolarization. Activators that slow deactivation gating (type I), such as RPR260243, may enhance repolarizing hERG current during the refractory period, thus ameliorating arrhythmogenicity with reduced early repolarization risk. Here, we show that, at physiological temperature, RPR260243 enhances hERG channel repolarizing currents conducted in the refractory period in response to premature depolarizations. This occurs with little effect on the resurgent hERG current during the action potential. The effects of RPR260243 were particularly evident in LQTS2-associated R56Q mutant channels, whereby RPR260243 restored WT-like repolarizing drive in the early refractory period and diastolic interval, combating attenuated protective currents. In silico kinetic modeling of channel gating predicted little effect of the R56Q mutation on hERG current conducted during the action potential and a reduced repolarizing protection against afterdepolarizations in the refractory period and diastolic interval, particularly at higher pacing rates. These simulations predicted partial rescue from the arrhythmic effects of R56Q by RPR260243 without risk of early repolarization. Our findings demonstrate that the pathogenicity of some hERG variants may result from reduced repolarizing protection during the refractory period and diastolic interval with limited effect on action potential duration, and that the hERG channel activator RPR260243 may provide targeted antiarrhythmic potential in these cases.     

Mitochondrial DNA differentiation during the speciation process in Peromyscus

We address the problem of the possible significance of biological speciation to the magnitude and pattern of divergence of asexually transmitted characters in bisexual species. The empirical data for this report consist of restriction endonuclease site variability in maternally transmitted mitochondrial DNA (mtDNA) isolated from 82 samples of Peromyscus polionotus and P. leucopus collected from major portions of the respective species' ranges. Data are analyzed together with previously published information on P. maniculatus, a sibling species to polionotus. Maps of restriction sites indicate that all of the variation observed can be reasonably attributed to base substitutions leading to loss or gain of particular recognition sites. Magnitude of mtDNA sequence divergence within polionotus (maximum approximately equal to 2%) is roughly comparable to that observed within any of five previously identified mtDNA assemblages in maniculatus. Sequence divergence within leucopus (maximum approximately equal to 4%) is somewhat greater than that within polionotus. Consideration of probable evolutionary links among mtDNA restriction site maps allowed estimation of matriarchal phylogenies within polionotus and leucopus. Clustering algorithms and qualitative Wagner procedures were used to generate phenograms and parsimony networks, respectively, for the between-species comparisons. Three simple graphical models are presented to illustrate some conceivable relationships of mtDNA differentiation to speciation. In theoretical case I, each of two reproductively defined species (A and B) is monophyletic in matriarchal genealogy; the common female ancestor of either species can either predate or postdate the speciation. In case II, neither species is monophyletic in matriarchal genotype. In case III, species B is monophyletic but forms a subclade within A which is thus paraphyletic with respect to B. The empirical results for mtDNA in maniculatus and polionotus appear to conform closely to case III. These theoretical and empirical considerations raise a number of questions about the general relationship of the speciation process to the evolution of uniparentally transmitted traits. Some of these considerations are presented, and it is suggested that the distribution patterns of mtDNA sequence variation within and among extant species should be of considerable relevance to the particular demographies of speciation.      

Cloning, expression, and characterization of the trout cardiac Na+/Ca2+ exchanger

Isoform 1 of the cardiacNa⁺/Ca²⁺exchanger (NCX1) is an important regulator of cytosolicCa²⁺ concentration in contractionand relaxation. Studies with trout heart sarcolemmal vesicles haveshown NCX to have a high level of activity at 7°C, and this uniqueproperty is likely due to differences in protein structure. In thisstudy, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAPII cDNA library constructed from rainbow trout(Oncorhynchus mykiss) heart RNA. TheNCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plotindicates the protein contains 12 hydrophobic segments (of which thefirst is predicted to be a cleaved leader peptide) and a largecytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have ninetransmembrane segments. The sequences demonstrated to be the exchangerinhibitory peptide site and the regulatoryCa²⁺ binding site in thecytoplasmic loop of mammalian NCX1 are almost completely conserved inNCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4days currents were measured by the giant excised patch technique.NCX-TR1 currents measured at ~23°C demonstratedNa⁺-dependent inactivation andCa²⁺-dependent activation in amanner qualitatively similar to that for NCX1 currents.

     

Active oxygen in neuromuscular disorders

Although muscle and nerve are reasonably well protected against active oxygen and related free radicals, environmental or inherited malfunctions can overpower their defences. Active oxygen is involved in many neuropathies and myopathies. In every case the damage is caused by agents which exert effects disproportionately greater than the quantites encountered, through a variety of amplification mechanisms. We can categorize these amplification mechanisms as follows: (a) non-replacement of targets (e.g. loss of genetic information, ataxia telangectasia being an hereditary ataxia in which an oxygen mediated chromosomal instability is apparent), (b) non-removal of unwanted materials (e.g. lipofuscin accumulation in brain and heart), (c) redox cycling, usually involving catalysis by trace-metal ions (e.g. some forms of Parkinsonism), (d) non-redox catalysis (e.g. toxicity in cardiac muscle or brain due to vanadium or aluminium respectively), (e) modification of ion transport (e.g. calcium ionophore or acrylamide induce histopathological changes in muscle, similar in some respects to those seen in Duchenne muscular dystrophy), (f) compromised defences (e.g. muscle and nerve become particularly susceptible to free radical damage after loss of the protective actions of vitamin E), and (g) amplification by inflammatory and immune responses (e.g. multiple sclerosis, reperfusion injury to brain and heart, and traumatic injury to nervous tissue). Unfortunately, a variety of therapeutic agents which might be expected to protect against almost every conceivable form of oxygen mediated damage have proved clinically ineffective in most of these disorders. The reasons for this will be explored with an emphasis on common features, differences, mechanisms, and potential therapeutic approaches.     

Activity of a hypochlorous acid-producing electrochemical bandage as assessed with a porcine explant biofilm model

The activity of a hypochlorous acid-producing electrochemical bandage (e-bandage) in preventing methicillin-resistant Staphylococcus aureus infection (MRSA) infection and removing biofilms formed by MRSA was assessed using a porcine explant biofilm model. e-Bandages inhibited S. aureus infection (p = 0.029) after 12 h (h) of exposure and reduced 3-day biofilm viable cell counts after 6, 12, and 24 h exposures (p = 0.029). Needle-type microelectrodes were used to assess HOCl concentrations in explant tissue as a result of e-bandage treatment; toxicity associated with e-bandage treatment was evaluated. HOCl concentrations in infected and uninfected explant tissue varied between 30 and 80 µM, decreasing with increasing distance from the e-bandage. Eukaryotic cell viability was reduced by an average of 71% and 65% in fresh and day 3-old explants, respectively, when compared to explants exposed to nonpolarized e-bandages. HOCl e-bandages are a promising technology that can be further developed as an antibiotic-free treatment for wound biofilm infections.