The ClC-1 chloride channel inhibitor NMD670 improves skeletal muscle function in rat models and patients with myasthenia gravis.

Myasthenia gravis (MG) is a neuromuscular disease that results in compromised transmission of electrical signals at the neuromuscular junction (NMJ) from motor neurons to skeletal muscle fibers. As a result, patients with MG have reduced skeletal muscle function and present with symptoms of severe muscle weakness and fatigue. ClC-1 is a skeletal muscle specific chloride (Cl-) ion channel that plays important roles in regulating neuromuscular transmission and muscle fiber excitability during intense exercise. Here, we show that partial inhibition of ClC-1 with an orally bioavailable small molecule (NMD670) can restore muscle function in rat models of MG and in patients with MG. In severely affected MG rats, ClC-1 inhibition enhanced neuromuscular transmission, restored muscle function, and improved mobility after both single and prolonged administrations of NMD670. On this basis, NMD670 was progressed through nonclinical safety pharmacology and toxicology studies, leading to approval for testing in clinical studies. After successfully completing phase 1 single ascending dose in healthy volunteers, NMD670 was tested in patients with MG in a randomized, placebo-controlled, single-dose, three-way crossover clinical trial. The clinical trial evaluated safety, pharmacokinetics, and pharmacodynamics of NMD670 in 12 patients with mild MG. NMD670 had a favorable safety profile and led to clinically relevant improvements in the quantitative myasthenia gravis (QMG) total score. This translational study spanning from single muscle fiber recordings to patients provides proof of mechanism for ClC-1 inhibition as a potential therapeutic approach in MG and supports further development of NMD670.

Feeding alters blood flow patterns in the American alligator (Alligator mississippiensis).

The crocodilian cardiovascular design with a four-chambered heart and a left aorta that emerge from the right ventricle allows blood to be shunted away from the lungs, a right-to-left (R-L) shunt. The adaptive significance of this R-L shunt remains both poorly understood and controversial with particular debate on its putative role during digestion. Here we measure blood flow patterns in the right aorta (RAo), left aorta (LAo) and the coeliac artery (CoA) of undisturbed American alligators (Alligator mississippiensis) during fasting and throughout most of the digestive period. Digestion doubled blood flow in the RAo (10.1±0.9 to 20.7±1.5mlmin-1kg-1), whereas LAo increased approximately 3-fold (3.8±0.6 to 12.2±2.1mlmin-1kg-1). Blood flow in the CoA increased more than four-fold during digestion (3.0±0.6 to 13.3±1.6mlmin-1kg-1). The rise in blood flows was achieved by a doubling of heart rate (18.5±3.3 to 37.8±3.6mlmin-1kg-1). Maximal flows measured in all arteries and heart rate occurred in the first hour of the postprandial period and continued for the next 7h.

Right-to-left shunt has modest effects on CO2 delivery to the gut during digestion, but compromises oxygen delivery.

By virtue of their cardiovascular anatomy, reptiles and amphibians can shunt blood away from the pulmonary or systemic circuits, but the functional role of this characteristic trait remains unclear. It has been suggested that right-to-left (R-L) shunt (recirculation of systemic blood within the body) fuels the gastric mucosa with acidified and CO2-rich blood to facilitate gastric acid secretion during digestion. However, in addition to elevating PCO2 , R-L shunt also reduces arterial O2 levels and would compromise O2 delivery during the increased metabolic state of digestion. Conversely, arterial PCO2  can also be elevated by lowering ventilation relative to metabolism (i.e. reducing the air convection requirement, ACR). Based on a mathematical analysis of the relative roles of ACR and R-L shunt on O2 and CO2 levels, we predict that ventilatory modifications are much more effective for gastric CO2 supply with only modest effects on O2 delivery. Conversely, elevating CO2 levels by means of R-L shunt would come at a cost of significant reductions in O2 levels. The different effects of altering ACR and R-L shunt on O2 and CO2 levels are explained by the differences in the effective blood capacitance coefficients.

Reduced L-type Ca2+ current and compromised excitability induce loss of skeletal muscle function during acute cooling in locust.

Low temperature causes most insects to enter a state of neuromuscular paralysis, termed chill coma. The susceptibility of insect species to chill coma is tightly correlated to their distribution limits and for this reason it is important to understand the cellular processes that underlie chill coma. It is known that muscle function is markedly depressed at low temperature and this suggests that chill coma is partly caused by impairment in the muscle per se. To find the cellular mechanism(s) underlying muscle dysfunction at low temperature, we examined the effect of low temperature (5°C) on several events in excitation-contraction coupling in the migratory locust (Locusta migratoria). Intracellular membrane potential recordings during single nerve stimulations showed that 70% of fibers at 20°C produced an action potential (AP), while only 55% of fibers were able to fire an AP at 5°C. Reduced excitability at low temperature was caused by an ∼80% drop in L-type Ca(2+) current and a depolarizing shift in its activation of around 20 mV, which means that a larger endplate potential would be needed to activate the muscle AP at low temperature. In accordance, we showed that intracellular Ca(2+) transients were largely absent at low temperature following nerve stimulation. In contrast, maximum contractile force was unaffected by low temperature in chemically skinned muscle bundles, which demonstrates that the function of the contractile filaments is preserved at low temperature. These findings demonstrate that reduced L-type Ca(2+) current is likely to be the most important factor contributing to loss of muscle function at low temperature in locust.

Cold-induced depolarization of insect muscle: differing roles of extracellular K+ during acute and chronic chilling.

Insects enter chill coma, a reversible state of paralysis, at temperatures below their critical thermal minimum (CTmin), and the time required for an insect to recover after a cold exposure is termed chill coma recovery time (CCRT). The CTmin and CCRT are both important metrics of insect cold tolerance that are used interchangeably, although chill coma recovery is not necessarily permitted by a direct reversal of the mechanism causing chill coma onset. Nevertheless, onset and recovery of coma have been attributed to loss of neuromuscular function due to depolarization of muscle fibre membrane potential (Vm). Here we test the hypothesis that muscle depolarization at chill coma onset and repolarization during chill coma recovery are caused by changes in extracellular [K(+)] and/or other effects of low temperature. Using Locusta migratoria, we measured in vivo muscle resting potentials of the extensor tibialis during cooling, following prolonged exposure to -2°C and during chill coma recovery, and related changes in Vm to transmembrane [K(+)] balance and temperature. Although Vm was rapidly depolarized by cooling, hemolymph [K(+)] did not rise until locusts had spent considerable time in the cold. Nonetheless, a rise in hemolymph [K(+)] during prolonged cold exposure further depressed muscle resting potential and slowed recovery from chill coma upon rewarming. Muscle resting potentials had a bimodal distribution, and with elevation of extracellular [K(+)] (but not temperature) muscle resting potentials become unimodal. Thus, a disruption of extracellular [K(+)] does depolarize muscle resting potential and slow CCRT following prolonged cold exposure. However, onset of chill coma at the CTmin relates to an as-yet-unknown effect of temperature on neuromuscular function.

Why do insects enter and recover from chill coma? Low temperature and high extracellular potassium compromise muscle function in Locusta migratoria.

When exposed to low temperatures, many insect species enter a reversible comatose state (chill coma), which is driven by a failure of neuromuscular function. Chill coma and chill coma recovery have been associated with a loss and recovery of ion homeostasis (particularly extracellular [K(+)], [K(+)]o) and accordingly onset of chill coma has been hypothesized to result from depolarization of membrane potential caused by loss of ion homeostasis. Here, we examined whether onset of chill coma is associated with a disturbance in ion balance by examining the correlation between disruption of ion homeostasis and onset of chill coma in locusts exposed to cold at varying rates of cooling. Chill coma onset temperature changed maximally 1°C under different cooling rates and marked disturbances of ion homeostasis were not observed at any of the cooling rates. In a second set of experiments, we used isolated tibial muscle to determine how temperature and [K(+)]o, independently and together, affect tetanic force production. Tetanic force decreased by 80% when temperature was reduced from 23°C to 0.5°C, while an increase in [K(+)]o from 10 mmol l(-1) to 30 mmol l(-1) at 23°C caused a 40% reduction in force. Combining these two stressors almost abolished force production. Thus, low temperature alone may be responsible for chill coma entry, rather than a disruption of extracellular K(+) homeostasis. As [K(+)] also has a large effect on tetanic force production, it is hypothesized that recovery of [K(+)]o following chill coma could be important for the time to recovery of normal neuromuscular function.

Feeding impairs chill coma recovery in the migratory locust (Locusta migratoria).

Low temperature causes loss of neuromuscular function in a wide range of insects, such that the animals enter a state known as chill coma. The ability to recover from chill coma (chill coma recovery time) is often a popular phenotype to characterise chill tolerance in insects. Chill coma in insects has been shown to be associated with a decrease in haemolymph volume and a marked increase in [K(+)], causing dissipation of K(+) equilibrium potential and resting membrane potential. High potassium diet (wheat) has also previously been shown to increase haemolymph [K(+)] in Locusta migratoria leading to sluggish behaviour. The present study combined these two independent stressors of ion and water homeostasis, in order to investigate the role of K(+)- and water-balance during recovery from chill coma, in the chill sensitive insect L. migratoria. We confirmed that cold shock elicits a fast increase in haemolymph [K(+)] which is likely caused by a water shift from the haemolymph to the muscles and other tissues. Recovery of haemolymph [K(+)] is however not only reliant on recovery of haemolymph volume, as the recovery of water and K(+) is decoupled. Chill coma recovery time, after 2h at -4 °C, differed significantly between fasted animals and those fed on high K(+) diet. This difference was not associated with an increased disturbance of haemolymph [K(+)] in the fed animals, instead it was associated with a slowed recovery of muscle [K(+)], muslce water, haemolymph [Na(+)] and K(+)equilibrium potential in the fed animals.

Rapid cold hardening improves recovery of ion homeostasis and chill coma recovery time in the migratory locust, Locusta migratoria.

Chill tolerance of insects is defined as the ability to tolerate low temperature under circumstances not involving freezing of intracellular or extracellular fluids. For many insects chill tolerance is crucial for their ability to persist in cold environments and mounting evidence indicates that chill tolerance is associated with the ability to maintain ion and water homeostasis, thereby ensuring muscular function and preventing chill injury at low temperature. The present study describes the relationship between muscle and haemolymph ion homeostasis and time to regain posture following cold shock (CS, 2 h at -4°C) in the chill-susceptible locust Locusta migratoria. This relationship was examined in animals with and without a prior rapid cold-hardening treatment (RCH, 2 h at 0°C) to investigate the physiological underpinnings of RCH. CS elicited a doubling of haemolymph [K(+)] and this disturbance was greater in locusts pre-exposed to RCH. Recovery of ion homeostasis was, however, markedly faster in RCH-treated animals, which correlated well with whole-organism performance as hardened individuals regained posture faster than non-hardened individuals following CS. The present study indicates that loss and recovery of muscular function are associated with the resting membrane potential of excitable membranes as attested by the changes in the equilibrium potential for K(+) (EK) following CS. Both hardened and non-hardened animals regained movement once K(+) homeostasis had recovered to a fixed level (EK≈-41 mV). RCH is therefore not associated with altered sensitivity to ion disturbance but instead is correlated to a faster recovery of haemolymph [K(+)].

Aerobic scope and cardiovascular oxygen transport is not compromised at high temperatures in the toad Rhinella marina.

Numerous recent studies convincingly correlate the upper thermal tolerance limit of aquatic ectothermic animals to reduced aerobic scope, and ascribe the decline in aerobic scope to failure of the cardiovascular system at high temperatures. In the present study we investigate whether this 'aerobic scope model' applies to an air-breathing and semi-terrestrial vertebrate Rhinella marina (formerly Bufo marinus). To quantify aerobic scope, we measured resting and maximal rate of oxygen consumption at temperatures ranging from 10 to 40°C. To include potential effects of acclimation, three groups of toads were acclimated chronically at 20, 25 and 30°C, respectively. The absolute difference between resting and maximal rate of oxygen consumption increased progressively with temperature and there was no significant decrease in aerobic scope, even at temperature immediately below the lethal limit (41-42°C). Haematological and cardiorespiratory variables were measured at rest and immediately after maximal activity at benign (30°C) and critically high (40°C) temperatures. Within this temperature interval, both resting and active heart rate increased, and there was no indication of respiratory failure, judged from high arterial oxygen saturation, P(O2) and [Hb(O2)]. With the exception of elevated resting metabolic rate for cold-acclimated toads, we found few differences in the thermal responses between acclimation groups with regard to the cardiometabolic parameters. In conclusion, we found no evidence for temperature-induced cardiorespiratory failure in R. marina, indicating that maintenance of aerobic scope and oxygen transport is unrelated to the upper thermal limit of this air-breathing semi-terrestrial vertebrate.

Minimizing collision risk between migrating raptors and marine wind farms: development of a spatial planning tool.

An increased focus on renewable energy has led to the planning and construction of marine wind farms in Europe. Since several terrestrial studies indicate that raptors are especially susceptible to wind turbine related mortality, a Spatial Planning Tool is needed so that wind farms can be sited, in an optimal way, to minimize risk of collisions. Here we use measurements of body mass, wingspan and wing area of eight European raptor species, to calculate their Best Glide Ratio (BGR). The BGR was used to construct a linear equation, which, by the use of initial take-off altitude, could be used to calculate a Theoretical Maximum Distance (TMD) from the coast, attained by these soaring-gliding raptor species. If the nearest turbine, of future marine wind farms, is placed farther away from the coast than the estimated TMD, the collision risk between the turbine blades and these gliding raptors will be minimized. The tool was demonstrated in a case study at the Rødsand II wind farm in Denmark. Data on raptor migration altitude were gathered by radar. From the TMD attained by registered soaring-gliding raptors in the area, we concluded that the Rødsand II wind farm is not sited ideally, from an ornithological point of view, as potentially all three registered species are at risk of gliding through the area swept by the turbine rotor blades, and thereby at risk of colliding with the wind turbines.