Alterations in fast-twitch muscle membrane conductance regulation do not explain decreased muscle function of SOD1G93A rats.

INTRODUCTION/AIMS: Both neuromuscular junction (NMJ) dysfunction and altered electrophysiological properties of muscle fibers have been reported in amyotrophic lateral sclerosis (ALS) patients. ALS-related preclinical studies typically use rodent SOD1G93A overexpression models, but translation to the human disease has been challenged. The present work explored NMJ function and cellular electrophysiological properties of muscles fibers in SOD1G93A overexpression rats. METHODS: Longitudinal studies of compound muscle action potentials (CMAPs) were performed in SOD1G93A rats. Cellular studies were performed to evaluate electrophysiological properties of muscle fibers, including the resting membrane conductance (Gm ) and its regulation during prolonged action potential (AP) firing. RESULTS: SOD1G93A rats showed a substantial loss of gastrocnemius CMAP amplitude (35.8 mV, P < .001) and a minor increase in CMAP decrement (8.5%, P = .002) at 25 weeks. In addition, SOD1G93A EDL muscle fibers showed a lower baseline Gm (wild-type, 1325 µS/cm2 ; SOD1G93A , 1137 µS/cm2 ; P < .001) and minor alterations in Gm regulation during repeated firing of APs as compared with wild-type rats. DISCUSSION: The current data suggest that loss of CMAP amplitude is largely explained by defects in either lower motor neuron or skeletal muscle with only minor indications of a role for neuromuscular transmission defects in SOD1G93A rats. Electrophysiological properties of muscle fibers were not markedly affected, and an elevated Gm , as has been reported in motor neuron disease (MND) patients, was not replicated in SOD1G93A muscles. Collectively, the neuromuscular pathology of SOD1G93A rats appears to differ from that of ALS/MND patients with respect to neuromuscular transmission defects and electrophysiological properties of muscle fibers.

Fatiguing stimulation increases curvature of the force-velocity relationship in isolated fast-twitch and slow-twitch rat muscles.

In skeletal muscles, the ability to generate power is reduced during fatigue. In isolated muscles, maximal power can be calculated from the force-velocity relationship. This relationship is well described by the Hill equation, which contains three parameters: (1) maximal isometric force, (2) maximum contraction velocity and (3) curvature. Here, we investigated the hypothesis that a fatigue-induced loss of power is associated with changes in curvature of the force-velocity curve in slow-twitch muscles but not in fast-twitch muscles during the development of fatigue. Isolated rat soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles were incubated in Krebs-Ringer solution at 30°C and stimulated electrically at 60 Hz (soleus) and 150 Hz (EDL) to perform a series of concentric contractions to fatigue. Force-velocity data were fitted to the Hill equation, and curvature was determined as the ratio of the curve parameters a/F0 (inversely related to curvature). At the end of the fatiguing protocol, maximal power decreased by 58±5% in the soleus and 69±4% in the EDL compared with initial values in non-fatigued muscles. At the end of the fatiguing sequence, curvature increased as judged from the decrease in a/F0 by 81±20% in the soleus and by 31±12% in the EDL. However, during the initial phases of fatiguing stimulation, we observed a small decrease in curvature in the EDL, but not in the soleus, which may be a result of post-activation potentiation. In conclusion, fatigue-induced loss of power is strongly associated with an increased curvature of the force-velocity relationship, particularly in slow-twitch muscles.

The anti-convulsants lacosamide, lamotrigine, and rufinamide reduce myotonia in isolated human and rat skeletal muscle.

INTRODUCTION: In myotonia congenita, loss of ClC-1 Cl- channel function results in skeletal muscle hyperexcitability and myotonia. Anti-myotonic treatment has typically targeted the voltage-gated sodium channel in skeletal muscle (Nav1.4). In this study we explored whether 3 sodium channel-modulating anti-epileptics can reduce myotonia in isolated rat and human muscle. METHODS: Dissected muscles were rendered myotonic by ClC-1 channel inhibition. The ability of the drugs to suppress myotonia was then assessed from subclinical to maximal clinical concentrations. Drug synergy was determined using isobole plots. RESULTS: All drugs were capable of abolishing myotonia in both rat and human muscles. Lamotrigine and rufinamide completely suppressed myotonia at submaximal clinical concentrations, whereas lacosamide had to be raised above the maximal clinical concentration to suppress myotonia completely. A synergistic effect of lamotrigine and rufinamide was observed. CONCLUSION: These findings suggest that lamotrigine and rufinamide could be considered for anti-myotonic treatment in myotonia congenita. Muscle Nerve 56: 136-142, 2017.

Olfactory testing in consecutive patients referred with suspected dementia.

BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia and early and accurate diagnosis is important. Olfactory dysfunction is an early sign of AD. The contribution by test of olfactory function has been surveyed in AD vs a line of conditions but remains to be settled in the workup of unselected patients referred with suspected dementia. METHODS: We performed a two-step investigation: first, a comparative study of healthy controls and probable AD patients to test the applicability of the chosen scents (cuisine study); second, a study of consecutive patients referred to our geriatric outpatient clinic for suspected dementia with the investigating personnel blinded to the results of the Olfactory Test (blinded study). RESULTS: The sum of scents detected discriminated patients with probable AD from controls in the cuisine study (n = 40; p < 0.001; area under ROC curve 0.94). In the blinded study (n = 50) the diagnosis was probable AD in 48%, minimal cognitive impairment in 24%, vascular dementia in 8%, alcohol induced impairment in 12%, depression in 4%, and Parkinson's disease and Lewy body dementia in 2%. Area under the ROC-curve was 0.67. The odds ratio for probable AD with 2+ smell errors was 12 (95%-CI: 1.3-101; p = 0.026 (reference 0-1 smell errors)) age adjusted. None in the AD group had zero smell errors (Negative Predictive Value 100%). CONCLUSION: Olfactory testing may support to dismiss the diagnosis of probable AD in the workup of a mixed group of patients referred with cognitive impairment. Still, it had a low sensitivity for probable AD.

Intrarater reproducibility and validity of Nintendo Wii balance testing in community-dwelling older adults.

The aims of the current study were to examine the intrarater intersession reproducibility of the Nintendo Wii agility and stillness tests and explore the concurrent validity in relation to gold-standard force-plate analysis. Within-day intersession reproducibility was examined in 30 older adults (age 71.8 ± 5.1 yr). No systematic test-retest differences were found for the Wii stillness test; however, the Wii agility test scores differed systematically between test sessions (p < .05). The Wii stillness test yielded a test-retest ICC of .86 (95% CI 0.74-0.93), CV of 6.4%, LOA of 11.0, and LOA% of 17.9%. Likewise for the Wii agility test ICC was .73 (95% CI 0.50-0.86), CV 5.3%, LOA 1.8, and LOA% of 14.6%. Wii stillness scores correlated to force plate measures (r = .65-.82, p < .01), reflecting moderate to excellent validity. In conclusion, it appears that the Wii stillness test represents a low-cost, objective, reproducible, and valid test of undisturbed postural balance in community-dwelling older adults.

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.

Force potentiation during eccentric contractions in rat skeletal muscle.

Postactivation potentiation refers to an acute enhancement of contractile properties following muscle activity. Previously, the effects of prior muscle activation on eccentric force at tetanic activation frequencies have only been sparsely reported. This paper aimed to study acute activity-induced effects on eccentric force of slow and fast-twitch muscles and characterize them in relation to postactivation potentiation. We elicited eccentric contractions in isolated rat extensor digitorum longus and soleus muscles by actively lengthening muscles at a constant velocity. We assessed contractile properties by measuring force over shortly interspaced, identical eccentric, and isometric contractions. We then analyzed stretch force, isometric peak force, rate of force development, and relaxation times. Finally, we compared the time courses for the development and cessation of changes in stretch force to known features of postactivation potentiation. In extensor digitorum longus, muscles stretch force consistently increased in a contraction-to-contraction manner by up to 49% [95% confidence interval (CI): 35-64%] whereas isometric peak force simultaneously showed minor declines (8%, 95% CI: 5-10%). The development and cessation of eccentric force potentiation coincided with the development of twitch potentiation and increases in rate of force development. In soleus muscles we found no consistent eccentric potentiation. Characterization of the increase in eccentric force revealed that force only increased in the very beginning of an active stretch. Eccentric force at tetanic activation frequencies potentiates substantially in extensor digitorum longus muscles over consecutive contractions with a time course coinciding with postactivation potentiation. Such eccentric potentiation may be important in sport performance.NEW & NOTEWORTHY Force during eccentric contractions can increase to a magnitude that may have profound consequences for our understanding of skeletal muscle locomotion. This increase in eccentric force occurs over consecutive, shortly interspaced, tetanic contractions in rat extensor digitorum longus muscles-not in rat soleus muscles-and coincides with well-known traits of postactivation potentiation. Eccentric force potentiation may significantly enhance muscle performance in activities involving stretch-shortening cycles.

Increased excitability of acidified skeletal muscle: role of chloride conductance.

Generation of the action potentials (AP) necessary to activate skeletal muscle fibers requires that inward membrane currents exceed outward currents and thereby depolarize the fibers to the voltage threshold for AP generation. Excitability therefore depends on both excitatory Na+ currents and inhibitory K+ and Cl- currents. During intensive exercise, active muscle loses K+ and extracellular K+ ([K+]o) increases. Since high [K+]o leads to depolarization and ensuing inactivation of voltage-gated Na+ channels and loss of excitability in isolated muscles, exercise-induced loss of K+ is likely to reduce muscle excitability and thereby contribute to muscle fatigue in vivo. Intensive exercise, however, also leads to muscle acidification, which recently was shown to recover excitability in isolated K(+)-depressed muscles of the rat. Here we show that in rat soleus muscles at 11 mM K+, the almost complete recovery of compound action potentials and force with muscle acidification (CO2 changed from 5 to 24%) was associated with reduced chloride conductance (1731 +/- 151 to 938 +/- 64 microS/cm2, P < 0.01) but not with changes in potassium conductance (405 +/- 20 to 455 +/- 30 microS/cm2, P < 0.16). Furthermore, acidification reduced the rheobase current by 26% at 4 mM K+ and increased the number of excitable fibers at elevated [K+]o. At 11 mM K+ and normal pH, a recovery of excitability and force similar to the observations with muscle acidification could be induced by reducing extracellular Cl- or by blocking the major muscle Cl- channel, ClC-1, with 30 microM 9-AC. It is concluded that recovery of excitability in K(+)-depressed muscles induced by muscle acidification is related to reduction in the inhibitory Cl- currents, possibly through inhibition of ClC-1 channels, and acidosis thereby reduces the Na+ current needed to generate and propagate an AP. Thus short term regulation of Cl- channels is important for maintenance of excitability in working muscle.

Effects of high-frequency stimulation and doublets on dynamic contractions in rat soleus muscle exposed to normal and high extracellular [K(+)].

The development of maximal velocity and power in muscle depends on the ability to transmit action potentials (AP) at very high frequencies up to about 400 Hz. However, for every AP there is a small loss of K(+) to the interstitium, which during intense exercise, may build up to a point where excitability is reduced, thus limiting the intensity of further exercise. It is still unknown how the muscle responds to high-frequency stimulation when exposed to high [K(+)]. Contractile parameters of the muscles (force [F], velocity [V], power [P], rate of force development [RFD], and work) were examined during dynamic contractions, performed in vitro using rat soleus muscles incubated in buffers containing 4 or 8 mmol/L K(+) and stimulated with constant trains of tetanic or supratetanic frequency or with trains initiated by a high-frequency doublet, followed by tetanic or subtetanic trains. At 4 mmol/L K(+), an increase in frequency increased P max when using constant train stimulation. When stimulating with trains containing high-frequency doublets an increase in 120-msec work was seen, however, no increase in P max was observed. At 8 mmol/L K(+), no differences were seen for either P max or 120-msec work when increasing frequency or introducing doublets. In all experiments where the frequency was increased or doublets applied, an increase in RFD was seen in both normal and high [K(+)]. The results indicate that stimulation with supratetanic frequencies can improve dynamic muscle contractility, but improvements are attenuated when muscles are exposed to high extracellular [K(+)].

Extracellular magnesium and calcium reduce myotonia in ClC-1 inhibited rat muscle.

Loss-of-function mutations in the ClC-1 Cl(-) channel trigger skeletal muscle hyperexcitability in myotonia congenita. For reasons that remain unclear, the severity of the myotonic symptoms can vary markedly even among patients with identical ClC-1 mutations, and may become exacerbated during pregnancy and with diuretic treatment. Since both these conditions are associated with hypomagnesemia and hypocalcemia, we explored whether extracellular Mg(2+) and Ca(2+) ([Mg(2+)]o and [Ca(2+)]o) can affect myotonia. Experimental myotonia was induced in isolated rat muscles by ClC-1 inhibition and effects of [Mg(2+)]o or [Ca(2+)]o on myotonic contractions were determined. Both cations dampened myotonia within their physiological concentration ranges. Thus, myotonic contractile activity was 6-fold larger at 0.3 than at 1.2 mM [Mg(2+)]o and 82-fold larger at 0.3 than at 1.27 mM [Ca(2+)]o. In intracellular recordings of action potentials, the threshold for action potential excitation was raised by 4-6 mV when [Mg(2+)]o was elevated from 0.6 to 3 mM, compatible with an increase in the depolarization of the membrane potential necessary to activate the Na(+) channels. Supporting this notion, mathematical simulations showed that myotonia went from appearing with normal Cl(-) channel function to disappearing in the absence of Cl(-) channel function when Na(+) channel activation was depolarized by 6 mV. In conclusion, variation in serum Mg(2+) and Ca(2+) may contribute to phenotypic variation in myotonia congenita patients.

Intracellular acidosis enhances the excitability of working muscle.

Intracellular acidification of skeletal muscles is commonly thought to contribute to muscle fatigue. However, intracellular acidosis also acts to preserve muscle excitability when muscles become depolarized, which occurs with working muscles. Here, we show that this process may be mediated by decreased chloride permeability, which enables action potentials to still be propagated along the internal network of tubules in a muscle fiber (the T system) despite muscle depolarization. These results implicate chloride ion channels in muscle function and emphasize that intracellular acidosis of muscle has protective effects during muscle fatigue.