Excitability properties of mouse and human skeletal muscle fibres compared by muscle velocity recovery cycles.

Mouse models of skeletal muscle channelopathies are not phenocopies of human disease. In some cases (e.g. Myotonia Congenita) the phenotype is much more severe, whilst in others (e.g. Hypokalaemic periodic paralysis) rodent physiology is protective. This suggests a species' difference in muscle excitability properties. In humans these can be measured indirectly by the post-impulse changes in conduction velocity, using Muscle Velocity Recovery Cycles (MVRCs). We performed MVRCs in mice and compared their muscle excitability properties with humans. Mouse Tibialis Anterior MVRCs (n = 70) have only one phase of supernormality (increased conduction velocity), which is smaller in magnitude (p = 9 × 10-21), and shorter in duration (p = 3 × 10-24) than human (n = 26). This abbreviated supernormality is followed by a period of late subnormality (reduced velocity) in mice, which overlaps in time with the late supernormality seen in human MVRCs. The period of late subnormality suggests increased t-tubule Na+/K+-pump activity. The subnormal phase in mice was converted to supernormality by blocking ClC-1 chloride channels, suggesting relatively higher chloride conductance in skeletal muscle. Our findings help explain discrepancies in phenotype between mice and humans with skeletal muscle channelopathies and potentially other neuromuscular disorders. MVRCs are a valuable new tool to compare in vivo muscle membrane properties between species and will allow further dissection of the molecular mechanisms regulating muscle excitability.

Associations between weight loss history and factors related to type 2 diabetes risk in the Stop Diabetes study.

BACKGROUND: Frequent weight loss attempts are related to maladaptive eating behaviours and higher body mass index (BMI). We studied associations of several type 2 diabetes (T2D) risk factors with weight loss history, defined as the frequency of prior weight loss attempts, among Finnish adults at increased risk for T2D. METHODS: This study (n = 2684, 80% women) is a secondary analysis of the 1-year StopDia lifestyle intervention with digital intervention group, digital intervention + face-to-face counselling group, or control group. The frequency of prior weight loss attempts was categorized into five groups: no attempts/no attempts to lose weight, but trying to keep weight stable/1-2 attempts/3 or more attempts/ continuous attempts. Data on emotional eating and social/emotional nutrition self-efficacy were collected with a digital questionnaire. We assessed baseline differences between categories of weight loss history as well as the intervention effects. RESULTS: Altogether 84% of participants had attempted weight loss. Those with one or more weight loss attempts had higher BMI, larger waist circumference, and more emotional eating compared to 'no attempts' and 'no attempts to lose weight, but trying to keep weight stable' categories. The 'no attempts' category had the highest baseline fasting insulin, whereas it showed the largest decrease in this measure with the intervention. This change in fasting insulin in the 'no attempts' category was significantly different from all the other categories. Emotional nutrition self-efficacy slightly improved in the 'no attempts' category, which was significantly different from its concomitant decrease in the categories '1-2 attempts' and '3 or more attempts'. The intervention group assignment did not affect the results. CONCLUSIONS: Multiple attempts to lose weight may unfavourably affect T2D risk factors as well as lifestyle intervention outcomes. More research is needed on how weight loss frequency could affect T2D risk factors and how to design lifestyle interventions for individuals with frequent previous weight loss attempts.

Clinical and genetic spectrum of a Chinese cohort with SCN4A gene mutations.

Skeletal muscle sodium channelopathies due to SCN4A gene mutations have a broad clinical spectrum. However, each phenotype has been reported in few cases of Chinese origin. We present detailed phenotype and genotype data from a cohort of 40 cases with SCN4A gene mutations seen in neuromuscular diagnostic service in Huashan hospital, Fudan University. Cases were referred from 6 independent provinces from 2010 to 2018. A questionnaire covering demographics, precipitating factors, episodes of paralysis and myotonia was designed to collect the clinical information. Electrodiagnostic studies and muscle MRI were retrospectively analyzed. The clinical spectrum of patients included: 6 Hyperkalemic periodic paralysis (15%), 18 Hypokalemic periodic paralysis (45%), 7 sodium channel myotonia (17.5%), 4 paramyotonia congenita (10%) and 5 heterozygous asymptomatic mutation carriers (12.5%). Review of clinical information highlights a significant delay to diagnosis (median 15 years), reports of pain and myalgia in the majority of patients, male predominance, circadian rhythm and common precipitating factors. Electrodiagnostic studies revealed subclinical myotonic discharges and a positive long exercise test in asymptomatic carriers. Muscle MRI identified edema and fatty infiltration in gastrocnemius and soleus. A total of 13 reported and 2 novel SCN4A mutations were identified with most variants distributed in the transmembrane helix S4 to S6, with a hotspot mutation p.Arg675Gln accounting for 32.5% (13/40) of the cohort. Our study revealed a higher proportion of periodic paralysis in SCN4A-mutated patients compared with cohorts from England and the Netherlands. It also highlights the importance of electrodiagnostic studies in diagnosis and segregation studies.

Pharmacological and electrophysiological characterization of AZSMO-23, an activator of the hERG K(+) channel.

BACKGROUND AND PURPOSE: We aimed to characterize the pharmacology and electrophysiology of N-[3-(1H-benzimidazol-2-yl)-4-chloro-phenyl]pyridine-3-carboxamide (AZSMO-23), an activator of the human ether-a-go-go-related gene (hERG)-encoded K(+) channel (Kv 11.1). EXPERIMENTAL APPROACH: Automated electrophysiology was used to study the pharmacology of AZSMO-23 on wild-type (WT), Y652A, F656T or G628C/S631C hERG, and on other cardiac ion channels. Its mechanism of action was characterized with conventional electrophysiology. KEY RESULTS: AZSMO-23 activated WT hERG pre-pulse and tail current with EC50 values of 28.6 and 11.2 µM respectively. At 100 µM, pre-pulse current at +40 mV was increased by 952 ± 41% and tail current at -30 mV by 238 ± 13% compared with vehicle values. The primary mechanism for this effect was a 74.5 mV depolarizing shift in the voltage dependence of inactivation, without any shift in the voltage dependence of activation. Structure-activity relationships for this effect were remarkably subtle, with close analogues of AZSMO-23 acting as hERG inhibitors. AZSMO-23 blocked the mutant channel, hERG Y652A, but against another mutant channel, hERG F656T, its activator activity was enhanced. It inhibited activity of the G628C/S631C non-inactivating hERG mutant channel. AZSMO-23 was not hERG selective, as it blocked hKv 4.3-hKChIP2.2, hCav 3.2 and hKv 1.5 and activated hCav 1.2/ß2/α2δ channels. CONCLUSION AND IMPLICATIONS: The activity of AZSMO-23 and those of its close analogues suggest these compounds may be of value to elucidate the mechanism of type 2 hERG activators to better understand the pharmacology of this area from both a safety perspective and in relation to treatment of congenital long QT syndrome.

Dietary associations with prediabetic states--the DR's EXTRA Study (ISRCTN45977199).

BACKGROUND/OBJECTIVES: Impaired fasting plasma glucose (IFG) and impaired glucose tolerance (IGT) predict development of type 2 diabetes (T2D), but display different pathophysiology for T2D. We studied the association of selected food items and nutrients with IFG, IGT and combined IFG and IGT (IFG+IGT), independent of cardiorespiratory fitness (VO(2max)). SUBJECTS/METHODS: In a population-based sample of 1261 individuals, aged 58-78 years, we identified 126 subjects with IFG, 97 with IGT and 49 with simultaneous IFG and IGT by an oral glucose tolerance test. Dietary intake was assessed by 4-day food records. Cardiorespiratory fitness was assessed by defining maximal oxygen uptake (VO(2max)) from respiratory gas analysis during a maximal symptom-limited exercise stress test on a bicycle ergometer. RESULTS: Increased intake of saturated fat was associated with higher odds for IFG (OR 1.07; 1.01-1.14) after adjustment for age, gender, VO(2max) and energy misreporting variable. Consumption of additional whole-grain bread (50 g/1000 kcal) and intake of dietary fiber (g/1000 kcal) were inversely associated with IGT (OR 0.61; 0.41-0.92, OR 0.91; CI 0.85-0.97, respectively). CONCLUSION: Dietary fiber and sources of cereal fiber are negatively associated with IGT, and saturated fat intake is positively associated with IFG, but not with IGT. The present data give practical dietary means at the population level for the elimination of prediabetic conditions.

Mice expressing a human K(ATP) channel mutation have altered channel ATP sensitivity but no cardiac abnormalities.

AIMS/HYPOTHESIS: Patients with severe gain-of-function mutations in the Kir6.2 subunit of the ATP-sensitive potassium (K(ATP)) channel, have neonatal diabetes, muscle hypotonia and mental and motor developmental delay-a condition known as iDEND syndrome. However, despite the fact that Kir6.2 forms the pore of the cardiac K(ATP) channel, patients show no obvious cardiac symptoms. The aim of this project was to use a mouse model of iDEND syndrome to determine whether iDEND mutations affect cardiac function and cardiac K(ATP) channel ATP sensitivity. METHODS: We performed patch-clamp and in vivo cine-MRI studies on mice in which the most common iDEND mutation (Kir6.2-V59M) was targeted to cardiac muscle using Cre-lox technology (m-V59M mice). RESULTS: Patch-clamp studies of isolated cardiac myocytes revealed a markedly reduced K(ATP) channel sensitivity to MgATP inhibition in m-V59M mice (IC(50) 62 µmol/l compared with 13 µmol/l for littermate controls). In vivo cine-MRI revealed there were no gross morphological differences and no differences in heart rate, end diastolic volume, end systolic volume, stroke volume, ejection fraction, cardiac output or wall thickening between m-V59M and control hearts, either under resting conditions or under dobutamine stress. CONCLUSIONS/INTERPRETATION: The common iDEND mutation Kir6.2-V59M decreases ATP block of cardiac K(ATP) channels but was without obvious effect on heart function, suggesting that metabolic changes fail to open the mutated channel to an extent that affects function (at least in the absence of ischaemia). This may have implications for the choice of sulfonylurea used to treat neonatal diabetes.

S4 charges move close to residues in the pore domain during activation in a K channel.

Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET(+)). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 A) to a position close (8 A) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling.