Muscle Sodium Research Articles

Abstract MP34: Muscle Sodium Content and Isolevuglandin Formation in Antigen Presenting Cells in Human Salt Sensitive Hypertension

Background: Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular mortality. However, the mechanisms by which salt exposure affects blood pressure in SSBP are still unclear. We have shown that under high sodium (Na + ) concentrations, Na + enters antigen-presenting cells (APCs) via epithelial Na + channel (ENaC), which leads to isolevuglandin (IsoLG) production and hypertension. Baseline %IsoLG+ APC correlates with SSBP. While the exact location where APCs are activated by salt and trigger an inflammatory response remains to be elucidated, muscle and skin are proposed as potential sites. We hypothesize that muscle and skin Na + levels are associated with SSBP. Methods: We assessed SSBP in 30 hypertensive subjects using an inpatient salt loading and depletion protocol. Flow cytometry was used to identify IsoLG-containing APCs; dendritic cells (DCs), classic (CD14+16-), non-classic (CD14-16+), and intermediate (CD14+16+) monocytes. Salt loading was achieved through saline infusion and a high-salt diet (460mmol/24 hours), while salt depletion was achieved via diuretic administration (furosemide 40mg x3) and a low-salt diet (10mmol/24hours). Baseline muscle and skin Na + levels were assessed with the 23 Na MRI technique. Spearman's correlation was used to analyze the associations. Demographic data are presented with mean and standard error of the mean. Results: Participants’ age was 53 ± 1.34 years, 53% female, baseline blood pressure was 140.2 ± 2.62 / 87.30 ± 1.77 mmHg, and salt sensitivity index was 3.72 ± 1.4 mmHg. We found an inverse correlation between baseline muscle Na + levels and the decrease in IsoLGs from salt loading to depletion (ΔIsoLGs) in DCs (r=-0.50, p=0.007), intermediate (r=-0.42 p=0.0284) and non-classic monocytes (r=-0.39 p=0.0432), but not in classical monocytes. Skin Na + levels were not correlated with any of these measures. There was a decreasing trend of salt sensitivity index with increasing muscle Na + , although this correlation did not reach statistical significance (r=-0.35 p=0.0686) Conclusion: This highlights the potential importance of muscle Na + in SSBP, such that patients with lower muscle Na + exhibited a higher trend of salt sensitivity index and increased variability in IsoLG+ APCs. Moreover, baseline tissue Na + assessment may emerge as a potential tool for evaluating the degree of salt sensitivity and IsoLG activation in SSBP.

Production of fish fillet analogues using novel fish muscle cell powder and sodium alginate-κ-carrageenan based bio-ink

Cellular aquaculture has emerged as a promising technology to address the challenges associated with traditional fishing and aquaculture practices. This study focuses on developing a biomimetic fish fillet structure using fish muscle cell powder (FMCP) and Alg-κ-Car-based bio-ink, exploring its potential for cultured seafood applications. Grouper muscle satellite cells (GMSCs) were isolated through enzymatic digestion and demonstrated robust growth and myotube differentiation with 2% horse serum (HS). FMCP, derived from the differentiated GMSCs, is characterized as a high-protein source with promising seafood applications. Cytotoxicity analysis reveals FMCP's biocompatibility and its ability to enhance cell proliferation. Alg-κ-Car is chosen as the bio-ink due to its favourable impact on bioink stability. The developed Alg-κ-Car-FMCP bio-ink, with favourable rheological properties, facilitates 3D bioprinting of scaffolds mimicking the texture of native fish fillets. These printed scaffolds retain structural integrity when subjected to boiling and frying. Texture profiles indicate that Alg-κ-Car-FMCP constructs possess enhanced hardness, springiness, gumminess, and chewiness compared to Alg-κ-Car, with cooking influencing textural changes. Taste sensory analysis using an electronic tongue demonstrates Alg-κ-Car-FMCP constructs present a pronounced umami taste, showcasing potential for desirable sensory attributes in cell-based seafood. This study significantly advances cellular aquaculture for cultured seafood production, offering insights for further research and development in sustainable and innovative food production.

Intermuscular adipose tissue accumulation is associated with higher tissue sodium in healthy individuals.

High tissue sodium accumulation and intermuscular adipose tissue (IMAT) are associated with aging, type 2 diabetes, and chronic kidney disease. In this study, we aim to investigate whether high lower-extremity tissue sodium accumulation relates to IMAT quantity and whether systemic inflammatory mediators and adipocytokines contribute to such association. Tissue sodium content and IMAT accumulation (percentage of IMAT area to muscle area) were measured in 83 healthy individuals using sodium imaging (23Na-MRI) and proton (1H-MRI) imaging of the calf. Insulin sensitivity was assessed by glucose disposal rate (GDR) measured with the hyperinsulinemic-euglycemic clamp. Median (interquartile range) muscle and skin sodium contents were 16.6 (14.9, 19.0) and 12.6 (10.9, 16.7) mmol/L, respectively. Median IMAT was 3.69 (2.80, 5.37) %. In models adjusted for age, sex, BMI, GDR, adiponectin, and high-sensitivity C-reactive protein, increasing tissue sodium content was significantly associated with higher IMAT quantity (p = 0.018 and 0.032 for muscle and skin tissue sodium, respectively). In subgroup analysis stratified by sex, skin sodium was significantly associated with IMAT only among men. In interaction analysis, the association between skin sodium and IMAT was greater with increasing levels of high-sensitivity C-reactive protein and interleukin-6 (p for interaction = 0.022 and 0.006, respectively). Leg muscle and skin sodium are associated with IMAT quantity among healthy individuals. The relationship between skin sodium and IMAT may be mediated by systemic inflammation.

HCN2-based biological pacing in a modified rat AV-block model

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Eurostars - Heartpace 114245 Background Electronic pacemakers have several important shortcomings which are difficult to overcome due to their hardware-based design. Gene therapy-based biological pacemakers may provide a different platform which can overcome these limitations. Such biological pacemaker strategies center around overexpression of the pacemaker ion channel HCN2, either alone or with other transgenes to potentiate pacemaker performance, such as the skeletal muscle sodium channel SkM1. In order to develop clinically applicable pacemaker gene therapies further engineering is needed, which would benefit from a reliable small animal model. At present it is unknown if the rat AV block model can be used to evaluate ion channel-based biological pacing. Aim In the present study, we aim to validate a modified rat AV-block model for the evaluation of HCN2 and SKM1-based biological pacing. Methods AV-block was generated in female rats by epicardially applied needle ablation of the AV-nodal region, as previously described (N.K. ). Complete AV-block was confirmed 7 days post-ablation by loss of P-wave and QRS-complex association on the body surface ECG. Seven days post-ablation, 3 groups of animals were injected into the left ventricular apex with Ad5-TNNT2-HCN2, Ad5-TNNT2-SkM1 or saline (control). 14 days post-ablation, ECG analysis was performed during anesthesia under baseline and after i.v. administration of isoprenaline. Upon completion of functional testing, tissue samples were harvested followed by gene expression analysis using qPCR. Results Injection of Ad5-TNNT2-HCN2, Ad5-TNNT2-SkM1 or saline did not result in ectopic activation patterns in baseline conditions 7 days after injection. Administration of isoprenaline results in reversal of the QRS complexes 90% of total measurement time in Ad5-TNNT2-HCN2 injected animals. Saline and Ad5-TNNT2-SkM1 showed no change in activation pattern upon isoprenaline administration. Gene expression analysis revealed expression of HCN2 in the injection site following Ad5-TNNT2-HCN2 injection, with minimal spread to non-target regions. However, this analysis also showed Ad5-TNNT2-SkM1 did not express well in this context. Conclusion Our modified rat AV-block model, when administered with isoprenaline, recapitulated expected biological pacemaker activity of HCN2 gene delivery. However, lack of effective expression of SkM1 renders direct comparison of these relevant transgenes difficult. Nevertheless, we expect this rat AV-block model can provide a valuable tool for the evaluation of novel ion channel-based biological pacemaker strategies.

#3063 MRI assessment of skin and muscle sodium (23Na) and fluid volume in haemodialysis patients

Abstract Background and Aims Haemodialysis (HD) is life sustaining for patients with end-stage kidney disease (ESKD). In healthy people, sodium balance is regulated by the kidneys; in ESKD this is achieved by sodium removal during HD. Recent evidence suggests non-osmotically stored sodium in the muscle and/or skin may be a critical factor impacting the development of hypertension and cardiovascular disease (CVD). Sodium (23Na) MRI allows skin and muscle sodium storage assessment and may provide a valuable tool in evaluating sodium storage in dialysis patients. Here, 23Na MRI is used to measure muscle and skin tissue sodium concentration (TSC) in HD participants. HD participants are scanned before and after a single haemodialysis session using 23Na MRI as well as 1H T2 relaxometry to study changes in fluid status. Method Data were collected on HD patients who underwent a pre-HD 23Na MRI calf scan, then had their usual dialysis session with a dialysate sodium of 137 mmol/L, followed by a repeat post-HD 23Na MRI scan. Patient dialysis vintage, residual renal function, and ultrafiltration volume, and blood measures pre- and post-HD were collected. 1H scans were acquired for localisation and muscle segmentation (mDIXON) along with T1 and T2 mapping. 23Na images were acquired for (TSC) quantification. Reference bottles (10, 20, 30 and 40 mmol/L NaCl) were placed above the leg to calibrate TSC muscle and skin maps. Regions-of-interest (ROIs) of each muscle group and the skin were manually segmented on mDIXON scans. In each muscle group the voxel-wise mode (to avoid influence of signals from vessels) of TSC and T2 was estimated. A paired-sample t-test was performed between metrics pre-HD and post-HD. Absolute measures of ΔTSC and ΔT2 were correlated with clinical measures. Results Ten people receiving HD were recruited (age 55-73 yrs, 5 M:5F). Figure 1 shows TSC maps of the HD patients pre-HD and post-HD, with a significant reduction (p < 0.05) in TSC in the extensor, peroneus and gastrocnemius muscles post-HD but skin sodium showed no detectable change. Post-HD TSC significantly positively correlated with post-HD Systolic Blood Pressure (SBP) (P < 0.004) but not with plasma sodium. 1H T2-maps (Fig. 2A) show a significant reduction (p < 0.01) in muscle T2 post-HD compared to pre-HD all muscle groups (Fig. 2B). There were no significant correlations between T2 and 23Na measures, or T2 and clinical measures. There was no significant detectable change in 1H T1. Conclusion Post-HD TSC values are consistent with published data from patients dialysed against a dialysate sodium of 137 mmol/L, and shows a significant correlation of post-HD TSC with post-HD SBP. 1H muscle T2 reduced post-HD, consistent with published data, as patients change from a pre-HD hypervolemia state closer to a post-HD to euvolemia state after fluid removal. There was no correlation of absolute or ΔTSC and 1H T2 and ΔT2 values, suggesting different mechanisms for equilibrium conditions of water and sodium ion concentrations. Future studies will study the effect of dialysate sodium on muscle TSC and mechanistic links to CVD, and include the use of a dedicated skin coil to study skin sodium alongside bioimpedance fluid measures.

#2651 Microwave tissue digestion and flame atomic absorption spectroscopy for measuring tissue sodium content in CKD patients

Abstract Background and Aims Skin and muscle sodium accumulation has been demonstrated in CKD patients and may contribute to progression of the disease. Various methods are available for measuring tissue sodium content. However, the accuracy and reproducibility of these methods remain unknown. Dry ashing is the most used digestion method, but is expensive, time consuming and has a risk of incomplete digestion. This study compared microwave digestion, dry ashing, and two electrolyte quantification methods for quantifying tissue sodium and potassium. Method We compared microwave digestion (Ethos LEAN Compact Microwave Digestion System, Milestone SRL) and dry ashing (Nabertherm S27) as sample preparation methods, and flame atomic absorption spectroscopy (FAAS, Model 425 Flame Photometer, Sherwood Scientific) and inductively coupled plasma optical emission spectrometry (ICP-OES, Varian 720-ES, Varian Inc.) as electrolyte quantification methods. Skin and muscle samples were obtained from rats and mice, and human skin samples were used. Tissues biopsies were cut in three pieces and digested separately. The methods were compared using interclass correlation coefficient (ICC) and Bland-Altman analysis. The third piece of tissue was microwave-digested by another operator to test inter-operator reproducibility by calculating the ICC and absolute overall mean difference. Results With microwave digestion and FAAS median (IQR) human skin concentration was 114.8 (99.2-157.0, n = 12) mmol/L, confirming tissue sodium storage. When including all tissues, FAAS and ICP-OES showed high agreement for sodium (ICC 0.98, bias –0.00008 mmol/g, n = 141 and potassium (ICC 0.98, bias –0.005 mmol/g, n = 141). Microwave digestion and ashing showed agreement for sodium (ICC 0.93, bias –0.002 mmol/g, n = 61) and potassium (ICC 0.93, bias –0.02 mmol/g, n = 61). The inter-operator agreement was good for sodium (ICC 0.95, absolute mean difference (SD) 0.044 mmol/g (0.073), n = 70) and potassium (ICC 0.96, absolute mean difference (SD) 0.031 mmol/g (0.032), n = 70). Conclusion Microwave digestion with FAAS is a reliable and consistent method for sodium and potassium trace analysis in animal and human tissues compared to ashing and ICP-OES, respectively. This method showed more quantification of cations than the most frequently reported method, representing an easier, and less time-consuming way to quantify tissue sodium in CKD research.

Development of Riluzole Analogs with Improved Use-Dependent Inhibition of Skeletal Muscle Sodium Channels.

Several commercially available and newly synthesized riluzole analogs were evaluated in vitro as voltage-gated skeletal muscle sodium-channel blockers. Data obtained from the patch-clamp technique demonstrated that potency is well correlated with lipophilicity and the introduction of a protonatable amino function in the benzothiazole 2-position enhances the use-dependent behavior. The most interesting compound, the 2-piperazine analog of riluzole (14), although slightly less potent than the parent compound in the patch-clamp assay as well as in an in vitro model of myotonia, showed greater use-dependent Nav1.4 blocking activity. Docking studies allowed the identification of the key interactions that 14 makes with the amino acids of the local anesthetic binding site within the pore of the channel. The reported results pave the way for the identification of novel compounds useful in the treatment of cell excitability disorders.

#4196 MAGNETIC RESONANCE IMAGING ASSESSMENT OF SKIN AND MUSCLE SODIUM IN HAEMODIALYSIS

Abstract Background and Aims Haemodialysis (HD) is life sustaining for patients with end-stage kidney disease (ESKD) but is associated with a marked increase in incidence of cardiovascular disease (CVD) and high annual mortality rates. Sodium balance is regulated by the kidneys in health but has to be achieved by sodium removal during HD for those with ESKD. Recent evidence suggests that non-osmotically stored sodium in the muscle and/or skin may be a critical factor impacting the development of hypertension and CVD. Sodium magnetic resonance imaging (23Na MRI) allows assessment of skin and muscle sodium storage and may provide a valuable tool in evaluating sodium storage in dialysis patients. In this study, we used 23Na MRI to measure muscle and skin sodium content in younger and older healthy individuals and people receiving HD, and investigated the effect of a single dialysis session on muscle and skin sodium content. Method 23Na MRI was acquired on a 3T Philips Ingenia scanner using a custom-made 23Na RF coil. 3D GRE 23Na scans (3 × 3 × 30mm3, 10 slices) were acquired in a 15-minute scan. Four reference bottles of increasing sodium concentration were placed in the RF coil above the leg to calibrate sodium concentration. In the same imaging session, 1H MR images were acquired to delineate muscle groups, skin structures and tissue water content. 23Na concentration maps were generated using custom software and regions of interest of each muscle and the skin were manually segmented. Data was collected on 14 younger (23-38yrs,7M:7F) and three older (66-77yrs,3M:1F) healthy volunteers (HVs), and 5 male HD patients (55-68 yrs) who had been on HD for more than 3-months. HD patients underwent a pre-dialysis 23Na MRI calf scan, then had their usual dialysis session with a dialysate Na prescription of 137 mmol/L, followed by a repeat 23Na MRI calf scan post dialysis. Patient demographics, dialysis vintage, residual renal function, and ultrafiltration volume were collected, along with blood samples at the start and end of the dialysis session, including serum sodium. Results Figure 1 shows 23Na images in the younger and older HV group and HD patients pre-dialysis. Muscle and skin sodium was increased in older HVs compared to younger, with HD patients pre-dialysis tending to be lower than older HVs (Fig. 2). HD patients’ demographics and clinical measures pre- and post-dialysis are shown in Table 1. HD treatment reduced muscle sodium whilst skin sodium showed little change (Fig. 3). Conclusion We have optimised methods for measuring muscle and skin sodium. 23Na concentration in muscle and skin is higher in older subjects. HD patients’ 23Na values fall between those observed in the older and younger HV groups. These values are consistent with published data from patients dialysed against 137mmol/l sodium dialysate, but lower than other studies in which dialysate sodium levels were higher. Further studies are planned to study the effect of dialysate sodium on skin and muscle sodium concentrations and mechanistic links to cardiovascular disease.

A KETOGENIC DIET REDUCES TISSUE SODIUM CONTENT: AN EXPLORATORY STUDY

Objective: Hypertension and diabetes are a substantial global public health problem. The prevalence of these diseases will further increase in the near future, leading to increased health and socioeconomic costs. Hence efficient therapeutic approaches are urgently needed. It has been previously shown that nutritional approaches including a very-low carbohydrate, or a ketogenic diet potentially decrease body weight, blood pressure and glucose levels in patients with type 2 diabetes along with an overall significant reduction in their pill burden. However, the detailed physiological mechanisms underlying the observed induction and maintenance of diabetes remission remain largely unknown. Previous studies have shown that the skin could be a buffer system for sodium and that skin sodium content could be both a pathogenic cause and a marker of hypertension and salt sensitivity. Since a nutritionally created metabolic milieu such as nutritional ketosis can lower blood pressure, we hypothesize that this dietary approach also reduces tissue sodium content. Design and method: By using 7-T 23Na MRI technique, we performed an exploratory study and analyzed skin and muscle sodium concentration before, and after a 6-week ketogenic diet in three healthy probands. Nutritional ketosis was verified by frequent check of blood ketone (beta-hydroxybutyrate; BOHB) levels greater than 0.3 mmol/L. Results: Preliminary data of our planned exploratory study (in patients with chronic kidney disease and type 2 diabetes mellitus and healthy probands) demonstrated that compared to baseline 6 weeks of ketogenic diet decreased tissue sodium content in the skin (16.6 ± 2.1 vs. 12.6 ± 2.1 mmol/L), in the medial gastrocnemius muscle (20.5 ± 0.9 vs. 16.7 ± 0.7 mmol/L), in the lateral gastrocnemius muscle (19.7 ± 1.0 vs. 16.0 ± 1.0 mmol/L) and in the soleus muscle (13.1 ± 0.8 vs. 11.5 ± 0.5 mmol/L). Conclusions: Our results indicate a massive reduction of tissue sodium concentration in healthy volunteers in response to a ketogenic diet which might be associated with the observed clinical benefits and be implicated in the anti-hypertensive effects of this powerful dietary intervention. Findings of this study could have an impact on future treatment strategies of various chronic diseases.

New insights from modelling studies and molecular dynamics simulations of the DIS5-S6 extracellular linker of the skeletal muscle sodium channel NaV1.4.

In the CryoEM-structure of the hSkMNaV1.4 ion channel (PDB:6AGF), the 59-residue DIS5-S6 linker peptide was omitted due to absence of electron density. This peptide is intriguing - comprised of unique sequence and found only in mammalian skeletal muscle sodium ion channels. To probe potential physiological and evolutionary significance, we constructed an homology model of the complete hSkMNaV1.4 channel. Rather than a flexible random coil potentiating drift across the channel, the linker folds into a compact configuration through self-assembling secondary structural elements. Analogous sequences from 48 mammalian organisms show hypervariability with between 40% and 100% sequence similarity. To investigate structural implications, sequences from 14 representative organisms were additionally modelled. All showed highly conserved N-and C-terminal residues closely superimposed, suggesting a critical functional role. An optimally located asparagine residue within the conserved region was investigated for N-linked glycosylation and MD simulations carried out. Results suggest a complex glycan added at this site in the linker may form electrostatic interactions with the DIV voltage sensing domain and be mechanistically involved in channel gating. The relationship of unique sequence, compact configuration, potential glycosylation and MD simulations are discussed relative to SkMNaV1.4 structure and function.

Development of a detection method for 10 non-steroidal anti-inflammatory drugs residues in four swine tissues by ultra-performance liquid chromatography with tandem mass spectrometry

The ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) detection method was developed for the residues of 10 NSAIDs (salicylic acid, acetylsalicylic acid, acetaminophen, diclofenac, tolfenamic acid, antipyrine, flunixin meglumine, aminophenazone, meloxicam, metamizole sodium) in swine muscle, liver, kidney, and fat. Swine tissue samples were extracted by phosphorylated acetonitrile with the addition of an appropriate amount of internal standard working solution, defatted with acetonitrile-saturated n-hexane, and purified by Hydrophile-Lipophile Balance (HLB) solid-phase extraction column, then separated by UPLC BEH shield RP18 column with 0.1% formic acid in water/0.1% formic acid in acetonitrile with gradient elution, which was detected in the multiple reaction monitoring (MRM) modes. The correlation coefficient of the standard curve equation is greater than 0.99, and the coefficient of variation within and between batches is less than 14.4%. We evaluated the analytical method using two green assessment tools. The method established in this study met the requirements of NSAID residue analysis and provides analytical tools for determining and confirming NSAIDs in swine tissue samples. This is the first report on the simultaneous determination of 10 NSAIDs in four swine tissues by the UPLC-MS/MS method and accurate quantification using deuterated internal standards.

High tissue-sodium associates with systemic inflammation and insulin resistance in obese individuals

Background and aimsHigh sodium intake is associated with obesity and insulin resistance, and high extracellular sodium content may induce systemic inflammation, leading to cardiovascular disease. In this study, we aim to investigate whether high tissue sodium accumulation relates with obesity-related insulin resistance and whether the pro-inflammatory effects of excess tissue sodium accumulation may contribute to such association. Methods and resultsIn a cross-sectional study of 30 obese and 53 non-obese subjects, we measured insulin sensitivity determined as glucose disposal rate (GDR) using hyperinsulinemic euglycemic clamp, and tissue sodium content using 23Na magnetic resonance imaging. Median age was 48 years, 68% were female and 41% were African American. Median (interquartile range) BMI was 33 (31.5, 36.3) and 25 (23.5, 27.2) kg/m2 in the obese and non-obese individuals, respectively. In obese individuals, insulin sensitivity negatively correlated with muscle (r = −0.45, p = 0.01) and skin sodium (r = −0.46, p = 0.01). In interaction analysis among obese individuals, tissue sodium had a greater effect on insulin sensitivity at higher levels of high-sensitivity C-reactive protein (p-interaction = 0.03 and 0.01 for muscle and skin Na+, respectively) and interleukin-6 (p-interaction = 0.024 and 0.003 for muscle and skin Na+, respectively). In interaction analysis of the entire cohort, the association between muscle sodium and insulin sensitivity was stronger with increasing levels of serum leptin (p-interaction = 0.01). ConclusionsHigher muscle and skin sodium are associated with insulin resistance in obese patients. Whether high tissue sodium accumulation has a mechanistic role in the development of obesity-related insulin resistance through systemic inflammation and leptin dysregulation remains to be examined in future studies.Clinicaltrials.gov registration: NCT02236520.

Potassium-Induced Changes in Muscle Free Amino Acid Concentrations in Chicks

Two experiments with purified crystalline amino acid diets were conducted to determine the effect of dietary potassium level on chick breast muscle free amino acid concentrations. In both experiments, concentrations of muscle free glutamine, lysine and arginine increased as dietary potassium increased from 0.12 to 0.18% and decreased as dietary potassium exceeded 0.18%. As dietary potassium increased, muscle potassium concentration increased linearly with an equivalent linear decrease in muscle sodium concentration. Peak concentrations of muscle free glutamine, lysine and arginine occurred in muscle with a K+:Na+ ratio of approximately 1.4. Concentrations of the three amino acids were less in muscle with K+:Na+ ratios either less than or greater than 1.4. Breast muscle concentrations of free histidine decreased slightly, while muscle free carnosine increased substantially when dietary potassium exceeded 0.18%.amino acids glutamine lysine arginine potassium

Muscle fat replacement and contractility in patients with skeletal muscle sodium channel disorders

Skeletal muscle sodium channel disorders give rise to episodic symptoms such as myotonia and/or periodic paralysis. Chronic symptoms with permanent weakness are not considered characteristic of the phenotypes. Muscle fat replacement represents irreversible damage that inevitably will impact on muscle strength. This study investigates muscle fat replacement and contractility in patients with pathogenic SCN4A variants compared to healthy controls. T1-weighted and 2-point Dixon MRI of the legs were conducted to assess fat replacement. Stationary dynamometry was used to assess muscle strength. Contractility was determined by maximal muscle contraction divided by cross-sectional muscle area. The average cross-sectional intramuscular fat fraction was greater in patients compared with controls by 2.5% in the calves (95% CI 0.74–4.29%, p = 0.007) and by 2.0% in the thighs (95% CI 0.75–3.2%, p = 0.003). Muscle contractility was less in patients vs. controls by 14–27% (p < 0.05). Despite greater fat fraction and less contractility, absolute strength was not significantly less. This study quantitatively documents greater fat fraction and additionally describes difference in muscle contractility in a large cohort of patients with skeletal muscle sodium channel disorders. The clinical impact of these abnormal findings is likely limited as muscle hypertrophy in the patients served to preserve absolute muscle strength. Subgroup analysis indicated significant difference in phenotype by genotype, however these findings lack statistical significance and serve as inspiration for future researchers to probe into the geno- phenotype relationship in these disorders.Trial registration: The study was registered at http://clinicaltrials.gov (identifier: NCT04808388).

Blockers of Skeletal Muscle Nav1.4 Channels: From Therapy of Myotonic Syndrome to Molecular Determinants of Pharmacological Action and Back.

The voltage-gated sodium channels represent an important target for drug discovery since a large number of physiological processes are regulated by these channels. In several excitability disorders, including epilepsy, cardiac arrhythmias, chronic pain, and non-dystrophic myotonia, blockers of voltage-gated sodium channels are clinically used. Myotonia is a skeletal muscle condition characterized by the over-excitability of the sarcolemma, resulting in delayed relaxation after contraction and muscle stiffness. The therapeutic management of this disorder relies on mexiletine and other sodium channel blockers, which are not selective for the Nav1.4 skeletal muscle sodium channel isoform. Hence, the importance of deepening the knowledge of molecular requirements for developing more potent and use-dependent drugs acting on Nav1.4. Here, we review the available treatment options for non-dystrophic myotonia and the structure-activity relationship studies performed in our laboratory with a focus on new compounds with potential antimyotonic activity.

Exploring the Pivotal Components Influencing the Side Effects Induced by an Analgesic-Antitumor Peptide from Scorpion Venom on Human Voltage-Gated Sodium Channels 1.4 and 1.5 through Computational Simulation.

Voltage-gated sodium channels (VGSCs, or Nav) are important determinants of action potential generation and propagation. Efforts are underway to develop medicines targeting different channel subtypes for the treatment of related channelopathies. However, a high degree of conservation across its nine subtypes could lead to the off-target adverse effects on skeletal and cardiac muscles due to acting on primary skeletal muscle sodium channel Nav1.4 and cardiac muscle sodium channel Nav1.5, respectively. For a long evolutionary process, some peptide toxins from venoms have been found to be highly potent yet selective on ion channel subtypes and, therefore, hold the promising potential to be developed into therapeutic agents. In this research, all-atom molecular dynamic methods were used to elucidate the selective mechanisms of an analgesic-antitumor β-scorpion toxin (AGAP) with human Nav1.4 and Nav1.5 in order to unravel the primary reason for the production of its adverse reactions on the skeletal and cardiac muscles. Our results suggest that the rational distribution of residues with ring structures near position 38 and positive residues in the C-terminal on AGAP are critical factors to ensure its analgesic efficacy. Moreover, the substitution for residues with benzene is beneficial to reduce its side effects.

Muscle sodium content in patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

BackgroundMuscle fatigue and pain are key symptoms of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Although the pathophysiology is not yet fully understood, there is ample evidence for hypoperfusion which may result in electrolyte imbalance and sodium overload in muscles. Therefore, the aim of this study was to assess levels of sodium content in muscles of patients with ME/CFS and to compare these to healthy controls.MethodsSix female patients with ME/CFS and six age, BMI and sex matched controls underwent 23Na-MRI of the left lower leg using a clinical 3T MR scanner before and after 3 min of plantar flexion exercise. Sodium reference phantoms with solutions of 10, 20, 30 and 40 mmol/L NaCl were used for quantification. Muscle sodium content over 40 min was measured using a dedicated plugin in the open-source DICOM viewer Horos. Handgrip strength was measured and correlated with sodium content.ResultsBaseline tissue sodium content was higher in all 5 lower leg muscle compartments in ME/CFS compared to controls. Within the anterior extensor muscle compartment, the highest difference in baseline muscle sodium content between ME/CFS and controls was found (mean ± SD; 12.20 ± 1.66 mM in ME/CFS versus 9.38 ± 0.71 mM in controls, p = 0.0034). Directly after exercise, tissue sodium content increased in gastrocnemius and triceps surae muscles with + 30% in ME/CFS (p = 0.0005) and + 24% in controls (p = 0.0007) in the medial gastrocnemius muscle but not in the extensor muscles which were not exercised. Compared to baseline, the increase of sodium content in medial gastrocnemius muscle was stronger in ME/CFS than in controls with + 30% versus + 17% to baseline at 12 min (p = 0.0326) and + 29% versus + 16% to baseline at 15 min (p = 0.0265). Patients had reduced average handgrip strength which was associated with increased average muscle tissue sodium content (p = 0.0319, R2 = 0.3832).ConclusionMuscle sodium content before and after exercise was higher in ME/CFS than in healthy controls. Furthermore, our findings indicate an inverse correlation between muscle sodium content and handgrip strength. These findings provide evidence that sodium overload may play a role in the pathophysiology of ME/CFS and may allow for potential therapeutic targeting.

The SGLT2 inhibitor empagliflozin reduces tissue sodium content in patients with chronic heart failure: results from a placebo-controlled randomised trial

IntroductionSodium-glucose co-transporter 2 (SGLT2) inhibitors have cardiovascular protective properties in addition to the metabolic effects and represent a cornerstone of treating patients with chronic heart failure (CHF). We hypothesised that empagliflozin reduces tissue sodium content in patients with CHF.MethodsIn a double-blind, randomised (2:1), placebo-controlled, parallel-group, clinical trial, 74 patients with NYHA class II–III CHF and an ejection fraction of 49% or less received empagliflozin 10 mg once daily or placebo for 3 months. In each patient, tissue sodium content of the lower leg was assessed non-invasively by sodium-MRI (23Na-MRI) at baseline, after 1 and 3 months of treatment.ResultsAfter 1 and 3 months treatment with empagliflozin (n = 48), a significant decrease in skin sodium content was observed (1 month: 22.8 ± 6.1 vs. 21.6 ± 6.0 AU, p = 0.039; 3 months: 22.9 ± 6.1 vs. 21.6 ± 6.1 AU, p = 0.013), while there was no change in muscle sodium and muscle water content. In direct comparison, the change in skin sodium content between baseline and 3 months was − 1.3 ± 3.5 AU in the empagliflozin group versus 0.6 ± 3.5 AU in the placebo group (p for between-group difference = 0.022). No significant difference regarding change in muscle sodium and in muscle water content was observed after 3 months treatment between the two groups.ConclusionThis trial showed a significant decrease in skin sodium content after 1 and 3 months of treatment with empagliflozin. The decrease in skin sodium content may reflect a decrease in subclinical micro-oedema or/and in non-osmotic bound tissue sodium, both reported to impair left ventricular function.Trial registration numberNCT03128528 (http://www.clinicaltrials.gov).Trial registration date25th April 2017.

Block of Voltage-Gated Sodium Channels by Aripiprazole in a State-Dependent Manner.

Aripiprazole is an atypical antipsychotic drug, which is prescribed for many psychiatric diseases such as schizophrenia and mania in bipolar disorder. It primarily acts as an agonist of dopaminergic and other G-protein coupled receptors. So far, an interaction with ligand- or voltage-gated ion channels has been classified as weak. Meanwhile, we identified aripiprazole in a preliminary test as a potent blocker of voltage-gated sodium channels. Here, we present a detailed analysis about the interaction of aripiprazole with the dominant voltage-gated sodium channel of heart muscle (hNav1.5). Electrophysiological experiments were performed by means of the patch clamp technique at human heart muscle sodium channels (hNav1.5), heterologously expressed in human TsA cells. Aripiprazole inhibits the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state is weak with an extrapolated Kr of about 55 µM. By contrast, the interaction with the inactivated state is strong. The affinities for the fast and slow inactivated state are in the low micromolar range (0.5-1 µM). Kinetic studies indicate that block development for the inactivated state must be described with a fast (ms) and a slow (s) time constant. Even though the time constants differ by a factor of about 50, the resulting affinity constants were nearly identical (in the range of 0.5 µM). Besides this, aripirazole also interacts with the open state of the channel. Using an inactivation deficit mutant, an affinity of about 1 µM was estimated. In summary, aripiprazole inhibits voltage-gated sodium channels at low micromolar concentrations. This property might add to its possible anticancer and neuroprotective properties.

Tissue Sodium in Patients With Early Stage Hypertension: A Randomized Controlled Trial.

BackgroundSodium (Na+) stored in skin and muscle tissue is associated with essential hypertension. Sodium magnetic resonance imaging is a validated method of quantifying tissue stores of Na+. In this study, we evaluated tissue Na+ in patients with elevated blood pressure or stage I hypertension in response to diuretic therapy or low Na+ diet.Methods and ResultsIn a double‐blinded, placebo‐controlled trial, patients with systolic blood pressure 120 to 139 mm Hg were randomized to low sodium diet (<2 g of sodium), chlorthalidone, spironolactone, or placebo for 8 weeks. Muscle and skin Na+ using sodium magnetic resonance imaging and pulse wave velocity were assessed at the beginning and end of the study. Ninety‐eight patients were enrolled to undergo baseline measurements and 54 completed randomization. Median baseline muscle and skin Na+ in 98 patients were 16.4 mmol/L (14.9, 18.9) and 13.1 mmol/L (11.1, 16.1), respectively. After 8 weeks, muscle Na+ increased in the diet and chlorthalidone arms compared with placebo. Skin sodium was decreased only in the diet arm compared with placebo. These associations remained significant after adjustment for age, sex, body mass index, systolic blood pressure, and urinary sodium. No changes were observed in pulse wave velocity among the different groups when compared with placebo.ConclusionsDiuretic therapy for 8 weeks did not decrease muscle or skin sodium or improve pulse wave velocity in patients with elevated blood pressure or stage I hypertension.RegistrationURL: https://www.clinicaltrials.gov; Unique identifier: NCT02236520.