Relevance of pathogenicity prediction tools in human RYR1 variants of unknown significance.

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle metabolism characterized by generalized muscle rigidity, increased body temperature, rhabdomyolysis, hyperkalemia and severe metabolic acidosis. The underlying mechanism of MH involves excessive Ca2+ release from myotubes via the ryanodine receptor type 1 (RYR1) and the voltage-dependent L-type calcium channel (CACNA1S). As more than 300 variants of unknown significance have been detected to date, we examined whether freely available pathogenicity prediction tools are able to detect relevant MH causing variants. In this diagnostic accuracy study, blood samples from 235 individuals with a history of a clinical malignant hyperthermia or their close relatives were genetically screened for RYR1 variants of all 106 RYR1 exons and additionally for known variants of CACNA1S. In vitro contracture tests were conducted on muscle biopsies obtained from all individuals, independently of whether a pathogenic variant, a variant of unknown significance or no variant was detected. Comparisons were made to three established bioinformatic pathogenicity detection tools to identify the clinical impact of the variants of unknown significance. All detected genetic variants were tested for pathogenicity by three in silico approaches and compared to the in vitro contracture test. Sensitivity and specificity of exon screening of all individuals listed in our MH database was analyzed. Exon screening identified 97 (41%) of the 235 individuals as carriers of pathogenic variants. Variants of unknown significance were detected in 21 individuals. Variants of unknown significance were subdivided into 19 malignant-hyperthermia-susceptible individuals and 2 non-malignant-hyperthermia-susceptible individuals. All pathogenic variants as well as the malignant-hyperthermia-suspectible variants were correctly identified by the bioinformatic prediction tools. Sensitivity of in silico approaches ranged between 0.71 and 0.98 (Polyphen 0.94 [CI 95% 0.75; 0.99]; Sift 0.98 [CI 95% 0.81; 0.99]; MutationTaster 0.92 [CI 95% 0.75; 0.99]). Specificity differed depending on the used tool (Polphen 0.98 [CI 95% 0.32; 0.99]; Sift 0.98 [CI 95% 0.32; 0.99]; MutationTaster 0.00 [CI 95% 0.00; 0.60]). All pathogenic variants and variants of unknown significance were scored as probably damaging in individuals, demonstrating a high sensitivity. Specificity was very low in one of the three tested programs. However, due to potential genotype-phenotype discordance, bioinformatic prediction tools are currently of limited value in diagnosing pathogenicity of MH-susceptible variants.

Paxilline Prevents the Onset of Myotonic Stiffness in Pharmacologically Induced Myotonia: A Preclinical Investigation.

Reduced Cl- conductance causes inhibited muscle relaxation after forceful voluntary contraction due to muscle membrane hyperexcitability. This represents the pathomechanism of myotonia congenita. Due to the prevailing data suggesting that an increased potassium level is a main contributor, we studied the effect of a modulator of a big conductance Ca2+- and voltage-activated K+ channels (BK) modulator on contraction and relaxation of slow- and high-twitch muscle specimen before and after the pharmacological induction of myotonia. Human and murine muscle specimens (wild-type and BK-/-) were exposed to anthracene-9-carboxylic acid (9-AC) to inhibit CLC-1 chloride channels and to induce myotonia in-vitro. Functional effects of BK-channel activation and blockade were investigated by exposing slow-twitch (soleus) and fast-twitch (extensor digitorum longus) murine muscle specimens or human musculus vastus lateralis to an activator (NS1608) and a blocker (Paxilline), respectively. Muscle-twitch force and relaxation times (T90/10) were monitored. Compared to wild type, fast-twitch muscle specimen of BK-/- mice resulted in a significantly decreased T90/10 in presence of 9-AC. Paxilline significantly shortened T90/10 of murine slow- and fast-twitch muscles as well as human vastus lateralis muscle. Moreover, twitch force was significantly reduced after application of Paxilline in myotonic muscle. NS1608 had opposite effects to Paxilline and aggravated the onset of myotonic activity by prolongation of T90/10. The currently used standard therapy for myotonia is, in some individuals, not very effective. This in vitro study demonstrated that a BK channel blocker lowers myotonic stiffness and thus highlights its potential therapeutic option in myotonia congenital (MC).

The Stiffness Comparison Test: A pilot study to determine inter-individual differences in palpatory skill related to gender, age, and occupation-related experience.

BACKGROUND: Manual palpation is a core skill in physical examination. Assessing elastic properties such as tissue stiffness has the potential for being an important diagnostics tool in the detection of cancer and other diseases. OBJECTIVE: The study describes the newly developed Stiffness Comparison Test (SCT). The aim of our study was to test the SCT as a tool to detect interindividual differences in palpation skill related to gender, age and occupational experience. METHODS: We used eight pairs of polyuterhane gel pads with the stiffness difference decreasing from the first to the last pair. Test subjects were asked to palpate each pair and determine stiffness differences. PARTICIPANTS: We recruited 25 osteopaths, 48 other manual therapists and 50 participants from other non-manual professions. RESULTS: As hypothesized there was no significant difference in SCT performance between the sexes (t(121) = 0.288, p = .774). To investigate if an age-related decline would have an effect on palpation skill, we carried out a linear regression. As hypothesized, the model did not predict any significant associations (F(1, 121) = 2.733, b = -0.149, p = .101, R2 = 0.022). To compare the effect of occupational groups on SCT performance a one-way ANOVA was conducted. There were no statistically significant differences between group means (F(2, 120) = 0.598, p = .552). CONCLUSIONS: The SCT can be used as simple and affordable tool for assessment, teaching and training in all disciplines of manual medicine. Further refinements of the tool are suggested to advance its discrimination power.

Correction to: Preclinical pharmacological in vitro investigations on low chloride conductance myotonia: effects of potassium regulation.

The original article contains an error during online publication. Table 2 was included during production round and now deleted. The Original article has been corrected.

Preclinical pharmacological in vitro investigations on low chloride conductance myotonia: effects of potassium regulation.

In myotonia, reduced Cl- conductance of the mutated ClC-1 channels causes hindered muscle relaxation after forceful voluntary contraction due to muscle membrane hyperexcitability. Repetitive contraction temporarily decreases myotonia, a phenomena called "warm up." The underlying mechanism for the reduction of hyperexcitability in warm-up is currently unknown. Since potassium displacement is known to reduce excitability in, for example, muscle fatigue, we characterized the role of potassium in native myotonia congenita (MC) muscle. Muscle specimens of ADR mice (an animal model for low gCl- conductance myotonia) were exposed to increasing K+ concentrations. To characterize functional effects of potassium ion current, the muscle of ADR mice was exposed to agonists and antagonists of the big conductance Ca2+-activated K+ channel (BK) and the voltage-gated Kv7 channel. Effects were monitored by functional force and membrane potential measurements. By increasing [K+]0 to 5 mM, the warm-up phenomena started earlier and at [K+]0 7 mM only weak myotonia was detected. The increase of [K+]0 caused a sustained membrane depolarization accompanied with a reduction of myotonic bursts in ADR mice. Retigabine, a Kv7.2-Kv7.5 activator, dose-dependently reduced relaxation deficit of ADR myotonic muscle contraction and promoted the warm-up phenomena. In vitro results of this study suggest that increasing potassium conductivity via activation of voltage-gated potassium channels enhanced the warm-up phenomena, thereby offering a potential therapeutic treatment option for myotonia congenita.

Structural and Functional Changes in the Coupling of Fascial Tissue, Skeletal Muscle, and Nerves During Aging.

Aging is a one-way process associated with profound structural and functional changes in the organism. Indeed, the neuromuscular system undergoes a wide remodeling, which involves muscles, fascia, and the central and peripheral nervous systems. As a result, intrinsic features of tissues, as well as their functional and structural coupling, are affected and a decline in overall physical performance occurs. Evidence from the scientific literature demonstrates that senescence is associated with increased stiffness and reduced elasticity of fascia, as well as loss of skeletal muscle mass, strength, and regenerative potential. The interaction between muscular and fascial structures is also weakened. As for the nervous system, aging leads to motor cortex atrophy, reduced motor cortical excitability, and plasticity, thus leading to accumulation of denervated muscle fibers. As a result, the magnitude of force generated by the neuromuscular apparatus, its transmission along the myofascial chain, joint mobility, and movement coordination are impaired. In this review, we summarize the evidence about the deleterious effect of aging on skeletal muscle, fascial tissue, and the nervous system. In particular, we address the structural and functional changes occurring within and between these tissues and discuss the effect of inflammation in aging. From the clinical perspective, this article outlines promising approaches for analyzing the composition and the viscoelastic properties of skeletal muscle, such as ultrasonography and elastography, which could be applied for a better understanding of musculoskeletal modifications occurring with aging. Moreover, we describe the use of tissue manipulation techniques, such as massage, traction, mobilization as well as acupuncture, dry needling, and nerve block, to enhance fascial repair.

Elevation of extracellular osmolarity improves signs of myotonia congenita in vitro: a preclinical animal study.

KEY POINTS: During myotonia congenita, reduced chloride (Cl- ) conductance results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Repetitive contraction of myotonic muscle decreases or even abolishes myotonic muscle stiffness, a phenomenon called 'warm up'. Pharmacological inhibition of low Cl- channels by anthracene-9-carboxylic acid in muscle from mice and ADR ('arrested development of righting response') muscle from mice showed a relaxation deficit under physiological conditions compared to wild-type muscle. At increased osmolarity up to 400 mosmol L-1 , the relaxation deficit of myotonic muscle almost reached that of control muscle. These effects were mediated by the cation and anion cotransporter, NKCC1, and anti-myotonic effects of hypertonicity were at least partly antagonized by the application of bumetanide. ABSTRACT: Low chloride-conductance myotonia is caused by mutations in the skeletal muscle chloride (Cl- ) channel gene type 1 (CLCN1). Reduced Cl- conductance of the mutated channels results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Exercise decreases muscle stiffness, a phenomena called 'warm up'. To gain further insight into the patho-mechanism of impaired muscle stiffness and the warm-up phenomenon, we characterized the effects of increased osmolarity on myotonic function. Functional force and membrane potential measurements were performed on muscle specimens of ADR ('arrested development of righting response') mice (an animal model for low gCl- conductance myotonia) and pharmacologically-induced myotonia. Specimens were exposed to solutions of increasing osmolarity at the same time as force and membrane potentials were monitored. In the second set of experiments, ADR muscle and pharmacologically-induced myotonic muscle were exposed to an antagonist of NKCC1. Upon osmotic stress, ADR muscle was depolarized to a lesser extent than control wild-type muscle. High osmolarity diminished myotonia and facilitated the warm-up phenomenon as depicted by a faster muscle relaxation time (T90/10 ). Osmotic stress primarily resulted in the activation of the NKCC1. The inhibition of NKCC1 with bumetanide prevented the depolarization and reversed the anti-myotonic effect of high osmolarity. Increased osmolarity decreased signs of myotonia and facilitated the warm-up phenomenon in different in vitro models of myotonia. Activation of NKCC1 activity promotes warm-up and reduces the number of contractions required to achieve normal relaxation kinetics.

Hypermetabolism in B-lymphocytes from malignant hyperthermia susceptible individuals.

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle metabolism which is characterized by generalized muscle rigidity, increased body temperature, rhabdomyolysis, and severe metabolic acidosis. The underlying mechanism of MH involves excessive Ca(2+) release in myotubes via the ryanodine receptor type 1 (RyR1). As RyR1 is also expressed in B-lymphocytes, this study investigated whether cellular metabolism of native B-lymphocytes was also altered in MH susceptible (MHS) individuals. A potent activator of RyR1, 4-chloro-m-cresol (4-CmC) was used to challenge native B-lymphocytes in a real-time, metabolic assay based on a pH-sensitive silicon biosensor chip. At the cellular level, a dose-dependent, phasic acidification occurred with 4-CmC. The acidification rate, an indicator of metabolic activation, was significantly higher in B-lymphocytes from MHS patients and required 3 to 5 fold lower concentrations of 4-CmC to evoke similar acidification rates to MHN. Native B-lymphocytes from MHS individuals are more sensitive to 4-CmC than those from MHN, reflecting a greater Ca(2+) turnover. The acidification response, however, was less pronounced than in muscle cells, presumably reflecting the lower expression of RyR1 in B-lymphocytes.

Effects of intravenous sulfide during resuscitated porcine hemorrhagic shock*.

OBJECTIVE: Controversial data are available on the effects of hydrogen sulfide during hemorrhage. Because the clinical significance of hydrogen sulfide administration in rodents may not be applicable to larger species, we tested the hypothesis whether intravenous Na2S (sulfide) would beneficially influence organ dysfunction during long-term, porcine hemorrhage and resuscitation. DESIGN: Prospective, controlled, randomized study. SETTING: University animal research laboratory. SUBJECTS: Forty-five domestic pigs of either gender. INTERVENTIONS: Anesthetized and instrumented animals underwent 4 hrs of hemorrhage (removal of 40% of the blood volume and subsequent blood removal/retransfusion to maintain mean arterial pressure at 30 mm Hg). Sulfide infusion was started 2 hrs before hemorrhage, simultaneously with blood removal or at the beginning of retransfusion of shed blood, and continued for 12 hrs. Resuscitation comprised hydroxyethyl starch and norepinenephrine infusion titrated to maintain mean arterial pressure at preshock values. MEASUREMENTS AND MAIN RESULTS: Before, immediately at the end of and 12 and 22 hrs after hemorrhage, we measured systemic and regional hemodynamics (portal vein, hepatic and right kidney artery ultrasound flow probes) and oxygen transport, nitric oxide and cytokine production (nitrate+nitrite, interleukin-6, tumor necrosis factor-α levels). Postmortem biopsies were analyzed for histomorphology (hematoxylin and eosin staining) and DNA damage (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling staining). The progressive kidney (creatinine levels, creatinine clearance), liver (transaminase activities, bilirubin levels), and cardiocirculatory (norepipnehrine requirements, troponin I levels) dysfunction was attenuated in the simultaneous treatment group only, which coincided with reduced lung, liver, and kidney histological damage. Sulfide reduced mortality, however, irrespective of the timing of its administration. CONCLUSIONS: While the sulfide-induced protection against organ injury was only present when initiated simultaneously with blood removal, it was largely unrelated to hypothermia. The absence of sulfide-mediated protection in the pretreatment protocol may be due to the accumulation of sulfide during low flow states. In conclusion, sulfide treatment can be effective in hemorrhagic shock, but its effectiveness is restricted to a narrow timing and dosing window.