Rare genetic variants impact muscle strength.

Muscle strength is highly heritable and predictive for multiple adverse health outcomes including mortality. Here, we present a rare protein-coding variant association study in 340,319 individuals for hand grip strength, a proxy measure of muscle strength. We show that the exome-wide burden of rare protein-truncating and damaging missense variants is associated with a reduction in hand grip strength. We identify six significant hand grip strength genes, KDM5B, OBSCN, GIGYF1, TTN, RB1CC1, and EIF3J. In the example of the titin (TTN) locus we demonstrate a convergence of rare with common variant association signals and uncover genetic relationships between reduced hand grip strength and disease. Finally, we identify shared mechanisms between brain and muscle function and uncover additive effects between rare and common genetic variation on muscle strength.

Modern understanding of intussusception and recent trends in management / A gyermekkori invaginatio modern értelmezése és kezelése

Intussusception is one of the most common abdominal emergencies in children. The understanding of its aetiology and management has changed significantly over the last decades. Earlier, the hypertrophic Peyer's patches and polyps were considered responsible, but with the knowledge obtained from the lipopolysaccharide-induced animal model of intussusception, the rotavirus vaccination, the seasonality and the postnatal changes of the enteric nervous system it became clear that the intestinal motility plays a key role in the aetiology. The efficacy of non-operative management is continuously improving. The radiologists initially moved from the hydrostatic X-ray-controlled reduction towards the air enema (pneumatic reduction), however, nowadays, there is a shift back to hydrostatic procedures but under ultrasound guidance to reduce radiation exposure. In many institutions, intussusception is managed as day-case rather than as an inpatient case. The role of medications like glucagon and cyclo-oxygenase inhibitors used during reduction manoeuvres and prevention of recurrence is still controversial. Surgical management is shifting towards laparoscopy. The authors herein reviewed the current literature to present recent insights into understanding the pathogenesis and management updates. Orv Hetil. 2020; 161(32): 1331-1338.

High Time Resolution Analysis of Voltage-Dependent and Voltage-Independent Calcium Sparks in Frog Skeletal Muscle Fibers.

In amphibian skeletal muscle calcium (Ca2+) sparks occur both as voltage-dependent and voltage-independent ligand-activated release events. However, whether their properties and their origin show similarities are still in debate. Elevated K+, constant Cl- content solutions were used to initiate small depolarizations of the resting membrane potential to activate dihydropyridine receptors (DHPR) and caffeine to open ryanodine receptors (RyR) on intact fibers. The properties of Ca2+ sparks observed under control conditions were compared to those measured on depolarized cells and those after caffeine treatment. Calcium sparks were recorded on intact frog skeletal muscle fibers using high time resolution confocal microscopy (x-y scan: 30 Hz). Sparks were elicited by 1 mmol/l caffeine or subthreshold depolarization to different membrane potentials. Both treatments increased the frequency of sparks and altered their morphology. Images were analyzed by custom-made computer programs. Both the amplitude (in ΔF/F0; 0.259 ± 0.001 vs. 0.164 ± 0.001; n = 24942 and 43326, respectively; mean ± SE, p < 0.001) and the full width at half maximum (FWHM, in µm; parallel with fiber axis: 2.34 ± 0.01 vs. 1.92 ± 0.01, p < 0.001; perpendicular to fiber axis: 2.08 ± 0.01 vs. 1.68 ± 0.01, p < 0.001) of sparks was significantly greater after caffeine treatment than on depolarized cells. 9.8% of the sparks detected on depolarized fibers and about one third of the caffeine activated sparks (29.7%) overlapped with another one on the previous frame on x-y scans. Centre of overlapping sparks travelled significantly longer distances between consecutive frames after caffeine treatment then after depolarization (in µm; 1.66 ± 0.01 vs. 0.95 ± 0.01, p < 0.001). Our results suggest that the two types of ryanodine receptors, the junctional RyRs controlled by DHPRs and the parajunctional RyRs are activated independently, using alternate ways, with the possibility of cooperation between neighboring release channels.

Capsaicin-induced rapid neutrophil leukocyte activation in the rat urinary bladder microcirculatory bed.

AIMS: This study was initiated to investigate the involvement of neutrophil leukocyte activation in neurogenic inflammation, a process also involved in human urinary pathologies, elicited in the rat urinary bladder by the local administration of capsaicin, the archetypal TRPV1 agonist. The contribution of afferent nerves and sensory neuropeptides to leukocyte activation in the urinary bladder microcirculatory bed was examined. METHODS: Following a 15-min topical application of capsaicin (50 µM), leukocyte-endothelial interactions were examined for an observation period of 45 min with intravital microscopy. Expression of adhesion molecules E-selectin and ICAM-1 implicated in these interactions was assessed by immunohistochemistry. Selective sensory denervation was performed by neonatal treatment with capsaicin. The role of the TRPV1 receptor and two sensory neuropeptides (CGRP and substance P [SP]) were studied using the selective antagonists capsazepine, CGRP8-37 and RP67580, respectively. RESULTS: Capsaicin induced rapid increases in leukocyte rolling and adhesion and increased the expression of E-selectin and ICAM-1 in the postcapillary venules. Sensory chemodenervation via capsaicin and also TRPV1 receptor antagonism effectively prevented these changes. A similar reduction was observed in leukocyte adhesion after topical application of CGRP8-34 or RP67580, but only CGRP8-34 reduced the capsaicin-evoked leukocyte rolling. CONCLUSIONS: Topical application of capsaicin induces early neurogenically mediated cellular microcirculatory inflammatory reactions via the activation of the TRPV1 receptor and the release of CGRP and SP from sensory nerves in the bladder. Co-administration of SP and CGRP receptor antagonists may ameliorate microcirculatory inflammatory changes elicited by capsaicin in the urinary bladder.

Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-ß1a subunit (CACNB1).

Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of ß1a subunit of the dihydropyridine receptor (DHPR), a channel essential for Ca2+ regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR ß1a subunit and assess its role on Ca2+ metabolism of cultured myotubes. Using differential scanning fluorimetry, we show that mutation V156A causes a significant reduction in thermal stability of the Src homology 3/guanylate kinase core domain of ß1a subunit. Expression of the variant subunit in ß1-null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-clamp analysis of ß1a-V156A-expressing myotubes revealed a -2 mV shift in voltage dependence of channel activation, but no changes in Ca2+ conductance, current kinetics, or sarcoplasmic reticulum Ca2+ load were observed. Measurement of resting free Ca2+ and Na+ concentrations shows that both cations were significantly elevated in ß1a-V156A-expressing myotubes and that these changes were linked to increased rates of plasmalemmal Ca2+ entry through Na+/Ca2+ exchanger and/or transient receptor potential canonical channels. Overall, our data show that mutant variant V156A results in instability of protein subdomains of ß1a subunit leading to a phenotype of Ca2+ dysregulation that partly resembles that of other MH-linked mutations of DHPR α1S subunit. These data prove that homozygous expression of variant ß1a-V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.

STIM-TRP Pathways and Microdomain Organization: Ca2+ Influx Channels: The Orai-STIM1-TRPC Complexes.

Ca2+ influx by plasma membrane Ca2+ channels is the crucial component of the receptor-evoked Ca2+ signal. The two main Ca2+ influx channels of non-excitable cells are the Orai and TRPC families of Ca2+ channels. These channels are activated in response to cell stimulation and Ca2+ release from the endoplasmic reticulum (ER). The protein that conveys the Ca2+ content of the ER to the plasma membrane is the ER Ca2+ sensor STIM1. STIM1 activates the Orai channels and is obligatory for channel opening. TRPC channels can function in two modes, as STIM1-dependent and STIM1-independent. When activated by STIM1, both channel types function at the ER/PM (plasma membrane) junctions. This chapter describes the properties and regulation of the channels by STIM1, with emphasis how and when TRPC channels function as STIM1-dependent and STIM1-independent modes and their unique Ca2+-dependent physiological functions that are not shared with the Orai channels.

Dietary selenium augments sarcoplasmic calcium release and mechanical performance in mice.

BACKGROUND: As an essential trace element selenium plays a significant role in many physiological functions of the organs. It is found within muscles as selenocystein in selenoprotein N, which is involved in redox-modulated calcium homeostasis and in protection against oxidative stress. METHODS: The effects of two different selenium compounds (selenate and NanoSe in 0.5 and 5 ppm concentration for two weeks) on muscle properties of mice were examined by measuring in vivo muscle performance, in vitro force in soleus (SOL) and extensor digitorum longus (EDL) muscles and changes in intracellular Ca2+ concentration in single fibers from flexor digitorum brevis (FDB) muscle.. Western-blot analysis on muscle lysates of EDL and SOL were used to measure the selenoprotein N expression. Control mice received 0.3 ppm Se. RESULTS: While the grip force did not change, 5 ppm selenium diets significantly increased the speed of voluntary running and the daily distance covered. Both forms of selenium increased significantly the amplitude of single twitches in EDL and SOL muscle in a concentration dependent manner. Selenate increased fatigue resistance in SOL. The amplitude of the calcium transients evoked by KCl depolarization increased significantly from the control of 343 ± 44 nM to 671 ± 51 nM in the presence of 0.5 ppm selenate in FDB fibers. In parallel, the rate of calcium release during short depolarizations increased significantly from 28.4 ± 2.2 to 45.5 ± 3.8 and 52.1 ± 1.9 µM/ms in the presence of 0.5 ppm NanoSe and selenate, respectively. In 0.5 ppm concentration both selenium compounds increased significantly the selenoprotein N expression only in EDL muscle. CONCLUSIONS: Selenium supplementation augments calcium release from the sarcoplasmic reticulum thus improves skeletal muscle performance. These effects are accompanied by the increased selenoprotein N expression in the muscles which could result in increased oxidative stress tolerance in case of long lasting contraction.

Cannabinoid signalling inhibits sarcoplasmic Ca2+ release and regulates excitation-contraction coupling in mammalian skeletal muscle.

KEY POINTS: Marijuana was found to cause muscle weakness, although the exact regulatory role of its receptors (CB1 cannabinoid receptor; CB1R) in the excitation-contraction coupling (ECC) of mammalian skeletal muscle remains unknown. We found that CB1R activation or its knockout did not affect muscle force directly, whereas its activation decreased the Ca2+ -sensitivity of the contractile apparatus and made the muscle fibres more prone to fatigue. We demonstrate that CB1Rs are not connected to the inositol 1,4,5-trisphosphate pathway either in myotubes or in adult muscle fibres. By contrast, CB1Rs constitutively inhibit sarcoplasmic Ca2+ release and sarcoplasmic reticulum Ca2+ ATPase during ECC in a Gi/o protein-mediated way in adult skeletal muscle fibres but not in myotubes. These results help with our understanding of the physiological effects and pathological consequences of CB1R activation in skeletal muscle and may be useful in the development of new cannabinoid drugs. ABSTRACT: Marijuana was found to cause muscle weakness, although it is unknown whether it affects the muscles directly or modulates only the motor control of the central nervous system. Although the presence of CB1 cannabinoid receptors (CB1R), which are responsible for the psychoactive effects of the drug in the brain, have recently been demonstrated in skeletal muscle, it is unclear how CB1R-mediated signalling affects the contraction and Ca²âº homeostasis of mammalian skeletal muscle. In the present study, we demonstrate that in vitro CB1R activation increased muscle fatigability and decreased the Ca2+ -sensitivity of the contractile apparatus, whereas it did not alter the amplitude of single twitch contractions. In myotubes, CB1R agonists neither evoked, nor influenced inositol 1,4,5-trisphosphate (IP3 )-mediated Ca2+ transients, nor did they alter excitation-contraction coupling. By contrast, in isolated muscle fibres of wild-type mice, although CB1R agonists did not evoke IP3 -mediated Ca2+ transients too, they significantly reduced the amplitude of the depolarization-evoked transients in a pertussis-toxin sensitive manner, indicating a Gi/o protein-dependent mechanism. Concurrently, on skeletal muscle fibres isolated from CB1R-knockout animals, depolarization-evoked Ca2+ transients, as well qas Ca2+ release flux via ryanodine receptors (RyRs), and the total amount of released Ca2+ was significantly greater than that from wild-type mice. Our results show that CB1R-mediated signalling exerts both a constitutive and an agonist-mediated inhibition on the Ca2+ transients via RyR, regulates the activity of the sarcoplasmic reticulum Ca2+ ATPase and enhances muscle fatigability, which might decrease exercise performance, thus playing a role in myopathies, and therefore should be considered during the development of new cannabinoid drugs.

A novel method for in vivo visualization of the microcirculation of the mandibular periosteum in rats.

OBJECTIVE: The periosteum plays an important role in bone physiology, but observation of its microcirculation is greatly limited by methodological constraints at certain anatomical locations. This study was conducted to develop a microsurgical procedure which provides access to the mandibular periosteum in rats. METHODS: Comparisons of the microcirculatory characteristics with those of the tibial periosteum were performed to confirm the functional integrity of the microvasculature. The mandibular periosteum was reached between the facial muscles and the anterior surface of the superficial masseter muscle at the external surface of the mandibular corpus; the tibial periosteum was prepared by dissecting the covering muscles at the anteromedial surface. Intravital fluorescence microscopy was used to assess the leukocyte-endothelial interactions and the RBCV in the tibial and mandibular periosteum. Both structures were also visualized through OPS and fluorescence CLSM. RESULTS: The microcirculatory variables in the mandibular periosteum proved similar to those in the tibia, indicating that no microcirculatory failure resulted from the exposure technique. CONCLUSION: This novel surgical approach provides simple access to the mandibular periosteum of the rat, offering an excellent opportunity for investigations of microcirculatory manifestations of dentoalveolar and maxillofacial diseases.

Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling.

Myostatin, a member of the transforming growth factor ß family, is a potent negative regulator of skeletal muscle growth, as myostatin-deficient mice show a great increase in muscle mass. Yet the physical performance of these animals is reduced. As an explanation for this, alterations in the steps in excitation-contraction coupling were hypothesized and tested for in mice with the 12 bp deletion in the propeptide region of the myostatin precursor (Mstn(Cmpt-dl1Abc) or Cmpt). In voluntary wheel running, control C57BL/6 mice performed better than the mutant animals in both maximal speed and total distance covered. Despite the previously described lower specific force of Cmpt animals, the pCa-force relationship, determined on chemically permeabilized fibre segments, did not show any significant difference between the two mouse strains. While resting intracellular Ca(2+) concentration ([Ca(2+)]i) measured on single intact flexor digitorum brevis (FDB) muscle fibres using Fura-2 AM was similar to control (72.0 ± 1.7 vs. 78.1 ± 2.9 nM, n = 38 and 45), the amplitude of KCl-evoked calcium transients was smaller (360 ± 49 vs. 222 ± 45 nM, n = 22) in the mutant strain. Similar results were obtained using tetanic stimulation and Rhod-2 AM, which gave calcium transients that were smaller (2.42 ± 0.11 vs. 2.06 ± 0.10 ΔF/F0, n = 14 and 13, respectively) on Cmpt mice. Sarcoplasmic reticulum (SR) calcium release flux calculated from these transients showed a reduced peak (23.7 ± 3.0 vs. 15.8 ± 2.1 mM s(-1)) and steady level (5.7 ± 0.7 vs. 3.7 ± 0.5 mM s(-1)) with no change in the peak-to-steady ratio. The amplitude and spatial spread of calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results suggest that reduced SR calcium release underlies the reduced muscle force in Cmpt animals.

Phosphoinositide substrates of myotubularin affect voltage-activated Ca²âº release in skeletal muscle.

Skeletal muscle excitation­contraction (E­C) coupling is altered in several models of phosphatidylinositol phosphate (PtdInsP) phosphatase deficiency and ryanodine receptor activity measured in vitro was reported to be affected by certain PtdInsPs, thus prompting investigation of the physiological role of PtdInsPs in E­C coupling. We measured intracellular Ca2+ transients in voltage-clamped mouse muscle fibres microinjected with a solution containing a PtdInsP substrate (PtdIns(3,5)P2 or PtdIns(3)P) or product (PtdIns(5)P or PtdIns) of the myotubularin phosphatase MTM1. No significant change was observed in the presence of either PtdIns(5)P or PtdIns but peak SR Ca2+ release was depressed by ~30% and 50% in fibres injected with PtdIns(3,5)P2 and PtdIns(3)P, respectively, with no concurrent alteration in the membrane current signals associated with the DHPR function as well as in the voltage dependence of Ca2+ release inactivation. In permeabilized muscle fibres, the frequency of spontaneous Ca2+ release events was depressed in the presence of the three tested phosphorylated forms of PtdInsP with PtdIns(3,5)P2 being the most effective, leading to an almost complete disappearance of Ca2+ release events. Results support the possibility that pathological accumulation of MTM1 substrates may acutely depress ryanodine receptor-mediated Ca2+ release. Overexpression of a mCherry-tagged form of MTM1 in muscle fibres revealed a striated pattern consistent with the triadic area. Ca2+ release remained although unaffected by MTM1 overexpression and was also unaffected by the PtdIns-3-kinase inhibitor LY2940002, suggesting that the 3-phosphorylated PtdIns lipids active on voltage-activated Ca2+ release are inherently maintained at a low level, inefficient on Ca2+ release in normal conditions.

[Experimental studies on microcirculatory inflammatory reactions of the urinary bladder]. / A húgyhólyag gyulladásos mikrokeringési reakcióinak kísérletes vizsgálata.

BACKGROUND: The vascular endothelium is a primary target of ischemia/reperfusion (IR) injury of the urinary bladder. In case of interstitial cystitis (painful bladder syndrome) or in cyclophosphamide-induced hemorrhagic cystitis, the injury is initiated at the epithelial/urothelial surface and propagates towards the interstitium, causing secondary involvement of the microvasculature. Hence the aim of our study was to assess and compare the microcirculatory aspects of the non-infectious forms of cystitis with that of IR-caused reactions. MATERIALS AND METHODS: In male Sprague-Dawley rats, interstitial cystitis was induced by intravesical instillation of protamine sulphate (2 mg in 200 µl saline for 30 min; n = 6). In another group, cyclophosphamide (75 mg/kg, ip) was administered 24 hr prior to the experiments (n = 5). In the third group, urinary bladder ischemia was induced by 60-min occlusion of the vessels supplying the bladder (n = 5). The microcirculatory inflammatory reactions were investigated by fluorescence intravital microscopy 60 min after reperfusion and 24 hr after protamine sulphate instillation or cyclophosphamide administration, respectively. In the control group, the bladder was instilled with saline (n = 5). RESULTS: Rolling of leukocytes increased ~3-fold in the postcapillary vessels in the protamine sulphate-treated group and the increase in this parameter was ~5 and ~6.5-fold in cyclophosphamide and IR groups, respectively. The increase in leukocyte adherence reached similar, approx. 7-fold increase in each of the challenged groups. The red blood cell velocity in the capillaries decreased in the protamine sulphate and IR groups, while the velocity increased moderately in the cyclophosphamide-treated group. CONCLUSIONS: Our results demonstrate that direct endothelial injury (caused by IR), as well as protamine sulphate and cyclophosphamide administrations induce inflammatory microcirculatory changes of the urinary bladder. These observations suggest a causative role for microcirculatory disturbances in the pathogenesis of interstitial cystitis and hemorrhagic cystitis as well.