Cholesterol-Depletion-Induced Membrane Repair Carries a Raft Conformer of P-Glycoprotein to the Cell Surface, Indicating Enhanced Cholesterol Trafficking in MDR Cells, Which Makes Them Resistant to Cholesterol Modifications.

The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane's raft and non-raft domains. One specific conformation of P-gp that binds to the monoclonal antibody UIC2 is primarily associated with raft domains and displays heightened internalization in cells overexpressing P-gp, such as in NIH-3T3 MDR1 cells. Our primary objective was to investigate whether the trafficking of this particular P-gp conformer is dependent on cholesterol levels. Surprisingly, depleting cholesterol using cyclodextrin resulted in an unexpected increase in the proportion of raft-associated P-gp within the cell membrane, as determined by UIC2-reactive P-gp. This increase appears to be a compensatory response to cholesterol loss from the plasma membrane, whereby cholesterol-rich raft micro-domains are delivered to the cell surface through an augmented exocytosis process. Furthermore, this exocytotic event is found to be part of a complex trafficking mechanism involving lysosomal exocytosis, which contributes to membrane repair after cholesterol reduction induced by cyclodextrin treatment. Notably, cells overexpressing P-gp demonstrated higher total cellular cholesterol levels, an increased abundance of stable lysosomes, and more effective membrane repair following cholesterol modifications. These modifications encompassed exocytotic events that involved the transport of P-gp-carrying rafts. Importantly, the enhanced membrane repair capability resulted in a durable phenotype for MDR1 expressing cells, as evidenced by significantly improved viabilities of multidrug-resistant Pgp-overexpressing immortal NIH-3T3 MDR1 and MDCK-MDR1 cells compared to their parents when subjected to cholesterol alterations.

Laser induced calcium oscillations in fluorescent calcium imaging.

Phototoxicity is the most common problem investigators may encounter when performing live cell imaging. It develops due to excess laser exposure of cells loaded with fluorophores and can lead to often overlooked but significant artifacts, such as massive increase of intracellular Ca2+ concentration, which would make data interpretation problematic. Because information about laser- and dye-related changes in cytoplasmic calcium concentration is very limited, we aimed to describe this phenomenon to help investigators using laser scanning confocal microscopy in a non-invasive way. Therefore, in the present study we evaluated fluorescent fluctuations, which evolved in Fluo-3/4/8 loaded mouse pancreatic acinar cells during very low intensity laser excitation. We demonstrate that after standard loading procedure (2 µM Fluo-3/4/8-AM, 30 min at room temperature), applying 488 nm laser at as low as ca. 10 µW incident laser power (0.18 µW/µm2) at 1 Hz caused repetitive, 2-3 fold elevations of the resting intracellular fluorescence. The first latency and the pattern of the fluorescence fluctuations were laser power dependent and were related to Ca2+-release from intracellular stores, as they were abolished by BAPTA-AM treatment in Ca2+-free medium, but were not diminished by the reactive oxygen species (ROS) scavenger DMPO. Worryingly enough, the qualitative and quantitative features of the Ca2+-waves were practically indistinguishable from the responses evoked by secretagogue stimulation. Since using similar imaging conditions, a number of other cell types were reported to display spontaneous Ca2+ oscillations, we propose strategies to distinguish the real signals from artifacts.

SOCE Is Important for Maintaining Sarcoplasmic Calcium Content and Release in Skeletal Muscle Fibers.

Store-operated Ca2+ entry (SOCE) is a Ca2+-entry process activated by the depletion of intracellular stores and has an important role in many cell types. In skeletal muscle, however, its role during physiological muscle activation has been controversial. To address this question, sarcoplasmic reticulum (SR) calcium release in a mouse strain with a naturally occurring mutation in the myostatin gene (Compact (Cmpt)) leading to a hypermuscular yet reduced muscle-force phenotype was compared to that in wild-type mice. To elicit Ca2+ release from the SR of flexor digitorum brevis (FDB) fibers, either a ryanodine receptor agonist (4-chloro-meta-cresol) or depolarizing pulses were used. In muscles from Cmpt mice, endogenous protein levels of STIM1 and Orai1 were reduced, and consequently, SOCE after 4-chloro-meta-cresol-induced store depletion was suppressed. Although the voltage dependence of SR calcium release was not statistically different between wild-type and Cmpt fibers, the amount of releasable calcium was significantly reduced in the latter, indicating a smaller SR content. To assess the immediate role of SOCE in replenishing the SR calcium store, the evolution of intracellular calcium concentration during a train of long-lasting depolarizations to a maximally activating voltage was monitored. Cmpt mice exhibited a faster decline in calcium release, suggesting a compromised ability to refill the SR. A simple model that incorporates a reduced SOCE as an important partner in regulating immediate calcium influx through the surface membrane readily accounts for the steady-state reduction in SR calcium content and its more pronounced decline after calcium release.

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.

HMGB1 expression and muscle regeneration in idiopathic inflammatory myopathies and degenerative joint diseases.

The High-Mobility Group Box 1 protein (HMGB1) is a known nuclear protein which may be released from the nucleus into the cytoplasm and the extracellular space. It is believed that the mobilized HMGB1 plays role in the autoimmune processes as an alarmin, stimulating the immune response. In addition, muscle regeneration and differentiation may also be altered in the inflammatory surroundings. Biopsy specimens derived from patients with idiopathic inflammatory myopathies (IIM) such as polymyositis or dermatomyositis were compared to muscle samples from patients undergoing surgical interventions for coxarthrosis. The biopsy and surgery specimens were used for Western blot analysis, for immunohistochemical detection of HMGB1 in histological preparations and for cell culturing to examine cell proliferation and differentiation. Our data show lower HMGB1 expression, impaired proliferation and slightly altered fusion capacity in the primary cell cultures started from IIM specimens than in cultures of coxarthrotic muscles. The ratio of regenerating muscle fibres with centralised nuclei (myotubes) is lower in the IIM samples than in the coxarthrotic ones but corticosteroid treatment shifts the ratio towards the coxarthrotic value. Our data suggest that the impaired regeneration capacity should also be considered to be behind the muscle weakness in IIM patients. The role of HMGB1 as a pathogenic signal requires further investigation.

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.

Activation of muscarinic receptors increases the activity of the granule neurones of the rat dorsal cochlear nucleus--a calcium imaging study.

Acetylcholine modulates the function of the cochlear nucleus via several pathways. In this study, the effects of cholinergic stimulation were studied on the cytoplasmic Ca(2+) concentration of granule neurones of the rat dorsal cochlear nucleus (DCN). Ca(2+) transients were recorded in Oregon-Green-BAPTA 1-loaded brain slices using a calcium imaging technique. For the detection, identification and characterisation of the Ca(2+) transients, a wavelet analysis-based method was developed. Granule cells were identified on the basis of their size and localisation. The action potential-coupled character of the Ca(2+) transients of the granule cells was established by recording fluorescence changes and electrical activity simultaneously. Application of the cholinergic agonist carbamyl-choline (CCh) significantly increased the frequency of the Ca(2+) transients (from 0.37 to 6.31 min(-1), corresponding to a 17.1-fold increase; n = 89). This effect was antagonised by atropine, whereas CCh could still evoke an 8.3-fold increase of the frequency of the Ca(2+) transients when hexamethonium was present. Using immunolabelling, the expression of both type 1 and type 3 muscarinic receptors (M1 and M3 receptors, respectively) was demonstrated in the granule cells. Application of 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (an M3-specific antagonist) prevented the onset of the CCh effect, whereas an M1-specific antagonist (pirenzepine) was less effective. We conclude that cholinergic stimulation increases the activity of granule cells, mainly by acting on their M3 receptors. The modulation of the firing activity of the granule cells, in turn, may modify the firing of projection neurones and may adjust signal processing in the entire DCN.

Fluvastatin-induced alterations of skeletal muscle function in hypercholesterolaemic rats.

Although statins, the most widely used drugs in the treatment of hyperlipidaemia, are generally accepted as efficient and safe drugs their side-effects on skeletal muscle have been reported with increasing frequency. The lack of an animal model in which these side effects would consistently be observed is one of the important drawbacks in studying statin associated myopathy. To overcome this and enable the studying of the effects of fluvastatin on skeletal muscles an animal model with high blood cholesterol levels was developed. In these animals cholesterol levels rose more than seven fold (from 1.5 ± 0.1 to 10.7 ± 2.0 mmol/l; n = 15 and 16) with a dramatic increase in low density lipoprotein/high density lipoprotein ratio (from 0.29 ± 0.02 to 1.56 ± 0.17). While the latter was reversed by statin treatment, an elevation in blood creatine kinase (CK) level indicated the presence of muscle wasting. Fibers from m. extensor digitorum longus (EDL) showed significant reduction in cross sectional area in the statin treated groups. Statin treatment also decreased the proliferation and fusion of skeletal myotubes in culture. In line with this, resting intracellular calcium concentration ([Ca(2+)](i)) was reduced in statin treated satellite cells and myotubes. On the other hand, in adult skeletal muscle fibers statin treatment increased resting [Ca(2+)](i) (116 ± 4 nM vs. 151 ± 5 nM; n = 33 and 34) and decreased both twitch and tetanic force both in EDL and m. soleus. In addition, in m. soleus the duration of twitch and tetanic force was shortened. These results clearly indicate that statin administration in these animals results in a myopathy characterized by decreased muscle force and elevated plasma CK level.

Improved spark and ember detection using stationary wavelet transforms.

Calcium sparks and embers are localized intracellular events of calcium release in muscle cells studied frequently by confocal microscopy using line-scan imaging. The large quantity of images and large number of events require automatic detection procedures based on signal processing methods. In the past decades these methods were based on thresholding procedures. Although, recently, wavelet transforms were also introduced, they have not become widespread. We have implemented a set of algorithms based on one- and two-dimensional versions of the à trous wavelet transform. The algorithms were used to perform spike filtering, denoising and detection procedures. Due to the dependence of the algorithms on user adjustable parameters, their effect on the efficiency of the algorithm was studied in detail. We give methods to avoid false positive detections which are the consequence of the background noise in confocal images. In order to establish the efficiency and reliability of the algorithms, various tests were performed on artificial and experimental images. Spark parameters (amplitude, full width-at-half maximum) calculated using the traditional and the wavelet methods were compared. We found that the latter method is capable of identifying more events with better accuracy on experimental images. Furthermore, we extended the wavelet-based transform from calcium sparks to long-lasting small-amplitude events as calcium embers. The method not only solved their automatic detection but enabled the identification of events with small amplitude that otherwise escaped the eye, rendering the determination of their characteristic parameters more accurate.

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.