Gelsolin Treatment Research Articles

Is there a renal protective role for gelsolin treatment in crush syndrome? An experimental study.

This study aims to investigate the impact of combining crush fluid resuscitation with gelsolin treatment on renal function in a rat model of crush syndrome. Twenty-four adult female Wistar albino rats were randomly assigned to one of three groups for crush syndrome treat-ment: Control (C) group, gelsolin + crush fluid (gel) group, and crush fluid only (CF) group, each containing eight rats. Sedated rats underwent unilateral hind limb compression of 2 kg using a compression device, maintained for five hours. The control group received no treatment post-compression. After removing the tourniquet, rats in the gelsolin group received an intravenous administration of recombinant human gelsolin at a dose of 2 mg/kg in 0.1 ml sterile saline, along with crush fluid. The CF group received only the crush solution. At 24 hours, creatine kinase (CK) levels in the CF group were lower compared to those in the control and gelsolin + CF groups (132 IU vs. 630 IU [p=0.004] and 519.5 IU [p=0.014], respectively). By 48 hours, CK levels in both CF and gelsolin + CF groups were lower than in the control group (p<0.001 and p=0.014, respectively), with no significant difference between the CF and gelsolin + CF groups (p=0.773). At 72 hours, CK levels in the gelsolin + CF group were lower than in the control group (p=0.023) but comparable to the CF group (p>0.05). Blood urea nitrogen (BUN) levels at 24 and 72 hours were similar in the control and gelsolin + CF groups (p>0.05). At 48 hours, BUN levels in both CF and gelsolin + CF groups were lower than in the control group (p=0.001 and p=0.003, respectively), with no significant difference between the CF and Gelsolin + CF groups (p>0.05). At 24 hours, creatinine levels in the gelsolin + CF group were lower than in the control group (p=0.017), while levels in the CF and gelsolin + CF groups were similar (p>0.05). By 48 and 72 hours, creatinine levels in both CF and gelsolin + CF groups were similar but lower than in the control group (p<0.05). Changes in creatinine levels were comparable across all groups (p>0.05). This study marks the first instance in literature where it has been demonstrated that administering gelsolin along with crush solution does not yield superior results compared to crush solution alone in treating crush syndrome. Nonetheless, further research utilizing varying doses of gelsolin is warranted.

Gelsolin expression in liver, spleen and blood plasma provides insights into its function in mice following acute insults

Background: Gelsolin (GSN) is an actin-binding plasma protein with a pivotal role in the systemic response to acute tissue damage. The present study investigated GSN expression in the liver, spleen and blood serum in mice after burn. Method: A murine model of thermal injury was selected, and the animals were sacrificed at 8, 24, 48 and 72 h after injury. Real-time quantitative polymerase chain reaction (RT-PCR) was performed to determine the messenger RNA (mRNA) expression of GSN, and GSN protein expression was determined by enzyme-linked immunosorbent assay (ELISA). Results: We found that GSN mRNA and protein were expressed in the liver, spleen and blood serum of the mice. GSN expression in these tissues was the lowest among the tested time points at 8 h after burn injury. The mortality within 72 h among the mice subjected to burn injury was significantly lower in those treated with GSN than in those not treated with GSN. Treatment with GSN markedly increased the GSN levels in the liver, spleen and blood serum after injury. Conclusion: These results indicated that GSN treatment may affect the outcome of thermal injury via changes in the GSN content of multiple tissues.

Regeneration of Actin Filaments in Actin-Extracted Bumblebee Flight Muscle Fibers as Probed by X-ray Diffraction

Asynchronous flight muscle enables the insects to beat their wings at high frequencies not achievable by ordinary twitch-type muscles. To understand the mechanism of action of asynchronous flight muscle, it is essential to know which of the constituent contractile proteins (many of them are expressed as flight muscle-specific isoforms) is/are indispensable or replaceable. For this purpose, we tried to extract myosin from the flight muscle and replace it with that of vertebrate skeletal muscle, by increasing ionic strength. However, this procedure seemed to extract other proteins important for maintaining the integrity of the myofibril (2015 annual meeting). Alternatively, we have established a protocol to extract actin from bumblebee flight muscle fibers by using gelsolin, an actin-severing protein. After treatment with gelsolin, actin layer line reflections disappeared from the X-ray diffraction pattern, but reflections from myosin filaments and their lattice remained basically intact. The results show that gelsolin treatment is a method suitable for extracting actin and its associated proteins without sacrificing the structural integrity of the myofibril. As a next step, we are currently trying to introduce exogenous actin (from vertebrate skeletal muscle) and to see how the contractile properties of the flight muscle fibers are affected.

Treatment with gelsolin reduces brain inflammation and apoptotic signaling in mice following thermal injury

BackgroundBurn survivors develop long-term cognitive impairment with increased inflammation and apoptosis in the brain. Gelsolin, an actin-binding protein with capping and severing activities, plays a crucial role in the septic response. We investigated if gelsolin infusion could attenuate neural damage in burned mice.MethodsMice with 15% total body surface area burns were injected intravenously with bovine serum albumin as placebo (2 mg/kg), or with low (2 mg/kg) or high doses (20 mg/kg) of gelsolin. Samples were harvested at 8, 24, 48 and 72 hours postburn. The immune function of splenic T cells was analyzed. Cerebral pathology was examined by hematoxylin/eosin staining, while activated glial cells and infiltrating leukocytes were detected by immunohistochemistry. Cerebral cytokine mRNAs were further assessed by quantitative real-time PCR, while apoptosis was evaluated by caspase-3. Neural damage was determined using enzyme-linked immunosorbent assay of neuron-specific enolase (NSE) and soluble protein-100 (S-100). Finally, cerebral phospho-ERK expression was measured by western blot.ResultsGelsolin significantly improved the outcomes of mice following major burns in a dose-dependent manner. The survival rate was improved by high dose gelsolin treatment compared with the placebo group (56.67% vs. 30%). Although there was no significant improvement in outcome in mice receiving low dose gelsolin (30%), survival time was prolonged against the placebo control (43.1 ± 4.5 h vs. 35.5 ± 5.0 h; P < 0.05). Burn-induced T cell suppression was greatly alleviated by high dose gelsolin treatment. Concurrently, cerebral abnormalities were greatly ameliorated as shown by reduced NSE and S-100 content of brain, decreased cytokine mRNA expressions, suppressed microglial activation, and enhanced infiltration of CD11b+ and CD45+ cells into the brain. Furthermore, the elevated caspase-3 activity seen following burn injury was remarkably reduced by high dose gelsolin treatment along with down-regulation of phospho-ERK expression.ConclusionExogenous gelsolin infusion improves survival of mice following major burn injury by partially attenuating inflammation and apoptosis in brain, and by enhancing peripheral T lymphocyte function as well. These data suggest a novel and effective strategy to combat excessive neuroinflammation and to preserve cognition in the setting of major burns.

Regulatory mechanism of smooth muscle contraction studied with gelsolin-treated strips of taenia caeci in guinea pig

The potential roles of the regulatory proteins actin, tropomyosin (Tm), and caldesmon (CaD), i.e., the components of the thin filament, in smooth muscle have been extensively studied in several types of smooth muscles. However, controversy remains on the putative physiological significance of these proteins. In this study, we intended to determine the functional roles of Tm and CaD in the regulation of smooth muscle contraction by using a reconstitution system of the thin filaments. At appropriate conditions, the thin (actin) filaments within skinned smooth muscle strips of taenia caeci in guinea pigs could be selectively removed by an actin-severing protein, gelsolin, without irreversible damage to the contractile apparatus, and then the thin filaments were reconstituted with purified components of thin filaments, i.e., actin, Tm, and CaD. We found that the structural remodeling of actin filaments or thin filaments was functionally linked to the Ca(2+)-induced force development and reduction in muscle cross-sectional area (CSA). That is, after the reconstitution of the gelsolin-treated skinned smooth muscle strips with pure actin, the Ca(2+)-dependent force development was partially restored, but the Ca(2+)-induced reduction in CSA occurred once. In contrast, the reconstitution with actin, followed by Tm and CaD, restored not only the force generation but also both its Ca(2+) sensitivity and the reversible Ca(2+)-dependent reduction in CSA. We confirmed that both removal of the thin filaments by gelsolin treatment and reconstitution of the actin (thin) filaments with Tm and CaD caused no significant changes in the level of myosin regulatory light chain phosphorylation. We thus conclude that Tm and CaD are necessary for the full regulation of smooth muscle contraction in addition to the other regulatory systems, including the myosin-linked one.

Multiple sources of passive stress relaxation in muscle fibres

The forces developed during stretch of nonactivated muscle consist of velocity-sensitive (viscous/viscoelastic) and velocity-insensitive (elastic) components. At the myofibrillar level, the elastic-force component has been described in terms of the entropic-spring properties of the giant protein titin, but entropic elasticity cannot account for viscoelastic properties, such as stress relaxation. Here we examine the contribution of titin to passive stress relaxation of isolated rat-cardiac myofibrils depleted of actin by gelsolin treatment. Monte Carlo simulations show that, up to ∼5 s after a stretch, the time course of stress relaxation can be described assuming unfolding of 1–2 immunoglobulin domains per titin molecule. For extended periods of stress relaxation, the simulations failed to correctly describe the myofibril data, suggesting that in situ, titin-Ig domains may be more stable than predicted in earlier single-molecule atomic-force-microscopy studies. The reasons behind this finding remain unknown; simply assuming a reduced unfolding probability of domains—an effect found here by AFM force spectroscopy on titin-Ig domains in the presence of a chaperone, alpha-B-crystallin—did not help correctly simulate the time course of stress relaxation. We conclude that myofibrillar stress relaxation likely has multiple sources. Evidence is provided that in intact myofibrils, an initial, rapid phase of stress relaxation results from viscous resistance due to the presence of actin filaments.

Molecular Basis of Passive Stress Relaxation in Human Soleus Fibers: Assessment of the Role of Immunoglobulin-Like Domain Unfolding

Titin (also known as connectin) is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is constructed of the PEVK domain and tandem-immunoglobulin segments comprising serially linked immunoglobulin (Ig)-like domains. It is unresolved whether under physiological conditions Ig domains remain folded and act as “spacers” that set the sarcomere length at which the PEVK extends or whether they contribute to titin's extensibility by unfolding. Here we focused on whether Ig unfolding plays a prominent role in stress relaxation (decay of force at constant length after stretch) using mechanical and immunolabeling studies on relaxed human soleus muscle fibers and Monte Carlo simulations. Simulation experiments using Ig-domain unfolding parameters obtained in earlier single-molecule atomic force microscopy experiments recover the phenomenology of stress relaxation and predict large-scale unfolding in titin during an extended period (>∼20 min) of relaxation. By contrast, immunolabeling experiments failed to demonstrate large-scale unfolding. Thus, under physiological conditions in relaxed human soleus fibers, Ig domains are more stable than predicted by atomic force microscopy experiments. Ig-domain unfolding did not become more pronounced after gelsolin treatment, suggesting that the thin filament is unlikely to significantly contribute to the mechanical stability of the domains. We conclude that in human soleus fibers, Ig unfolding cannot solely explain stress relaxation.

Titin elasticity and mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction

Titin (also known as connectin) is a giant filamentous protein whose elastic properties greatly contribute to the passive force in muscle. In the sarcomere, the elastic I-band segment of titin may interact with the thin filaments, possibly affecting the molecule's elastic behavior. Indeed, several studies have indicated that interactions between titin and actin occur in vitro and may occur in the sarcomere as well. To explore the properties of titin alone, one must first eliminate the modulating effect of the thin filaments by selectively removing them. In the present work, thin filaments were selectively removed from the cardiac myocyte by using a gelsolin fragment. Partial extraction left behind approximately 100-nm-long thin filaments protruding from the Z-line, whereas the rest of the I-band became devoid of thin filaments, exposing titin. By applying a much more extensive gelsolin treatment, we also removed the remaining short thin filaments near the Z-line. After extraction, the extensibility of titin was studied by using immunoelectron microscopy, and the passive force-sarcomere length relation was determined by using mechanical techniques. Titin's regional extensibility was not detectably affected by partial thin-filament extraction. Passive force, on the other hand, was reduced at sarcomere lengths longer than approximately 2.1 microm, with a 33 +/- 9% reduction at 2.6 microm. After a complete extraction, the slack sarcomere length was reduced to approximately 1.7 microm. The segment of titin near the Z-line, which is otherwise inextensible, collapsed toward the Z-line in sarcomeres shorter than approximately 2.0 microm, but it was extended in sarcomeres longer than approximately 2.3 microm. Passive force became elevated at sarcomere lengths between approximately 1.7 and approximately 2.1 microm, but was reduced at sarcomere lengths of >2.3 microm. These changes can be accounted for by modeling titin as two wormlike chains in series, one of which increases its contour length by recruitment of the titin segment near the Z-line into the elastic pool.

Improved Procedures for Electron Microscopic Visualization of the Cytoskeleton of Cultured Cells

We have developed an improved electron microscopic procedure appropriate for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, critical point drying, and platinum/carbon coating of cultured cells and the improvements consist of modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyethylene glycol into the extraction medium; cell lysis at room temperature; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydration and drying; and uranyl acetate treatment during dehydration. As a result, we have obtained a greatly improved quality of electron microscopic images together with a high consistency of results. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia. Their polarity was distinctly revealed by decoration with myosin subfragment 1. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin, intermediate filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreported millipede-like appearance. The suggested procedure was compatible with immunogold labeling as demonstrated with an antibody to tubulin. Correlative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, producing a close resemblance between the two kinds of images.

Microscopic analysis of the elastic properties of nebulin in skeletal myofibrils

The elastic properties of nebulin were studied by measuring the elasticity of single skeletal myofibrils, from which the portion of the thin filament located at the I band had been selectively removed by treatment with plasma gelsolin under rigor conditions. In this myofibril model, a portion of each nebulin molecule at the I band was expected to be free of actin filaments and exposed. The length of the exposed portion of the nebulin molecule was controlled by performing the gelsolin treatment at various sarcomere lengths. The relation between the passive tension and extension of the exposed portion of the nebulin showed a convex curve starting from a slack length, apparently in a fashion similar to that of wool. The slack sarcomere length shifted depending on the length of the exposed portion of the nebulin, however, the relation being represented by a single master curve. The elastic modulus of nebulin was estimated to be two to three orders of magnitude smaller than that of an actin filament. Based on these results, we conclude that nebulin attaches to an actin filament in a side-by-side fashion and that it does not significantly contribute to the elastic modulus of thin filaments. The relation between the passive tension and extension of connectin (titin) was obtained for a myofibril from which thin filaments had been completely removed with gelsolin under contracting conditions; this showed a concave curve, consistent with the previous results obtained in single fibers.

Exogenous gelsolin binds to sarcomeric thin filaments without severing.

We have investigated the binding of gelsolin to thin myofilaments in situ and their stability against severing. Differentiated myotubes from chicken skeletal muscle containing cross-striated myofibrils were permeabilized with Triton X-100 and incubated with gelsolin. Immunofluorescence microscopy localized both endogenous and exogenous gelsolin in the I-Z-I-regions of the sarcomers. The staining pattern suggested a binding of the exogenous gelsolin along the entire length of the thin filaments. This binding was Ca2+ dependent, but gelsolin was not removed after subsequent addition of EGTA. The fluorescence staining for actin remained unchanged after gelsolin incubation, indicating that thin filaments in cross-striated myofibrils were resistant to the severing action of gelsolin, in contrast to the microfilaments in stress fibers. After extraction of the permeabilized cells with high ionic strength to remove tropomyosin and myosin, gelsolin still bound along the entire thin filament and the actin pattern also remained unchanged. After Triton X-100 permeabilization and high ionic strength extraction, the giant protein nebulin was found to be still present as a myofibrillar component. Gelsolin treatment after high salt extraction affected neither actin nor nebulin in the thin filaments. We therefore conclude that nebulin confers the gelsolin resistance to the sarcomeric actin filaments.

Non-sarcomeric mode of myosin II organization in the fibroblast lamellum.

The organization of myosin in the fibroblast lamellum was studied by correlative fluorescence and electron microscopy after a novel procedure to reveal its underlying morphology. An X-rhodamine analog of conventional smooth muscle myosin (myosin II) that colocalized after microinjection with endogenous myosin was used to trace myosin distribution in living fibroblasts. Then, the same cells were examined by EM of platinum replicas. To visualize the structural arrangement of myosin, other cytoskeletal fibrillar structures had to be removed: microtubules were depolymerized by nocodazole treatment of the living cells before injection of myosin; continued nocodazole treatment also induced the intermediate filaments to concentrate near the nucleus, thus removing them from the lamellar region; actin filaments were removed after lysis of the cells by incubation of the cytoskeletons with recombinant gelsolin. Possible changes in myosin organization caused by this treatment were examined by fluorescence microscopy. No significant differences in myosin distribution patterns between nocodazole-treated and control cells were observed. Cell lysis and depletion of actin also did not induce reorganization of myosin as was shown by direct comparison of myosin distribution in the same cells in the living state and after gelsolin treatment. EM of the well-spread, peripheral regions of actin-depleted cytoskeletons revealed a network of bipolar myosin mini-filaments, contracting each other at their terminal, globular regions. The morphology of this network corresponded well to the myosin distribution observed by fluorescence microscopy. A novel mechanism of cell contraction by folding of the myosin filament network is proposed.

Interplay between passive tension and strong and weak binding cross-bridges in insect indirect flight muscle. A functional dissection by gelsolin-mediated thin filament removal.

The interplay between passive and active mechanical properties of indirect flight muscle of the waterbug (Lethocerus) was investigated. A functional dissection of the relative contribution of cross-bridges, actin filaments, and C filaments to tension and stiffness of passive, activated, and rigor fibers was carried out by comparing mechanical properties at different ionic strengths of sarcomeres with and without thin filaments. Selective thin filament removal was accomplished by treatment with the actin-severving protein gelsolin. Thin filament, removal had no effect on passive tension, indicating that the C filament and the actin filament are mechanically independent and that passive tension is developed by the C filament in response to sarcomere stretch. Passive tension increased steeply with sarcomere length until an elastic limit was reached at only 6-7% sarcomere extension, which corresponds to an extension of 350% of the C filament. The passive tension-length relation of insect flight muscle was analyzed using a segmental extension model of passive tension development (Wang, K, R. McCarter, J. Wright, B. Jennate, and R Ramirez-Mitchell. 1991. Proc. Natl. Acad. Sci. USA. 88:7101-7109). Thin filament removal greatly depressed high frequency passive stiffness (2.2 kHz) and eliminated the ionic strength sensitivity of passive stiffness. It is likely that the passive stiffness component that is removed by gelsolin is derived from weak-binding cross-bridges, while the component that remains is derived from the C filament. Our results indicate that a significant number of weak-binding cross-bridges exist in passive insect muscle at room temperature and at an ionic strength of 195 mM. Analysis of rigor muscle indicated that while rigor tension is entirely actin based, rigor stiffness contains a component that resists gelsolin treatment and is therefore likely to be C filament based. Active tension and active stiffness of unextracted fibers were directly proportional to passive tension before activation. Similarly, passive stiffness due to weak bridges also increased linearly with passive tension, up to a limit. These correlations lead us to propose a stress-activation model for insect flight muscle in which passive tension is a prerequisite for the formation of both weak-binding and strong-binding cross-bridges.

Nebulin as a length regulator of thin filaments of vertebrate skeletal muscles: correlation of thin filament length, nebulin size, and epitope profile.

Nebulin, a family of giant proteins with size-variants from 600 to 900 kD in various skeletal muscles, have been proposed to constitute a set of inextensible filaments anchored at the Z line (Wang, K., and J. Wright. 1988. J. Cell Biol. 107:2199-2212). This newly discovered filament of the skeletal muscle sarcomere is an attractive candidate for a length-regulating template of thin filaments. To evaluate this hypothesis, we address the question of coextensiveness of nebulin and the thin filament by searching for a correlation between the size of nebulin variants and the length distribution of the thin filaments in several skeletal muscles. A positive linear correlation indeed exists for a group of six skeletal muscles that display narrow thin filament length distributions. To examine the molecular and architectural differences of nebulin size-variants, we carried out immunoelectron microscopic studies to map out epitope profiles of nebulin variants in these muscles. For this purpose, a panel of mAbs to distinct nebulin epitopes was produced against rabbit nebulin purified by an improved protocol. Epitope profiles of nebulin variants in three skeletal muscles revealed that (a) nebulin is inextensible since nebulin epitopes maintain a fixed distance to the Z line irrespective of the degree of sarcomere stretch; (b) a single nebulin polypeptide spans a minimal distance of 0.9 microns from the Z line; (c) nebulin contains repeating epitopes that are spaced at 40 nm or its multiples; (d) nebulin repeats coincide with thin filament periodicity; (e) nebulin variants differ mainly at either or both ends; and (f) nebulin remains in the sarcomere in actin-free sarcomeres produced by gelsolin treatment. Together, these data suggest that nebulin is an inextensible full-length molecular filament that is coextensive with thin filaments in skeletal muscles. We propose that nebulin acts as a length-regulating template that determines thin filament length by matching its large number of 40-nm repeating domains with an equal number of helical repeats of the actin filaments.