CERAMIDE ENRICHED MEMBRANE DOMAINS IN RAT SKELETAL MUSCLE EXPOSED TO SHORT-TERM HYPOGRAVITATIONAL UNLOADING
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1
Izhevsk State Medical Academy, Russia
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2
Udmurt State University, Russia
Introduction
Since the 90th, many studies confirmed the important role of sphingolipid messengers including ceramide in regulation of skeletal muscle function. We have previously found that ceramide accumulates in disused soleus muscle of rodents subjected to 12 h, 4, 14 and 30 days of hindlimb unloading (HU), and one of the possible mechanisms of this effect is acid sphingomyelinase (aSMase) upregulation (Bryndina et al., 2014, 2017). We also revealed that aSMase inhibitor, clomipramine partially prevented membrane lipid raft disassembly (Bryndina et al., 2018), shown previously to be induced by 6-12 h HU (Petrov et al., 2017). In the present work we studied the impact of short-term HU on the amount and distribution of ceramide in rat soleus muscle.
Materials and methods
Adult male Wistar rats (n=12) were subjected to 4-day hindlimb unloading (HU) by tail-suspension method of Novikov & Il’in in Morey-Holton modification. Some of these rats were clomipramine treated (1.25 mg/g/day, intramuscularly, for 5 days before HU and every other day during HU, n=6), another part of the animals (n=6) was treated with vehicle (0.9% saline solution). Intact rats were used as a control group (n=6).
Ceramide amount and distribution in soleus muscle fibers were studied biochemically and immunohistochemically. Biochemical analysis was performed by thin layer chromatography of detergent resistant membrane fraction (DRM) isolated from muscle homogenates by ultracentrifugation in sucrose gradient (Radeva, Sharom, 2004). Lipids extracted from the DRM by the Folch reagent were developed on HPTLC Silica gel 60 F254 plates (Merck) according to Boath et al. (2008) together with a standard chloroform solution of ceramide (Avanti). Spots were visualized by iodine vapor. Quantitative evaluation of ceramide amount was performed with Sorbfil UV Videodensitometer (Sorbpolymer). Western-blot analysis (Laemmli, 1970) was applied to detect the amount of aSMase and nSMase proteins in DRM.
For immunohistochemical staining of longitudinal and transverse muscle cryosections, anti-ceramide antibodies (murine IgG, 1: 300, Abcam) and anti-mouse biotinylated antibodies (goat IgG, 1: 200, Abcam) were used. Analysis of the images obtained with the Nikon Epsilon E200 was performed using the Image-Pro Plus 6.0 and Image-Pro Insight programs. The expression of immunoreactive Cer was estimated by the fluorescence intensity of the test section on a standard area (0.1mm2). Each 5th section was taken and 10 measurements of intensity were made (at least 150 measurements per animal).
Statistical analysis was performed using SPSS 6.0 program. Depending on the distribution, one-way ANOVA with post hoc test or Mann-Whitney U test were used, the differences between groups considered significant at p<0.05.
Results
Biochemical study of DRM revealed the increase of ceramide and sphingomyelinases amount after 4d HU. Ceramide was higher 4-fold (p<0.05) after HU, but did not differ significantly from the control level in rats suspended with clomipramine treatment. The levels of aSMase and nSMase proteins were increased by 75.2 ± 9.4% and 161.6 ± 27.3% respectively (p<0.05). Clomipramine treatment completely abolished aSMase increase and did not restore nSMase content.
Immunoreactive labeling of Cer in m. soleus fibers showed a substantial rearrangement of its distribution in unloaded muscle compared with the intact one. The anti-Cer staining of intact muscle fibers revealed very weak Cer labeling. Immunoreactive Cer looked like a diffuse fluorescence of an adjucent to membrane sarcoplasm, a low amount of lightly colored small size granules on the surface of the cytoplasmic membrane (Fig. 1A-C), and small fibrillar and globular structures within the fibers (not shown). Even in the visual assessment, a significant increase in both the number and volume of Cer immunoreactive structures were observed after 4 days of HU. Unloading was accompanied by the increase in total intensity of immunoreactive Cer staining, which became higher by 84.6 ± 12.1% (p<0.05) in the sarcolemma and by 55.7 ± 12.7% (p<0.05) in sarcoplasmic compartment. In addition, we observed a significant rearrangement of the immunopositive to Cer surface structures which looked like intensely luminescent fused large granules and irregular fibrils that formed a kind of big clusters or platforms. Clomipramine pretreatment decreased the overall luminescent immunoreactivity to Cer (by 14.3 ± 4.5%, p<0.05), but its value was still significantly higher than in the control animals (p<0.01).
3D reconstruction of Cer membrane domains allowed us to evaluate their volumes (fig.1D-F). It has been shown that, in the membrane of control animals, there are two principal populations of Cer structures, "small" and "medium" in size. Unloading leads to the emergence of two additional groups of clusters: "large" and "very large," and a number of such clusters becomes more than "small" and "medium" ones. Clomipramine treatment abolished the population of "very large" clusters, significantly reduced the amount of "large" and "medium" ones, associated with the parallel formation of the group “intermediate” in size. Although the number and volume of Cer immunopositive clusters reduced their amount still kept higher than in control animals.
Discussion
The present data confirm our previous findings of the intensive Cer formation in postural muscle disused with unloading (Bryndina et al., 2014), and expand the knowledge about the membrane effects of Cer in short-term disuse (Bryndina et al., 2018). We demonstrated for the first time that ceramide forms the peculiar clusters in the plasma membrane of unloaded soleus muscle, and we believe that these clusters are similar to the previously described Cer enriched membrane domains (Grassme et al., 2001). Cer microdomaines can spontaneously merge into large clusters known to serve as signaling platforms for a number of receptors, channels and other proteins. The main mechanism of their formation is heightened sphingomyelinase hydrolysis activated in response to some environmental stimuli (Grassme et al., 2001) including muscle disuse (Bryndina et al., 2014, 2017). In the present study we have demonstrated the increase of aSMase and nSMase availability in DRM of soleus muscle, and this is the main mechanism of Cer accumulation in the plasma membrane of unloaded muscle. Clomipramine attenuates Cer and aSMase upregulation without affecting nSMase. New results obtained in the present study support our previous data about efficacy of clomipramine pretreatment in the prevention of lipid raft disorders caused by 12h disuse (Bryndina et al., 2018).
Acknowledgements
This work was supported by Russian Science Foundation (research grant # 16-15-10220)
Keywords:
skeletal muscle,
unloading,
acid and neutral sphingomyelinase,
ceramide enriched membrane domains,
Clomipramine
Conference:
39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018.
Presentation Type:
Extended abstract
Topic:
Bones and Muscles
Citation:
Bryndina
IG,
Protopopov
VA,
Sergeev
VG,
Shalagina
MN,
Ovechkin
SV and
Yakovlev
AA
(2019). CERAMIDE ENRICHED MEMBRANE DOMAINS IN RAT SKELETAL MUSCLE EXPOSED TO SHORT-TERM HYPOGRAVITATIONAL UNLOADING.
Front. Physiol.
Conference Abstract:
39th ISGP Meeting & ESA Life Sciences Meeting.
doi: 10.3389/conf.fphys.2018.26.00028
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Received:
02 Dec 2018;
Published Online:
16 Jan 2019.
*
Correspondence:
Prof. Irina G Bryndina, Izhevsk State Medical Academy, Izhevsk, Russia, i_bryndina@mail.ru