Application of nile blue and nile red, two fluorescent probes, for detection of lipid droplets in human skeletal muscle.

1987 ◽  
Vol 35 (5) ◽  
pp. 619-621 ◽  
Author(s):  
E Bonilla ◽  
A Prelle
Keyword(s):  
Skeletal Muscle ◽  
Fluorescent Probes ◽  
Lipid Droplets ◽  
Nile Blue ◽  
Muscle Fibers ◽  
Nile Red ◽  
Normal Muscle ◽  
Mitochondrial Myopathies ◽  
Muscle Lipid ◽  
Muscle Biopsies

Using frozen sections from human muscle biopsies, we assessed the value of Nile blue and Nile red, two fluorescent probes, as stains for lipid droplets in normal and pathological skeletal muscle fibers. In normal muscle, lipid storage disorders, and mitochondrial myopathies, Nile blue stained the lipid droplets as yellow-gold fluorescent structures. The lipid droplets were also seen as yellow-gold fluorescent structures in Nile red-stained sections, but the outstanding feature in these preparations was the staining of the membrane network of the muscle fibers and membrane proliferations in pathological muscle as red-orange fluorescent structures. These results suggest that both Nile blue and Nile red stains are useful for visualization of lipid droplets and membrane proliferations in pathological muscle biopsies.

scholarly journals Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse.

1990 ◽  
Vol 111 (6) ◽  
pp. 2437-2449 ◽  
Author(s):  
J E Morgan ◽  
E P Hoffman ◽  
T A Partridge
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Mdx Mouse ◽  
Newborn Mouse ◽  
Normal Muscle ◽  
Newborn Mice ◽  
X Ray ◽  
Dystrophin Deficiency ◽  
Regenerated Fibers ◽  
Muscle Precursor Cells

Dystrophin deficiency in skeletal muscle of the x-linked dystrophic (mdx) mouse can be partially remedied by implantation of normal muscle precursor cells (mpc) (Partridge, T. A., J. E. Morgan, G. R. Coulton, E. P. Hoffman, and L. M. Kunkel. 1989. Nature (Lond.). 337:176-179). However, it is difficult to determine whether this biochemical "rescue" results in any improvement in the structure or function of the treated muscle, because the vigorous regeneration of mdx muscle more than compensates for the degeneration (Coulton, G. R., N. A. Curtin, J. E. Morgan, and T. A. Partridge. 1988. Neuropathol. Appl. Neurobiol. 14:299-314). By using x-ray irradiation to prevent mpc proliferation, it is possible to study loss of mdx muscle fibers without the complicating effect of simultaneous fiber regeneration. Thus, improvements in fiber survival resulting from any potential therapy can be detected easily (Wakeford, S., D. J. Watt, and T. A. Patridge. 1990. Muscle & Nerve.) Here, we have implanted normal mpc, obtained from newborn mice, into such preirradiated mdx muscles, finding that it is far more extensively permeated and replaced by implanted mpc than is nonirradiated mdx muscle; this is evident both from analysis of glucose-6-phosphate isomerase isoenzyme markers and from immunoblots and immunostaining of dystrophin in the treated muscles. Incorporation of normal mpc markedly reduces the loss of muscle fibers and the deterioration of muscle structure which otherwise occurs in irradiated mdx muscles. Surprisingly, the regenerated fibers are largely peripherally nucleated, whereas regenerated mouse skeletal muscle fibers are normally centrally nucleated. We attribute this regeneration of apparently normal muscle to the tendency of newborn mouse mpc to recapitulate their neonatal ontogeny, even when grafted into 3-wk-old degenerating muscle.

Nile-red and Nile-blue-based near-infrared fluorescent probes for in-cellulo imaging of hydrogen sulfide

2014 ◽  
Vol 406 (28) ◽  
pp. 7059-7070 ◽  
Author(s):  
Xiao-Dong Liu ◽  
Chen Fan ◽  
Ru Sun ◽  
Yu-Jie Xu ◽  
Jian-Feng Ge
Keyword(s):  
Hydrogen Sulfide ◽  
Fluorescent Probes ◽  
Near Infrared ◽  
Nile Blue ◽  
Nile Red

Subcellular localization of skeletal muscle lipid droplets and PLIN family proteins OXPAT and ADRP at rest and following contraction in rat soleus muscle

2012 ◽  
Vol 302 (1) ◽  
pp. R29-R36 ◽  
Author(s):  
Rebecca E. K. MacPherson ◽  
Eric A. F. Herbst ◽  
Erica J. Reynolds ◽  
Rene Vandenboom ◽  
Brian D. Roy ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Lipid Droplet ◽  
Protein Interactions ◽  
Lipid Droplets ◽  
Droplet Surface ◽  
Protein Protein Interactions ◽  
Muscle Lipid ◽  
Droplet Distribution ◽  
Associated Proteins ◽  
Skeletal Muscle Lipid

Skeletal muscle lipid droplet-associated proteins (PLINs) are thought to regulate lipolysis through protein-protein interactions on the lipid droplet surface. In adipocytes, PLIN2 [adipocyte differentiation-related protein (ADRP)] is found only on lipid droplets, while PLIN5 (OXPAT, expressed only in oxidative tissues) is found both on and off the lipid droplet and may be recruited to lipid droplet membranes when needed. Our purpose was to determine whether PLIN5 is recruited to lipid droplets with contraction and to investigate the myocellular location and colocalization of lipid droplets, PLIN2, and PLIN5. Rat solei were isolated, and following a 30-min equilibration period, they were assigned to one of two groups: 1) 30 min of resting incubation and 2) 30 min of stimulation ( n = 10 each). Immunofluorescence microscopy was used to determine subcellular content, distribution, and colocalization of lipid droplets, PLIN2, and PLIN5. There was a main effect for lower lipid and PLIN2 content in stimulated compared with rested muscles ( P < 0.05). Lipid droplet distribution declined exponentially from the sarcolemma to the fiber center in the rested muscles ( P = 0.001, r2= 0.99) and linearly in stimulated muscles (slope = −0.0023 ± 0.0006, P < 0.001, r2= 0.93). PLIN2 distribution declined exponentially from the sarcolemma to the fiber center in both rested and stimulated muscles ( P < 0.0001, r2= 0.99 rest; P = 0.0004, r2= 0.98 stimulated), while PLIN5 distribution declined linearly (slope = −0.0085 ± 0.0009, P < 0.0001, r2= 0.94 rest; slope=−0.0078 ± 0.0010, P = 0.0003, r2= 0.91 stimulated). PLIN5-lipid droplets colocalized at rest with no difference poststimulation ( P = 0.47; rest r2= 0.55 ± 0.02, stimulated r2= 0.58 ± 0.03). PLIN2-lipid droplets colocalized at rest with no difference poststimulation ( P = 0.48; rest r2= 0.66 ± 0.02, stimulated r2= 0.65 ± 0.02). Contrary to our hypothesis, these results show that PLIN5 is not recruited to lipid droplets with contraction in isolated skeletal muscle.

Muscle-nerve communication and the molecular assessment of human skeletal muscle denervation with aging

2021 ◽  
Author(s):  
Casper Søndenbroe ◽  
Jesper L. Andersen ◽  
Abigail L. Mackey
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fiber ◽  
Protein Profile ◽  
Muscle Fibers ◽  
Active Role ◽  
Tissue Samples ◽  
Culture Models ◽  
Muscle Nerve ◽  
Muscle Biopsies ◽  
Cell Culture Models

Muscle fiber denervation is a major contributor to the decline in physical function observed with aging. Denervation can occur through breakdown of the NMJ itself, affecting only that particular fiber, or through the death of a motoneuron, which can lead to a loss of all the muscle fibers in that motor unit. In this review we discuss the muscle-nerve relationship, where signaling from both the motor neuron and the muscle fiber is required for maximal preservation of neuromuscular function in old age. Physical activity is likely to be the most important single factor that can contribute to this preservation. Furthermore, we propose that inactivity is not an innocent bystander, but plays an active role in denervation through the production of signals hostile to neuron survival. Investigating denervation in human muscle tissue samples is challenging due to the shared protein profile of regenerating and denervated muscle fibers. In this review we provide a detailed overview of the key traits observed in immunohistochemical preparations of muscle biopsies from healthy young and elderly individuals. Overall, a combination of assessing tissue samples, circulating biomarkers, and electrophysiological assessments in humans will prove fruitful in the quest to gain more understanding of denervation of skeletal muscle. In addition, cell culture models represent a valuable tool in the search for key signaling factors exchanged between muscle and nerve, and which exercise has the capacity to alter.

scholarly journals Muscle Lipid Metabolism: Role of Lipid Droplets and Perilipins

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Pablo Esteban Morales ◽  
Jose Luis Bucarey ◽  
Alejandra Espinosa
Keyword(s):  
Skeletal Muscle ◽  
Lipid Metabolism ◽  
Lipid Droplets ◽  
Specific Cell ◽  
Muscle Lipid ◽  
Cell Compartments ◽  
Carbohydrate And Lipid Metabolism ◽  
Common Causes ◽  
Specific Lipid

Skeletal muscle is one of the main regulators of carbohydrate and lipid metabolism in our organism, and therefore, it is highly susceptible to changes in glucose and fatty acid (FA) availability. Skeletal muscle is an extremely complex tissue: its metabolic capacity depends on the type of fibers it is made up of and the level of stimulation it undergoes, such as acute or chronic contraction. Obesity is often associated with increased FA levels, which leads to the accumulation of toxic lipid intermediates, oxidative stress, and autophagy in skeletal fibers. This lipotoxicity is one of the most common causes of insulin resistance (IR). In this scenario, the “isolation” of certain lipids in specific cell compartments, through the action of the specific lipid droplet, perilipin (PLIN) family of proteins, is conceived as a lifeguard compensatory strategy. In this review, we summarize the cellular mechanism underlying lipid mobilization and metabolism inside skeletal muscle, focusing on the function of lipid droplets, the PLIN family of proteins, and how these entities are modified in exercise, obesity, and IR conditions.

Serum carnosinase activities in patients with alcoholic chronic skeletal muscle myopathy

1988 ◽  
Vol 75 (2) ◽  
pp. 185-190 ◽  
Author(s):  
P. Duane ◽  
T. J. Peters
Keyword(s):  
Skeletal Muscle ◽  
Enzyme Activity ◽  
Enzyme Activities ◽  
Muscle Fibres ◽  
Significant Activity ◽  
Type Ii ◽  
Normal Muscle ◽  
Serum Enzyme ◽  
Chronic Alcoholic ◽  
Muscle Biopsies

1. Serum carnosinase activity was assayed in a group of alcoholic patients with and without histologically proven atrophy of type II skeletal muscle fibres, and in control subjects. No significant activity was detected in muscle biopsy samples or washed erythrocytes. 2. Serum carnosinase activity was significantly lower in chronic alcoholic patients compared with a group of age-matched controls. Alcoholics with abnormal muscle biopsies had significantly lower enzyme activities than either those patients with normal muscle biopsies or the controls. Serum enzyme activities in patients with normal muscle biopsies were not significantly different from controls. 3. Serum carnosinase activity was inversely correlated with the degree of muscle atrophy as measured by the type II fibre atrophy factor. There was a positive correlation between the enzyme activity and skeletal muscle mass as reflected by the creatinine-height index. Furthermore, the enzyme activity significantly increased, with resolution or improvement in the myopathy, in patients who abstained from alcohol. 4. Kinetic studies showed that the reduced carnosinase activity was due mainly to a decrease in the apparent Vmax. The apparent Km was significantly higher in the myopathic compared with non-myopathic alcoholics. Mixing serum from controls and patients with myopathy gave the expected values, indicating the absence of a serum enzyme inhibitory factor. Acute alcohol loading had no effect on the serum carnosinase activity. 5. The decrease in serum carnosinase activity in alcoholics was not related to the severity of their liver disease. Assays of serum carnosinase in chronic alcoholics can thus be used as a marker of their associated myopathy.

scholarly journals Deep muscle-proteomic analysis of freeze-dried human muscle biopsies reveals fiber type-specific adaptations to exercise training

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
A. S. Deshmukh ◽  
D. E. Steenberg ◽  
M. Hostrup ◽  
J. B. Birk ◽  
J. K. Larsen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Exercise Training ◽  
Fiber Type ◽  
Muscle Fibers ◽  
Human Muscle ◽  
Fiber Types ◽  
Freeze Dried ◽  
Fast Twitch Muscle ◽  
Fast Twitch ◽  
Muscle Biopsies

AbstractSkeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca2+ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

scholarly journals Bed rest increases the amount of mismatched fibers in human skeletal muscle

1999 ◽  
Vol 86 (2) ◽  
pp. 455-460 ◽  
Author(s):  
J. L. Andersen ◽  
T. Gruschy-Knudsen ◽  
C. Sandri ◽  
L. Larsson ◽  
S. Schiaffino
Keyword(s):  
Skeletal Muscle ◽  
Protein Level ◽  
Human Skeletal Muscle ◽  
Muscle Fibers ◽  
Rest Period ◽  
Bed Rest ◽  
Load Bearing ◽  
Transitional State ◽  
Muscle Biopsies

The effects of a 37-day period of bed rest on myosin heavy chain (MHC) expression on both mRNA and protein level in human skeletal muscle fibers were studied. Muscle biopsies from vastus lateralis muscle were obtained from seven healthy young male subjects before and after the bed-rest period. Combined in situ hybridization, immunocytochemistry, and ATPase histochemistry analysis of serial sections of the muscle biopsies demonstrated that fibers showing a mismatch between MHC isoforms at the mRNA and protein level increased significantly after the bed-rest period, suggesting an increase in the amount of muscle fibers in a transitional state. Accordingly, fibers showing a match in expression of MHC-1 and of MHC-2A at the mRNA and protein level decreased, whereas fibers showing a match between MHC-2X mRNA and protein increased after bed rest. Overall, there was an increase in fibers in a transitional state from phenotypic type 1 → 2A and 2A → 2X. Furthermore, a number of fibers with unusual MHC mRNA and isoprotein combinations were observed after bed rest (e.g., type 1 fibers with only mRNA for 2X and type 1 fibers negative for mRNA for MHC-β/slow, 2A, and 2X). In contrast, no changes were revealed after an examination at the protein level alone. These data suggest that the reduced load-bearing activity imposed on the skeletal muscles through bed rest will alter MHC gene expression, resulting in combinations of mRNA and MHC isoforms normally not (or only rarely) observed in muscles subjected to load-bearing activity. On the other hand, the present data also show that 37 days of bed rest are not a sufficient stimulus to induce a similar change at the protein level, as was observed at the gene level.

Ca2+-dependent slow action potentials in normal and dystrophic mouse skeletal muscle

1983 ◽  
Vol 245 (5) ◽  
pp. C415-C422 ◽  
Author(s):  
L. M. Kerr ◽  
N. Sperelakis
Keyword(s):  
Skeletal Muscle ◽  
Action Potentials ◽  
Osmotic Shock ◽  
Muscle Fibers ◽  
Normal Muscle ◽  
Dystrophic Muscle ◽  
Recording Technique ◽  
Mammalian Muscle ◽  
Dystrophic Mice ◽  
Slow Action Potentials

Slowly rising action potentials (APs), previously described in amphibian skeletal muscle, were examined in skeletal muscle of normal and dystrophic mice (129/ReJ strain). A standard two-microelectrode recording technique was used. Muscles were bathed in a solution that was Cl- free (methanesulfonate substituted), high in K+ (20 mM), and contained 15 mM tetraethylammonium. The slow APs were elicited under conditions in which the fast Na+ channels were voltage inactivated (by partial depolarization) and in which the external Na+ concentration was only 10 mM. Increases in external Ca2+ concentration produced increases in slow AP amplitude and duration. Mn2+ (4 mM), La3+ (4 mM), and detubulation with osmotic shock blocked the slow APs. When slow APs were generated at 30-s intervals, their amplitude stayed constant. When they were generated at 15-s intervals, their amplitude decreased progressively and then fell to zero by the 11th stimulus. The Ca antagonists verapamil (10(-5) M) and bepridil (10(-5) M) caused this decrease in amplitude to occur more quickly. Voltage inactivation of the slow APs occurred between -45 and -10 mV. Slow APs recorded from dystrophic muscle fibers were decreased in amplitude and duration compared with those in normal fibers, and there was a reduced incidence of occurrence; 96% of the fibers in normal muscle exhibited slow APs compared with only 46% of dystrophic muscle fibers. In summary, slow Ca2+ APs in mammalian muscle are similar to those in cardiac and amphibian skeletal muscle, and these slow APs are depressed in dystrophic skeletal muscle.

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