scholarly journals A mouse model of Huntington’s disease shows altered ultrastructure of transverse tubules in skeletal muscle fibers

2021 ◽  
Vol 153 (4) ◽  
Author(s):  
Shannon H. Romer ◽  
Sabrina Metzger ◽  
Kristiana Peraza ◽  
Matthew C. Wright ◽  
D. Scott Jobe ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Muscle Fibers ◽  
Membrane Capacitance ◽  
Mrna Levels ◽  
Primary Role ◽  
Ec Coupling ◽  
Skeletal Muscle Fibers

Huntington’s disease (HD) is a fatal and progressive condition with severe debilitating motor defects and muscle weakness. Although classically recognized as a neurodegenerative disorder, there is increasing evidence of cell autonomous toxicity in skeletal muscle. We recently demonstrated that skeletal muscle fibers from the R6/2 model mouse of HD have a decrease in specific membrane capacitance, suggesting a loss of transverse tubule (t-tubule) membrane in R6/2 muscle. A previous report also indicated that Cav1.1 current was reduced in R6/2 skeletal muscle, suggesting defects in excitation–contraction (EC) coupling. Thus, we hypothesized that a loss and/or disruption of the skeletal muscle t-tubule system contributes to changes in EC coupling in R6/2 skeletal muscle. We used live-cell imaging with multiphoton confocal microscopy and transmission electron microscopy to assess the t-tubule architecture in late-stage R6/2 muscle and found no significant differences in the t-tubule system density, regularity, or integrity. However, electron microscopy images revealed that the cross-sectional area of t-tubules at the triad were 25% smaller in R6/2 compared with age-matched control skeletal muscle. Computer simulation revealed that the resulting decrease in the R6/2 t-tubule luminal conductance contributed to, but did not fully explain, the reduced R6/2 membrane capacitance. Analyses of bridging integrator-1 (Bin1), which plays a primary role in t-tubule formation, revealed decreased Bin1 protein levels and aberrant splicing of Bin1 mRNA in R6/2 muscle. Additionally, the distance between the t-tubule and sarcoplasmic reticulum was wider in R6/2 compared with control muscle, which was associated with a decrease in junctophilin 1 and 2 mRNA levels. Altogether, these findings can help explain dysregulated EC coupling and motor impairment in Huntington’s disease.

scholarly journals Phenotypic Modulation of Skeletal Muscle Fibers in LPIN1-Deficient Lipodystrophic (fld) Mice

2018 ◽  
Vol 56 (2) ◽  
pp. 322-331
Author(s):  
Rani S. Sellers ◽  
S. Radma Mahmood ◽  
Geoffrey S. Perumal ◽  
Frank P. Macaluso ◽  
Irwin J. Kurland
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Fiber Type ◽  
Periodic Acid ◽  
Muscle Fibers ◽  
Myosin Atpase ◽  
Hybrid Fibers ◽  
Periodic Acid Schiff ◽  
Skeletal Muscle Fibers ◽  
Lipin 1

Lipin-1 ( Lpin1)–deficient lipodystrophic mice have scant and immature adipocytes and develop transient fatty liver early in life. Unlike normal mice, these mice cannot rely on stored triglycerides to generate adenosine triphosphate (ATP) from the β-oxidation of fatty acids during periods of fasting. To compensate, these mice store much higher amounts of glycogen in skeletal muscle and liver than wild-type mice in order to support energy needs during periods of fasting. Our studies demonstrated that there are phenotypic changes in skeletal muscle fibers that reflect an adaptation to this unique metabolic situation. The phenotype of skeletal muscle (soleus, gastrocnemius, plantaris, and extensor digitorum longus [EDL]) from Lpin1-/- was evaluated using various methods including immunohistochemistry for myosin heavy chains (Myh) 1, 2, 2a, 2b, and 2x; enzyme histochemistry for myosin ATPase, cytochrome-c oxidase (COX), and succinyl dehydrogenase (SDH); periodic acid–Schiff; and transmission electron microscopy. Fiber-type changes in the soleus muscle of Lpin1-/- mice were prominent and included decreased Myh1 expression with concomitant increases in Myh2 expression and myosin-ATPase activity; this change was associated with an increase in the presence of Myh1/2a or Myh1/2x hybrid fibers. Alterations in mitochondrial enzyme activity (COX and SDH) were apparent in the myofibers in the soleus, gastrocnemius, plantaris, and EDL muscles. Electron microscopy revealed increases in the subsarcolemmal mitochondrial mass in the muscles of Lpin1-/- mice. These data demonstrate that lipin-1 deficiency results in phenotypic fiber-specific modulation of skeletal muscle necessary for compensatory fuel utilization adaptations in lipodystrophy.

scholarly journals Skeletal muscle regeneration is altered in the R6/2 mouse model of Huntington's disease

2022 ◽  
Author(s):  
Sanzana Hoque ◽  
Marie Sjogren ◽  
Valerie Allamand ◽  
Kinga Gawlik ◽  
Naomi Franke ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Muscle Damage ◽  
Satellite Cell ◽  
Mouse Models ◽  
Satellite Cells ◽  
Muscle Fibers ◽  
Cag Repeat ◽  
Skeletal Muscle Regeneration

Huntington's disease (HD) is caused by CAG repeat expansion in the huntingtin (HTT) gene. Skeletal muscle wasting alongside central pathology is a well-recognized phenomenon seen in patients with HD and HD mouse models. HD muscle atrophy progresses with disease and affects prognosis and quality of life. Satellite cells, progenitors of mature skeletal muscle fibers, are essential for proliferation, differentiation, and repair of muscle tissue in response to muscle injury or exercise. In this study, we aim to investigate the effect of mutant HTT on the differentiation and regeneration capacity of HD muscle by employing in vitro mononuclear skeletal muscle cell isolation and in vivo acute muscle damage model in R6/2 mice. We found that, similar to R6/2 adult mice, neonatal R6/2 mice also exhibit a significant reduction in myofiber width and morphological changes in gastrocnemius and soleus muscles compared to WT mice. Cardiotoxin (CTX)-induced acute muscle damage in R6/2 and WT mice showed that the Pax7+ satellite cell pool was dampened in R6/2 mice at 4 weeks post-injection, and R6/2 mice exhibited an altered inflammatory profile in response to acute damage. Our results suggest that, in addition to the mutant HTT degenerative effects in mature muscle fibers, expression of mutant HTT in satellite cells might alter developmental and regenerative processes to contribute to the progressive muscle mass loss in HD. Taken together, the results presented here encourage further studies evaluating the underlying mechanisms of satellite cell dysfunction in HD mouse models.

scholarly journals Mechanisms of altered skeletal muscle action potentials in the R6/2 mouse model of Huntington’s disease

2020 ◽  
Vol 319 (1) ◽  
pp. C218-C232 ◽  
Author(s):  
Daniel R. Miranda ◽  
Eric Reed ◽  
Abdulrahman Jama ◽  
Michael Bottomley ◽  
Hongmei Ren ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Decay Time ◽  
Motor Dysfunction ◽  
Peak Amplitude ◽  
Repetitive Firing ◽  
Muscle Pathology ◽  
Mrna Levels ◽  
Electrode Current

Huntington’s disease (HD) patients suffer from progressive and debilitating motor dysfunction for which only palliative treatment is currently available. Previously, we discovered reduced skeletal muscle Cl− channel (ClC-1) and inwardly rectifying K+ channel (Kir) currents in R6/2 HD transgenic mice. To further investigate the role of ClC-1 and Kir currents in HD skeletal muscle pathology, we measured the effect of reduced ClC-1 and Kir currents on action potential (AP) repetitive firing in R6/2 mice using a two-electrode current clamp. We found that R6/2 APs had a significantly lower peak amplitude, depolarized maximum repolarization, and prolonged decay time compared with wild type (WT). Of these differences, only the maximum repolarization was accounted for by the reduction in ClC-1 and Kir currents, indicating the presence of additional ion channel defects. We found that both KV1.5 and KV3.4 mRNA levels were significantly reduced in R6/2 skeletal muscle compared with WT, which explains the prolonged decay time of R6/2 APs. Overall, we found that APs in WT and R6/2 muscle significantly and progressively change during activity to maintain peak amplitude despite buildup of Na+ channel inactivation. Even with this resilience, the persistently reduced peak amplitude of R6/2 APs is expected to result in earlier fatigue and may help explain the motor impersistence experienced by HD patients. This work lays the foundation to link electrical changes to force generation defects in R6/2 HD mice and to examine the regulatory events controlling APs in WT muscle.

scholarly journals Anatomical distribution of voltage-dependent membrane capacitance in frog skeletal muscle fibers.

1989 ◽  
Vol 93 (3) ◽  
pp. 565-584 ◽  
Author(s):  
C L Huang ◽  
L D Peachey
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Fiber Surface ◽  
Membrane Capacitance ◽  
Hypertonic Solution ◽  
Charge Movement ◽  
Frog Skeletal Muscle ◽  
Transverse Tubule ◽  
Skeletal Muscle Fibers ◽  
Voltage Dependent

Components of nonlinear capacitance, or charge movement, were localized in the membranes of frog skeletal muscle fibers by studying the effect of 'detubulation' resulting from sudden withdrawal of glycerol from a glycerol-hypertonic solution in which the muscles had been immersed. Linear capacitance was evaluated from the integral of the transient current elicited by imposed voltage clamp steps near the holding potential using bathing solutions that minimized tubular voltage attenuation. The dependence of linear membrane capacitance on fiber diameter in intact fibers was consistent with surface and tubular capacitances and a term attributable to the capacitance of the fiber end. A reduction in this dependence in detubulated fibers suggested that sudden glycerol withdrawal isolated between 75 and 100% of the transverse tubules from the fiber surface. Glycerol withdrawal in two stages did not cause appreciable detubulation. Such glycerol-treated but not detubulated fibers were used as controls. Detubulation reduced delayed (q gamma) charging currents to an extent not explicable simply in terms of tubular conduction delays. Nonlinear membrane capacitance measured at different voltages was expressed normalized to accessible linear fiber membrane capacitance. In control fibers it was strongly voltage dependent. Both the magnitude and steepness of the function were markedly reduced by adding tetracaine, which removed a component in agreement with earlier reports for q gamma charge. In contrast, detubulated fibers had nonlinear capacitances resembling those of q beta charge, and were not affected by adding tetracaine. These findings are discussed in terms of a preferential localization of tetracaine-sensitive (q gamma) charge in transverse tubule membrane, in contrast to a more even distribution of the tetracaine-resistant (q beta) charge in both transverse tubule and surface membranes. These results suggest that q beta and q gamma are due to different molecules and that the movement of q gamma in the transverse tubule membrane is the voltage-sensing step in excitation-contraction coupling.

scholarly journals Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse, a model of Huntington’s disease

2014 ◽  
Vol 144 (5) ◽  
pp. 393-413 ◽  
Author(s):  
Peter Braubach ◽  
Murat Orynbayev ◽  
Zoita Andronache ◽  
Tanja Hering ◽  
Georg Bernhard Landwehrmeyer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Voltage Clamp ◽  
Trinucleotide Repeat ◽  
Transport Model ◽  
Muscle Fibers ◽  
Muscle Pathology ◽  
Release Flux ◽  
Inward Currents

Huntington’s disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor–based Ca2+ signaling, which is crucial for skeletal muscle excitation–contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca2+ transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca2+ inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca2+ transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca2+ removal from the myoplasm and Ca2+ release flux from the sarcoplasmic reticulum were characterized using a Ca2+ binding and transport model, which indicated a significant reduction in slow Ca2+ removal activity and Ca2+ release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca2+ channel conductance. These results indicate profound changes of Ca2+ turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD.

scholarly journals Measurement of force and calcium release using mechanically skinned fibers from mammalian skeletal muscle

2018 ◽  
Vol 125 (4) ◽  
pp. 1105-1127 ◽  
Author(s):  
Graham D. Lamb ◽  
D. George Stephenson
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fiber ◽  
Calcium Release ◽  
Muscle Fibers ◽  
Mammalian Skeletal Muscle ◽  
Ec Coupling ◽  
Skeletal Muscle Fibers ◽  
Intact Muscle ◽  
Key Variables ◽  
Coupling Sequence

The mechanically skinned (or “peeled”) skeletal muscle fiber technique is a highly versatile procedure that allows controlled examination of each of the steps in the excitation-contraction (EC)-coupling sequence in skeletal muscle fibers, starting with excitation/depolarization of the transverse tubular (T)-system through to Ca2+ release from sarcoplasmic reticulum (SR) and finally force development by the contractile apparatus. It can also show the overall response of the whole EC-coupling sequence together, such as in twitch and tetanic force responses. A major advantage over intact muscle fiber preparations is that it is possible to set and rapidly manipulate the “intracellular” conditions, allowing examination of the effects of key variables (e.g., intracellular pH, ATP levels, redox state, etc.) on each individual step in EC coupling. This Cores of Reproducibility in Physiology (CORP) article describes the rationale, procedures, and experimental details of the various ways in which the mechanically skinned fiber technique is used in our laboratory to examine the physiological mechanisms controlling Ca2+ release and contraction in skeletal muscle fibers and the aberrations and dysfunction occurring with exercise and disease.

Physiological aspects of the subcellular localization of glycogen in skeletal muscle

2013 ◽  
Vol 38 (2) ◽  
pp. 91-99 ◽  
Author(s):  
Joachim Nielsen ◽  
Niels Ørtenblad
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Subcellular Localization ◽  
Quantitative Methods ◽  
Muscle Fibers ◽  
Physiological Role ◽  
Glycogen Metabolism ◽  
Spherical Particles ◽  
Chain Polymer ◽  
Skeletal Muscle Fibers

Glucose is stored in skeletal muscle fibers as glycogen, a branched-chain polymer observed in electron microscopy images as roughly spherical particles (known as β-particles of 10–45 nm in diameter), which are distributed in distinct localizations within the myofibers and are physically associated with metabolic and scaffolding proteins. Although the subcellular localization of glycogen has been recognized for more than 40 years, the physiological role of the distinct localizations has received sparse attention. Recently, however, studies involving stereological, unbiased, quantitative methods have investigated the role and regulation of these distinct deposits of glycogen. In this report, we review the available literature regarding the subcellular localization of glycogen in skeletal muscle as investigated by electron microscopy studies and put this into perspective in terms of the architectural, topological, and dynamic organization of skeletal muscle fibers. In summary, the distribution of glycogen within skeletal muscle fibers has been shown to depend on the fiber phenotype, individual training status, short-term immobilization, and exercise and to influence both muscle contractility and fatigability. Based on all these data, the available literature strongly indicates that the subcellular localization of glycogen has to be taken into consideration to fully understand and appreciate the role and regulation of glycogen metabolism and signaling in skeletal muscle. A full understanding of these phenomena may prove vital in elucidating the mechanisms that integrate basic cellular events with changing glycogen content.

scholarly journals Elevated nuclear Foxo1 suppresses excitability of skeletal muscle fibers

2013 ◽  
Vol 305 (6) ◽  
pp. C643-C653 ◽  
Author(s):  
Erick O. Hernández-Ochoa ◽  
Tova Neustadt Schachter ◽  
Martin F. Schneider
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Atrophy ◽  
Action Potentials ◽  
Muscle Fibers ◽  
Calcium Transients ◽  
Skeletal Muscle Atrophy ◽  
Nuclear Activity ◽  
Ec Coupling ◽  
Skeletal Muscle Fibers

Forkhead box O 1 (Foxo1) controls the expression of proteins that carry out processes leading to skeletal muscle atrophy, making Foxo1 of therapeutic interest in conditions of muscle wasting. The transcription of Foxo1-regulated proteins is dependent on the translocation of Foxo1 to the nucleus, which can be repressed by insulin-like growth factor-1 (IGF-1) treatment. The role of Foxo1 in muscle atrophy has been explored at length, but whether Foxo1 nuclear activity affects skeletal muscle excitation-contraction (EC) coupling has not yet been examined. Here, we use cultured adult mouse skeletal muscle fibers to investigate the effects of Foxo1 overexpression on EC coupling. Fibers expressing Foxo1-green fluorescent protein (GFP) exhibit an inability to contract, impaired propagation of action potentials, and ablation of calcium transients in response to electrical stimulation compared with fibers expressing GFP alone. Evaluation of the transverse (T)-tubule system morphology, the membranous system involved in the radial propagation of the action potential, revealed an intact T-tubule network in fibers overexpressing Foxo1-GFP. Interestingly, long-term IGF-1 treatment of Foxo1-GFP fibers, which maintains Foxo1-GFP outside the nucleus, prevented the loss of normal calcium transients, indicating that Foxo1 translocation and the atrogenes it regulates affect the expression of proteins involved in the generation and/or propagation of action potentials. A reduction in the sodium channel Nav1.4 expression in fibers overexpressing Foxo1-GFP was also observed in the absence of IGF-1. We conclude that increased nuclear activity of Foxo1 prevents the normal muscle responses to electrical stimulation and that this indicates a novel capability of Foxo1 to disable the functional activity of skeletal muscle.

Involvement of calpains in Ca2+-induced disruption of excitation-contraction coupling in mammalian skeletal muscle fibers

2009 ◽  
Vol 296 (5) ◽  
pp. C1115-C1122 ◽  
Author(s):  
Esther Verburg ◽  
Robyn M. Murphy ◽  
Isabelle Richard ◽  
Graham D. Lamb
Keyword(s):  
Skeletal Muscle ◽  
Surface Membrane ◽  
Muscle Fibers ◽  
Mammalian Skeletal Muscle ◽  
Skinned Muscle ◽  
Ec Coupling ◽  
Calpain 3 ◽  
Skeletal Muscle Fibers ◽  
Longus Muscle ◽  
Rate Of Decline

In skeletal muscle fibers, the coupling between excitation of the surface membrane and the release of Ca2+ from the sarcoplasmic reticulum is irreversibly disrupted if cytoplasmic Ca2+ concentration ([Ca2+]) is raised to micromolar levels for a prolonged period. This excitation-contraction (EC) uncoupling may contribute to muscle weakness after some types of exercise and in certain muscle diseases and has been linked to structural alteration of the triad junctions, but its molecular basis is unclear. Both μ-calpain, a ubiquitous Ca2+-activated protease, and muscle-specific calpain-3 become autolytically activated at micromolar Ca2+ and have been suggested to be responsible for the uncoupling. This study used controlled Ca2+ exposure in mechanically skinned fibers from extensor digitorum longus muscle to show that EC uncoupling still occurs in muscle fibers of calpain-3-deficient mice, with a Ca2+ dependence indistinguishable from that in normal mice and rats. Western blotting of muscle fibers that had been partially EC uncoupled by exposure to an intermediate Ca2+ level (∼5 μM Ca2+ for 3 min, no ATP) showed the presence of autolytic activation of a proportion of the μ-calpain present, but with little or no activation of calpain-3. Homogenates of normal and calpain-3-deficient muscles exposed to micromolar Ca2+ displayed similar levels of diffusible proteolytic activity, as gauged by the rate of decline of passive force in stretched, skinned muscle fibers. Exogenously added μ-calpain, preactivated by elevated [Ca2+] and applied in the presence of 1 μM Ca2+, disrupted EC coupling in a manner similar to raised [Ca2+]. We conclude that calpain-3 is not responsible for Ca2+-induced disruption of EC coupling, but that μ-calpain is a plausible candidate.

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