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Disuse-induced morphological changes in muscle. (A) Changes in muscle size (left) and MHC expression (middle and right). Bar, 1 cm. Same animal was used in left and middle figures. Muscle bundles (∼30 mg) were solubilized and run for gel electrophoresis. EDL muscle was used as control for separation of MHC IIA/X and IIB. Cont., control without disuse; Dis., disused. MHC was run on 8% gel. In bar graph, ND indicates “not detectable” for densitometric analysis. *, P < 0.05, compared with the corresponding value for control soleus muscle. (B) Electron micrographs of longitudinal sections of intact soleus muscle, with (right) and without (left) disuse (taken from the same animal). SL ∼ 3.2 μm. Black arrows indicate the positions of the Z-line. Bar, 1 μm. Yellow arrows indicate mitochondria. Orange arrows indicate small granule vesicles. (C) Electron micrographs of longitudinal sections of sarcomeres in skinned single fibers, with (bottom) and without (top) disuse. Images at short (∼2.6 μm; type I) and long (∼3.2 μm; type I/II) SLs are shown (same animal used for each SL). Red dotted arrows indicate the position of the A/I junction in control sarcomere. Note similar thickness of the M-line in control and disused sarcomeres (indicated by thin blue lines) (see Fig. 4 B). Bar, 0.5 μm. Graph shows average thick filament length in control vs. disused fibers at various SLs (from eight animals; type I and I/II fibers pooled for disused group). *, P < 0.05, compared with control. (D) Fluorescence analyses of sarcomeres in skinned fibers, with and without disuse. Typical images and corresponding intensity profiles are shown. Top, A-band width measurement. Images: black arrows indicate the positions of the Z-line. Intensity profiles: horizontal dashed lines indicate the highest and lowest intensity values and vertical lines the midpoint of fluorescent intensity. The distance between vertical lines was defined as the average A-band width (noted as A). Bar graph summarizes the data from four animals (n = 10; type I and I/II fibers pooled for disused group). SL was 2.70 ± 0.07 and 2.78 ± 0.08 (P > 0.05) in control and disused muscle, respectively. Bottom, I-Z-I brush width measurement. Images: black arrows indicate the positions of the Z-line and green arrows the positions of the pointed end of the thin filament (Yasuda et al., 1994). The thin filament length was obtained by dividing the I-Z-I brush width (noted as I-Z-I) by a factor of 2. Bar graph summarizes the data from four animals (n = 6; type I and I/II fibers pooled for disused group). The value of the thin filament length in control muscle was similar to that reported previously with electron microscopy (Herzog et al., 1992). SL was 2.86 ± 0.05 and 2.73 ± 0.03 (P > 0.05) in control and disused muscle, respectively. *, P < 0.05.
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Persistent muscle weakness due to disuse-associated skeletal muscle atrophy limits the quality of life for patients with various diseases and individuals who are confined to bed. Fibers from disused muscle exhibit a marked reduction in active force production, which can exacerbate motor function, coupled with the well-known loss of muscle quantity....
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... inactivity. 30 These may have contributed to the increased muscle hardness. Thus, the eccentric exercise was performed by weaker and stiffer muscles by the participants in the IM than the control group. ...
The present study investigated the effects of a 3‐week immobilization (IM) on muscle damage induced by maximal eccentric exercise (MaxEC) to test the hypothesis that the IM would make muscles prone to muscle damage. Young healthy sedentary men were pseudo‐randomly assigned to IM or control group (n=12/group). Non‐dominant arms of the IM group participants were immobilized at 90° elbow flexion by a cast for 21 days. All participants performed MaxEC consisting of five sets of six elbow flexor contractions by lowering a dummbell set at 100% of pre‐exercise maximal voluntary isometric contraction (MVC) strength of the non‐dominant arm. This was performed at two days after the cast removal for the IM group. MVC torque, range of motion (ROM), muscle thickness (MT), muscle hardness, position sense (PS) and joint reaction angle (JRA) of the elbow flexors were measured at baseline, post‐immobilization, and before, immediately after, and one to five days after MaxEC. The IM decreased MVC torque (‐17±2%), ROM (‐2±1%), MT (‐7±3%), and JRA (‐12±6%), and increased in muscle hardness (20±6%) and PS (11±2%) (P<0.05). Changes in MVC (e.g., 2 days: ‐40±5 vs ‐30±9%), ROM (2 days: ‐11±2 vs ‐9±3%), muscle soreness (peak: 63±22 vs 48±14 mm), plasma CK activity (peak: 7,820±4,011 vs 4,980±1,363 IU/L), PS (maximal change: ‐23±2 vs ‐18±3%) and JRA (maximal change: ‐37±4 vs 26±3%) after MaxEC were greater (P<0.05) for the IM than control group. These results supported the hypothesis and showed that the IM made the muscles more vulnerable to muscle damage induced by eccentric exercise.
... If this is the case, SL in living R2509C-Hom myocytes can be calculated within the ∼1.3-1.7 μm range, which is the physiologically relevant A-band length in vertebrate striated muscles (cf. Suzuki et al., 2005;Udaka et al., 2008;Shimomura et al., 2016). The short SL in R2509C-Hom myocytes indicates that abnormal contractions occur in these cells, even in the resting state. ...
Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum (SR) of the skeletal muscle and plays a critical role in excitation–contraction coupling. Mutations in RYR1 cause severe muscle diseases, such as malignant hyperthermia, a disorder of Ca2+-induced Ca2+ release (CICR) through RYR1 from the SR. We recently reported that volatile anesthetics induce malignant hyperthermia (MH)-like episodes through enhanced CICR in heterozygous R2509C-RYR1 mice. However, the characterization of Ca2+ dynamics has yet to be investigated in skeletal muscle cells from homozygous mice because these animals die in utero. In the present study, we generated primary cultured skeletal myocytes from R2509C-RYR1 mice. No differences in cellular morphology were detected between wild type (WT) and mutant myocytes. Spontaneous Ca2+ transients and cellular contractions occurred in WT and heterozygous myocytes, but not in homozygous myocytes. Electron microscopic observation revealed that the sarcomere length was shortened to ∼1.7 µm in homozygous myocytes, as compared to ∼2.2 and ∼2.3 µm in WT and heterozygous myocytes, respectively. Consistently, the resting intracellular Ca2+ concentration was higher in homozygous myocytes than in WT or heterozygous myocytes, which may be coupled with a reduced Ca2+ concentration in the SR. Finally, using infrared laser-based microheating, we found that heterozygous myocytes showed larger heat-induced Ca2+ transients than WT myocytes. Our findings suggest that the R2509C mutation in RYR1 causes dysfunctional Ca2+ dynamics in a mutant-gene dose-dependent manner in the skeletal muscles, in turn provoking MH-like episodes and embryonic lethality in heterozygous and homozygous mice, respectively.
... These findings suggest that calpain-dependent degradation of titin may play a significant role in the mechanical unloading-induced reduction in the intrinsic soleus muscle stiffness. The obtained data on the decreased content of titin in rat soleus under unloading conditions are consistent with previously published reports [47,55,58,60]. It is well established that a giant protein titin significantly contributes to the intrinsic passive stiffness/tension of muscle fibers [18,55]. ...
In mammals, prolonged mechanical unloading results in a significant decrease in passive stiffness of postural muscles. The nature of this phenomenon remains unclear. The aim of the present study was to investigate possible causes for a reduction in rat soleus passive stiffness after 7 and 14 days of unloading (hindlimb suspension, HS). We hypothesized that HS-induced decrease in passive stiffness would be associated with calpain-dependent degradation of cytoskeletal proteins or a decrease in actomyosin interaction. Wistar rats were subjected to HS for 7 and 14 days with or without PD150606 (calpain inhibitor) treatment. Soleus muscles were subjected to biochemical analysis and ex vivo measurements of passive tension with or without blebbistatin treatment (an inhibitor of actomyosin interactions). Passive tension of isolated soleus muscle was significantly reduced after 7- and 14-day HS compared to the control values. PD150606 treatment during 7- and 14-day HS induced an increase in alpha-actinin-2 and -3, desmin contents compared to control, partly prevented a decrease in intact titin (T1) content, and prevented a decrease in soleus passive tension. Incubation of soleus muscle with blebbistatin did not affect HS-induced reductions in specific passive tension in soleus muscle. Our study suggests that calpain-dependent breakdown of cytoskeletal proteins, but not a change in actomyosin interaction, significantly contributes to unloading-induced reductions in intrinsic passive stiffness of rat soleus muscle.
... Thus, an increase in individual sarcomere lengths caused by sarcomere length nonuniformity would increase the Ca 2 þ sensitivity of the myofibrils. Second, the titininduced passive force is an important contributor to the increase in Ca 2 þ sensitivity at long sarcomere lengths (59)(60)(61). Although there are no other studies investigating the Ca 2 þ sensitivity of the contractile apparatus in myofibrils, it has been shown that the level of muscle activation influences directly the RFE; stretches invoked in low Ca 2 þ concentrations lead to a larger RFE than large Ca 2 þ concentrations (3). Figure 10. ...
When muscle fibers from limb muscles are stretched while activated, the force increases to a steady-state level that is higher than that produced during isometric contractions at a corresponding sarcomere length, a phenomenon known as residual force enhancement (RFE). The mechanisms responsible for the RFE are an increased stiffness of titin molecules which may lead to an increased Ca ²⁺ sensitivity of the contractile apparatus, and the development of sarcomere length non-uniformities. RFE is not observed in cardiac muscles, which makes this phenomenon specific to certain preparations. The aim of this study was to investigate if the RFE is present in the diaphragm, and its potential association with an increased Ca ²⁺ sensitivity and the development of sarcomere length non-uniformities. We used two preparations: single intact fibers and myofibrils isolated from the diaphragm from mice. We investigated RFE in a variety of lengths across the force-length relationship. RFE was observed in both preparations at all lengths investigated, and was larger with increasing magnitudes of stretch. RFE was accompanied by an increased Ca ²⁺ sensitivity as shown by a change in the force-pCa ²⁺ -curve, and increased sarcomere length non-uniformities. Therefore, RFE is a phenomenon commonly observed in skeletal muscles, with mechanisms that are similar across preparations.
... Shorter thick filament length due to remodeling could also reduce active force generation because fewer myosin heads would be available to strongly bind to actin. Myosin and/or actin loss, titin modifications, and/or thick filament shortening have been observed in disuse and disease (53,54,55); however, direct data on these potential mechanisms in prehemodialysis CKD patients are lacking. CKD may additionally cause post-translational modifications of sarcomeric proteins, leading to altered properties. ...
Background:
Patients with chronic kidney disease (CKD) frequently have compromised physical performance, which increases their mortality; however, their skeletal muscle dysfunction has not been characterized at the single-fiber and molecular levels. Notably, interventions to mitigate CKD myopathy are scarce.
Methods:
The effect of CKD in the absence and presence of iron supplementation on the contractile function of individual skeletal muscle fibers from the soleus and extensor digitorum longus muscles was evaluated in 16-week-old mice. CKD was induced by the adenine diet, and iron supplementation was by weekly iron dextran injections.
Results:
Maximally activated and fatigued fiber force production was decreased 24%-52% in untreated CKD, independent of size, by reducing strongly bound myosin/actin cross-bridges and/or decreasing myofilament stiffness in myosin heavy chain (MHC) I, IIA, and IIB fibers. Additionally, myosin/actin interactions in untreated CKD were slower for MHC I and IIA fibers and unchanged or faster in MHC IIB fibers. Iron supplementation improved anemia and did not change overall muscle mass in CKD mice. Iron supplementation ameliorated CKD-induced myopathy by increasing strongly bound cross-bridges, leading to improved specific tension, and/or returning the rate of myosin/actin interactions toward or equivalent to control values in MHC IIA and IIB fibers.
Conclusions:
Skeletal muscle force production was significantly reduced in untreated CKD, independent of fiber size, indicating that compromised physical function in patients is not solely due to muscle mass loss. Iron supplementation improved multiple aspects of CKD-induced myopathy, suggesting that timely correction of iron imbalance may aid in ameliorating contractile deficits in CKD patients.
... Loss of passive stiffness has been observed in human tendons after long periods (90-180 days) of simulated microgravity 38,39 , but also after 20 days of bed rest 40 . Muscle disuse also produces a decrease of intrinsic muscle passive tension by reducing the amount of titin, a noncontractile protein that is important for the structural stability of the sarcomere [41][42][43] . ...
Chronic obstructive pulmonary disease (COPD) produces skeletal muscle atrophy and weakness, leading to impairments of exercise performance. The mechanical work needed for movement execution is also provided by the passive tension developed by musculoarticular connective tissue. To verify whether COPD affects this component, the passive viscoelastic properties of the knee joint were evaluated in 11 patients with COPD and in 11 healthy individuals. The levels of stiffness and viscosity were assessed by means of the pendulum test, consisting in a series of passive leg oscillations. In addition, to explore the contribution of passive tension in the mechanical output of a simple motor task, voluntary leg flexion–extension movements were performed. Patients with COPD showed a statistically significant reduction in stiffness and viscosity compared to controls. Voluntary execution of flexion–extension movements revealed that the electromyographic activity of the Rectus Femoris and Biceps Femoris was lower in patients than in controls, and the low viscoelastic tension in the patients conditioned the performance of active movements. These results provide novel insights on the mechanism responsible for the movement impairments associated with COPD.
... Pressure was applied to the muscle fibers under relaxing solution or rigor solution with the pressure pump used in the above "Optical microscopic observation of combining myofibrils under atmospheric pressure", and a sample was removed for use as an observation sample. This sample was fixed with 2% (vol/vol) glutaraldehyde, embedded in epoxy resin, sliced to a thickness of approximately 50 nm, and then observed with a transmission electron microscope (H-7500, Hitachi) [30]. ...
The effects of high pressure (40–70 MPa) on the structure and function of myofibrils were investigated by high pressure microscopy. When this pressure was applied to myofibrils immersed in relaxing solution, the sarcomere length remained almost unchanged, and the A band became shorter and wider. The higher the applied pressure, the faster the change. However, shortening and widening of the A band were not observed when pressure was applied to myofibrils immersed in a solution obtained by omitting ATP from the relaxing solution. However, even under these conditions, structural loss, such as loss of the Z-line structure, occurred. In order to evaluate the consequences of this pressure-treated myofibril, the oscillatory movement of sarcomere (sarcomeric oscillation) was evoked and observed. It was possible to induce sarcomeric oscillation even in pressure-treated myofibrils whose structure was destroyed. The pressurization reduced the total power of the sarcomeric oscillation, but did not change the average frequency. The average frequency did not change even when a pressure of about 40 MPa was applied during sarcomeric oscillation. The average frequency returned to the original when the pressure was returned to the original value after applying stronger pressure to prevent the sarcomere oscillation from being observed. This result suggests that the decrease in the number of myosin molecules forming the crossbridge does not affect the average frequency of sarcomeric oscillation. This fact will help build a mechanical hypothesis for sarcomeric oscillation. The pressurization treatment is a unique method for controlling the structure of myofibrils as described above.
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... Thus, a biomarker to assess muscle atrophy is urgently needed. In a rat study, Udaka et al. found that six weeks of immobilization caused titin loss in the soleus muscle, which was associated with muscle dysfunction [35]. Thus, it is theoretically reasonable to expect levels of urinary titin to be elevated in the urine of patients with muscle atrophy. ...
Titin is a giant protein that functions as a molecular spring in sarcomeres. Titin interconnects the contraction of actin-containing thin filaments and myosin-containing thick filaments. Titin breaks down to form urinary titin N-fragments, which are measurable in urine. Urinary titin N-fragment was originally reported to be a useful biomarker in the diagnosis of muscle dystrophy. Recently, the urinary titin N-fragment has been increasingly gaining attention as a novel biomarker of muscle atrophy and intensive care unit-acquired weakness in critically ill patients, in whom titin loss is a possible pathophysiology. Furthermore, several studies have reported that the urinary titin N-fragment also reflected muscle atrophy and weakness in patients with chronic illnesses. It may be used to predict the risk of post-intensive care syndrome or to monitor patients’ condition after hospital discharge for better nutritional and rehabilitation management. We provide several tips on the use of this promising biomarker in post-intensive care syndrome.
... Further possible explanation of reduced muscle function could be the marked disarray in the sarcomere structure. 48 As a result of the above-mentioned reasons, such an analysis was not feasible. Similarly, quantitative 3-dimensional mapping of the human skeletal muscle mitochondrial might be a better method to describe mitochondrial function. ...
BACKGROUND: Reduced exercise capacity in patients with heart failure (HF) could be partially explained by skeletal muscle dysfunction. We compared skeletal muscle function, structure, and metabolism among clinically stable outpatients with HF with preserved ejection fraction, HF with reduced ejection fraction, and healthy controls (HC). Furthermore, the molecular, metabolic, and clinical profile of patients with reduced muscle endurance was described.
METHODS: Fifty-five participants were recruited prospectively at the University Hospital Jena (17 HF with preserved ejection fraction, 18 HF with reduced ejection fraction, and 20 HC). All participants underwent echocardiography, cardiopulmonary exercise testing, 6-minute walking test, isokinetic muscle function, and skeletal muscle biopsies. Expression levels of fatty acid oxidation, glucose metabolism, atrophy genes, and proteins as well as inflammatory biomarkers were assessed. Mitochondria were evaluated using electron microscopy.
RESULTS: Patients with HF with preserved ejection fraction showed compared with HF with reduced ejection fraction and HC reduced muscle strength (eccentric extension: 13.3±5.0 versus 18.0±5.9 versus 17.9±5.1 Nm/kg, P=0.04), elevated levels of MSTN-2 (myostatin-2), FBXO-32 (F-box only protein 32 [Atrogin1]) gene and protein, and smaller mitochondrial size (P<0.05). Mitochondrial function and fatty acid and glucose metabolism were impaired in HF- patients compared with HC (P<0.05). In a multiple regression analysis, GDF-15 (growth and differentiation factor 15), CPT1B (carnitine palmitoyltransferase IB)-protein and oral anticoagulation were independent factors for predicting reduced muscle endurance after adjusting for age (log10 GDF-15 [pg/mL] [B, −54.3 (95% CI, −106 to −2.00), P=0.043], log10 CPT1B per fold increase [B, 49.3 (95% CI, 1.90–96.77), P=0.042]; oral anticoagulation present [B, 44.8 (95% CI, 27.90–61.78), P<0.001]).
CONCLUSIONS: Patients with HF with preserved ejection fraction have worse muscle function and predominant muscle atrophy compared with those with HF with reduced ejection fraction and HC. Inflammatory biomarkers, fatty acid oxidation, and oral anticoagulation were independent factors for predicting reduced muscle endurance.
... It has been shown that the development of muscle atrophy under conditions of gravitational unloading for 7 days or more (up to 6 weeks) is attended by increased proteolysis of intact titin-1 (T1), a decrease in its content and an increase in the content of proteolytic T2 fragments of this protein [1,[11][12][13][14]. These changes disrupt highly ordered sarcomere structure and impair muscle contractility. ...
We studied the effect of histone deacetylase 1 (HDAC1) inhibition on titin content and expression of TTN gene in rat m. soleus after 3-day gravitational unloading. Male Wistar rats weighing 210±10 g were randomly divided into 3 groups: control, 3-day hindlimb suspension, and 3-day hindlimb suspension and injection of HDAC1 inhibitor CI-994 (1 mg/kg/day). In hindlimb-suspended rats, the muscle weight/animal body weight ratio was reduced by 13.8% (p<0.05) in comparison with the control, which attested to the development of atrophic changes in the soleus muscle. This was associated with a decrease in the content of NT-isoform of intact titin-1 by 28.6% (p˂0.05) and an increase in TTN gene expression by 1.81 times (p˂0.05) in the soleus muscle. Inhibition of HDAC1 by CI-994 during 3-day hindlimb suspension prevented the decrease in titin content and development of atrophy in rat soleus muscle. No significant differences in the TTN gene expression from the control were found. These results can be used when finding the ways of preventing or reducing the negative changes in the muscle caused by gravitational unloading.
