CAS3D: 3D quantitative morphometry on Second Harmonic Generation image volumes from single skeletal muscle fibers

The purpose of the present study was to test the hypothesis that a preceding contractile period in isolated single skeletal muscle fibers would attenuate the rise in cytosolic [H+] ([H+]cyt) during a subsequent contractile period, thereby reducing the rate of fatigue. Intact single skeletal muscle fibers (n = 9) were isolated from Xenopus lumbrical muscle, and incubated with the [H+]cyt indicator BCECF AM for 30 min. Following incubation, fibers were mounted in a glass chamber on the stage of an inverted microscope configured for epl-illumination. Two identical contractile periods were performed in each fiber, separated by a 1hr recovery period. Force and [H+cyt were measured simultaneously while fibers were stimulated (tetanic contractions of 350 ms trains with 70 Hz stimuli at 9 V) at progressively increasing frequencies (0.25, 0.33, 0.5 and 1 contraction/sec) until significant fatigue developed (50% initial force). No significant difference (P < 0.05) was observed between the first and second contractile periods in initial force development or in resting [H+]cyt. Similarly, no significant difference was observed in the time to fatigue between the first (341 ± 36 sec) and second (333 ± 38 sec) contractile periods. However, the increase in [H+]cyt from rest to fatigue was significantly greater (P < 0.05) during the first contractile period (175 ± 36 nmol) when compared to the second (70 ± 18 nmol). The results of the present study demonstrated in contracting single skeletal muscle fibers that when preceded by an initial fatiguing contractile period, the subsequent increase in [H+]cyt during a second contractile period was reduced. However, this did not attenuate the fatigue process in the second contractile period, suggesting that factors other than H+ ion accumulation compromised force development under these conditions. Supported by NIH Grant No. AR-40155

The purpose of the present study was to test the hypothesis that a preceding contractile period in isolated single skeletal muscle fibers would attenuate the decrease in pH during an identical, subsequent contractile period, thereby reducing the rate of fatigue. Intact single skeletal muscle fibers (n = 9) were isolated from Xenopus lumbrical muscle and incubated with the fluorescent cytosolic H+ indicator 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) AM for 30 min. Two identical contractile periods were performed in each fiber, separated by a 1-h recovery period. Force and intracellular pH (pHi) fluorescence were measured simultaneously while fibers were stimulated (tetanic contractions of 350-ms trains with 70-Hz stimuli at 9 V) at progressively increasing frequencies (0.25, 0.33, 0.5, and 1 contraction/s) until the development of fatigue (to 60% initial force). No significant difference (P < 0.05) was observed between the first and second contractile periods in initial force development, resting pHi, or time to fatigue (5.3 +/- 0.5 vs. 5.1 +/- 0.6 min). However, the relative decrease in the BCECF fluorescence ratio (and therefore pHi) from rest to the fatigue time point was significantly greater (P < 0.05) during the first contractile period (to 65 +/- 4% of initial resting values) compared with the second (77 +/- 4%). The results of the present study demonstrated that, when preceded by an initial fatiguing contractile period, the rise in cytosolic H+ concentration in contracting single skeletal muscle fibers during a second contractile period was significantly reduced but did not attenuate the fatigue process in the second contractile period. These results suggest that intracellular factors other than H+ accumulation contribute to the fall in force development under these conditions.

The rising and falling phases of the active state were analyzed, by means of quick‐release technique, during a series of 4 incompletely fused isometric twitches in single semitendinosus muscle fibres of the frog. The analysis was carried out at different sarcomere lengths within the range 1.7–2.8 μ. The rate of rise of the active state was independent of the sarcomere length. A reduction in fibre length was associated with an earlier onset of the decay of the active state leading to a decrease in duration of the activity at the shorter length. The duration of the active state at 1.9 μ sarcomere spacing was 69 (range: 51–40) per cent of the duration existing at 2.6 μ sarcomere length as determined in 7 different fibres. Movement (active shortening) per se, apart from the change in sarcomere length, affected the kinetics of the active state. An interval of free shortening interposed during an isometric contraction reduced the fibre's capacity to produce tension for the rest of the contraction period without changing the total duration of the mechanical activity substantially. The movement produced a relatively small effect as long as the active state was maximum; the depressant effect became greater and greater, however, the later the movement occurred during the decay phase of the active state. The nature of the length and movement dependence of the active state is discussed in the light of the calcium‐activator mechanism of the excitation‐contraction coupling.

Laser diffraction and photomicrography have been used to monitor sarcomere length changes in single muscle fibres of the frog, at long sarcomere lengths, during fixed end tetani. In the central 90% of all fibres, changes in sarcomere length were consistently less than 0.25 micron. Sarcomere length showed an initial rapid change, followed by a progressively slower increase, which persisted throughout a 4s tetanus. Sarcomere length in the terminal 200-400 microns segment at each end of a fibre decreased rapidly by up to 1 micron in the first second of a tetanus. This shortening was accompanied by a marked increase in disorder of the striation pattern. Maximum isometric tensions in fixed end tetani were much greater than those predicted by crossbridge theory over the entire range of sarcomere lengths studied. An analysis of the intersarcomere dynamics suggests that this extra tension may be explained by known phenomena on the basis of a progressive increase in sarcomere length dispersion along the fibre.

Dietary nitrate supplementation increases plasma nitrite concentration, which provides an oxygen-independent source of nitric oxide and can delay skeletal muscle fatigue. Nitrate supplementation has been shown to increase myofibre calcium release and force production in mouse skeletal muscle during contractions at a supra-physiological oxygen tension, but it is unclear whether nitrite exposure can delay fatigue development and improve myofibre calcium handling at a near-physiological oxygen tension. Single mouse muscle fibres acutely treated with nitrite had a lower force and cytosolic calcium concentration during single non-fatiguing contractions at a near-physiological oxygen tension. Nitrite treatment delayed fatigue development during repeated fatiguing isometric contractions at near-physiological, but not at supra-physiological, oxygen tension in combination with better maintenance of myofilament calcium sensitivity and sarcoplasmic reticulum calcium pumping. These findings improve understanding of the mechanisms by which increased skeletal muscle nitrite exposure might be ergogenic and imply that this is related to improved calcium handling. Dietary nitrate (NO3- ) supplementation, which increases plasma nitrite (NO2- ) concentration, has been reported to attenuate skeletal muscle fatigue development. Sarcoplasmic reticulum (SR) calcium (Ca2+ ) release is enhanced in isolated single skeletal muscle fibres following NO3- supplementation or NO2- incubation at a supra-physiological but it is unclear whether NO2- incubation can alter Ca2+ handling and fatigue development at a near-physiological . We hypothesised that NO2- treatment would improve Ca2+ handling and delay fatigue at a physiological in intact single mouse skeletal muscle fibres. Each muscle fibre was perfused with Tyrode solution pre-equilibrated with either 20% ( ∼150Torr) or 2% O2 ( =15.6Torr) in the absence and presence of 100µM NaNO2 . At supra-physiological (i.e. 20% O2 ), time to fatigue was lowered by 34% with NaNO2 (control: 257±94 vs. NaNO2 : 159±46s, Cohen's d=1.63, P<0.05), but extended by 21% with NaNO2 at 2% O2 (control: 308±217 vs. NaNO2 : 368±242s, d=1.14, P<0.01). During the fatiguing contraction protocol completed with NaNO2 at 2% O2 , peak cytosolic Ca2+ concentration ([Ca2+ ]c ) was not different (P>0.05) but [Ca2+ ]c accumulation between contractions was lower, concomitant with a greater SR Ca2+ pumping rate (P<0.05) compared to the control condition. These results demonstrate that increased exposure to NO2- blunts fatigue development at near-physiological, but not at supra-physiological, through enhancing SR Ca2+ pumping rate in single skeletal muscle fibres. These findings extend our understanding of the mechanisms by which increased NO2- exposure can mitigate skeletal muscle fatigue development.

The purpose of the present study was to examine the effects of aging, inactivity and weight-bearing exercise on fast-twitch single Type IIB skeletal muscle fibers from the superficial region of the lateral head of the gastrocnemius (Type IIB fibers). Specifically, this study compared the biochemical properties of Type IIB fibers after 7 days of hindlimb unweighting (HU), 7 days of HU with intermittent weight-bearing (HU-Ex), and cage control (C) from adult and aged Fischer 344 Brown Norway F1 Hybrid rats (12- and 30-month old). Biochemical measurements included total lactate dehydrogenase (LDH) and beta-hydroxy-acyl-coenzyme A dehydrogenase activities (BHAD), expressed in nmoles/microg/hr dry weight. Fiber-typing for myosin heavy chain isoform was determined by SDS-PAGE. With age, LDH activity in Type IIB fibers decreased from 52.0 +/- 3.4 nmoles/microg/hr (12-month old) to 39.5 +/- 2.9 nmoles/microg/hr (30-month old). Following HU, LDH activity of single Type IIB fibers increased by 22% (52.0 +/- 3.4 to 66.4 +/- 3.2 nmoles/microg/hr) in the 12-month-old animals, whereas no difference was observed with HU in the Type IIB fibers of the 30-month-old animals. Following HU-Ex, LDH activity of Type IIB fibers in the 12-month-old animals was not significantly different from that of Type IIB fibers from HU animals, whereas a significant increase was observed (38.1 +/- 2.9 to 51.8 +/- 3.1 nmoles/microg/hr) in Type IIB fibers of 30-month-old animals, for HU and HU-Ex, respectively. Analysis of variance revealed an interaction between age and condition, indicating that Type IIB fibers from adult and aged animals have a different biochemical response to inactivity. The enzyme activities for BHAD were not different between the experimental conditions. The results demonstrate that the total LDH enzyme activities of the Type IIB fibers decrease with age, suggesting an age-related shift in the biochemical profile. Further, single skeletal muscle fiber adaptation is age-dependent.

The intrinsic activating factors that induce transcription of heat shock protein 72 (HSP72) in skeletal muscle following exercise remain unclear. We hypothesized that the cytosolic Ca(2+) transient that occurs with depolarization is a determinant. We utilized intact, single skeletal muscle fibers from Xenopus laevis to test the role of the cytosolic Ca(2+) transient and several other exercise-related factors (fatigue, hypoxia, AMP kinase, and cross-bridge cycling) on the activation of HSP72 transcription. HSP72 and HSP60 mRNA levels were assessed with real-time quantitative PCR; cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was assessed with fura-2. Both fatiguing and nonfatiguing contractions resulted in a significant increase in HSP72 mRNA. As expected, peak [Ca(2+)]cyt remained tightly coupled with peak developed tension in contracting fibers. Pretreatment with N-benzyl-p-toluene sulfonamide (BTS) resulted in depressed peak developed tension with stimulation, while peak [Ca(2+)]cyt remained largely unchanged from control values. Despite excitation-contraction uncoupling, BTS-treated fibers displayed a significant increase in HSP72 mRNA. Treatment of fibers with hypoxia (Po2: <3 mmHg) or AMP kinase activation had no effect on HSP72 mRNA levels. These results suggest that the intermittent cytosolic Ca(2+) transient that occurs with skeletal muscle depolarization provides a sufficient activating stimulus for HSP72 transcription. Metabolic or mechanical factors associated with fatigue development and cross-bridge cycling likely play a more limited role.

The protein substructure of skeletal muscle fibers forms a diffraction grating with repeating units, termed 'sarcomeres'. A laser scanning system is described that maps the lengths of sarcomeres (SL) and the widths of the first-order diffraction lines (DLW) of permeabilized single fibers in real-time. The apparatus translates a laser beam (λ = 670 nm and w0 = ~75 μm) along the length of a fiber segment through 20 contiguous regions per sweep at 500 sweeps/s. The fiber segments (~1 mm long) were obtained from vastus lateralis muscles of humans by needle biopsy. During both passive stretches and maximum fixed-end activations, the mappings of SL and DLW of the fibers were extracted from the diffraction spectra. Heterogeneity of SLs was evaluated by computing the standard deviation ( σSL) of the 20 SLs measured during a single sweep. Compared with the σSL before a passive stretch, the increase of 5±0.5% in σSL after the passive stretch, indicated differences in passive length-tension relationships along the fiber. In contrast, no change, ~0.5±0.1%, was observed in DLW. Within 10s after the fiber was returned to its initial length, the shape of the SL profile returned close to pre-stretch conditions ( σSL = 1± 0.2%). Following maximum Ca2+ - activation of the fiber, the heterogeneity of the steady state SLs increased greatly (DLW up by ~300% and σSL up by ~100%). The scanning system provided high resolution tracking of sarcomere behavior single muscle fibers. Potential applications are for studies of the mechanisms of muscle fiber injury and injury propagation.

Accurate and robust detection of myonuclei in single muscle fiber is required to calculate myonuclear domain size. However, this task is challenging because: 1) The myonuclei have a variety of sizes and shapes. 2) Imaging techniques exhibit myonuclei that are often overlapping. 3) Inhomogeneous intensity due to DAPI concentration in heterochromatin, abundant in mouse nuclei, results in a speckled appearance inside each myonucleus. In this paper, we propose a novel automatic approach to robustly detect the myonuclei in isolated single skeletal muscle fibers. The original z-stack images are first fused into one all-in-focus image. A sufficient number of ellipse fitting hypotheses are then generated using the myonuclei contour segments. A set of morphological features are calculated from the ellipses and utilized to train a support vector machine (SVM) classifier to choose the best candidates. A modified inner geodesic distance based clustering algorithm is used to produce the final results. The proposed method was extensively tested using 42 sets of z-stack images containing about 1500 myonuclei. The method demonstrates excellent results outperforming current state-of-the-arts.

Skeletal muscle is an archetypal organ whose structure is tuned to match function. The magnitude of order in muscle fibers and myofibrils containing motor protein polymers determines the directed force output of the summed force vectors and, therefore, the muscle’s power performance on the structural level. Structure and function can change dramatically during disease states involving chronic remodeling. Cellular remodeling of the cytoarchitecture has been pursued using noninvasive and label-free multiphoton second harmonic generation (SHG) microscopy. Hereby, structure parameters can be extracted as a measure of myofibrillar order and thus are suggestive of the force output that a remodeled structure can still achieve. However, to date, the parameters have only been an indirect measure, and a precise calibration of optical SHG assessment for an exerted force has been elusive as no technology in existence correlates these factors. We engineered a novel, automated, high-precision biomechatronics system into a multiphoton microscope allows simultaneous isometric Ca2+-graded force or passive viscoelasticity measurements and SHG recordings. Using this MechaMorph system, we studied force and SHG in single EDL muscle fibers from wt and mdx mice; the latter serves as a model for compromised force and abnormal myofibrillar structure. We present Ca2+-graded isometric force, pCa-force curves, passive viscoelastic parameters and 3D structure in the same fiber for the first time. Furthermore, we provide a direct calibration of isometric force to morphology, which allows noninvasive prediction of the force output of single fibers from only multiphoton images, suggesting a potential application in the diagnosis of myopathies.

Sarcomere length dependence of the force-velocity relation in single frog muscle fibers

Skeletal muscle fatigue is a multi-factorial process that involves Ca2+ release and uptake from sarcoplasmic reticulum (SR), Ca2+ sensitivity in myofilaments, cross-bridge activation, and ATP supply from anaerobic and aerobic metabolism. PURPOSE: Investigate the manner in which Ca2+ release and uptake by the SR is dependent on cross-bridge activity during fatiguing contractions. METHODS: In order to determine Ca2+ transients during contractions, intact Xenopus single muscle fibers were microinjected with FURA-2, followed by a fatigue protocol (increasing frequency of contractions until force reduced to 50% of peak) in the presence or absence of the specific cross-bridge cycling inhibitor N-benzyl-p-toluene sulfonamide (BTS, 12.5 μM, ∼98% force inhibition). RESULTS: In the absence of BTS at the fatigue time-point when force was reduced to 50% of peak, peak intracellular Ca2+ was reduced (46 ± 4 % at 0.33 Hz), Ca2+ accumulated in the cytosol (∼2.3x), and there was a progressive slower rate of Ca2+ uptake into the SR during the relaxation period (T0.5= 30 ± 5 ms during first contraction to 230 ± 5 ms at fatigue, P<0.01). Compared to the same frequency that promoted fatigue during control (0.33 Hz), BTS treatment resulted in increased peak intracellular Ca2+ (42 ± 8%) and reduced Ca2+ accumulation in the cytosol (44 ± 17% at 0.33 Hz). Interestingly, with BTS the rate of SR Ca2+ uptake was unchanged at the fatigue time-point (T0.5= 13 ± 7 ms during first contraction to 44 ± 20 ms at fatigue, P>0.05). CONCLUSION: These results suggest that factors related to cross-bridge cycling limit Ca2+ uptake during fatiguing contractions, and that this may be due to limited ATP availability for SERCA activity or accumulation of metabolites. Supported by NIH grant 5R01AR040155-17.

In vitro experiments indicate a non-metabolic role of muscle glycogen in contracting skeletal muscles. Since the sequence of events in excitation\#8211;contraction (E\#8211;C) coupling is known to be located close to glycogen granules, at specific sites on the fibre, we hypothesized that the distinct compartments of glycogen have specific effects on muscle fibre contractility and fatigability. Single skeletal muscle fibres (n = 19) from fed and fasted rats were mechanically skinned and divided into two segments. In one segment glycogen localization and volume fraction were estimated by transmission electron microscopy. The other segment was mechanically skinned and, in the presence of high and constant myoplasmic ATP and PCr, electrically stimulated (10 Hz, 0.8 s every 3 s) eliciting repeated tetanic contractions until the force response was decreased by 50% (mean +/- S.E.M., 81 +/- 16, range 22-252 contractions). Initially the total myofibrillar glycogen volume percentage was 0.46 +/- 0.07%, with 72 +/- 3% in the intermyofibrillar space and 28 +/- 3% in the intramyofibrillar space. The intramyofibrillar glycogen content was positively correlated with the fatigue resistance capacity (r(2) = 0.32, P = 0.02). Intermyofibrillar glycogen was inversely correlated with the half-relaxation time in the unfatigued tetanus (r(2) = 0.25, P = 0.03). These results demonstrate for the first time that two distinct subcellular populations of glycogen have different roles in contracting single muscle fibres under conditions of high myoplasmic ATP.

BackgroundWhile aging causes muscle weakness, type 2 diabetes mellitus (T2DM) is also considered a high-risk factor for the induction of skeletal muscle weakness. Previous studies have reported increased collagen content in insulin-resistant skeletal muscles. Here, we studied the mechanical properties of aged skeletal muscle in patients with T2DM to investigate whether aged skeletal muscles with T2DM induce higher passive tension due to the abundance of extracellular matrix (ECM) inside or outside of the muscle fibers. MethodsSamples from the gluteus maximus muscles of older adults with diabetes (T2DM) and non-diabetic (non-DM) older adults who underwent elective orthopedic surgery were collected. Permeabilized single muscle fibers from these samples were used to identify their mechanical properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to quantify titin and fiber type distributions in these samples. ResultsWe confirmed a significant predominance of type I fiber ratio in both T2DM and non-DM aged muscles. While the average cross-sectional area and maximal active tension of the single fibers were smaller in the T2DM group than those in the non-DM group, the difference was not statistically significant. T2DM subjects showed significantly greater passive tension and lower titin-/ECM-based passive tension ratios than those in non-DM subjects, which indicated that more ECM but less titin contributed to the total passive tension. ConclusionBased on our findings, we concluded that T2DM may cause increased passive stiffness of single skeletal muscle fibers in older adults because of an excessive accumulation of ECM in and around single muscle fibers due to increased insulin resistance.

At sarcomere lengths above the plateau region of the length-tension diagram, it has been found that isometric tetanic tension is proportional to the amount of thick and thin filament overlap. This finding has been questioned recently and is reinvestigated here. Central segments of single frog skeletal muscle fibres were held at constant length during contractions at various sarcomere lengths above those associated with the plateau region. Tension records showed little or no creep, and the tetanic tensions measured at 0 and 20°C were inversely proportional to sarcomere length. These results extend and substantiate earlier findings. In contrast, when a stretched fibre had only its ends fixed during a tetanus, a different tension response was observed. The tension rise was initially very rapid but soon slowed to a gradual upward creep as stimulation was continued. This was followed by a tension decline. These tension phases were correlated with large decreases in sarcomere length at the fibre ends, while sarcomeres in the middle were extended a small amount. This tetanic tension response can be explained using the sarcomere length-tension relation and the force-velocity properties of muscle. These results strongly support the sliding filament, cross-bridge theory of muscle contraction.