Use of the intact mouse skeletal-muscle preparation for metabolic studies. Evaluation of the model.

1. We examined the isolated mouse skeletal-muscle model in vitro, commonly used by many investigators, for its suitability for metabolic studies. 2. Despite the fact that pH, O2 saturation, osmolality and the release of the enzyme creatine kinase remained stable, histochemical studies showed large cores devoid of glycogen, suggesting that the incubated muscle had lost its viability. 3. This study indicates that caution should be exercised when interpreting the results of studies with intact isolated mouse muscles.     

Electron spin resonance studies of intact mammalian skeletal muscle

Samples of skeletal muscle from mice, rats and man have been examined by conventional electron spin resonance techniques. One major free-radical signal with g value 2.0036-2.004 was detected in all intact muscle samples and homogenates at 77 K whereas this signal was not seen at room temperature. Other less prominant signals were also detected. Thirty minutes of excessive contractile activity of rat hind limb muscles was found to result in a leakage of intracellular creatine kinase enzyme into the blood plasma and also produced an average 70% increase in the amplitude of the major electron spin resonance signal. These data support the hypothesis that increased free-radical activity may play some role in muscle damage caused by extensive muscular activity.     

Use of papain in the preparation of adult mammalian skeletal muscle for tissue culture

Summary This report describes in detail a method of enzymatic separation of adult mammalian muscle using papain. The procedure has proved valuable in the preparation of suspensions of muscle cell pieces from normal human skeletal muscle obtained from patients of all ages, from 3 months to 79 years. Muscle cultures have been successfully growth from biopsy material from boys with Duchenne’s muscular dystrophy and from their mothers. The procedure was initially established with adult canine skeletal muscle and has also been used for monkey muscle. Small pieces of skeletal muscle are chopped in a solution of 0.05% papain and 0.01% cysteine hydrochloride in Ca²⁺-and Mg²⁺-free balanced salt solution and transferred in the papain solution to a flask, in which they are incubated at 37°C for 10 min with occasional agitation. The resulting cell suspension is collected and the remaining pieces are treated with further portions of fresh papain until only connective tissue remains. The cell pellets obtained by centrifugation are resuspended in Eagle’s minimum essential medium (supplemented with 20% fetal calf serum) and transferred to culture chambers. The muscle can be observed at all times, during the separation procedure and subsequently in culture. The events occurring during skeletal muscle regeneration can be followed. Using the same papain preparation, myoblasts and myotubes may be subcultured and collected for indefinite frozen storage in dimethylsulfoxide. This work was supported by a grant-in-aid from the American Heart Association with funds contributed in part by the Chicago Heart Association, the Pharmaceutical Manufactures Association, and National Institutes of Health Research Grant NS 10385 from the Institute of Neurological Diseases and Stroke.     

Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies

It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production. respiration rate; parallel activation; oxidative phosphorylation; metabolic control analysis; flux control coefficient; muscle contraction     

Regional skeletal muscle measurement: evaluation of new dual-energy X-ray absorptiometry model.

Although there is growing interest in studying muscle distribution, regional skeletal muscle (SM) mass measurement methods remain limited. The aim of the present study was to develop a new dual-energy X-ray absorptiometry (DEXA) model for estimating regional adipose tissue-free skeletal muscle mass (AT-free SM). Relationships were derived from Reference Man data between tissue-system- level components (i.e., AT-free SM, AT, skeleton, and skin) and molecular-level components including fat-free soft tissue, fat, and bone mineral. The proposed DEXA-SM model was evaluated by multiscan computerized axial tomography (CT). Twenty-seven male subjects [age, 36 +/- 12 (SD) yr; body mass, 73.2 +/- 12.4 kg; 20 were healthy, and 7 had acquired immunodeficiency syndrome] completed DEXA and CT studies. Identical landmarks for DEXA and CT measurements were selected in three regions, including calves, thighs, and forearms. There was a strong correlation for AT-free SM estimates between the new DEXA and CT methods (e.g., sum of three regions, r = 0.86, P < 0.001). Regional AT-free SM measured in the 27 subjects by DEXA and CT, respectively, were 3.44 +/- 0.60 and 3. 47 +/- 0.55 kg (difference 0.9%, P > 0.05) for calves, 10.49 +/- 1. 77 and 10.05 +/- 1.79 kg (difference 4.4%, P < 0.05) for thighs, 1. 36 +/- 0.49 and 1.20 +/- 0.41 kg (difference 13.3%, P < 0.01) for forearms, and 15.29 +/- 2.33 and 14.72 +/- 2.33 kg (difference 3.9%, P < 0.05) for the sum all three regions. Although the suggested DEXA-SM model needs minor refinements, this is a promising in vivo approach for measurement of regional SM, because DEXA is widely available, relatively inexpensive, and radiation exposure is low.     

A rapid preparation of primary cultures of mouse skeletal muscle cells

We describe a rapid and reproducible technique for establishing primary cultures of skeletal muscle cells from mouse origin. This method was aimed at avoiding extensive enzymatic proteolysis which is commonly used for preparation of primary skeletal muscle cultures. It relies on a Stomacher^® blender that allows a rapid and regular mechanical dissociation of muscle samples by repeated shocks. Cultures have been compared to those obtained by a modification of the method of Yaffé (1993) based on tryptic dissociation of rat muscle thighs. The time of preparation was reduced to 1 h and 15 min as compared to 4 h with the technique of Yaffé. Both cultures displayed similar morphologies and exhibited comparable myogenesis processes. Cellular yield, rate of myotube formation and myotube numbers were similar. The expression of myogenesis markers were identical as assessed by determination of acetylcholine receptor number, creatine kinase activity and level of myosin light chain.Abbreviations AChR Acetylcholine receptor - CK creatine kinase (EC 2.7.3.2) - PBS Phosphate buffered saline-free of Ca²⁺ and Mg²⁺     

Calcium sparks in intact skeletal muscle fibers of the frog.

Calcium sparks were studied in frog intact skeletal muscle fibers using a home-built confocal scanner whose point-spread function was estimated to be approximately 0.21 microm in x and y and approximately 0.51 microm in z. Observations were made at 17-20 degrees C on fibers from Rana pipiens and Rana temporaria. Fibers were studied in two external solutions: normal Ringer's ([K(+)] = 2.5 mM; estimated membrane potential, -80 to -90 mV) and elevated [K(+)] Ringer's (most frequently, [K(+)] = 13 mM; estimated membrane potential, -60 to -65 mV). The frequency of sparks was 0.04-0.05 sarcomere(-1) s(-1) in normal Ringer's; the frequency increased approximately tenfold in 13 mM [K(+)] Ringer's. Spark properties in each solution were similar for the two species; they were also similar when scanned in the x and the y directions. From fits of standard functional forms to the temporal and spatial profiles of the sparks, the following mean values were estimated for the morphological parameters: rise time, approximately 4 ms; peak amplitude, approximately 1 DeltaF/F (change in fluorescence divided by resting fluorescence); decay time constant, approximately 5 ms; full duration at half maximum (FDHM), approximately 6 ms; late offset, approximately 0.01 DeltaF/F; full width at half maximum (FWHM), approximately 1.0 microm; mass (calculated as amplitude x 1.206 x FWHM(3)), 1.3-1.9 microm(3). Although the rise time is similar to that measured previously in frog cut fibers (5-6 ms; 17-23 degrees C), cut fiber sparks have a longer duration (FDHM, 9-15 ms), a wider extent (FWHM, 1.3-2.3 microm), and a strikingly larger mass (by 3-10-fold). Possible explanations for the increase in mass in cut fibers are a reduction in the Ca(2+) buffering power of myoplasm in cut fibers and an increase in the flux of Ca(2+) during release.     

Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers from the mouse

The myoplasmic free Ca²⁺concentration([Ca²⁺]_i)was measured in intact single fibers from mouse skeletal muscle withthe fluorescent Ca²⁺ indicatorindo 1. Some fibers were perfused in a solution in which theconcentration of Na⁺ was reducedfrom 145.4 to 0.4 mM (low-Na⁺solution) in an attempt to activate reverse-modeNa⁺/Ca²⁺exchange (Ca²⁺ entry in exchangefor Na⁺ leaving the cell). Undernormal resting conditions, application oflow-Na⁺ solution only increased[Ca²⁺]_iby 5.8 ± 1.8 nM from a mean resting[Ca²⁺]_iof 42 nM. In other fibers,[Ca²⁺]_iwas elevated by stimulating sarcoplasmic reticulum (SR)Ca²⁺ release with caffeine (10 mM)and by inhibiting SR Ca²⁺ uptakewith2,5-di(tert-butyl)-1,4-benzohydroquinone(TBQ; 0.5 µM) in an attempt to activate forward-modeNa⁺/Ca²⁺exchange (Ca²⁺ removal from thecell in exchange for Na⁺ influx).These two agents caused a large increase in[Ca²⁺]_i,which then declined to a plateau level approximately twice the baseline[Ca²⁺]_iover 20 min. If the cell was allowed to recover between exposures tocaffeine and TBQ in a solution in whichCa²⁺ had been removed, theincrease in[Ca²⁺]_iduring the second exposure was very low, suggesting thatCa²⁺ had left the cell during theinitial exposure. Application of caffeine and TBQ to a preparation inlow-Na⁺ solution produced a large,sustained increase in[Ca²⁺]_iof ~1 µM. However, when cells were exposed to caffeine and TBQ in alow-Na⁺ solution in whichCa²⁺ had been removed, a sustainedincrease in[Ca²⁺]_iwas not observed, although[Ca²⁺]_iremained higher and declined slower than in normalNa⁺ solution. This suggests thatforward-modeNa⁺/Ca²⁺exchange contributed to the fall of[Ca²⁺]_iin normal Na⁺ solution, but whenextracellular Na⁺ was low, aprolonged elevation of[Ca²⁺]_icould activate reverse-modeNa⁺/Ca²⁺exchange. The results provide evidence that skeletal muscle fibers possess aNa⁺/Ca²⁺exchange mechanism that becomes active in its forward mode when [Ca²⁺]_iis increased to levels similar to that obtained during contraction.

     

Development of an infusion bioreactor for the accelerated preparation of decellularized skeletal muscle scaffolds

The implantation of decellularized tissue has shown effectiveness as a strategy for the treatment of volumetric muscle loss (VML) injuries. The preparation of decellularized tissue typically relies on the diffusion driven removal of cellular debris. For bulky tissues like muscle, the process can be lengthy, which introduces opportunities for both tissue contamination and degradation of key ECM molecules. In this study we report on the accelerated preparation of decellularized skeletal muscle (DSM) scaffolds using a infusion system and examine scaffold performance for the repair of VML injuries. The preparation of DSM scaffolds using infusion was dramatically accelerated. As the infusion rate (1% SDS) was increased from 0.1 to 1 and 10ml/hr, the time needed to remove intracellular myoglobin and actin decreased from a maximum of 140 ± 3hrs to 45 ± 3hrs and 10 ± 2hrs respectively. Although infusion appeared to remove cellular debris more aggressively, it did not significantly decrease the collagen or glycosaminoglycan composition of DSM samples when compared to un‐infused controls. Infusion prepared DSM samples retained the aligned network structure and mechanical integrity of control samples. Infusion prepared DSM samples supported the attachment and in‐vitro proliferation of myoblast cells and was well tolerated by the host when examined in‐vivo. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:745–755, 2016     

Reaction of intact skeletal muscle on the transplantation of minced muscle tissue

The experiments on rats have revealed that grafting of minced muscle tissue to unaffected gastrochemical muscle of the same animal leads to active regeneration in the graft accompanied by the formation of myogenic elements. In the zone of unaffected muscle contact with the graft the plastic state of muscle tissue was observed: sarcoplasm basophily of muscle fibers, groups and chains of round nuclei in them, the presence of satellite cells, ultrastructural changes, indicative of metabolic activity of the muscle fibers. With the resorption of the graft, these phenomena in the unaffected muscle gradually disappeared.     

A model for the active site of skeletal muscle myosin.

A model for a main element of the active site of skeletal muscle myosin is presented that relates directly to the 92 amino acid fragment (p₁₀) of myosin recently described by Elzinga &; Collins (1977). In this model, the substrate, an eight-membered cyclic complex of MgATP, fits tightly into a 16 amino acid segment of p₁₀ and interacts with seven of its amino acids. A main feature of the model is the important role played by the one molecule of N^τ-methylhistidine² that is present in each myosin heavy chain. At the site, it is postulated that this rare amino acid functions as a donor ligand to Mg²⁺. Once N^τ-methylhistidine is put in place next to the metal, the other amino acids that appear to form a pocket come easily into position around the MgATP. These amino acids with their postulated functions are: tyrosine 72, which through a Mg-bound water, or perhaps directly, is attached to the Mg; histidine 76, which donates a proton to the P_γ of ATP; lysine 78, which binds electrostatically to P_β of ATP; phenylalanines 80 and 81, which flank the purine ring of ATP; and aspartate 66, which forms a hydrogen bond to the 6-amino group of adenine. The Mg-coordination role ascribed to N^τ-methylhistidine 69 in skeletal muscle myosin could be taken by histidine 69 in cardiac myosin and in other muscle myosins that do not contain the methylated amino acid.The choice of p₁₀ to contain a main element of the active site is based on: (a) the presence in p₁₀ of the essential sulfhydryl groups, SH1 and SH2, whose modification affects the ATPase activity of myosin; (b) the presence in ρ₁₀ of N^τ-methylhistidine, an unusual amino acid whose methylation in skeletal muscle we take as an indicator for a special function at the active site; (c) the position of p₁₀ in the primary structure near the junction between subfragment 1 and subfragment 2 (the hinge region) where, we postulate, enzymatic events at the active site are coupled to movements of the hinge that occur during contraction; (d) indications that the DTNB light chain, probably involved in regulation, is also near the hinge; (e) the effects of MgATP at the active site on the chemical reactivity of three SH groups (SH1, SH2 and SH3) located near the hinge; and (f) the effect of hinge cleavage on the oxygen exchange reaction catalyzed at the active site. The correlation of all these observations forms the basis for our placement of part of the active site on p₁₀ near the subfragment 1-subfragment 2 hinge.     

Endogenous vascular remodeling in ischemic skeletal muscle: a role for nitric oxide.

To test the hypothesis that nitric oxide (NO) production is essential for endogenous vascular remodeling in ischemic skeletal muscle, 22 New Zealand White rabbits were chronically instrumented with transit-time flow probes on the common iliac arteries and underwent femoral ligation to produce unilateral hindlimb ischemia. Iliac blood flow and arterial pressure were recorded at rest and during a graded exercise test. An osmotic pump connected to a femoral arterial catheter continuously delivered N-nitro-l-arginine methyl ester (a NO synthase inhibitor) or a control solution (N-nitro-d-arginine methyl ester or phenylephrine) to the ischemic limb over a 2-wk period. At 1, 3, and 6 wk after femoral ligation, maximal treadmill exercise blood flow in the ischemic limb was reduced compared with baseline in each group. However, maximal exercise blood flow was significantly (P < 0.05) lower in the l-NAME-treated group than in controls for the duration of the study: 48 +/- 4 vs. 60 +/- 5 ml/min at 6 wk. Consistent with the reduction in maximal blood flow response, the duration of voluntary exercise was also substantially (P < 0.05) shorter in the l-NAME-treated group: 539 +/- 67 vs. 889 +/- 87 s. Resting blood flow was unaffected by femoral ligation in either group. The results of this study show that endogenous vascular remodeling, which partially alleviated the initial deficit in blood flow, was interrupted by NO synthase inhibition. Therefore, we conclude that NO is essential for endogenous collateral development and angiogenesis in ischemic skeletal muscle in the rabbit.     

Effects of hyperosmotic solutions on the filament lattice of intact frog skeletal muscle.

The effect of increasing the osmotic strength of the extracellular solution on the fifament lattice of living frog sartorius and semitendinosus muscle has been studied using low-angle x-ray diffraction to measure the lattice spacing. As the extracellular osmotic strength is increased, the filament lattice shrinks like an osmometer until a minimal spacing between the thick filaments is reached. This minimal spacing varies from 20 to 31 nm, depending on the sarcomere length. Further increase in the osmotic strength produces little further shrinkage. The osmotic shrinkage curve indicates, for both muscles, an osmotically-inactive volume of approximately 30% of the volume in normal Ringer's solution. Shrinkage appears to be independent of temperature and the type of particle used to increase the osmotic strength (glucose, sucrose, small ions). The rate at which osmotic equilibruim is reached depends on muscle size, being slower for greater muscle diameters. Equilibrium spacings are approached exponentially with time constants ranging from 20 to 60 min. Independent of osmotic equilibrium, the lattice tends to shrink slowly by approximately 3% over the first few hours after dissection, probably because of a leakage of K+ ions from inside the muscle cells. This can be partly prevented by using an extracellular solution which contains a higher concentration of K+ ions or which is hypoosmotic. The volume of the muscle filament lattice (1.155d10(2) . S) is constant over a very wide range of sarcomere lengths, and is equal to approximately 3.6 x 10(6) nm3 for a range of amphibian muscle types.     

Development of an adult mouse model for studies on protection against rotavirus.

Although mice have been used as an animal model for studies on rotavirus disease, these studies have been limited by the short time period after birth during which mice are susceptible to rotavirus illness (i.e., approximately 15 days). To overcome this limitation, an adult mouse model was developed in which the endpoint was infection rather than illness. The model developed utilized a strain of mouse rotavirus (EDIM) adapted to grow in culture by multiple passages in MA104 cells. The second cell culture passage of EDIM caused severe diarrhea in neonatal BALB/c mice, and little or no amelioration of disease was observed after nine cell culture passages, even when this preparation was plaque purified. Oral administration of 2 x 10(3) PFU of passage 9 also consistently caused infection of mice 4, 10, 15, 30, 60, 120, and 180 days of age as determined by viral shedding and seroconversion. Reinoculation of these mice with the same virus preparation at 2, 3, or 4 months after the first inoculation produced no evidence of reinfection. In contrast, infection of neonatal mice with the heterotypic WC3 bovine rotavirus did not prevent reinfection with culture-adapted EDIM. Thus, this strain of EDIM caused consistent infection of previously uninoculated neonatal and adult BALB/c mice and produced homotypic but not heterotypic protection against reinfection.