Regulation of binding of subfragment 1 in isolated rigor myofibrils.

A steric-hindrance model has been used to explain the regulation of muscle contraction by tropomyosin-troponin complex. The regulation of binding was studied by microscopic observation of mixtures of fluorescent subfragment 1 (S1) with rigor myofibrils at different actin-to-S1 ratios and in the presence and absence of calcium. Procedures were adapted to protect the critical thiols of S1 before conjugation to thiol-specific fluorochromes, this giving fluorescent S1 with unaltered enzyme activity. S1 binding was greatest in the I band (except at the Z-lines) in the presence of calcium regardless of the [S1]. The patterns in the absence of calcium depended on the actin-to-S1 ratios: low [S1], binding in the myosin-actin overlap region; intermediate [S1], highest binding at the A-I junction; high [S1], greatest binding in the I-band. The two distinct binding patterns observed at low [S1] were demonstrated by dual-channel fluorescence microscopy when myofibrils were sequentially incubated with fluorescent S1 without calcium followed by a different fluorescent S1 with calcium. These observations support the concept of rigor activation of actin sites. The change in the pattern upon increasing [S1] without calcium demonstrate cooperative interactions along the thin filament. However, these interactions (under the conditions used without calcium) do not appear to extend over greater than 2-3 tropomyosin-troponin-7 actin functional units.

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers.

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.

Studies on cardiac myofibrillogenesis with antibodies to titin, actin, tropomyosin, and myosin.

Cardiac myofibrillogenesis was examined in cultured chick cardiac cells by immunofluorescence using antibodies against titin, actin, tropomyosin, and myosin. Primitive cardiomyocytes initially contained stress fiber-like structures (SFLS) that stained positively for alpha actin and/or muscle tropomyosin. In some cases the staining for muscle tropomyosin and alpha actin was disproportionate; this suggests that the synthesis and/or assembly of these two isoforms into the SFLS may not be stoichiometric. The alpha actin containing SFLS in these myocytes could be classified as either central or peripheral; central SFLS showed developing sarcomeric titin while peripheral SFLS had weak titin fluorescence and a more uniform stain distribution. Sarcomeric patterns of titin and myosin were present at multiple sites on these structures. A pair of titin staining bands was clearly associated with each developing A band even at the two or three sarcomere stage, although occasional examples of a titin band being associated with a half sarcomere were noted. The appearance of sarcomeric titin patterns coincided or preceded sarcomere periodicity of either alpha actin or muscle tropomyosin. The early appearance of titin in myofibrillogenesis suggests it may have a role in filament alignment during sarcomere assembly.

Myofibrillar calcium sensitivity of isometric tension is increased in human dilated cardiomyopathies: role of altered beta-adrenergically mediated protein phosphorylation.

To examine the role of alterations in myofibrillar function in human dilated cardiomyopathies, we determined isometric tension-calcium relations in permeabilized myocytesized myofibrillar preparations (n = 16) obtained from left ventricular biopsies from nine patients with dilated cardiomyopathy (DCM) during cardiac transplantation or left ventricular assist device implantation. Similar preparations (n = 10) were obtained from six normal hearts used for cardiac transplantation. Passive and maximal Ca2+-activated tensions were similar for the two groups. However, the calcium sensitivity of isometric tension was increased in DCM compared to nonfailing preparations ([Ca2+]50=2.46+/-0.49 microM vs 3.24+/-0.51 microM, P < 0.001). In vitro treatment with the catalytic subunit of protein kinase A (PKA) decreased calcium sensitivity of tension to a greater degree in failing than in normal preparations. Further, isometric tension-calcium relations in failing and normal myofibrillar preparations were similar after PKA treatment. These findings suggest that the increased calcium sensitivity of isometric tension in DCM may be due at least in part to a reduction of the beta-adrenergically mediated (PKA-dependent) phosphorylation of myofibrillar regulatory proteins such as troponin I and/or C-protein.

Identifying constituents of whey protein concentrates that reduce the pink color defect in cooked ground turkey.

Whey protein concentrate constituents were tested for their ability to reduce naturally occurring pink color defect and pink cooked color induced by sodium nitrite (10ppm) and nicotinamide (1.0%) in ground turkey. ß-lactoglobulin (1.8%), α-lactalbumin (0.8%), bovine serum albumin (0.15-0.3%), lactose (1.0-3.0%), potassium chloride (500-1500ppm), and ferrous iron chloride (0.3-30ppm) had no effects on cooked pink color. Lactoferrin (30-5000ppm) increased or decreased pink color depending on its concentration in samples without added sodium nitrite or nicotinamide. Annatto (0.1-1.0ppm) reduced pink color whereas the higher concentration of magnesium chloride (22-88ppm) and ferric iron chloride (0.3-30ppm) increased pink color in samples with added nicotinamide. Calcium chloride (160-480ppm) was the only tested constituent that consistently reduced pink cooked color in samples with and without added nitrite and nicotinamide. Due to the variability of whey protein concentrates and the number of constituents that do not reduce pink cooked color, the addition of calcium alone or dried milk minerals containing calcium, phosphate, and citrate, represents a better means to regularly prevent the pink color defect in cooked ground turkey.

Investigation of mechanisms by which sodium citrate reduces the pink color defect in cooked ground turkey.

The principal mechanism by which sodium citrate reduces the pink color defect in cooked ground turkey was investigated. Sodium citrate (SC; 0, 0.125, 0.25, 0.5, 1.0, 2.0M), sodium nitrite (0.01, 0.1M), and nicotinamide (0.5, 0.75M) were combined in solutions of bovine hemin to determine SCs ability to bind heme iron and competitively inhibit pink-color-generating ligands from binding. Additionally, the effects of sodium erythorbate (0, 275, 550ppm), ferrous iron chloride (0, 0.3, 3.0, 30ppm), and ferric iron chloride (0, 0.3, 3.0, 30ppm) on SCs ability to reduce pink cooked color was examined. Absorbance curves of hemin+nitrite and hemin+nicotinamide were relatively unaffected by SC, therefore whether or not SC bound heme iron, that did not appear to be a mechanism for inhibiting the pink color defect. Both ferrous and ferric iron chloride had minimal effects on color values, possibly due to sodium tripolyphosphate chelation ability in the meat system and thus their presence did not enhance SCs ability to reduce the pink color defect. However, sodium erythorbate, a reducing agent, inhibited SCs ability to decrease the pink color defect in samples induced pink with sodium nitrite and nicotinamide. Therefore, it appears SC requires the presence of oxygen and may participate in oxidative processes to reduce the pink color defect.

The effects of partial extraction of TnC upon the tension-pCa relationship in rabbit skinned skeletal muscle fibers.

The activation of contraction in vertebrate skeletal muscle involves the binding of Ca2+ to low-affinity binding sites on the troponin C (TnC) subunit of the regulatory protein troponin. The present study is an investigation of possible cooperative interactions between adjacent functional groups, composed of seven actin monomers, one tropomyosin, and one troponin, along the same thin filament. Single skinned fibers were obtained from rabbit psoas muscles and were then placed in an experimental chamber containing relaxing solution maintained at 15 degrees C. Isometric tension was measured in solutions containing maximally and submaximally activating levels of free Ca2+ (a) in control fiber segments, (b) in the same segments after partial extraction of TnC, and finally (c) after recombination of TnC into the segments. The extraction was done at 11-13 degrees C in 20 mM Tris, 5 mM EDTA, pH 7.85 or 8.3, a procedure derived from that of Cox et al. (1981. Biochem. J. 195:205). Extraction of TnC was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the control and experimental samples. Partial extraction of TnC resulted in reductions in tension during maximal Ca activation and in a shift of the relative tension-pCa (i.e., -log[Ca2+]) relationship to lower pCa's. The readdition of TnC to the extracted fiber segments resulted in a recovery of tension to near-control levels and in the return of the tension-pCa relation to its original position. On the basis of these findings, we conclude that the sensitivity to Ca2+ of a functional group within the thin filament may vary depending upon the state of activation of immediately adjacent groups.

Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle.

The Ca2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83:3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC2) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (less than 1 ms) released to a shorter length and then reextended by approximately 200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 - e-kt) to obtain the value of ktr. In control fibers, ktr increased sigmoidally with increases in [Ca2+]; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 +/- 1.2 s-1 at 15 degrees C) than in rabbit psoas fibers (18.7 +/- 1.0 s-1). Phosphorylation of LC2 was accomplished by repeated Ca2+ activations (pCa 4.5) of the fibers in solutions containing 6 microM calmodulin and 0.5 microM myosin light chain kinase, a protocol that resulted in an increase in LC2 phosphorylation from approximately 10% in the control fibers to greater than 80% after treatment. After phosphorylation, ktr was unchanged at maximum or very low levels of Ca2+ activation. However, at intermediate levels of Ca2+ activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC2 phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology. 86:585-600), the effect of LC2 phosphorylation to increase the Ca2+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of partial extraction of light chain 2 on the Ca2+ sensitivities of isometric tension, stiffness, and velocity of shortening in skinned skeletal muscle fibers.

Various functional roles for myosin light chain 2 (LC2) have been suggested on the basis of numerous and predominantly in vitro biochemical studies. Using skinned fibers from rabbit psoas muscle, the present study examines the influence of partial removal of LC2 on isometric tension, stiffness, and maximum velocity of shortening at various levels of activation by Ca2+. Isometric tension, stiffness, and velocity of shortening were measured at pCa values between 6.6 and 4.5 (a) in a control fiber segment, (b) in the same fiber segment after partial removal of LC2, and (c) after recombination with LC2. The extraction solution contained 20 mM EDTA, 20 or 50 mM KCl, and either imidazole or PO4(2-) as a pH buffer (pH 7.0). The amount of LC2 extracted varied with the temperature, duration of extraction, and whether or not troponin C (0.5 mg/ml) was added to the extraction solution. Extraction of 20-40% LC2 resulted in increased active tensions in the range of pCa's between 6.6 and 5.7, but had no effect upon maximum tension. The tension-pCa relationship was left-shifted to lower [Ca2+] by as much as 0.2 pCa units after LC2 extraction. At low concentrations of Ca2+, an increase in stiffness proportional to the increase in tension was observed. Readdition of LC2 to these fiber segments resulted in a return of tension and stiffness to near control values. Stiffness during maximal activation was unaffected by partial extraction of LC2. LC2 extraction was shown to uniformly decrease (by 25-30%), the velocity of shortening during the high velocity phase but it did not significantly affect the low velocity phase of shortening. This effect was reversed by readdition of purified LC2 to the fiber segments. On the basis of these findings we conclude that LC2 may modulate the number of cross-bridges formed during Ca2+ activation and also the rate of cross-bridge detachment during shortening. These results are consistent with the idea that LC2 may modulate contraction via an influence upon the conformation of the S1-S2 hinge region of myosin.

Effects of partial extraction of troponin complex upon the tension-pCa relation in rabbit skeletal muscle. Further evidence that tension development involves cooperative effects within the thin filament.

Partial extraction of troponin C (TnC) decreases the Ca2+ sensitivity of tension development in mammalian skinned muscle fibers (Moss, R. L., G. G. Giulian, and M. L. Greaser. 1985. Journal of General Physiology. 86:585), which suggests that Ca2+-activated tension development involves molecular cooperativity within the thin filament. This idea has been investigated further in the present study, in which Ca2+-insensitive activation of skinned fibers from rabbit psoas muscles was achieved by removing a small proportion of total troponin (Tn) complexes. Ca2+-activated isometric tension was measured at pCa values (i.e., -log[Ca2+]) between 6.7 and 4.5: (a) in control fiber segments, (b) in the same fibers after partial removal of Tn, and (c) after recombination of Tn. Tn removal was accomplished using contaminant protease activity found in preparations of LC2 from rabbit soleus muscle, and was quantitated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning densitometry. Partial Tn removal resulted in the development of a Ca2+-insensitive active tension, which varied in amount depending on the duration of the extraction, and concomitant decreases in maximal Ca2+-activated tensions. In addition, the tension-pCa relation was shifted to higher pCa values by as much as 0.3 pCa unit after Tn extraction. Readdition of Tn to the fiber segments resulted in the reduction of tension in the relaxing solution to control values and in the return of the tension-pCa relation to its original position. Thus, continuous Ca2+-insensitive activation of randomly spaced functional groups increased the Ca2+ sensitivity of tension development in the remaining functional groups along the thin filament. In addition, the variation in Ca2+-insensitive active tension as a function of Tn content after extraction suggests that only one-third to one-half of the functional groups within a thin filament need to be activated for complete disinhibition of that filament to be achieved.

Quantitative determination of myosin and actin in rabbit skeletal muscle.

The myosin and actin content of muscle tissue and purified myofibrils from rabbit psoas muscle has been determined. Myofibrils were purified using Percoll gradients, which allowed rapid separation from nuclei and connective tissue proteins. Myosin and actin were quantitated by amino acid analysis of the appropriate bands from sodium dodecyl sulfate/polyacrylamide gels. Muscle tissue contained 94 and 619 nmol/g wet weight of myosin and actin, respectively, while myofibrils had 0.82 and 5.37 mumol/g protein. Thus myosin contributed 43% and actin 22% of the myofibril protein mass. The value of 2.5 myosins per 14.3 nm repeat as calculated from these results suggests that thick filament models with mixtures of two and three crossbridges per repeat should be considered.

Actin filaments in diabetic fibrovascular preretinal membrane.

A vitrectomy specimen from a diabetic patient was studied by light and electron microscopy using myosin subfragment 1 to decorate and identify actin filaments. The patient had proliferative diabetic retinopathy, a shrinking fibrovascular preretinal membrane associated with retraction of the thickened posterior hyaloid, and a localized traction retinal detachment. The fibrovascular tissue comprised normal mature collagen, few cells, and occasional blood vessels. The cells contained numerous thick bundles of the contractile protein, actin. We suggest that actin may have been involved in the contraction phenomena observed clinically and that membrane contraction might be blocked by pharmacologic treatment.

Actin filaments in contracting preretinal membranes.

In 14 patients, preretinal membranes, causing retinal traction and severe visual impairment, were removed by vitrectomy and evaluated by light and electron microscopy using myosin subfragment-1 to stain actin filaments. Eight membranes were of vascular origin, six of nonvascular origin. All but one contained bundles of oriented actin filaments within a number of their nonvascular stroma cells, suggesting that the contractile protein action may have been involved in their clinically observed contraction.

Myogenin, MyoD, and myosin expression after pharmacologically and surgically induced hypertrophy.

The relationship between myogenin or MyoD expression and hypertrophy of the rat soleus produced either by clenbuterol and 3,3', 5-triiodo-L-thyronine (CT) treatment or by surgical overload was examined. Mature female rats were subjected to surgical overload of the right soleus with the left soleus serving as a control. Another group received the same surgical treatment but were administered CT. Soleus muscles were harvested 4 wk after surgical overload and weighed. Myosin heavy chain isoforms were separated by using polyacrylamide gel electrophoresis while myogenin and MyoD expression were evaluated by Northern analysis. CT and functional overload increased soleus muscle weight. CT treatment induced the appearance of the fast type IIX myosin heavy chain isoform, depressed myogenin expression, and induced MyoD expression. However, functional overload did not alter myogenin or MyoD expression in CT-treated or non-CT-treated rats. Thus pharmacologically and surgically induced hypertrophy have differing effects on myogenin and MyoD expression, because their levels were associated with changes in myosin heavy chain composition (especially type IIX) rather than changes in muscle mass.

Myosin heavy chain composition in the rat diaphragm: effect of age and exercise training.

Increases in aerobic capacity in both young and senescent rats consequent to endurance exercise training are now known to occur not only in locomotor skeletal muscle but also in diaphragm. In the current study the effects of aging and exercise training on the myosin heavy chain (MHC) composition were determined in both the costal and crural diaphragm regions of female Fischer 344 rats. Exercise training [treadmill running at 75% maximal oxygen consumption (1 h/day, 5 day/wk, x 10 wk)] resulted in similar increases in plantaris muscle citrate synthase activity in both young (5 mo) and old (23 mo) trained animals (P < 0.05). Computerized densitometric image analysis of fast and slow MHC bands revealed the ratio of fast to slow MHC to be significantly higher (P < 0.005) in the crural compared with costal diaphragm region in both age groups. In addition, a significant age-related increase (P < 0.05) in percentage of slow MHC was observed in both diaphragm regions. However, exercise training failed to change the relative proportion of slow MHC in either the costal or crural region.

Sequence and mechanical implications of titin's PEVK region.

A widely used titin monoclonal antibody (9D10) was epitope mapped to the PEVK region in the I-band portion of titin. Sequence analysis of the titin PEVK region revealed a large number of 28 amino acid modules (termed "PPAK" repeats) alternating with glutamic acid rich segments. Species differences in cardiac rest tension could not be ascribed to differences in the PEVK length of the N2B titin isoform. The low rest tension generated by dog cardiac muscle also does not appear to be explained by the N2 and PEVK segment lengths in the N2A titin isoform.

Quantitation of satellite cell proliferation in vivo using image analysis.

A nonisotopic, double fluorescence technique was developed to study myogenic satellite cell proliferation in posthatch turkey skeletal muscle. Labeled satellite cell nuclei were identified on enzymatically isolated myofiber segments using a mouse monoclonal antibody (anti-BrdU) followed by fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG secondary antibody. Myofiber nuclei (myonuclei+satellite cell nuclei) were counterstained with propidium iodide (PI). The myofiber segment length, myofiber segment diameter, and the number of PI and FITC labeled nuclei contained in each segment was determined using a Nikon fluorescence microscope, a SIT video camera and Image-1 software. Data collected by three different operators of the image analysis system revealed 5.0 +/- 1.4 satellite cell nuclei per 1000 myofiber nuclei and 5284 +/- 462 microns3 of cytoplasm surrounding each myofiber nucleus in the pectoralis thoracicus of 9-week-old tom turkeys. BrdU immunohistochemistry coupled with the new approach of PI staining of whole myofiber mounts is an effective combination to allow the use of an efficient semi-automated image analysis protocol.

A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis.

A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

Effect of postmortem storage on the Z-line region of titin in bovine muscle.

Myofibrils were prepared from bovine muscles (cutaneous trunci, rectus abdominis, psoas major, and masseter) and compared between different aging periods at 4 degrees C (0, 1, 2, 4, 8, and 16 d). Myofibrils were stained with an antibody directed against a 56-kDa fragment (FE-RE) of titin located in the Z-line region. Unaged myofibrils from all four muscles showed a single stained band at the Z-line with similar intensities. Postmortem time did not significantly affect the total amount of fluorescence in the sarcomere, suggesting the titin FE-RE epitope was not degraded nor were titin fragments containing this epitope released during storage. However, the fluorescence patterns were altered. The relative fluorescence intensity at the Z-line decreased but that in the I-band increased gradually, showing the translocation of some titin FE-RE epitopes during the aging period. This suggested that a cleavage occurred in a region of titin very close to the Z-line during postmortem storage. Usually the position of maximum fluorescence remained at the Z-line, although about 1/3 of the myofibrils from rectus abdominis showed a two-band pattern around the Z-line after 16 d of aging. The titin changes observed may be related to the increased fragility of the myofibril and the improvement of meat tenderness during postmortem storage.

Effect of rapid rigor mortis processes on protein functionality in pectoralis major muscle of domestic turkeys.

The pale, soft, exudative (PSE) phenomenon in turkey pectoralis major (breast) muscle was studied using a combination of biochemical, meat quality, microscopic, and gel electrophoresis techniques. Breast muscle samples were collected from turkeys characterized by slow vs fast postmortem glycolysis assessed by muscle pH at 20 min after death. The PSE group was characterized by lower muscle ATP (P < .05) and higher lactate levels (P < .05) compared with the normal group. Excess water-holding capacity and cooking yield were significantly lower (P < .05) in the PSE group than in normal turkeys. Breast muscle of the PSE group was also lighter (P < .05) than that in the normal group as determined by Minolta L* values. The SDS-PAGE, Western blotting, and immunofluorescence microscopy revealed that phosphorylase, a soluble enzyme, became tightly associated with the myofibrils in muscle from the PSE group. Also, less myosin could be solubilized from PSE vs normal myofibril samples. The results indicate that irreversible myosin insolubility due to low pH and high-temperature conditions is decisive in the development of PSE turkey breast muscle.