Rat soleus muscle ultrastructure after hindlimb suspension

The aim of the present investigation was to determine, by quantitative electron microscopy, the effects of a 5-wk tail-suspension period on rat soleus muscle ultrastructure. A marked decline (-60%) in muscle mass occurred. The mean fiber cross-sectional area decreased to a greater extent (-75%) than the capillary-to-fiber ratio (-37%), leading to a higher capillary density (+148%) after hypokinesia. The total mitochondrial volume density remained unchanged, whereas the volume density of myofibrils was slightly but significantly reduced (-6%). A shift from subsarcolemmal to interfibrillar mitochondria occurred. Interfibrillar mitochondrial volume density was highest near the fiber border and decreased toward the fiber center. An increase in volume density of satellite cells suggested muscle regenerative events. Soleus atrophy with tail suspension greatly decreases the muscular volume but leaves the ultrastructural composition of muscle fibers relatively unaffected.     

Histologic organization of the myocardium of the rat during hypokinesia (stereologic study)

For 30 days Wistar rats have been kept under conditions of hypokinesia. Using stereological methods, the volume and surface density of muscle fibers, microcirculatory bed and connective tissue components have been defined, and volumetric and surface--volume ratios of parenchymal and stromal structures have been calculated. Intracellular stereological parameters of the cardiac myocytes have been studied. Three-dimensional parameters in the parenchymatous-stromal interrelations of the atrophying myocardium accompanied with a deep rearrangement of the intracellular architectonics have been determined, as well as certain destructive changes of the cardiac myocyte ultrastructures. Such a treatment as a decreased functional loading is demonstrated to be the cause of cardiomyocytic lesions.     

The features of mitochondria of cardiomyocytes from rats with chronic heart failure

Electron-microscopy study of rat myocardium 2 weeks after a heart attack revealed significant alterations in the ultrastructure of cardiomyocytes than for the control. The location of myofibrils was less regular than for normal cells. The population of interfibrillar mitochondria decreased. Mitochondrial cristae were located less densely and formed cellated structures. Swollen mitochondria were observed in the periinfarction and intact areas, indicating the development of ischemia in the myocardium as a whole. Six months after the occlusion of coronary vessels alterations in the location of myofibrils and mitochondria were mainly observed in the peri-infarction area. Mitochondria also formed cellated structures. A 30% decrease in the density of the arrangement of the inner membranes of mitochondria on an area unit was found in the periinfarction zone. The ratio between the relative volumes of mitochondria and myofibrils in the cardiomyocytes of the peri-infarction area was increased by 20%. The area of mitochondria in the intact zone of the left ventricle was 30% greater than for the control. A study of isolated living cardiomyocytes revealed that the mitochondrial- membrane potential in the rats subjected to myocardial infarction half a year ago previously was significantly lower than for the mitochondrial-membrane potential in the control rats. Thus, cardiomyocytes that were similar to healthy cardiomyocytes in their morphology exhibited lower total mitochondrial-membrane potential, indicating their decreased energy state.     

An ultrastructural stereological analysis of the myocardium in homoiothermal animals during cooling]

By methods of electron microscopy and stereology the ultrastructural cardiomyocyte reorganization of rats exposed to influence of low temperatures (exposure during 16 days at -7 degrees C) was studied. It was shown, that with this regime of cooling the disturbance of intracellular regeneration in cardiomyocytes occurred and the complex of morphological changes typical for the syndrome of regenerative-plastic deficiency was developed. The most essential changes were seen in myofibrils and mitochondria. According to the stereologic data a change in spatial reorganization of cardiomyocytes was connected largely with these organelles. With the increase in cooling duration an increase in the volume density of myofibrils and a decrease in this parameter for mitochondria were marked. As a result of these changes the mitochondria/myofibrils volume ratio was essentially decreased. As a whole, ultrastructural reorganization of cardiomyocytes under the influence of low temperatures on rats was characterized by a decrease in the ratio of the organelle volume to myofibrillar volume. The same quantitative reorganization was revealed in atrophied cardiomyocytes under the conditions of the decreased inflow of plastic substances to cells.     

Physiological Cost of Physical Exercise and Mitochondrial Volume in Working Muscles of Subjects Exposed to Long-Term Hypokinesia: Effects of Local Resistance Exercise

The results of changes in the physiological cost of 30-min submaximal aerobic bicycle ergometric exercise and characteristics of the mitochondrial apparatus of m. vastus lateralis were assessed comparatively during 120-day (–6°) antiorthostatic hypokinesia either without prophylactic measures or with low-intensity resistance exercise training for 60 days using a Penguin exercise suit. Hypokinesia was accompanied by an increase in the working heart rate and lactate accumulation in the blood during the test exercise, as well as by a decrease in the myofibril size and the volume density of mitochondria in the m. vastus lateralis fibers. The patterns of dynamic changes in the lactate concentration in the blood during exercise training and in the volume density of central mitochondria were found to be similar. A correlation between the rate of lactate accumulation in the blood during the test exercise and the volume density of mitochondria in the working muscle appeared after long-term (60 days) exposure to hypokinesia. The use of the Penguin exercise suit in dynamic mode during prolonged (60-day) exposure to hypokinesia completely prevented the following effects: atrophy of slow-type fibers, a decrease in the volume density of central mitochondria, and an increase in the level of lactate accumulation in the blood under conditions of a standard submaximal aerobic exercise load. The correlation links between the oxidative potential of working muscle and the energy supply of muscular work are discussed.     

Ultrastructural stereological analysis of acute myocardial hypertrophy of hypertensive etiology

The myocardium of Wistar rats was studied by electron microscopy after ligation of the renal artery resulting in a stable elevation of blood pressure. The ultrastructural data on the development of heart hypertrophy were described within 5 to 35 days after operation using the morphometric and stereological methods and correlation analysis. The hypertrophied cardiomyocytes showed a reduction in the ratio of the total volume density of the mitochondria, sarcoplasmic reticulum and T system to the volume density of myofibrils. It was discovered that hypertrophy of cardiomyocytes is marked by an increase in the sarcoplasmic reticulum rather than in myofibrils and other cell compartments, whereas the relative volume of mitochondria decreases.     

Cold acclimation induces proliferation of sarcoplasmic reticulum without increase in Ca2+-ATPase activity in white axial muscle of striped bass (Morone saxatilis)

The effects of acclimation of striped bass to cold (5 degrees C) and warm (25 degrees C) temperatures upon ultrastructural features of white axial skeletal muscle are quantified. Surface density of sarcoplasmic reticulum (SR) increased by almost 30%, and SR volume density increased by about 20% during cold acclimation. Proliferation of SR suggests an increase in available SR surface for re-sequestration of Ca2+ and a decrease in diffusion path length for Ca2+ during cold acclimation. Average cross-sectional areas and cross-sectional perimeters of myofibrils situated in the center of muscle fibers decreased during cold acclimation by approximately 20% and 11%, respectively. Additionally, average major and minor axes of ellipses fit to central myofibrillar cross-sections decreased by approximately 12% and 8%, respectively, during cold acclimation. These measurements define a decrease in average myofibrillar diameter and suggest a decrease in diffusion path length for Ca2+ to and from myofibrillar activation sites. Measurements of peripheral myofibrils that had elongated profiles in cross-sections indicate that maximum profile length of these myofibrils decreases by about 17%. Peripheral myofibrils may break up into smaller myofibrils with more rounded cross-sectional profiles during cold acclimation. SR Ca2+-ATPase of white axial muscle was also measured in unfractionated homogenates and in crude SR-enriched subcellular fractions from cold- and warm-acclimated striped bass. No difference in SR Ca2+-ATPase activity per g wet weight was observed between cold- and warm-acclimated animals. Lack of increase in SR Ca2+-ATPase per g wet weight, despite a significant proliferation of SR, probably results in a decrease in average Ca2+-ATPase pump density within the SR membrane during cold acclimation. Thus, compensation for decreased diffusion coefficient of Ca2+ during cold acclimation appears due to the combined effects of proliferation of SR surface density and a decrease in average myofibrillar diameter.     

Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease.

The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.     

Possible correlation between binding of muscle type AMP deaminase to myofibrils and ammoniagenesis in rat skeletal muscle on electrical stimulation

Contraction of rat skeletal muscle by electrical stimulation of the sciatic nerve caused remarkable increase in binding of AMP deaminase (EC 3.5.4.6) to myofibrils, but did not change the total enzyme activity. After 30 sec stimulation, the ratio of bound to free enzyme was about 5 times that in resting muscle. This treatment also increased the ammonia content of the muscle to 5 times that in resting state. From these findings, we suggest that there is a correlation between the binding of muscle type AMP deaminase to myofibrils and ammoniagenesis in the muscle.     

Structure of the thyroid gland in the rat during hypokinesia and after its elimination

The aim of the investigation is to objectively ascertain and study stages of the processes taking into account the zonal structure of the thyroid gland, that is along the whole distance from the center up to the surface of the lobe. Hypokinesia lasts from 1 to 56 days; rehabilitation begins after 28 days of hypokinesia and lasts 28 days. Morphometric analysis of 8 parameters demonstrating the state of the gland are used. In control animals the structural changes from the center towards the surface of the gland lobe occur gradually, the degree of the changes is different and diversily directed [increasing volumetric part (Vv) of the follicular epithelium, decreasing Vv of the colloid, stable Vv of the vascular component]. When both hypokinesia and rehabilitation develop, all signs change wavily. Natural borders of the waves are defined--5, 16, 28 and 51 days for hypokinesia and 13 and 23 days for readaptation. Different structural components of the gland respond variously to the factors mentioned. Changes of every sign in different zones are also not equal and they can be diversily directed. In the course of rehabilitation for 1 month, there is no return to the normal state.     

Troponin subunit stoichiometry and content in rabbit skeletal muscle and myofibrils

The quantity and molar ratio of the three troponin subunits to actin were determined in rabbit psoas muscle, muscle homogenates (800 X g pellet), and purified myofibrils. Proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The quantities of the separated proteins were determined directly from the gel slices by amino acid analysis after correction for losses and background. The molar ratio of actin, troponin T, troponin I, and troponin C was found to be 6.99:1:05:1:04:0.92 in purified myofibrils and was not significantly different (p greater than 0.05) from those obtained from 800 X g pellets of muscle homogenates or intact muscle tissue. Isolated troponin purified by several different procedures also had a 1:1:1 subunit ratio although the variability was much greater than that found in myofibrils. The troponin content of rabbit psoas muscle and myofibrils was 91 +/- 16 and 770 +/- 110 pmol/mg, respectively.     

Stress-induced hypokinesia is facilitated by ACTH-(7-10)

Subcutaneous treatment with the neuropeptide ACTH-(4-10) induced hypokinesia in rats subjected to a mild stress induced by placing the animals on a non-functional "hot" plate (21 degrees C) for 30 sec, but not in control animals not exposed to this stress-inducing environment. The lowest effective dose of ACTH-(4-10) was 5 micrograms/kg, administered 50 min before testing. The combination of peptide treatment and the mild stress-inducing procedure mimicked the effect of a short intense stress induced by placing the rats on a hot plate (57 degrees C) for 30 sec, suggesting that this stress-induced hypokinesia is mediated by ACTH neuropeptides. Structure-activity relationship studies revealed that the active core for the ACTH-(4-10)-induced hypokinesia is located in the C-terminal tetrapeptide Phe-Arg-Try-Gly (ACTH-(7-10)). Pretreatment with the opioid antagonist naltrexone did not influence the effect of ACTH-(4-10) indicating that activation of opioid systems is not implicated in this behavioral effect of the peptide.     

Combined Stereological and Biochemical Analysis of Storage and Release of Catecholamines in the Adrenal Medulla of the Rat

Adrenal medullae from rats injected with insulin 1 h prior to decapitation were analyzed by stereology and their catecholamine content was determined. In control animals the ratio between adrenaline- and noradrenaline- containing cells was 4.06 whereas the ratio between adrenaline and noradrenaline contents was 4.11. The glands from insulin-treated animals showed a 45% reduction in adrenaline content and a 42% decrease in the volumetric density of the adrenaline-containing granules. However, neither the noradrenaline content nor the volumetric density of noradrenaline-containing granules was modified in glands from insulin-treated animals. From these data, the amount and concentration of adrenaline or noradrenaline in a single granule have been calculated. The results are consistent with the view that chromaffin granules are the source of the released catecholamines. The present paper demonstrates that stereology combined with biochemistry is a useful tool for resolution of problems at the cellular and subcellular levels.     

Seasonal changes in the cardiomyocytes of heterothermal animals

In 20 Citellus erythrogenys the volumetric and surface densities of myofibrils, mitochondria, sarcoplasmic reticulum, T-system and lipid drops were estimated in cardiomyocytes by means of stereological methods before, during and after hibernation. The volumetric and surface-volumetric ratios for the main intracellular compartments were calculated. Certain morphological criteria were found for the seasonal peculiarities in the structural organization of cardiomyocytes in the hibernating rodents. During hibernation, myocardial atrophy (seasonal regression of the heart weight and decreasing diameter of cardiomyocytes) takes place. It is accompanied with an adaptive rearrangement of the cardiomyocyte ultrastructure. The latter facilitates to myocardial contractility at low body temperatures at the expense of activation of the cellular transport-energic interaction system.     

Aggregation of isolated myofibrils stimulated by their contraction. I. Origin of the second phase of optical changes during myofibril contraction

Aggregation of contracted myofibrils was shown to be responsible for spontaneous: slow reduction of optical density of myofibril suspension on the final stage of their contraction. This effect faded with an increase in light wavelength, with increased angle of view of the photocell and diminished sizes of myofibrils.     

EPR study of nitric oxide production in rat tissues under hypokinesia

EPR spectroscopy was used to study the intensity of nitric oxide (NO) production upon modeling 60-day progressive hypokinesia (restriction of motor activity) in rats and estimating the content of (DETC)₂-Fe²⁺-NO complexes in heart and liver tissues. In 30 days of hypokinesia, there was a 2–3-fold increase in tissue NO. Administration of a nonspecific inhibitor of NO synthases, L-NAME, to hypokinetic rats prior to measurement decreased their NO level even below the untreated control. Our results show that the intensified NO production in hypokinesia is mainly due to NO synthases, rather than to the nitrite reductase pathway.     

FITC-labeled I-protein specifically binds to A-bands and/or Z-lines of glycerinated myofibrils of chicken breast muscle

Ion-exchange column-purified I-protein was labeled by fluorescein isothiocyanate (FITC) at an equimolar ratio. When FITC-labeled I-protein was reacted with glycerinated myofibrils of chicken breast muscle in a phosphate-buffered saline, fluorescence was observed at the A-band and/or the Z-line of the sarcomere. However, FITC-labeled I-protein did not stain freshly prepared myofibrils. When FITC-I-protein was reacted with a nitrocellulose paper sheet on which muscle proteins were blotted after SDS-polyacrylamide gel electrophoresis, some peptide bands, including connectin and nebulin, were fluorescent. These facts can explain why anti-I-protein antibodies stain the A-I junctional region of fresh myofibrils and A-bands and/or Z-lines of glycerinated myofibrils. It is very likely that I-protein is transferred from the A-I junctions of myofibrils and translocates to A-bands and Z-lines, where some components that can bind to I-protein are localized, as myofibrils are degraded during the glycerination.     

Effect of Trypsin Treatment on the ATPase Activity of Myofibrils from the Stored Rabbit Muscle

The extent of activation of myofibrillar ATPase activity by trypsin treatment has been measured.

When myofibril (5 mg/ml) was treated with a low concentration of trypsin (2.5 μg/ml), the Mg-modified ATPase activity of myofibrils at a low ionic strength increased appreciably, while the EDTA-enhanced ATPase activity of myofibrils at a high ionic strength did not change with the progress of trypsin digestion.

The dependence of myofibrillar ATPase activity on KCl concentration also became greater with the progress of trypsin digestion.

Trypsin treatment caused 5-fold increase in the Mg-modified ATPase activity of 0-myofibril, when treated with trypsin in a ratio of 1 to 2000 myofibril for 80 min. Under the same condition, the ATPase activity of 1-myofibril increased by about 150%, whereas that of 8-myofibril increased by approximately 50%.

When myofibrils were treated with trypsin in a ratio of 1 to 200 myofibril, the Mg-ATPase activity of 8-myofibril decreased earlier than that of 1-myofibril did by about 20 min.

Experimental results obtained in this study were enough to confirm that the myofibrils from the aged muscle are more susceptible to tryptic action.

An assumption was made that the structural alteration of myofibrils during aging might be attributed to the change in thin filament of myofibrils, including Z-lines, which are mainly due to the change in the native tropomyosin of thin filaments.     

A quantitative analysis of the incidence of certain cytoplasmic structures in the ovum of the rat during cleavage

Summary The volumetric density of most cytoplasmic organelles in the segmenting ovum of the rat was determined by morphometric techniques described by Weibel, at the 1-, 2-, 4-, and 8-cell stages, and at the early and late blastocyst stages.During the course of cleavage, the volumetric density of mitochondria remained practically unchanged. The volumetric density of the Golgi complex was too small to permit assessment of the differences between the individual stages of development. Significant changes were found in both granular and agranular endoplasmic reticulum, secondary lysosomes, multivesicular bodies, lamellar structures and lipid droplets. Granular endoplasmic reticulum was first observed as late as the 8-cell ovum stage and its volumetric density increased further in the early and late blastocysts. The relative volume of agranular endoplasmic reticulum was highest in the 1-cell ovum and decreased during the course of cleavage. The same is true for multivesicular bodies. The volumetric density of secondary lysosomes increased during cleavage, reaching the highest values in the 8-cell ovum. Lamellar structures were the most voluminous part of the cytoplasm of the segmenting ovum at all stages. Their volumetric density, however, decreased during the course of cleavage. Lipid droplets occur in very small quantities in the 1- to 4-cell ova, but at later stages their volumetric density increased.Our findings underline the importance of acquiring quantitative information about changes in cell organelle populations for assessing morphological and functional relationships during the early stages of cleavage of the ovum.