Enzyme activity analyses along ragged-red and normal single muscle fibres.

Mitochondrial myopathies are morphologically characterized by ragged-red fibres (RRF). Serial cross-section revealed that the ragged-red appearance was only focal. This is in agreement with a partial cytochrome c oxidase (COX) deficiency in chronic progressive external ophthalmoplegia (CPEO). Since most of these patients show deletions of the mitochondrial genome single fibre analyses were performed determining COX and succinate dehydrogenase (SDH) in serial muscle sections from two patients with CPEO. High SDH activity was demonstrated in RRF; in contrast COX activity was lower in RRF in a patient, possibly representing a focal assembly of mitochondria with deletions in their genomes. The variation of enzyme activities along the muscle fibre was especially high in RRF. This study presents the first quantitative evidence that enzyme activities vary considerably along fibres in muscle from patients with a mitochondrial myopathy.     

Enzyme activity analyses along ragged-red and normal single muscle fibres

Summary Mitochondrial myopathies are morphologically characterized by ragged-red fibres (RRF). Serial cross-section revealed that the ragged-red appearance was only focal. This is in agreement with a partial cytochromec oxidase (COX) deficiency in chronic progressive external ophthalmoplegia (CPEO). Since most of these patients show deletions of the mitochondrial genome single fibre analyses were performed determining COX and succinate dehydrogenase (SDH) in serial muscle sections from two patients with CPEO. High SDH activity was demonstrated in RRF; in contrast COX activity was lower in RRF in a patient, possibly representing a focal assembly of mitochondria with deletions in their genomes. The variation of enzyme activities along the muscle fibre was especially high in RRF. This study presents the first quantitative evidence that enzyme activities vary considerably along fibres in muscle from patients with a mitochondrial myopathy.     

Earliest mechanical evidence of cross-bridge activity after stimulation of single skeletal muscle fibers.

The stiffness of single fibers from frog skeletal muscle was measured by the application of small 2-kHz sinusoidal length oscillations during twitch and tetanic contractions at a range of initial sarcomere lengths. The earliest mechanical signs of activation were a fall in tension (latency relaxation) and a rise in stiffness. The earliest stiffness increase and the earliest tension fall occurred simultaneously at all sarcomere lengths. This suggests a cross-bridge origin for the latency relaxation. The lead of stiffness over tension seen during the rise of tension was substantially established during the latent period. Reducing the size of the twitch by reducing calcium release with D-600 (methoxyverapamil) reduced the latency relaxation and the stiffness development during latency much less than it reduced the twitch tension. For very small twitches the peak of the stiffness response occurred during the latent period and the times of onset of both latency relaxation and stiffness rise were delayed, but remained coincident. This suggests a strong connection between the latency relaxation and the rise of stiffness during the latent period, whereas the connection between these events and positive tension generation appears to be less strong.     

Quasi-elastic light-scattering studies of single skeletal muscle fibers.

Measurements were made of the intensity autocorrelation function, g(2)[tau], of light scattered from intact frog muscle fibers. During the tension plateau of an isometric tenanus, scattered field statistics were approximately Gaussian and intensity fluctuations were quasi-stationary. The half time, tau 1/2, for the decay of g(2)[tau] was typically 70 ms at a scattering angle of 30 degrees. The decay rate, 1/tau 1/2, of g(2)[tau] varied roughly linearly with the projection of the scattering vector on the fiber axis. 1/tau 1/2 was greater during the tension creep phase of tetani of highly stretched fibers, but was roughly independent of sarcomere length during the tension plateau. g(2)[tau] measured during rest or on diffraction pattern maxima during isometric contraction were flat with low amplitudes. These results are consistent with a model of a 200-mu m segment of an isometrically contracting fiber in which scattering material possesses relative axial velocities of 1-2 mu m/s accompanied by relative axial displacements greater than 0.1 mu m. The slow (1-2 mu m/s) motion of one portion of the fiber relative to another observed under the microscope (500X) during isometric contraction is consistent with the light-scattering results. Structural fluctuations on the scale of the myofibrillar sarcomere which may arise from asynchronous cycling of cross-bridges must involve relative axial velocities less than 3 mu m/s or relative axial displacements less than 0.05 mu m.     

Membrane potential of muscle fibers exposed to corticotropin and hydrocortisone]

The effect of a single intraperitoneal administration of corticotropin (1 unit) and hydrocortisone (5 mg) per 100 g of a body weight on the membrane potential (MP) as well as on the response rate of miniature end plate potentials (RRMEPP) of musculus soleus fibres of various polarization levels has been investigated in rats. It is shown that administration of corticotropin does not change the MP value, while that of hydrocortisone elicits its increase at the low initial polarization level of the muscle fibre membrane and its decrease at the high level. Hydrocortisone administration does not change the MP value at normal levels of fibre polarization. Corticotropin having been administered, RRMEPP of fibres both with high MP levels and with low ones has increased. Fibres with normal polarization also show a tendency to increase. Administration of hydrocortisone has induced a substantial increase of RRMEPP in fibres with high polarization levels within 45 min, while PRMEPP of fibres with normal polarization levels increased within first 5 min., and that of fibres with low levels of polarization remained unchanged.     

Potassium efflux from single skinned skeletal muscle fibers.

The efflux of 42K from single, skinned (sarcolemma removed) skeletal muscle fibers has been determined. Isotope washout curves are kinetically complex and can be fit as the sum of three exponentials, including a fast component (k = 0.25 s-1) with a pool size equivalent to 91% of the fiber volume, an intermediate component (k = 0.08 s-1) equivalent to 6% of the fiber volume, and a slow component (k = 0.008 s-1) equivalent to 0.5% of fiber volume. Only the intermediate kinetic component is significantly affected by pretreatment of fibers with detergent. Efflux curves from detergent-treated fibers could be fit as the sum of two exponentials with coefficients and rate constants comparable to those of the fast and slow component of washout of untreated controls. Similarly the washout of [14C]sucrose can be described as the sum of two exponentials. We conclude that the intermediate component of 42K washout results from the movement of ions from a membrane bound space within the skinned fiber. Because of its relative volume, the sarcoplasmic reticulum seems to be a reasonable choice as a structural correlate for this component. Our estimate of the potassium permeability for the sarcoplasmic reticulum (SR) based on the efflux data is 10(-7) cm/s. This value is less than previous estimates from isolated preparations.     

Recruitment of single muscle fibers during submaximal cycling exercise.

In literature, an inconsistency exists in the submaximal exercise intensity at which type II fibers are activated. In the present study, the recruitment of type I and II fibers was investigated from the very beginning and throughout a 45-min cycle exercise at 75% of the maximal oxygen uptake, which corresponded to 38% of the maximal dynamic muscle force. Biopsies of the vastus lateralis muscle were taken from six subjects at rest and during the exercise, two at each time point. From the first biopsy single fibers were isolated and characterized as type I and II, and phosphocreatine-to-creatine (PCr/Cr) ratios and periodic acid-Schiff (PAS) stain intensities were measured. Cross sections were cut from the second biopsy, individual fibers were characterized as type I and II, and PAS stain intensities were measured. A decline in PCr/Cr ratio and in PAS stain intensity was used as indication of fiber recruitment. Within 1 min of exercise both type I and, although to a lesser extent, type II fibers were recruited. Furthermore, the PCr/Cr ratio revealed that the same proportion of fibers was recruited during the whole 45 min of exercise, indicating a rather constant recruitment. The PAS staining, however, proved inadequate to fully demonstrate fiber recruitment even after 45 min of exercise. We conclude that during cycling exercise a greater proportion of type II fibers is recruited than previously reported for isometric contractions, probably because of the dynamic character of the exercise. Furthermore, the PCr/Cr ratio method is more sensitive in determining fiber activation than the PAS stain intensity method.     

Theory of light diffraction by single skeletal muscle fibers.

A theoretical discussion is presented describing the diffraction of laser light by a single fiber of striated muscle. The complete three-dimensional geometry of the fiber has been taken into consideration. The basic repeated unit is taken as the sarcomere of a single myofibril, including its cylindrical geometry. The single fiber is considered as the sum of myofibrils up to the fiber dimensions. When proper phasing is taken into account, three cases of interest are analyzed. (a) When the adjacent myofibrils are totally aligned with respect to their index of refraction regions (e.g., A and I bands), then the diffraction pattern reflects that of a larger striated cylinder with the dimensions of the fiber. (b) When a particular skew plane develops for the myofibril elements, additional Bragg reflection occurs at certain specific sarcomere lengths, and intensity asymmetry amongst the diffracted orders occurs. (c) When the myofibril phasing changes in a random fashion, while all sarcomeres remain at the same length, then intensity decrease is directly related to the phase deviation from a reference phase point. This condition may well describe a fiber undergoing active isometric contraction.     

Fluctuations in tension during contraction of single muscle fibers.

We have searched for fluctuations in the steady-state tension developed by stimulated single muscle fibers. Such tension "noise" is expected to be present as a result of the statistical fluctuations in the number and/or state of myosin cross-bridges interacting with thin filament sites at any time. A sensitive electro-optical tension transducer capable of resolving the expected fluctuations in magnitude and frequency was constructed to search for the fluctuations. The noise was analyzed by computing the power spectra and amplitude of stochastic fluctuations in the photomultiplier counting rate, which was made proportional to muscle force. The optical system and electronic instrumentation together with the minicomputer software are described. Tensions were measured in single skinned glycerinated rabbit psoas muscle fibers in rigor and during contraction and relaxation. The results indicate the presence of fluctuations in contracting muscles and a complete absence of tension noise in eith rigor or relaxation. Also, a numerical method was developed to simulate the power spectra and amplitude of fluctuations, given the rate constants for association and dissociation of the cross-bridges and actin. The simulated power spectra and the frequency distributions observed experimentally are similar.     

A-band shortening in single fibers of frog skeletal muscle.

The question of whether A-bands shorten during contraction was investigated using two methods: high-resolution polarization microscopy and electron microscopy. During shortening from extended sarcomere lengths in the passive state, sarcomere-length changes were essentially accounted for by I-band shortening. During active shortening under otherwise identical conditions, the sarcomere length change was taken up approximately equally by A- and I-bands. Several potential artifacts that could give rise to apparent A-band shortening were considered and judged unlikely. Results obtained with polarization microscopy were similar to those obtained with electron microscopy. Thus, modest but significant thick filament shortening appears to occur during active sarcomere shortening under physiological conditions.     

Longitudinal impedance of single frog muscle fibers

The longitudinal impedance of single skeletal muscle fibers has been measured from1 to 10,000 Hz in an oil gap apparatus which forces current to flow longitudinally down the fiber. The impedance observed is purely resistive in some fibers from the semitendinosus muscle and in two fibers from the sartorius muscle. In other fibers from the semitendinosus muscle a small phase shift is observed. The mean value of the maximum phase shift observed from all fibers is 1.07 degrees. The artifacts associated with the apparatus and method are examined theoretically and it is shown that one of the likely artifacts could account for the small phase observed. It is concluded that the longitudinal impedance of skeletal muscle fibers is essentially resistive and that little, if any, longitudinal current crosses the membranes of the sarcoplasmic reticulum.     

Correlation between myofibrillar ATPase activity and myosin heavy chain composition in single human muscle fibers.

Single human muscle fibers were analysed using a combination of histochemical and biochemical techniques. Routine myofibrillar adenosine triphosphatase (mATPase) histochemistry revealed a continuum of staining intensities between the fast fiber types IIA and IIB (type IIAB fibers) after preincubation at pH 4.6. Electrophoretic analysis of single, histochemically-identified fibers demonstrated a correlation between the staining intensity and the myosin heavy chain (MHC) composition. All fibers classified as type I contained exclusively MHCI and all type IIA fibers contained only MHCIIa. Type IIAB fibers displayed variable amounts of both MHCIIa and MHCIIb; the greater the staining intensity of these fibers after preincubation at pH 4.6, the greater the percentage of MHCIIb. Those fibers histochemically classified as type IIB contained either entirely MHCIIb or, in addition to MHCIIb, a small amount of MHCIIa. These data establish a correlation between the mATPase activity and MHC content in single human muscle fibers.