Interaction between series compliance and sarcomere kinetics determines internal sarcomere shortening during fixed-end contraction

The interaction between contractile force and in-series compliance was investigated for the intact skeletal muscle-tendon unit (MTU) of Rana pipiens semitendinosus muscles during fixed-end contraction. It was hypothesized that internal sarcomere shortening is a function of the length-force characteristics of contractile and series elastic components. The MTUs (n=18) were dissected, and, while submerged in Ringer's solution, muscles were activated at nine muscle lengths (-2 to +6 mm relative to optimal length in 1 mm intervals), while measuring muscle force and sarcomere length (SL) by laser diffraction. The MTU was clamped either at the bone (n=6), or at the proximal and distal ends of the aponeuroses (n=6). Muscle fibers were also trimmed along with aponeuroses down to 5-20 fibers and identical measurements were performed (n=6). The magnitude of shortening decreased as MTU length increased. The magnitude of shortening ranged from -0.08 to 0.3 microm, and there was no significant difference between delta SL as a function of clamp location. When aponeuroses were trimmed, sarcomere shortening was not observed at L(0) and longer. These results suggest that the aponeurosis is the major contributor to in-series compliance. Results also support our hypothesis but there also appear to be other factors affecting internal sarcomere shortening. The functional consequence of internal sarcomere shortening as a function of sarcomere length was to skew the muscle length-tension relationship to longer sarcomere lengths.     

Uniform sarcomere shortening behavior in isolated cardiac muscle cells

We have observed the dynamics of sarcomere shortening and the diffracting action of single, functionally intact, unattached cardiac muscle cells enzymatically isolated from the ventricular tissue of adult rats. Sarcomere length was measured either (a) continuously by a light diffraction method or (b) by direct inspection of the cell's striated image as recorded on videotape or by cinemicroscopy (120--400 frames/s). At physiological levels of added CaCl2 (0.5--2.0 mM), many cells were quiescent (i.e., they did not beat spontaneously) and contracted in response to electrical stimulation (less than or equal to 1.0-ms pulse width). Sarcomere length in the quiescent, unstimulated cells (1.93 +/- 0.10 [SD] micrometers), at peak shortening (1.57 +/- 0.13 micrometers, n = 49), and the maximum velocity of sarcomere shortening and relengthening were comparable to previous observations in intact heart muscle preparations. The dispersion of light diffracted by the cell remained narrow, and individual striations remained distinct and laterally well registered throughout the shortening- relengthening cycle. In contrast, appreciable nonuniformity and internal buckling were seen at sarcomere lengths < 1.8 micrometers when the resting cell, embedded in gelatin, was longitudinally compressed These results indicate (a) that shortening and relengthening is characterized by uniform activation between myofibrils within the cardiac cell and (b) that physiologically significant relengthening forces in living heart muscle originate at the level of the cell rather than in extracellular connections. First-order diffracted light intensity, extremely variable during sarcomere shortening, was always greatest during midrelaxation preceding the onset of a very slow and uniform phase of sarcomere relengthening.     

Stiffness, force, and sarcomere shortening during a twitch in frog semitendinosus muscle bundles

The time course of force and stiffness during a twitch was determined at 6 and 26 degrees C in frog semitendinosus muscle bundles using the transmission time technique of Schoenberg, M., J.B. Wells, and R.J. Podolsky, 1974, J. Gen. Physiol. 64:623-642. Sarcomere shortening due to series compliance was also measured using a laser light diffraction technique. Following stimulation, stiffness developed more rapidly than force, but had a slower time course than published Ca2+ transients determined from light signals using Ca2+ sensitive dyes (Baylor, S.M., W.K. Chandler, and M.W. Marshall, 1982, J. Physiol. (Lond.). 331:139-177). Stiffness (S) did not reach its tetanic value during a twitch at 6 or 26 degrees C, although at 6 degrees C, it approached close to this value with S-twitch/S-tetanus = 0.82 +/- 0.07 (+/- SEM). During relaxation, force fell more rapidly than stiffness both for a twitch and also a tetanus. Also in this paper, several of the assumptions inherent in using the transmission time technique for the measurement of stiffness are considered in detail.     

X-ray interference studies of crossbridge action in muscle contraction: evidence from muscles during steady shortening

During normal muscle shortening, the myosin heads must undergo many cycles of interaction with the actin filaments sliding past them. It is important to determine what range of configurations is found under these circumstances, and, in terms of the tilting lever arm model, what range of orientations the lever arms undergo. We have studied this using the X-ray interference technique described in the previous article, focusing mainly on the changes in the first order meridional reflection (M3) as compared to isometric. The change in ratio of the heights of the interference peaks indicates how far the mean lever arm angle has moved towards the end of the working stroke; the total intensity change depends on the angle change, on the number of heads now attached at any one time, and on the dispersion of lever arm angles. The latter provides a measure of the distance over which myosin heads remain attached to actin as they go through their working strokes. Surprisingly, the mean position of the attached heads moves only about 1 nm inwards (towards the center of the A-band) at low velocity shortening (around 0.9 T0): their dispersion changes very little. This shows that they must be detaching very early in the working stroke. However, at loads around 0.5 T0, the mean lever arm angle is about half way towards the end of the working stroke, and the dispersion of lever arm angles (with a uniform dispersion) is such as to distribute the heads throughout the whole of the working stroke. At higher velocities of shortening (at 0.3 T0), the mean position shifts further towards the end of the stroke, and the dispersion increases further. The details of the measurements, together with other data on muscle indicate that the force-generating mechanism within the myosin heads must have some unexpected properties.     

Measurement of sarcomere shortening in skinned fibers from frog muscle by white light diffraction.

A new optical-electronic method has been developed to detect striation spacing of single muscle fibers. The technique avoids Bragg-angle and interference-fringe effects associated with laser light diffraction by using polychromatic (white) light. The light is diffracted once by an acousto-optical device and then diffracted again by the muscle fiber. The double diffraction reverses the chromatic dispersion normally obtained with polychromatic light. In frog skinned muscle fibers, active and passive sarcomere shortening were smooth when observed by white light diffraction, whereas steps and pauses occurred in the striation spacing signals obtained with laser illumination. During active contractions skinned fibers shortened at high rates (3-5 microns/s per half sarcomere, 0-5 degrees C) at loads below 5% of isometric tension. Compression of the myofibrillar lateral filament spacing using osmotic agents reduced the shortening velocity at low loads. A hypothesis is presented that high shortening velocities are observed with skinned muscle fibers because the cross-bridges cannot support compressive loads when the filament lattice is swollen.     

Force-velocity relationship and biochemical-to-mechanical energy conversion by the sarcomere

The intracellular control mechanism leading to the well-known linear relationship between energy consumption by the sarcomere and the generated mechanical energy is analyzed here by coupling calcium kinetics with cross-bridge cycling. A key element in the control of the biochemical-to-mechanical energy conversion is the effect of filament sliding velocity on cross-bridge cycling. Our earlier studies have established the existence of a negative mechanical feedback mechanism whereby the rate of cross-bridge turnover from the strong, force-generating conformation to the weak, non-force-generating conformation is a linear function of the filament sliding velocity. This feedback allows the analytic derivation of the experimentally established Hill's equation for the force-velocity relationship. Moreover, it allows us to derive the transient length response to load clamps and the transient force response to sarcomere shortening at constant velocity. The results are in agreement with experimental studies. The mechanical feedback regulates the generated power, maintains the linear relationship between energy liberated by the actomyosin-ATPase and the generated mechanical energy, and determines the efficiency of biochemical-to-mechanical energy conversion. The mechanical feedback defines three elements of the mechanical energy: 1) external work done; 2) pseudopotential energy, required for cross-bridge recruitment; and 3) energy dissipation caused by the viscoelastic property of the cross bridge. The last two elements dissipate as heat.     

Modulation of sarcomere organization during embryonic stem cell-derived cardiomyocyte differentiation

Myofibrillogenesis - sarcomeres - mouse embryonic stem cells - cardiomyocytes - beta1 integrin Mouse embryonic stem (ES) cells, when cultivated as embryoid bodies, differentiate in vitro into cardiomyocytes of ventricle-, atrium- and pacemaker-like cell types characterized by developmentally controlled expression of cardiac-specific genes, structural proteins and ion channels. Using this model system, we show here, (I) that during cardiac myofibrillogenesis sarcomeric proteins are organized in a developmentally regulated manner following the order: titin (Z-disk), alpha-actinin, myomesin, titin (M-band), myosin heavy chain, alpha-actin, cardiac troponin T and M-protein, recapitulating the sarcomeric organization in the chicken embryonal heart in vivo. Our data support the view that the formation of I-Z-I complexes is developmentally delayed with respect to A-band assembly. We show (2) that the process of cardiogenic differentiation in vitro is influenced by medium components: Using a culture medium supplemented with glucose, amino acids, vitamins and selenium ions, we were able to increase the efficiency of cardiac differentiation of wild-type, as well as of beta1 integrin-deficient (beta1-/-) ES cells, and to improve the degree of organization of sarcomeric structures in wild-type and in beta1-/- cardiac cells. The data demonstrate the plasticity of cardiogenesis during the differentiation of wild-type and of genetically modified ES cells.     

Magnitude of sarcomere extension correlates with initial sarcomere length during lengthening of activated single fibers from soleus muscle of rats

A laser-diffraction technique was developed that rapidly reports the lengths of sarcomeres (L_s) in serially connected sectors of permeabilized single fibers. The apparatus translates a laser beam along the entire length of a fiber segment within 2 ms, with brief stops at each of 20 contiguous sectors. We tested the hypothesis that during lengthening contractions, when maximally activated fibers are stretched, sectors that contain the longer sarcomeres undergo greater increases in L_s than those containing shorter sarcomeres. Fibers (n = 16) were obtained from the soleus muscles of adult male rats and the middle portions (length = 1.05 ± 0.11 mm; mean ± SD) were investigated. Single stretches of strain 27% and a strain rate of 54% s⁻¹ were initiated at maximum isometric stress and resulted in a 19 ± 9% loss in isometric stress. The data on L_s revealed that 1), the stretch was not distributed uniformly among the sectors, and 2), during the stretch, sectors at long L_s before the stretch elongated more than those at short lengths. The findings support the hypothesis that during stretches of maximally activated skeletal muscles, sarcomeres at longer lengths are more susceptible to damage by excessive strain.     

Visual localization during eye tracking on steady background and during steady fixation on moving background

Experiments were performed to clarify the role of the background motion on the retina in the phenomenon of mislocation of brief visual stimuli during smooth eye tracking. It was found that these visual stimuli were mislocated also relative to a moving background during steady eye fixation. The magnitude of mislocation during pursuit eye movements and during steady fixation was influenced by the stimulus intensity, the background/eye velocity and the place of stimulus presentation in respect to the background; the influence having the same features in both cases. However, the magnitudes of mislocation under the two conditions were quantitatively different. The validity of a hypothesis that the eye movement itself plays no role in the process of localization, and, that this process is based on retinal information only, is considered.     

Correlated reduction of velocity of shortening and the rate of energy utilization in mouse fast-twitch muscle during a continuous tetanus

Isometric tetani of slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles of the mouse were studied at 20 degrees C. The total energy cost for 3- and 9-s isometric tetani was measured as a function of length above L0 and partitioned into a filament overlap-dependent fraction and a smaller filament overlap-independent fraction. In both muscles, the rate of filament overlap-independent energy cost did not change with tetanic duration. In the EDL, but not in the soleus, the rate of filament overlap-dependent energy utilization was greater in a 3-s tetanus than in a 9-s tetanus. The force-velocity relationships were studied after 3 and 9 s of isometric tetanus. In the soleus, Vmax was 2 fiber lengths/s and was not dependent on the duration of isometric tetanus. In contrast, in the EDL, Vmas decreased from 5.9 fiber lengths/s at 3 s to 3.9 fiber lengths/s at 9 s. The velocity of unloaded shortening (Vus) was examined by the slack test method as a function of the duration of isometric tetanus duration over the range of 1-15 s. In the soleus, Vus did not change, whereas in the EDL, Vus declined progressively from 6.4 to 3.2 fiber lengths/s after an isometric tetanus of increasing duration from 1 to 15 s. These results cannot exclude the hypothesis that in a maintained tetanus there is a decrease in the intrinsic cross- bridge turnover rate in the fast-twitch EDL, but not in the slow-twitch soleus muscle.     

Axial shortening during pachynema unrelated to nonhomologous synapsis

The pachytene behavior of chromosomes participating in quadrivalent formation in male mice heterozygous for T(X;4)7Rl or T(X;4)8Rl was analyzed in electron micrographs of microspread spermatocytes. In each population of nuclei from the translocation heterozygotes, the longest 4X axes were approximately the proportional length expected from the respective contributions of the 4 and the X estimated from breakpoint positions in mitotic chromosomes. However, the 4X axis of these translocation quadrivalents undergoes extensive shortening. In both R7 and R8 the shortest 4X axis observed in the population of nuclei was approximately the length of the normal 4 axis. This equalization of axial lengths suggests that there may be an interchromosomal interaction between synapsed chromosomes. In R8, axial shortening of the 4X occurs as pachynema progresses. In both translocations, shortening is accompanied by twisting of the 4X around the 4. Both axial shortening and twists are characteristics exhibited by chromosomal axes of unequal length as part of the meiotic phenomenon described as "synaptic adjustment" (Moses, 1977). Synaptic adjustment involves, in addition, nonhomologous synapsis, which is delayed until the latter part of pachynema. However, axial shortening in R7 and R8 is not accompanied by nonhomologous synapsis. In R7, nonhomologous synapsis does not occur; in R8, it is confined to quadrivalents in which the 4X axis is near its maximum length (i.e., early). This behavior suggests that axial shortening and nonhomologous synapsis during the progression of pachynema (previously considered collectively under the term "synaptic adjustment") are not necessarily coupled events.     

Regulation of myocardial substrate metabolism during increased energy expenditure: insights from computational studies

In response to exercise, the heart increases its metabolic rate severalfold while maintaining energy species (e.g., ATP, ADP, and Pi) concentrations constant; however, the mechanisms that regulate this response are unclear. Limited experimental studies show that the classic regulatory species NADH and NAD+ are also maintained nearly constant with increased cardiac power generation, but current measurements lump the cytosol and mitochondria and do not provide dynamic information during the early phase of the transition from low to high work states. In the present study, we modified our previously published computational model of cardiac metabolism by incorporating parallel activation of ATP hydrolysis, glycolysis, mitochondrial dehydrogenases, the electron transport chain, and oxidative phosphorylation, and simulated the metabolic responses of the heart to an abrupt increase in energy expenditure. Model simulations showed that myocardial oxygen consumption, pyruvate oxidation, fatty acids oxidation, and ATP generation were all increased with increased energy expenditure, whereas ATP and ADP remained constant. Both cytosolic and mitochondrial NADH/NAD+ increased during the first minutes (by 40% and 20%, respectively) and returned to the resting values by 10-15 min. Furthermore, model simulations showed that an altered substrate selection, induced by either elevated arterial lactate or diabetic conditions, affected cytosolic NADH/NAD+ but had minimal effects on the mitochondrial NADH/NAD+, myocardial oxygen consumption, or ATP production. In conclusion, these results support the concept of parallel activation of metabolic processes generating reducing equivalents during an abrupt increase in cardiac energy expenditure and suggest there is a transient increase in the mitochondrial NADH/NAD+ ratio that is independent of substrate supply.     

Model of calcium movements during activation in the sarcomere of frog skeletal muscle

A model of calcium movement during activation of frog skeletal muscle is described. The model was based on the half sarcomere of a myofibril and included compartments representing the terminal cisternae, the longitudinal sarcoplasmic reticulum, the extramyofibrillar space, and the myofibrillar space. The calcium-binding proteins troponin, parvalbumin, and calsequestrin were present in appropriate locations and with realistic binding kinetics. During activation a time-dependent permeability in the terminal cisternal wall led to calcium release into the myoplasm and its diffusion through the myoplasm longitudinally and radially was computed. After adjustment of three parameters, the model produced a myoplasmic free-calcium concentration that was very similar to those recorded experimentally with calcium indicators. The model has been used to demonstrate the importance of parvalbumin in the relaxation of skeletal muscle, to describe the time course and magnitude of calcium gradients associated with diffusion across the sarcomere, and to estimate the errors associated with the use of aequorin as an intracellular calcium indicator in muscle.     

Regulation of energy metabolism in liver

Energy metabolism in liver has to cope with the special tasks of this organ in intermediary metabolism. Main ATP-generating processes in the liver cell are the respiratory chain and glycolysis, whereas main ATP-consuming processes are gluconeogenesis, urea synthesis, protein synthesis, ATPases and mitochondrial proton leak. Mitochondrial respiratory chain in the intact liver cell is subject to control mainly by substrate (hydrogen donors, ADP, oxygen) transport and supply and proton leak/slip. Whereas hormonal control is mainly on substrate supply to mitochondria, proton leak/slip is supposed to play an important role in the modulation of the efficiency of oxidative phosphorylation.     

Regulation of energy metabolism in yeast

Summary The recessive, nuclear gene mutation glc1, which causes glycogen deficiency in Saccharomyces cerevisiae, is highly plciotropic. Studies of the inheritance of glc1 revealed two classes of phenotypic characteristics: I. Traits invariably associated with the mutant gene and II. Traits whose expressions require the presence of glc1 and one or more additional genes. Class I traits include glycogen deficiency and the loss of capacity to accumulate trehalose in nonproliferating conditions. Traits in the second class include a decreased rate of growth on ethanol medium, a deficiency in cytochrome a.a ₃ and an enhanced accumulation of pigment, probably a metalloporphyrin. Constructed strains containing both glc1 and the constitutive maltose fermentation gene MAL4 ⁰ can accumulate trehalose but not glycogen during growth on glucose. However, accumulated trehalose is degraded when cells are exposed to nonproliferating conditions. It is proposed that the glc1 mutation affects a regulatory system, probably involving a protein kinase and/or protein phosphatase, which regulates glycogen synthase and trehalase. Independent regulation of trehalose synthesis by a system controlled by MAL4 ⁰ is indicated.