F J Julian

1. The variation of isometric tetanus tension with sarcomere length in single fibres from frog striated muscle has been re-investigated with special precautions to ensure uniformity of sarcomere length within the part of the fibre being studied.2. In most respects the results of Ramsey & Street (1940) were confirmed, but (a) the peak of the curve was found to consist of a plateau between sarcomere lengths of 2.05 and 2.2 mu, (b) the decline of tension above this plateau is steeper than found by Ramsey & Street, and (c) the decline of tension below the plateau becomes suddenly steeper at a sarcomere length of about 1.67 mu.3. Many features of this length-tension relation are simply explained on the sliding-filament theory.4. It is concluded that, in the plateau and at greater lengths, the tension on each thin filament is made up of equal contributions from each bridge which it overlaps on adjacent thick filaments.5. Internal resistance to shortening is negligible in this range but becomes progressively more important with shortening below the plateau.

1. An apparatus is described by which the length of a selected part of an isolated muscle fibre can be held constant, giving isometric conditions, or alternatively its length can be measured while tension is held constant (isotonic). Control can be switched between length and tension so as to carry out afterloaded contractions with a shortening stop.2. When a part of a fibre with uniform striation spacing is stretched so far that there is presumably no overlap of filaments, the tension developed during an isometric tetanus with this apparatus is very small (not more than 3-5% of the tension developed at optimum length).3. If the tendon ends are held stationary, a fibre with the same initial length develops a large amount of tension (order of 30-40% of tension at optimum length) with a slow time course. This additional tension is due to shortening of the end parts of the fibre, where the striation spacing is smaller and overlap of filaments still exists.4. The resistance to elongation of a part of a fibre where there is no overlap is only slightly increased on stimulation.5. To a first approximation, the results are in good agreement with expectations based on the sliding filament theory. The development of detectable amounts of tension, and of a slight increase of stiffness, on stimulation, are however not expected on the simplest form of this theory; possible explanations are discussed.

1. The stability of sarcomere lengths along single frog twitch fibres was examined, during lengthening and shortening, using a spot follower appparatus to monitor or control the length of a central segment. 2. During active shortening from sarcomere lengths beyond 2.2 micrometer the end sarcomeres shortened dramatically, while much of the fibre did not shorten at all. It is proposed that this is the cause of the tension failing to recover, after the shortening ceased, to the value of isometric tension at the shorter length. 3. During active lenghtening from sarcomere lengths beyond 2.2 micrometer, non-uniformity of stretch was seen, with the middle stretching more than the ends. Some maintained extra tension after stretch above that appropriate to the longer length was found, as were consistent changes in internal movement, and in the shape of the tension record during relaxation. 4. Measurements of stiffness during and after a lengthening suggest that no increased activation is involved. Observation of internal movement during the raised tension after a lengthening contradicts theories involving 'locked on' bridges. 5. From these and other observations, an explanation for the extra tension in terms of non-uniformity of sarcomeres is proposed. The explanation is in accord with that previously suggested for the creep phase of tension rise seen at these lengths.

1. Twitch fibres were isolated from the semitendinosus muscles of frogs. The sarcolemma was made more permeable by a 30 min soak in a solution containing 47.3% glycerine (v/v), 2 mM-EGTA and 10 mM phosphate buffer, pH 7. This was followed by a 30-60 min soak in a solution containing the non-ionic detergent Lubrol-WX. The fibres were then placed in a relaxing medium containing (in mM): KCl, 100; MgCl(2), 1; ATP, 4; EGTA, 2; imidazole buffer, 10; pH 7.0.2. A piece of fibre about 1-2 mm long treated as described in (1) was attached to a servo apparatus. This apparatus made it possible either to hold fibre length constant giving isometric conditions, or alternatively to hold the force constant while measuring isotonic length changes. A special network made it possible to switch control from isometric to isotonic conditions so that afterloaded contractions with a shortening stop could be carried out.3. Contractions were induced at about 4 degrees C by lowering the pCa in the relaxing solution to various levels determined by the ratio of calcium and EGTA added. Contractions were never observed above pCa 7. The steady force generated reached a maximum over the range of pCa 6.09 to 5.49. The relationship between steady force generated and pCa is S-shaped and very steep, implying that multiple interacting binding sites for calcium are involved in the force generating process.4. The relative force-velocity relation is the same at pCa 6.09 and 5.49 where the steady force is at a maximum. The data points can be well fitted by a hyperbola in which the extrapolated value for V(max) is 2.39 muscle lengths/sec. The values obtained for the Hill parameters a/P(0) and b are within the range of those reported for living electrically excited frog muscle.5. The relative force-velocity points obtained at higher pCa values at which the steady force was on average 37% of that developed at pCa 5.49 can also be fitted by a hyperbola. However, the extrapolated value for V(max) is only 1.12 muscle lengths/sec. The value for a/P(0) is increased slightly and the value for b is markedly decreased.6. Evidence is presented against the possibility that an unrecognized fixed internal load is responsible for the change in the relative force-velocity relation obtained at high pCa.7. The relative force-velocity relation does not change appreciably over at least part of the range of sarcomere lengths in which the force generated varies linearly with overlap provided the pCa is held constant.8. The results support the view that lowering the pCa produces a mechanical state equivalent to that produced by tetanic electrical stimulation.9. Some models for calcium activation are discussed. It is concluded that a model based on calcium binding to troponin on the thin filaments is difficult to reconcile with all of the experimental evidence. There is additional evidence for believing that activating calcium may directly influence the cross-bridges.

Single frog skeletal muscle fibers were attached to a servo motor and force transducer by knotting the tendons to pieces of wire at the fiver insertions. Small amplitude, high frequency sinusoidal length changes were then applied during tetani while fibers contracted both isometrically and isotonically at various constant velocities. The amlitude of the resulting force oscillation provides a relative measure of muscle stiffness. It is shown from an analysis of the transient force responses observed after sudden changes in muscle length applied both at full and reduced overlap and during the rising phase of short tetani that these responses can be explained on the basis of varying numbers of cross bridges attached at the time of the length step. Therefore, the stiffness measured by the high frequency legth oscillation method is taken to be directly proportional to the number of cross bridges attached to thin filament sittes. It is found that muscle stiffness measured in this way falls with increasing shortening velocity, but not as rapidly as the force. The results suggest that at the maximum velocity of shortening, when the external force is zero, muscle stiffness is still substantial. The findings are interpreted in terms of a specific model for muscle contraction in which the maximum velocity of shortening under zero external load arises when a force balance is attained between attached cross bridges somr interpretations of these results are also discussed.