The primary purpose of this study was to determine if there was a fundamental difference in the length-dependence of staircase and posttetanic twitch contractions. The results of the experiments reported in this paper confirm that both staircase and the posttetanic response have a length-dependence, and that the activity-dependent change in twitch amplitude is proportional to sarcomere length with a negative slope in both cases. These observations are consistent with previous reports at 35°C [6, 7, 9, 10]. and one report of frog muscle at room temperature , but are in contradiction to the report of Moore and Persechini  who studied mouse skeletal muscle posttetanic potentiation at room temperature.
The force-length relation
In order to discuss the results regarding the length-dependence of potentiation, we had to first define the force-length relation for the muscle preparation used in this study. The force-length relation for tetanic contractions had a descending limb, which was deviated to the right when compared to the theoretical force-length relation. It also showed an extended plateau where the force did not decrease until well beyond estimated optimal filament overlap. This extended plateau and deviation from the theoretical force-length relation (Gordon et al.,  has been observed in other studies that used fixed-end contractions [15–17]. This augmented force observed at a given sarcomere length is due either to inhomogeneity of sarcomere lengths or to a compliance within the muscle or measurement system.
Forces that are higher than predicted could be explained if there is an inhomogeneity of sarcomere lengths during the contraction, as proposed by Gordon et al. . Inhomogeneity has been shown to occur during tetanic contractions elicited on the descending limb of the force-length relation [18, 19]., where some sarcomeres are being stretched while others are shortening. The sarcomeres that are shortening are likely situated near the end of the fibers. In our study, sarcomeres near the tendons may have been operating at a shorter sarcomere length during the contraction, when compared to the passive sarcomere length that we actually measured.
Any compliance in the muscle or measuring system would also cause the sarcomere length during the contractions to be shorter than that before (or after) the contraction. Assuming all of the discrepancy between the theoretical and actual length tension relation is due to compliance, a correction factor can be generated. The data in Figure 1 would center around the line representing the theoretical length-tension relationship if sarcomere length for each contraction was corrected by subtracting 0.19 μm. This correction could relate to a short range compliance that could be due to movement of the aluminum clips during each contraction.
The force-length relationship observed in this study is consistent with previous investigations on the force-length relation for fixed-end tetanic contractions of skeletal muscle. Therefore, our measurements of sarcomere length in the experiments on the length-dependence of potentiation are a reasonable expression of the actual (initial) sarcomere length for these conditions.
Length-dependence of potentiation
The observation that the magnitude of the posttetanic twitch response (Pt*/Pt) was proportional to length but with a negative slope is in direct contrast with a previous report . However, this observation is consistent with data of Roszek et al. , who reported enhanced low-frequency responses when these were preceded by tetanic stimulation in the medial rat gastrocnemius muscle (at 37°C). They observed that this enhancement was greater at short muscle lengths. Our results provide evidence that the discrepancy between  and those of  is not a function of temperature. Furthermore, it would appear that it is unnecessary to propose different mechanisms for posttetanic potentiation and staircase.
There are other differences between the study by  and this one. They used a whole mouse EDL, while we used a fiber bundle. While it is tempting to suggest that they may have had a central core of fatigued or damaged cells, it is not clear how this could account for the reversal of the relationship between length and potentiation. Another difference is the apparent length range which was studied. In the paper by Moore & Persechini , length is expressed relative to a reference length which appears to be the length at which tetanic developed tension is greatest. Contractile response was measured at this length, and up to 25% longer, at which length tetanic developed tension was apparently less than 40% of the peak value at the reference length. At this same length, twitch tension was just over 30% of its respective peak value at the reference length. In contrast, in our experiments, the length range studied was from a sarcomere length of 2.3 μm to 3.5 μm. The relative length increase here is over 50%. We would appear to be studying a greater range of lengths, but the range of twitch amplitudes is much less. In fact twitch amplitude at the longest length studied in this paper, has a developed tension that is not substantially different from that at 2.5 μm. The greatest twitch amplitude occurred at an intermediate length. Of interest, our tetanic developed tensions were about 40% of the maximum at a sarcomere length of 3.5 μm. This difference in the length-dependence of tetanic and twitch contractions has been reported previously and is attributed to length dependence of activation .
It is interesting that the twitch contraction after a brief tetanic contraction at 22°C in our experiments demonstrated potentiation when the response was measured at short sarcomere lengths, and depression when the response was measured at long sarcomere lengths. This is consistent with the observation of Krarup  that in the poststimulation period at room temperature, there are both factors which enhance subsequent contractile response and factors that diminish the contractile response. Krarup  reported that the dissipation of the depression of force was faster than the dissipation of enhancement, such that after a period of time, the enhancement became evident. In the case of the posttetanic contractions reported here, the enhancement apparently predominated at a short length, while depression was evident at long sarcomere length. This indicates that the factor(s) resulting in depression may have a mechanism that is length-dependent. It is also possible that the depression may be similar at all lengths, and the potentiating effects may be the only factor that is length-dependent. It is important to point out that our posttetanic twitch contractions were obtained at a variety of lengths after a tetanic contraction at optimal length. The metabolic disturbance, or level of regulatory light chain phosphorylation would not have been different between these lengths.
The depression of twitch amplitude after a tetanic contraction may be a consequence of hydrogen ion or inorganic phosphate (Pi) accumulation. These factors are likely to be more evident after the tetanic contraction (75 pulses per s) than after staircase (10 pulses per s). It is known that decreased pH or increased Pi results in a rightward shift in the force-pCa2+ relation [23–25]. It has been shown for both pH and Pi that the shift is more evident at room temperature than at physiological temperature [26–28]. This factor may also explain why potentiation is less during staircase at 22°C than at 35°C – in fact Martyn and Gordon  have shown an interdependence of changes in Ca2+ sensitivity associated with pH, and length.
The mechanism for the length-dependence of potentiation may be related to length-dependence of activation, as suggested previously . It was argued that the rightward shift in the control force-length relation during twitch (in comparison with tetanic) contractions is apparently due to enhanced sensitivity to Ca2+ as muscle length is increased [13, 30]. Since regulatory light chain phosphorylation also results in increased sensitivity to Ca2+ , then it may be that there is a ceiling effect, and the two mechanisms are not simply additive. Light chain phosphorylation may be less effective in enhancing the contractile response when it is already enhanced by length-dependent activation.
This interpretation relies on the assumption that the magnitude of light chain phosphorylation was similar after staircase elicited at different lengths. Myosin light chain kinase, the enzyme responsible for phosphorylating the regulatory light chain, is activated by Ca2+ bound to calmodulin. A similar level of light chain phosphorylation would be expected if Ca2+ transients during this stimulation were not affected by length. Balnave and Allen  have shown that Ca2+ transients in mouse muscle are independent of length. Furthermore, a length-dependence of potentiation similar to that reported here has been reported for rat muscle after staircase at one length , an approach similar to that used in this study for the posttetanic potentiation. It seems unlikely that the length-dependence of potentiation described here is due to length-dependent differences in regulatory light chain phosphorylation.
The data available in this study cannot discern the possible mechanisms that are operating to result in a length-dependence of Pt*/Pt. However, the observations in this paper clearly demonstrate that this length-dependence is operative at the sarcomere level, and Pt*/Pt decreases as sarcomere length increases whether it is studied after high frequency (tetanic contraction) or after low frequency (staircase) stimulation. These observations are consistent with the proposed mechanism by which light chain phosphorylation enhances developed tension. It has been proposed that light chain phosphorylation causes individual cross-bridges to swing out from the myosin back-bone , thereby bringing the actin binding site of the cross-bridge in close proximity to the actin filament. This would increase the probability of cross-bridge binding to actin, resulting in a greater number of cross-bridges exerting force during the twitch contraction. This mechanism is apparently ineffective when the myofilaments are in close proximity as would be the case at a long sarcomere length. There is no need to invoke a different explanation for posttetanic potentiation than for staircase.