Reply to “Letter to the editor: Comments on Cornachione et al. (2016): “The increase in non-cross-bridge forces after stretch of activated striated muscle is related to titin isoforms”

reply: In a letter to the Editor of the American Journal of Physiology-Cell Physiology, Prof. Walter Herzog (6a) chose and edited two figures displaying raw data from our paper (2) to challenge the conclusions drawn from our study. Herzog disregards all the remaining data and results that base the conclusions of our paper, including the figure (Fig. 4) and table with a summary of multiple experiments containing statistical information (Table 1), and fails to recognize that individual experiments may present variability, which makes his letter improperly biased. However, it still deserves clarification.

Herzog is concerned with one of our figures (Fig. 2B) showing active forces produced by a soleus myofibril in sarcomere lengths of 2.8 μm and 3.0 μm. Herzog cites a classic study performed in 1966 by Gordon et al. (5) to suggest that the force produced in a sarcomere length of 3.0 μm should be significantly smaller than the force produced in a sarcomere length of 2.8 μm. The reference is inappropriate: Gordon et al. (5) performed experiments with single, intact fibers from the frog (instead of permeabilized myofibrils from the rabbit) with a length-control method, as opposed to fixed-end contractions in which the sarcomere or fiber length is not clamped during activation. They measured forces at long lengths with an extrapolation method to build the force-length relation, and the resulting force was lower than the actual, total force produced by the fibers. Since then, it is well known that fixed-end contractions, like the contractions used in our study, produce a force-length relation with an extended plateau in which forces vary considerably, and do not necessarily drop significantly until sarcomere lengths of ∼3.0–3.2 μm (1, 6, 18). Without constructing a proper force-length relation for individual preparations, it is impossible to ascertain that the region between 2.8 μm and 3.0 μm in our study corresponds to a descending limb of the force-length relation derived in the study by Gordon et al. (5). Indeed, in several papers it does not (refs. 1, 6, 18, to cite a few). According to Herzog's letter, we have pointed out that myofibrils in our study were on the descending limb of the force-length relation. His comment is baffling, as we never made such statement anywhere in our paper. Nor did we make such a statement in a previous study investigating static stiffness using similar methods and with results consistent with the current study (3).

Fig. 2.

Four superimposed contractions produced by myofibrils isolated from the psoas (A), soleus (B), and ventricle (C) muscles. Black traces: isometric contractions and passive stretches performed in pCa 4.5 and 9.0, respectively. Red traces: stretches produced after full development in pCa 4.5. Blue traces: stretches applied in pCa 4.5 after myofibrils were treated with rigor-EDTA, gelsolin, and BB. Note the high signal-to-noise ratio in the force traces, which allows the detection of small force differences during the experiments. During the stretches in pCa 4.5, the force increased substantially, and after the stretch the force decreased to attain a steady state that was higher than the force obtained during isometric contractions at a similar length. During the stretches in pCa 4.5 after the myofibrils were treated with rigor-EDTA, gelsolin, and BB, the force was higher than that produced during stretches in pCa 9.0 for the psoas and soleus muscles, but not for the ventricle muscle.

Herzog mentions that the residual force enhancement shown in Fig. 2, B and C, should be larger than the “passive force enhancement,” i.e., an increase in passive force observed after deactivation. The comment is inappropriate, as we never measured the passive force enhancement. The passive forces after deactivation in our study are still decreasing when we shortened the myofibrils back to their original lengths— the passive force in the figures depicts the force decay when Ca²⁺ was quickly removed from the preparation. In order to study passive force enhancement in myofibrils, we would need to ascertain that the passive force is not a result of a slow decay of active force. Studies with myofibrils looking at passive force enhancement have typically waited for at least 15 seconds after deactivation and averaged data over 10 s for measurements of passive forces (9, 10). Instead, we investigated the Ca²⁺-induced increase in passive forces by measuring the static stiffness, which can be visualized in the traces that were unfortunately deleted from our original figure (shown in this Reply) by Herzog in his edited figure. These traces show clearly that the increase in passive force (when present) is significantly lower than the residual force enhancement (when present).

Herzog suggests that the myofibrils used in our study may be damaged or incompletely activated. As stated in our paper, we checked for sarcomere length nonuniformity throughout the experiments to make sure that the myofibrils were not damaged, in which case they would be discarded from analysis. If there were any decreases in force during the experiments, the myofibrils would also have been discarded from analysis. Finally, the myofibrils could not be incompletely deactivated: using a fast perfusion system similar to that used commonly in laboratories with expertise with myofibrils (e.g., refs. 15, 19), we activated myofibrils with pCa of 4.5, which has been shown to fully activate muscle fibers and myofibrils in any single study that we are aware in the literature—a truism in the muscle physiology field.

On the basis of his concerns with Fig. 2, B and C, Herzog suggests that our results are inconsistent with the known properties of skeletal muscle and the behavior of single myofibrils and sarcomeres. The suggestion is incorrect. The isometric forces, the rates of force development, and the rates of relaxation produced by myofibrils from psoas, soleus, and ventricle myofibrils (Table 1) are all very similar to values reported in the literature; we actually cite several studies in our paper to prove this point [refs. 25, 45, 58, 59, 64, 65 in the paper (2)]. The values for the residual force enhancement and the static stiffness (Table 1) are also well within range published by others (e.g., refs. 4, 11, 17, to cite just a few). Finally, the force-sarcomere length relation summarizing data from the three muscles investigated in this study (Fig. 4) are very similar in shape and magnitude to studies performed in well-known laboratories looking into passive properties of skeletal and cardiac muscles, in low or high pCa (ref. 16, their Fig. 6; ref. 12, their Fig. 5). The only results highly inconsistent with ours are presented in studies from Herzog's laboratory, in which they observe 1) a residual force enhancement in myofibrils of 285% (8) and 386% (13) of the isometric reference forces, 2) an increase in myofibril forces when tested in a pCa of 4.5 of ∼350% in a sarcomere length of 6 μm when compared to a pCa of 9.0 in a sarcomere length of 6 µm (14), and 3) an increase in force of ∼700% in a sarcomere length of 6 μm when compared to 2.2–2.4 μm (conceptually the optimal length for force development), after myofibrils are stretched in a pCa of 4.5 (14). These numbers represent large anomalies in the literature. These results are not only incompatible with known properties of skeletal muscles and the behavior of myofibrils and sarcomeres, but they have never been repeated in any other laboratory and cannot be conciliated into the current knowledge of muscle contraction. They have instead been used to form a “new theory of contraction” (7), which has not been embraced by the scientific community. The results of our paper are consistent with virtually all the reproducible scientific literature up to date.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

D.E.R. drafted manuscript; D.E.R. edited and revised manuscript; D.E.R. approved final version of manuscript.