reply: In a letter to the Editor, Prof. Kiisa Nishikawa (8) expressed concerns with our recent paper (1) published in American Journal of Physiology-Cell Physiology. Although her concerns are secondary to our main results and conclusion, they deserve clarification.
Nishikawa is concerned that we used phalloidin to image myofibrils in order to show the effects of gelsolin in extraction of thin filaments. She cites a study that showed that phalloidin bound to the Z-lines in the sarcomeres and pointed ends of the thin filaments was randomly oriented in glycerol-extracted myofibrils (12). However, when the myofibrils were relaxed during imaging (as in our study) the thin filaments were properly visualized, even though phalloidin was not distributed homogeneously when the myofibrils were imaged in short average sarcomere lengths. Although not perfect for filament quantification, phalloidin was effective for visualizing thin filaments. The goal of our experiments was to test the effects of Ca2+ in myofibrils without thin filaments. The use of gelsolin for thin filaments extraction is common in the field of muscle biophysics; it has been used successfully in several laboratories with large experience in thin filaments extraction [(3-5, 10) to cite just a few] and also in a previous study in our laboratory (2). It is worth noting that when the myofibrils were exposed to pCa 4.5 in our study, they did not produce any active force. Thus, we are confident that gelsolin treatment resulted in thin filament extraction in the myofibrils that were used in our experiments.
Nishikawa questions our conclusion that Ca2+ increases the static stiffness of myofibrils by increasing the stiffness of titin, because “several recent papers have suggested that a titin-actin interaction could be responsible for titin activation upon Ca2+ influx in muscle.” Two papers are cited in her letter (6, 7). The first one (7) is a highly speculative paper in which Nishikawa and collaborators propose a “new twist on muscle contraction” with a model that titin works as a “winding filament” that is activated upon contraction by myosin cross-bridges working as rotors. The paper presents no concrete evidence that the model may work, and to our knowledge it has never been tested experimentally. The second paper cited in the letter (6) was discussed in our paper and shows results of experiments conducted with myofibrils stretched to sarcomere lengths of ∼6.0 μm producing forces that are ∼350% higher in the presence of Ca2+ when compared to nonactivated myofibrils. As stated in the Discussion in our paper, the results are arguably incompatible with the current knowledge of titin and muscle contraction. Although the paper introduces new ideas about muscle contraction, the results have yet to be repeated in other laboratories. We failed to reproduce similar results in our laboratory and discussed possible reasons for this in our paper. Unfortunately, Nishikawa fails to cite studies performed in laboratories with recognized knowledge and extensive experience with titin (4, 5, 9, 11) that have reached conclusions similar to ours (i.e., Ca2+ does not strengthen titin-actin interactions).
Finally, we never intended to “report the death” of titin-actin interactions. We objectively reported that 1) the static tension in striated myofibrils correlates with titin isoforms, and 2) the static tension is present after thin filament extraction, suggesting that it is not associated with titin-actin interaction. These evidence-based claims are not premature as suggested in the letter. It is certainly premature to suggest new models of contraction without solid evidence and experimental work that is repeatable in different laboratories.
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