Inter-sarcomere coordination in muscle revealed through individual sarcomere response to quick stretch Yuta Shimamotoa,1, Madoka Suzukib, Sergey V. Mikhailenkoa,2, Kenji Yasudac, and Shin’ichi Ishiwataa,b,3 aDepartment of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan; bConsolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan; and cInstitute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan Edited by James A Spudich, Stanford University School of Medicine, Stanford, CA, and approved April 27, 2009 (received for review January 25, 2009) The force generation and motion of muscle are produced by the Fabiato in skinned cardiac cells at fixed concentrations of free Ca2ϩ, collective work of thousands of sarcomeres, the basic structural units suggesting the existence of Ca2ϩ-independent regulatory mecha- of striated muscle. Based on their series connection to form a myo- nism in sarcomeres (15). One decade later, we found that the steady fibril, it is expected that sarcomeres are mechanically and/or struc- periodic oscillation, named SPOC (SPontaneous Oscillatory Con- turally coupled to each other. However, the behavior of individual traction), can be well reproduced at partial activation by adding sarcomeres and the coupling dynamics between sarcomeres remain exogenous ADP and inorganic phosphate (Pi) to the relaxing elusive, because muscle mechanics has so far been investigated solution (in the presence of ATP and the absence of Ca2ϩ) (16), mainly by analyzing the averaged behavior of thousands of sarco- allowing the quantitative analysis of sarcomere behavior. During meres in muscle fibers. In this study, we directly measured the SPOC, each sarcomere repeats slow-shortening and rapid- length-responses of individual sarcomeres to quick stretch at partial lengthening phases with a period of a few seconds, lasting from activation, using micromanipulation of skeletal myofibrils under a minutes to hours. In addition, the lengthening phase propagates to phase-contrast microscope. The experiments were performed at ADP- adjacent sarcomeres along the long axis of myofibrils (SPOC wave) (activation (1 mM MgATP and 2 mM MgADP in the absence of Ca2؉ at a faster rate than the diffusion of chemical components. These BIOPHYSICS AND and also at Ca2؉-activation (1 mM MgATP at pCa 6.3) conditions. We observations strongly suggest that the cooperative behavior of COMPUTATIONAL BIOLOGY show that under these activation conditions, sarcomeres exhibit 2 distinct types of responses, either ‘‘resisting’’ or ‘‘yielding,’’ which are sarcomeres is not attributable to artificial non-uniform activation, clearly distinguished by the lengthening distance of single sarcomeres but is an intrinsic property of striated muscle. Moreover, such in response to stretch. These 2 types of sarcomeres tended to coexist dynamic properties of sarcomeres show a possible link to physio- within the myofibril, and the sarcomere ‘‘yielding’’ occurred in clus- logical functions. For example, the period of SPOC strongly cor- ters composed of several adjacent sarcomeres. The labeling of Z-line relates with that of heart beat in various animal species (17), and the with anti-␣-actinin antibody significantly suppressed the clustered relaxation of single sarcomeres propagates along the myofibril after 2ϩ sarcomere ‘‘yielding.’’ These results strongly suggest that the con- the rapid removal of Ca (12). These reports imply that the tractile system of muscle possesses the mechanism of structure-based cooperative behavior of individual sarcomeres, which is concealed inter-sarcomere coordination. by the ensemble averaging, plays a significant role for the efficient work and motion in muscle. Hence, the analysis at the single myofibril ͉ sarcomere ‘‘yielding’’ ͉ Z-line ͉ partial activation sarcomere level must be indispensable for fully understanding the regulatory mechanism that striated muscle possesses. he periodic architecture of striated muscle relies on the series In this study, we investigated the length-response of individual Tconnection of the basic contractile units, called sarcomeres, sarcomeres to externally applied load, using mechanical manipu- which are connected through the Z-line to form a myofibril. Each lation of skeletal myofibrils with microneedles. The characteristic sarcomere is composed of a bipolar array of myofilaments, i.e., the aspects of this study are: 1) The behavior of individual sarcomeres thick (myosin) and thin (actin) filaments. The cyclic interaction of was observed under a phase-contrast microscope simultaneously myosin with actin coupled to ATP hydrolysis produces force and with measuring tension response (18). 2) The myofibrils were motion along the long axis of the myofibril (1, 2). Such character- activated, in almost all cases, by adding MgADP to the relaxing istics of striated muscle imply that individual sarcomeres are conditions (19), which results in the regulation of sarcomeric structurally and mechanically interconnected and interact with each activity by strong-binding (or ADP-bound) cross-bridges indepen- other. Tension and length responses of sarcomeres to the external dently of Ca2ϩ. 3) The mechanical measurements were done at perturbations have revealed various mechanochemical properties partial activation, between contraction and relaxation, where the of striated muscle, particularly those relating to the mechanism of dynamic properties of sarcomeres may prominently emerge as force generation (3–6) and to the attachment/detachment kinetics exemplified by SPOC. These features ensure that the mechanical of cross-bridges (7–9). On the other hand, the interaction between sarcomeres remains unclear, primarily because the widely used technique for measuring sarcomere response is laser light diffrac- Author contributions: Y.S., M.S., K.Y., and S.I. designed research; Y.S. performed research; tion, where the individual sarcomere dynamics are obscured by the Y.S. analyzed data; and Y.S., S.V.M., and S.I. wrote the paper. ensemble averaging of thousands of sarcomeres connected in The authors declare no conflict of interest. parallel and in series in muscle fibers. This article is a PNAS Direct Submission. Recent developments in microscopic analysis using skeletal and Freely available online through the PNAS open access option. cardiac myofibrils revealed various dynamic properties of individual 1Present address: Laboratory of Chemistry and Cell Biology, The Rockefeller University, New sarcomeres (or even half-sarcomeres) upon force generation and York, NY 10065. relaxation of striated muscle (10–14). One of the particular char- 2Present address: Department of Physics, Gakushuin University, Tokyo 171-8588, Japan. acteristics found by this approach is the spontaneous oscillation of 3To whom correspondence should be addressed. E-mail: [email protected]. sarcomeres, which is observed at partial activating conditions. The This article contains supporting information online at www.pnas.org/cgi/content/full/ myofilament-generated oscillation was first reported by Fabiato and 0813288106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813288106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 28, 2021 Fig. 1. Overview of the stretch experiments. (A) Sche- matic diagram of the mechanical measurements in myo- fibrils under an inverted phase-contrast microscope. (B) The phase-contrast image of a myofibril. (C) The inten- sity profile of phase-contrast image. The profiles along the long axis of the myofibril in B, before stretch (upper trace) and 100 ms after stretch (lower trace) at ADP activation are shown. The position of the flexible needle (right) in the upper and the lower traces corresponds to those at P0 and P2, respectively, as defined in Fig. 2B. (Scale bar, 10 ␮m.) and structural interactions between sarcomeres are highlighted 10 myofibrils) at a sarcomere length (SL) 2.0–2.5 ␮m, which is independent of the regulation by Ca2ϩ. 5–6-fold smaller than at maximal Ca2ϩ-activation (19), indicating Here we report that at partial activation conditions the sarco- that the myofibrils were partially activated. At the activation meres show 2 distinct types of response to stretch: they either plateau, the myofibril was quickly stretched (0.01–0.04 P0/ms) by ‘‘resist’’ (only slightly elongate and then maintain the constant moving the stiff needle away from the flexible needle, followed by length, exhibiting a rectangular waveform) or ‘‘yield’’ (significantly holding it for 5 s and then slowly, over more than 5 s, returning it elongate and then gradually shorten, exhibiting a saw-tooth wave- to the original position (Ͻ0.00003 P0/ms). Such stretch protocol was form). This characteristic bimodal response allows us to analyze the repeated every 20 s, 5 times for each myofibril, with various load properties of sarcomere ‘‘yielding’’ depending on the applied load amplitudes. The length-responses of individual sarcomeres were and the mechanical properties of the Z-line structure, which shows analyzed from the phase-contrast image simultaneously with the how sarcomeres cooperatively regulate their force-generating ac- tension-response (Fig. 1B and C). The analysis was performed only tivity with each other without the master regulator, Ca2ϩ ions. when the active isometric force was Ͼ95% of that during the first stretch. The striation