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Microfluidic Perfusion Shows Intersarcomere Dynamics Within Single Skeletal Muscle Myofibrils

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Microfluidic Perfusion Shows Intersarcomere Dynamics Within Single Skeletal Muscle Myofibrils Microfluidic perfusion shows intersarcomere dynamics within single skeletal muscle myofibrils Felipe de Souza Leitea, Fabio C. Minozzoa, David Altmanb, and Dilson E. Rassiera,c,d,1 aDepartment of Kinesiology and Physical Education, McGill University, Montreal, QC, Canada, H2W 1S4; bDepartment of Physics, Willamette University, Salem, OR 97301; cDepartment of Physiology, McGill University, Montreal, QC, Canada, H3G 1Y6; and dDepartment of Physics, McGill University, Montreal, QC, Canada H3A 2T8 Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved July 5, 2017 (received for review January 30, 2017) The sarcomere is the smallest functional unit of myofibrils in passive force production, which enables the myofibrils to stabilize in striated muscles. Sarcomeres are connected in series through a a given activation condition. Because of technical limitations, the network of elastic and structural proteins. During myofibril activa- mechanisms governing the interactionofsarcomeresinamyofibril tion, sarcomeres develop forces that are regulated through complex and its consequences for force production remain unclear (11). dynamics among their structures. The mechanisms that regulate In this study, we tested the hypotheses that the mechanical work intersarcomere dynamics are unclear, which limits our understand- of one sarcomere effectively communicates with other sarcomeres ing of fundamental muscle features. Such dynamics are associated in series through the passive work of titin and that myofibril me- with the loss in forces caused by mechanical instability encountered chanics are largely governed by intersarcomere dynamics. To test in muscle diseases and cardiomyopathy and may underlie potential these hypotheses, we used microfluidic perfusions to locally control target treatments for such conditions. In this study, we developed a one sarcomere or a predetermined group of sarcomeres within an microfluidic perfusion system to control one sarcomere within a isolated myofibril. In this way, we could manipulate the activation myofibril, while measuring the individual behavior of all sarco- and relaxation of a target sarcomere while measuring the behavior meres. We found that the force from one sarcomere leads to of the other sarcomeres in a myofibril. Our data show that inter- adjustments of adjacent sarcomeres in a mechanism that is de- sarcomere dynamics are regulated through a mechanism that pendent on the sarcomere length and the myofibril stiffness. We combines the work of the contractile apparatus and intermediate concluded that the cooperative work of the contractile and the filament systems along myofibrils. elastic elements within a myofibril rules the intersarcomere dynam- ics, with important consequences for muscle contraction. Results Force Produced by Myofibrils After Point Activation of One Sarcomere. muscle contraction | sarcomere | intersarcomere dynamics | We tested isolated single sarcomeres and groups of sarcomeres force development | microfluidic perfusion within a rabbit myofibril in three different ranges of initial sarco- mere length (SLi), which were chosen so as to induce different – μ “ ” he smallest contractile unit of animal striated muscles is the initial passive tension: 2.4 2.65 m (very short; called here slack ), – μ > μ sarcomere, which is formed from a bipolar array of thick and 2.65 2.9 m (short), and 2.9 m (long). A local flow of activation T ∼ μ thin filaments composed mostly of myosin and actin proteins, re- solution, which surrounded 1 m of the preparation, resulted in spectively. The cyclic interaction between myosin and actin driven by the contraction of one sarcomere, whereas the other sarcomeres in A ATP hydrolysis drives sarcomere shortening and ultimately, produces series were maintained in a relaxed state (Fig. 1 and Movies S1 force (1, 2). In addition, the sarcomeres are structurally inter- and S2). The striation pattern of the nonactivated sarcomeres connected through the Z disks to form myofibrils (1, 3). Therefore, remained clear over the repeated activation/relaxation cycles of the individual sarcomeres are continuously interacting with each other. The sarcomeres contain titin, an elastic protein that spans the half- Significance sarcomere (4, 5). Titin molecules link all of the different areas of a single sarcomere as well as adjoining sarcomeres, creating an elastic The sarcomere contains a variety of molecules responsible for network throughout the length of a myofibril (6, 7). Because the force generation in striated muscles. During muscle contrac- sarcomeres are connected in series in a myofibril, changes in the tion, many sarcomeres work cooperatively to produce force. length of one sarcomere on activation may affect the length of ad- The mechanisms behind the interaction among sarcomeres jacent sarcomeres. Such phenomenon is hereupon referred to as during muscle activation have puzzled scientists for decades. intersarcomere dynamics, which may affect force in ways that are To investigate intersarcomere dynamics, we used microfluidic difficult to predict based solely on the sliding filament theory. perfusions to activate a single sarcomere within isolated The classic force–sarcomere length (FSL) relationship is widely myofibrils. Using computational sarcomere tracking, we ob- used to predict force from the overlap between thick and thin fil- served that the contraction of one sarcomere affects other + aments in a sarcomere during Ca2 activation, because different sarcomeres in series. Studying the interactions between sar- sarcomere lengths (SLs) lead to different degrees of filament comeres is crucial, because sarcomere nonuniformity has been overlap. However, the presence of intersarcomere dynamics and long associated with several phenomena in muscle contraction nonuniformity of SLs (8) provide additional complexity to the sys- that cannot be easily understood. tem during myofibril activation. It has been known that the lengths Author contributions: F.d.S.L. and D.E.R. designed research; F.d.S.L. and F.C.M. performed of different sarcomeres change and differ significantly during acti- research; F.d.S.L. and F.C.M. analyzed data; F.d.S.L. developed an experimental system; vation, with consequences for force production. Studies in- D.A. created a mathematical model requested by the reviewer; and F.d.S.L., D.A., and vestigating spontaneous oscillatory contractions (9) and the residual D.E.R. wrote the paper. force enhancement observed after myofibrils are stretched during The authors declare no conflict of interest. activation (10) suggest that changes in individual SLs are the result This article is a PNAS Direct Submission. of links between the contractile apparatus and intermediate fila- Freely available online through the PNAS open access option. ments composed mostly of titin molecules. Accordingly, sarcomere 1To whom correspondence should be addressed. Email: [email protected]. nonuniformity during activation may lead to the extension of some This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sarcomeres with the consequent engagement of titin molecules for 1073/pnas.1700615114/-/DCSupplemental. 8794–8799 | PNAS | August 15, 2017 | vol. 114 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1700615114 Downloaded by guest on September 24, 2021 A Microperfusion Standard precalibrated needles decreased with increasing numbers of Perfusion sarcomeres in series. These results were reproduced in our me- chanical model, in which the elements that compose a myofibril are modeled as either linear or nonlinear spring elements. We initiated the model calculations using the same average SLi that we used for the experiments with the myofibrils. When we acti- vated one sarcomere, the force developed by a myofibril de- creased nonlinearly as a function of the number of adjacent Sarcomere inactive sarcomeres and was higher at longer SLi (Fig. 1 C and F Z-disks ). At the end of the contraction, the forces developed in the model were also similar to the experimental forces. In our system, the measured force is a function of the displace- A-band 70 70 ment and stiffness of the microneedles used during mechanical ) BC) 2 Short SL 60 i 2 60 testing. Thus, assuming that the stiffness of sarcomeres within a Slack SLi 50 50 single myofibril is similar, our data suggest that the measured force 40 40 is dependent on the magnitude of shortening of the target sarco- 30 30 mere, which is greater at longer SLi (Fig. 1D)(P < 0.001). The force 20 20 sensed by the precalibrated needles results from the active and the Force (nN/ m Force (nN/ m 10 10 passive forces components in the myofibrils. Based on the charac- 0 0 0 5 10 15 20 25 30 35 40 0 1020304050607080 teristics of titin, it is likely that, at the beginning of activation, in- Number of Sarcomeres Number of Sarcomeres dividual sarcomere shortening results in an increased strain over the 3.5 *** 40 40 titin molecules of the nonactivated sarcomeres. Because passive D *** E F ) ) 2 2 3.0 *** 30 30 force develops in a nonlinear manner (13), contractions starting at longer SL will lead to a greater force development than contrac- 2.5 20 20 i p = 0.05 tions produced at shorter lengths (for a given change in length). Sarcomere 10 Length (µm) 2.0 10 Force (nN/ m Force (nN/ m Furthermore, the higher passive tension may cause the activated 0 1.5 0 Slack Short Long Slack Short Long 0 5 10 15 20 0.0 0.5 1.0 1.5 2.0 2.5 sarcomere to cease shortening at slightly longer SLf. Although our Inital Length Final Length Time (s) Time (s) data show a lack of statistical difference (Fig. 1C) in the range of μ Fig. 1. Experimental system used to control myofibril subareas and force SLf below 2.00 m, there is a steep region of the ascending limb of measured by precalibrated flexible needles in response to the activation. the FSL relation, and thus, even very small differences in SLf (that (A, Upper) Scheme of the experimental system showing the microfluidic we may have failed to detect) may strongly affect the force. We perfusion tip (blue), which is built to be smaller than the sarcomere A-band observed that ≥40 sarcomeres in a myofibril can adjust their length length.
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