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 dynamics, 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 sarcomere 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 mechanical 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 activated one sarcomere, the force developed by a myofibril decreased 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. Microperfusion was controlled by a pressure system that allowed after a point activation, a result that was confirmed with our local flow of solutions (black dashed arrow). The red dashed arrows repre- mechanical model. sent the flow from the regular perfusion system coming from a double- barreled pipette, which allows rapid changes of solutions. (A, Lower)A Nonactivated Sarcomeres Adjust Their Lengths After Target Activation of typical myofibril suspended between two flexible needles. The contrast and One Sarcomere. To evaluate (i) how an activated target sarcomere brightness of the image were adjusted to improve visibility and clarity. Be- ii cause the experiments were performed using phase contrast microscopy, some dynamically interacts with adjacent sarcomeres and ( ) how the areas around the needles appear brighter than other areas in the myofibrils. passive forces in adjacent sarcomeres are adjusted along the myo- (Scale bar: 5 μm.) (B) Force produced by experiments with single sarcomeres fibril, we measured the SLs (SLs; Z disk to Z disk distances) (Fig. (colored circles at 1 in x axis) and experiments in which activation of one sar- S1A) and tracked the A-band displacements of all sarcomeres in comere was performed within myofibrils with different numbers of sarcomeres response to the activation of a sarcomere in the center of the – = (colored circles at 5 40 on the x axis) at three different SL :slack(n 34; red), BIOPHYSICS AND i myofibril (Fig. 2 A and B). After activation, the SLfs were not dif- short (n = 73; green) and long (n = 71; blue) lengths. All data were fit using a ferent among nonactivated sarcomeres (Fig. 2C); all of these sar- COMPUTATIONAL BIOLOGY second-order polynomial. (C) Model simulation of the experiments shown in B, D showing the final force produced by activation of a single sarcomere as a comeres responded by slightly stretching (Fig. 2 and Fig. S1)(i.e., there is a mechanical linkage among the sarcomeres). To further function of the number of adjacent inactive sarcomeres. SLi was set at 2.5, 2.8, and 3.15 μm. (D) Average SL and SL of the target sarcomere for the prepara- investigate the processes leading to these SL adjustments, we i f B tions tested at slack, short, and long SLi.(E) Typical traces obtained from a tracked the A bands of the myofibrils (Fig. 2 and Fig. S2). The myofibril tested at different SLs. (F) Simulation of the first seconds of force de- absolute displacement of the A bands was larger in nonactivated velopmentafteractivationofasinglesarcomere adjacent to 10 inactive sarco- sarcomeres located closer to the activation point than in sarcomeres meres. Error bars represent SEM. ***P < 0.001. located farther from the activation point. The absolute A-band displacement was dependent on SLi and therefore, the initial pas- B target sarcomeres. The isometric forces produced by isolated single sive tension of myofibrils (Fig. 2 ). When the A-band displacements were normalized, this dependence of SL was not observed (Fig. sarcomeres (Fig. 1B) were between ∼10 and 60 nN/μm2 within the i S3A). These experimental results were well-captured in our me- range previously published in the literature (12). chanical model, which shows a decrease in the displacement of A At all SL tested, the targeted sarcomere could overcome the i bands when they are located in sarcomeres away from the activated overall myofibril passive tension. As long as there was overlap B ∼ μ sarcomere (Fig. S3 ). between the thick and thin filaments (SL up to 3.6 m), all It has been shown that the elastic network of intermediate fila- sarcomeres reached a similar final sarcomere length (SLf)on ments can link all force-bearing structures of the sarcomere (14). B activation (Fig. S1 and Movie S3). The force produced by an This mechanism is mainly governed by titin molecules, which are isolated sarcomere was higher than the force produced by one the proteins responsible for passive force development in myofibrils. + target sarcomere that was activated within a myofibril (Fig. 1B). During our experiments, the local increase in Ca2 leads the target Furthermore, when we compared preparations with the same sarcomere to contract and stretch the adjacent sarcomeres, likely number of sarcomeres in series, the force was higher at longer enhancing the strain over titin filaments. This strain is passively SLi, suggesting a SL dependence for the force development (Fig. transmitted across the myofibril via the intermediate filament sys- 1 B and E). For the same SLi group, force transmitted to the tem, affecting several sarcomeres in series.

de Souza Leite et al. PNAS | August 15, 2017 | vol. 114 | no. 33 | 8795 Downloaded by guest on September 24, 2021 C C and D), strengthening the interpretation that intersarcomere A 3.5 dynamics are not necessarily dependent on changes in total 3.0 myofibril length. 2.5 2.0 Half-Sarcomeres Are Displaced During Activation. During the ex- 1.5 -12 -9 -6 -3 Act. 3 6 9 12 periments described above, we noticed a small displacement of Sarcomere Length (µm) Sarcomere Position the target A band in the activated sarcomere, which may have D 75 been caused by half-sarcomere nonuniformity during activation. (-) Displacement Displacement (+) B Direction Direction 50 Moreover, previous studies have suggested that the stabilization 1.50 25 of the A band in the center of sarcomeres during muscle con-

1.25 traction is regulated by titin (15, 16). Consequently, we evaluated Frequency (%) 0 E -0.75-0.50-0.250.000.0 0.25 0.50 0.75 1.00 A-band displacements in sarcomeres by changing the position of 1.00 Sarcomere Length (m) 1.25 the point activation to measure how it affected intersarcomere 0.75 1.00 dynamics. Point activation with the microperfusion system 0.50 0.75 placed at the Z disk led to the activation of two adjoining half- 0.50 sarcomeres, because two A bands were collapsed together at the 0.25 Short SLi Long SLi +RIGOR Slack SL 0.25 Very Long SL end of the activation (Movie S4). A-Band Displacement ( m) i i 0.00 Long SLi -12-9-6-3Activation 3 6 912 0.00 Fig. 3B shows that activation of two half-sarcomeres led to a 1 ±1 ±2 ±3 ±4 ±5 ±6 ±7 ±8 ±9 Sarcomere Position Act. ±10 ±1 ±12 A-Band Displacement ( m) ± Sarcomeres Position relatively large displacement from the zero position of at least one of the A bands compared with one target sarcomere acti- Fig. 2. Response of sarcomeres after point activation. (A, Upper) A typical vation. However, in this situation, the adjoining sarcomeres × view of a myofibril (100 magnification) before, during, and after point displaced less than in conditions where one entire sarcomere was activation. For clarity, the side to the right of the activated sarcomere is activated. This change in position of the thick filament has been defined as the positive side, whereas the side to the left of the activated sarcomere is defined as the negative side. Vertical arrows represent the di- seen before at the beginning of myofibril contraction and is at- rection of the solution flow (red arrows indicate the large flow; the black tributed to half-sarcomere mechanics (17), leading us to in- arrow indicates the flow delivered via microperfusion). When the micro- vestigate how the myofibril responds to point activation of just perfusion flow is applied to the target sarcomere, it contracts and produces one half-sarcomere. We hypothesized that the asymmetrical force. (A, Lower) A-band tracking used for data analysis. Each white circle behavior results from differential half-sarcomere mechanics. We represents one A band tracked, and each color overlapping the A bands tested this hypothesis by activating half-sarcomeres using mi- represents the displacement (x, y coordinates for every frame). (Scale bar: μ 5 μm.) (B) Absolute displacement of A bands in the myofibrils after activation cropipettes with diameters of 0.5 m to reach a small flow area of one sarcomere. The activated sarcomere is represented at the center [slack (n = 31), short (n = 47), and long (n = 67) lengths] of the figure (dotted line). The A bands of the adjacent sarcomeres are displaced toward the activated sarcomere. Activation of the target sarcomere creates a quasisym- Half-Sarcomere Two Half-Sarcomeres B A 1.50 metrical displacement of the nonactivated sarcomeres in the plus and minus Z-disks A-band sides. (C) SL changes after activation of one sarcomere. Starting from dif- 1.25 1.00 ferent SLi (circles), the final SLf (diamonds) of the nonactivated sarcomeres do not change substantially after activation. (D) Histogram showing the 0.75 Δ change in SL ( SL) of the nonactivated sarcomeres caused by point activa- 0.50 tion of one sarcomere. The orange bars represent control measurements 0.25 One Sarcomere - Two Half-Sarcomeres that we performed by taking two images from the same myofibril in a re- Short SLi activation

∼ A-Band Displacement (0.00 m) laxed state ( 5 s apart). The continuous line shows the Gaussian fit for these -12-9-6-3Act. 36912 control experiments. The dotted line shows the Gaussian fit when three Sarcomere Position experimental groups are merged into the same analysis. Note that the cure is CD slightly skewed to the right, suggesting a small stretch of the nonactivated sarcomeres. (E) A-band displacements of myofibrils tested in three condi- 1.50 1.50 – μ = = 1.25 1.25 tions: long SLi (3 3.3 m; n 26), long SLi in rigor solution (n 26), and very A-Band remains A-Band shifts at zero postion from zero postion long SL (3.35 to ∼3.6 μm; n = 22). Error bars represent SEM. Act., activation. 1.00 1.00 i *** ** * 0.75 0.75 To further test the myofibril adjustment to local activation, we 0.50 0.50 repeated the previous experiments using inflexible needles, so 0.25 0.25

0.00 A-Band Displacement (0.00 m) that the total length of the myofibrils would not change signifi- A-Band Displacement ( m) -12 -9 -6 -3Act. 3 6 9 12 -12 -9 -6 -3Act. 3 6 9 12 cantly as a result of activation, removing partially the effects of Sarcomere Position Sarcomere Position – μ end compliance. Myofibrils were tested at long SLi (2.9 3.3 m), Fig. 3. Half-sarcomere activation. A, Left shows a sarcomere in three dif- very long SLi (3.4–3.6 μm), and long SLi in rigor solution (i.e., in ferent conditions: (Top) before point activation, (Middle) half-sarcomere + the absence of ATP and Ca2 ), a condition that substantially activated at the positive side, and (Bottom) half-sarcomere activated at increases the stiffness of the preparation, because it induces a the negative side. A, Top Right shows two half-sarcomeres activated to- large number of cross-bridges. For all of these conditions, gether. A, Middle Right shows the start of the activation; note that one half- sarcomere was contracted. A, Bottom Right shows that, after the second myofibrils remained able to contract in response to the point half-sarcomere was contracted, both half-sarcomeres come close together. activation, suggesting that sarcomeres in series can adapt their (Scale bar: 5 μm.) (B) A-band displacement of all sarcomeres in a myofibril in 2+ lengths in response to a local increase in Ca concentration response to activation of two half-sarcomeres by targeting the micro- without significant changes in total myofibril length (Fig. 2E). perfusion flow to a Z disk (n = 30). (C) A-band displacement in response to There was some shortening observed at each end of the myofi- activation of one half-sarcomere (n = 37). (D) Response of adjacent sarco- = brils as indicated by A-band displacements. However, our math- meres to the activation of one half-sarcomere (Z-disk displacement, n 20; A-band displacement, n = 17). The data were adjusted based on the A-band ematical model that incorporates the end compliances was able displacement (displacements of the target A band were significantly different; to predict sarcomere contraction and shortening even with P < 0.0001). The diagrams display the behavior observed by the A bands. Error probes of very high stiffness and myofibrils in rigor state (Fig. S3 bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Act., activation.

8796 | www.pnas.org/cgi/doi/10.1073/pnas.1700615114 de Souza Leite et al. Downloaded by guest on September 24, 2021 (Movie S5). A-band tracking showed that one side of the myo- A 50

fibril was displaced more than the other side (Fig. 3C). Sur- ) 2 prisingly, the side that was displaced more was not always the 40 one adjacent to the activated half-sarcomere. Instead, two be- 30 haviors were observed: either (i) the A band did not move (and 20 the Z disk was pulled toward the A band) or (ii) the A band was D 10 displaced toward the Z disk (Fig. 3 ), explaining the asymmetry Force (nN/ m

observed when two half-sarcomeres are activated together. For 0 Myofibril Merged simplicity, we will consider one sarcomere that has one half- Number of Sarcomeres Tested Data

sarcomere activated. If the A band is displaced from its zero 20 BC250 60 D position toward the Z disk of this half-sarcomere, it drags the ) 2 D 200 15 other half-sarcomere (Fig. 3 , blue). If the A band does not 40 150 change its position, it pulls the Z disk and the half-sarcomeres 10 100 D 20 (Fig. 3 , orange). In practice, both situations happen in our 5 50 Force (nN/ m

experiments. Note that these movements do not result in dif- Shortening ( m) 0 0 0

ferential SLf between the two sides of the myofibril. 11020304050 Force per Sarcomere 11020304050 11020304050 EFNumber of Sarcomeres Number of Sarcomeres GNumber of Sarcomeres 100 4.0 Sarcomeres and Myofibrils May Produce Different Forces. Sarco- 40 3.5 35 meres comprising a myofibril have a similar structure and cross- 75 27 3.0 25 section. Thus, if all sarcomeres were able to reach the same SLf, 18 50 2.5 they should hypothetically produce similar active forces. To test 17 2.0 12 this hypothesis, we point-activated all sarcomeres within a 25 11 1.5 10

Normalized Force (%) 8 myofibril (one at a time) and used the average displacement of 0 Sarcomere Length (µm)

1.0 Number of sarcomeres 1.0 1.5 2.0 2.5 3.0 3.5 4.0 01020304050 1.0 1.5 2.0 2.5 3.0 3.5 4.0 the ±1 and ±2 A bands in relation to the target sarcomere to es- Sarcomere Length ( m) Number of Sarcomeres Sarcomere Length ( m) timate the individual sarcomere forces. Although an indirect mea- surement of the force in this situation, this procedure was based on Fig. 4. Force of several sarcomeres within a myofibril and full activation of our observation that the displacement of the A bands surrounding myofibrils with different numbers of sarcomeres in series. The average displacement of ±1 and ±2 sarcomeres was multiplied by the needle’s stiffness an activated sarcomere is very similar to the displacement of the to estimate the force of the majority of sarcomeres in a myofibril. (A) Blue needles when we activated one sarcomere (mechanically isolated box and whiskers plots represent the 25th percentile, the median (line), and from the myofibril). In general, we found forces varying from 8 to the 75th percentile of different myofibrils, and the error bars show the 2 45 nN/μm when comparing all sarcomeres tested in different minimum and maximum values. The dots represent the force produced by myofibrils (merged data in Fig. 4A), similar to the forces produced sarcomeres within one myofibril. The dotted line shows the average force by isolated sarcomeres (dotted line in Fig. 1A). We observed a small considering all sarcomeres tested. The plot in Right merges the force of all range of forces among the different sarcomeres within a single preparations. (B) Isometric force of myofibrils ranging from 4 to 48 sarcomeres in series compared with forces produced by one sarcomere. (C) Force myofibril caused by small variations in SLi (Fig. 4A). When myofibrils were analyzed individually, it was possible to observe differ- normalized by the number of sarcomeres in series in a myofibril. (D) Re- lationship between the shortening magnitude during myofibril contraction ences in forces produced by sarcomeres in some myofibrils. These and the number of sarcomeres in series in a myofibril. (E) FSL relationship differences are likely related to different changes in length caused produced during full activation of isolated myofibrils (n = 12). The force was by nonuniform SLi or SLf. normalized by the highest value for a given myofibril. Data were fit using a 2 According to our data, sarcomeres reach similar SLf when third-order polynomial regression (r = 0.83). The blue area represents the pla- point-activated, but this activation affects other sarcomeres in teau of the FSL relationship. The green and yellow areas represent the ascending series. Therefore, during full myofibril activation, the dynamic work and descending limbs of the FSL relationship, respectively. Note that the green area covers lengths in which filament overlap is between 70 and 90% of the of sarcomeres in series and the differences in SLi among sarcomeres may influence how a myofibril develops force to reach a steady optimum overlap. The dotted line was created based on theoretical predictions BIOPHYSICS AND state. In such a state, myofibril internal motion is close to equilib- (20) and previous experimental data (18). (F) Average SLf of all myofibrils plotted COMPUTATIONAL BIOLOGY as a function of the number of sarcomeres and the amount (percentage) of rium. If, during myofibril activation, every sarcomere shortened to optimum overlap between myosin and actin filaments according to the FSL re- thesameSLf, the force would be a function of the number of lationship (n = 95). (G) Length of all sarcomeres (excluding the ones at the edges)

sarcomeres in series and the SLi of each sarcomere. We, therefore, of 10 randomly chosen myofibrils that contracted to an SLf within the plateau tested the hypothesis that a greater number of sarcomeres in series regions (blue) of the FSL relationship. Green and yellow areas represent the allows a greater shortening magnitude and hence, higher forces. We ascending and descending limbs of the FSL, respectively. Note that the green performed full activation of myofibrils with different numbers of area covers lengths in which filament overlap is 70–90% of the optimum. Gray box and whiskers plots represent a different myofibril with the 25th percentile, sarcomeres in series at an SLi of ∼2.8 μm. We first activated myofibrils with ∼25 sarcomeres, and after the first activation, we the median (line), and the 75th percentile; error bars show the minimum and reduced the number of sarcomeres in series in the preparation by maximum values. one-half by positioning the holding needle close to the center of the myofibril. We found that myofibrils with a greater number of sar- higher forces (Fig. 4 B and D). However, the increase in force in comeres produced higher forces, regardless of the average SLf (Fig. longer myofibrils was not linearly related to the number of sar- S4 A and D). These results may be attributed to cumulative short- comeres in series (Fig. 4B). When the force was normalized by ening when more sarcomeres are activated (i.e., a greater shorten- the number of sarcomeres in the myofibrils, they produced less ing of the myofibril may be possible when more sarcomeres are C activated) (Fig. S4 B and C). force per sarcomere than single sarcomeres (Fig. 4 ). To further test our hypothesis that more sarcomeres in series To evaluate whether the final SL reached during the contractions allow for a greater shortening and to remove the potential was in the zone where maximal force is produced, we performed confounding effect of myofibril damage during manipulation, we separate experiments to derive an FSL relation for the myofibrils measured the forces produced by the first contraction of used in this study (Movie S6). We observed an FSL relation similar 95 myofibrils and compared them with single sarcomeres. We to that reported previously in other preparations (18), with a pla- confirmed that myofibrils with more sarcomeres in series short- teau between SLfsof∼2.1 and 2.7 μm(Fig.4E). Based on these ened more than myofibrils with fewer sarcomeres and produced measurements, we evaluated if the SLf of the myofibrils reached

de Souza Leite et al. PNAS | August 15, 2017 | vol. 114 | no. 33 | 8797 Downloaded by guest on September 24, 2021 optimal filament overlap. In fact, the majority of the myofibrils not affect force development (Fig. 5A). Second, in another series contracted to the plateau of the FSL relationship or in an area of experiments, nonuniformity was induced either after con- + within 70% of the optimum filament overlap (Fig. 4E). There were traction or before activation by using a low-Ca2 solution to relax some sarcomeres in these myofibrils that reached a length longer one sarcomere in a fully activated myofibril (Movie S8). We than those at the plateau of the FSL, likely because of an increased noticed a sudden reduction in the total myofibril force (Fig. 5B) SL nonuniformity (Fig. 4F).Basedonthesefindings,wedecidedto that was reversed after removal of the microperfusion pipette. 2+ test the hypothesis that a larger degree of nonuniformity in SLs We then repeated this experiment but used a high-Ca solution along the descending limb of the FSL leads to a decrease in the with 40 mM 2,3-butanedione monoxime (BDM), which led to a C total force produced by the myofibrils. similar reduction in force (Fig. 5 ). A reduction in the total myofibril force was also observed by delaying the activation of a Induced Nonuniformity/Deactivation Reduces Total Myofibril Force. small portion of the myofibril (approximately one sarcomere) Over the years, SL nonuniformity has been observed to happen during a contraction (Fig. 5D). For all of these situations, the naturally during muscle contraction. To test how nonuniformity target sarcomere was able to contract, although both sides of the of SLs affects the final force reached by the myofibrils, we used myofibril were inducing sarcomere stretching. This finding sug- the microperfusion system to induce nonuniformity in activated gests that one sarcomere is able to pull several activated sarco- myofibrils. We performed five different sets of experiments. meres and produce a force that is close to its maximum level. First, we measured the myofibril force when three to five sar- Finally, we used a high-strength ionic solution that depletes the comeres were activated before full myofibril activation (pre- preparation from thick myosin filaments (19) (Movie S9). Extrac- activation group) and compared the results with those of regular tion of the A band results in a sarcomere with only the intermediate full activation (control) (Movie S7). Two segments of the same filaments that is able to communicate with other sarcomeres through passive forces. However, A-band extraction may also lead myofibril were tested for preactivation. The protocol consisted of to more compliant titin because of the loss of the interaction be- three cycles of full myofibril activation followed by two cycles of tween titin and myosin filaments. We compared forces developed by preactivation and a control contraction. When preactivated, the the myofibrils before and after the extraction of the A band of one myofibril generated a small increase in force before a larger A sarcomere. We found a significant reduction in the isometric force increase caused by full activation (dashed square in Fig. 5 ). compared with experiments in which the A band was intact (Fig. Interestingly, we did not observe significant differences in the 5E), supporting the idea that the number of sarcomeres is an im- final forces measured, showing that initial nonuniformities did portant factor for total force development. The extraction of one A band had a smaller effect on the total force in myofibrils with a large number of sarcomeres than in myofibrils with a small number of sarcomeres (Fig. 5F). In longer myofibrils, force dropped after the AB150 150 Control Re-Activation ) ) 2 *** additional extraction of A bands. We performed a few experiments Pre-Activation 2 *** Control 100 100 in which we applied a small stretch after extraction of the A band from myofibrils to evaluate if force could be recovered back to the One Sarcomere 50 50 Relaxing (Deact.) original values. However, the force remained lower than it was Force (nN/ m Force (nN/ m 0 0 before extraction of the A band (Fig. S5 and Movie S10). 0 1020304050 Control Deact. Re-act. 020406080 Time (s) Time (s) Overall, our data suggest that, during muscle contraction and force ControlPre-ActControlPre-ActControl D 150

C ) 150 *** development, events that create nonuniform distribution of SL along Control Re-Activation 2

) *** Re-Activation

2 ** ** myofibrils have an impact on the final force that they produce. 100 One Sarcomere 100 Clamping

One Sarcomere 50 Discussion 50 BDM Force (nN/ m In this study, we assessed intersarcomere dynamics in isolated Force (nN/ m 0 0 Control Clamp Re-act. 0 102030myofibrils using microfluidic perfusion, a technique that allows Control BDM React. 0 20 40 60 80 100 Time (s) Time (s) F E 1.1 the point activation of one (half-) sarcomere within a myofibril 150 Control 1.0 ) ** One while measuring the response of all other sarcomeres. We triggered 2 Extraction 0.9 100 cross-bridge formation and contraction of target sarcomeres by lo- 0.8 2+ 0.7 cally increasing the Ca concentration. We showed that the 50 0.6 shortening of an activated sarcomere leads to an internal regulation

% Isometric Force 0.5 Force (nN/ m 11 12 13 18 18 20 22 22 23 24 24 31 33 36 42 in the remaining sarcomeres in the myofibril through passive forces. 0 Number of Sarcomeres Control One 0560 70 Extraction Time (s) 1 2 3 4 6 7 A-Bands Extracted This result shows that intersarcomere dynamics arise from the cooperative work of the contractile and elastic systems, enabling the Fig. 5. Controlling nonuniformity. (A) Preactivation of a short area of the action of one sarcomere to be transmitted to the others. myofibril does not affect the total force. A subarea of the myofibril was We found that single sarcomeres are able to contract as long as activated before full activation using microperfusion. In a typical experi- there is myofilament overlap (20), reaching similar SLf independent ment, the first contraction induced full myofibril contraction, and the second ∼ μ contraction was induced after preactivation. The increase in force caused by of the SLi (with small differences when SLi is 3.6 m). This behavior is not observed during full myofibril activation (Figs. 1D and preactivation is shown in the dashed square. Note that the total forces were G similar in both contractions. (B) One sarcomere is deactivated during full 4 ), where the SLf is highly nonuniform during activation (17). + myofibril contraction using relaxing solution (pCa2 9.0) within the micro- Although passive and active forces have been investigated sepa- perfusion system (n = 21). (C) The same protocol used in A, but this time, the rately (21, 22), we showed that the passive force component is re- + solution (pCa2 4.5) used to deactivate the sarcomere contained 40 mM BDM sponsible for the interaction of sarcomeres in series, facilitating the (n = 8). (D) A small area of a myofibril was kept relaxed, whereas the rest of transmission of the force of one sarcomere to others. the myofibril was activated and contracted (n = 12). (E, Left) Myofibril force Recent studies have shown that titin controls important as- before and after one A-band extraction using high-strength ionic solution in pects of thick filament structure, playing an important role in the microperfusion system (n = 15). E, Right show typical experimental traces. (F) Extraction of one A band had a smaller effect or a lack of effect in muscle activation (23, 24). In the future, it will be important to longer myofibrils. We performed additional A-band extractions in three consider how the activation of one sarcomere may be sensed by a myofibrils (with 31, 33, and 36 sarcomeres), which reduced the force sig- thick filament from the neighboring sarcomeres. Moreover, it nificantly. The straight line represents the normalized isometric force before has been suggested that titin can assist muscle contraction at A-band extraction. Error bars represent SEM. **P < 0.01; ***P < 0.001. physiological SLs using the stored elastic energy from unfolded

8798 | www.pnas.org/cgi/doi/10.1073/pnas.1700615114 de Souza Leite et al. Downloaded by guest on September 24, 2021 Ig domains (25). The importance of the elastic system was shown 7.0. We used two precalibrated needles to pierce the myofibrils parallel to in our experiments, in which a few sarcomeres were activated the Z disks (28). The stiffness of all needles (43.40 ± 0.76 nN/μm) was mea- before full myofibril activation (Fig. 5A). There was not a dif- sured using the same atomic force cantilever. All procedures were imaged ference in force compared with myofibrils that were not pre- using a Hamamatsu Orca-ER digital camera. Movies were recorded at activated, showing that the passive force components hold the 43.3 frames per 1 s. Myofibrils were pierced and lifted from the coverslip, and the SLi was adjusted. Sarcomeres within myofibrils were point-perfused nonactivated area of the myofibril at the optimum length for by a microperfusion system, in which glass micropipettes filled with the contraction. desired solution were controlled by a pressure system and micromanipula- Our microfluidic perfusion system allowed the control of a tors (NT88-V3; Nikon). To avoid diffusion of the microperfusion solution to long known phenomenon of muscle contraction, the nonuniform undesired areas, myofibrils were constantly perfused by a larger perfusion lengths of sarcomeres during activation (26, 27). From the pro- flow that controlled the remaining sarcomeres in the preparation. Micro- posal of the sliding filament theory (1, 2), nonuniform behavior pipettes and needles were pulled using a capillary puller (Kopf Model 720; of sarcomeres and its effects in force production during con- David Kopf Instruments). Micropipette tip diameter was adjusted to 1 μmor traction have puzzled scientists. We found that, in short myofi- less (<0.5 μm for half-sarcomeres) to keep it smaller than the sarcomere A brils, enabling the relaxation of one sarcomere either during or bands. All experimental procedures were recorded and analyzed using the beforecontractionleadstoareductioninthetotalforce(Fig.5B– TrackMate plugin for ImageJ software (NIH). Absolute sarcomere displace- D). This result can be explained by the reduction in the total ment was defined as the difference between the zero position (relaxed state) and the position of the tracked A band during the experiment. The shortening caused by the relaxed sarcomeres. The same reduction experiments comparing the active force development of myofibrils before was observed when we extracted the A bands from myofibrils (Fig. E and after reduction in the number of sarcomeres in series were performed 5 ). However, this reduction in force was attenuated in myofibrils using atomic force microscopy as previously described (28). Because the with more sarcomeres in series (Fig. 5F). It is possible that the lack forces produced by myofibrils are proportional to their cross-sectional areas of one sarcomere was compensated for by an increase in shortening and because we wanted to compare different conditions irrespective of this of other sarcomeres. Such a mechanism may have important im- variable, all measured forces were normalized and reported in units of plications, because the structure of the muscle cell may be designed stress, assuming a circular geometry for the myofibril (nanonewtons to balance the presence of weaker or damaged sarcomeres along per micrometer2). the myofiber. This study uses a microfluidic perfusion to show the impor- Statistical Analyses. We used a two-way ANOVA for repeated measures to compare displacement of A bands. For nonuniformity experiments, we used tance of intersarcomere dynamics and the cooperative work of – – the contractile and elastic proteins in the myofibril, opening one-way ANOVA for repeated measures. We used a Student Newman Keuls test when significant changes were observed. A level of significance of P ≤ possibilities for new venues of investigation in muscle biophysics. 0.05 was set for all analyses. Values are presented as means ± SEM. Methods Mechanical Model. We developed a mechanical model of a half-myofibril to Experimental Solutions and Experimental System. Myofibril preparation and better interpret our experimental data (SI Text, Figs. S6–S8, Table S1,and imaging (SI Text) were done as described before (28). The protocol was Movie S11) using the framework of Campbell (29). approved by the McGill University Animal Care Committee and complied with the guidelines of the Canadian Council on Animal Care. Rigor solution ACKNOWLEDGMENTS. We thank Dr. Kenneth S. Campbell for helpful discus- was 50 mM Tris, 100 mM KCl, 4 mM MgCl2, and 10 mM EGTA, pH 7.0. sions during the writing of this paper. This study was funded by the Canadian Relaxing solution was 70 mM KCl, 20 mM imidazole, 5 mM MgCl2, 5 mM ATP, Institutes for Health Research and the Natural Science and Engineering Research 2+ 2+ 14.5 mM creatine phosphate, 7 mM EGTA, and pCa (equal to –log[Ca ]) 9.0, Council of Canada. F.d.S.L. was a recipient of a scholarship from the National pH 7.0. Activation solution was 50 mM KCl, 20 mM imidazole, 5 mM MgCl2, Counsel of Technological and Scientific Development (CNPq, Brazil). D.E.R. is a 5 mM ATP, 14.5 mM creatine phosphate, 7.2 mM EGTA, and pCa2+ 4.5, pH Canada Research Chair (Tier I) in Muscle Biophysics.

1. Huxley AF, Niedergerke R (1954) Structural changes in muscle during contraction; 16. Horowits R, Kempner ES, Bisher ME, Podolsky RJ (1986) A physiological role for titin interference microscopy of living muscle fibres. Nature 173:971–973. and nebulin in skeletal muscle. Nature 323:160–164. 2. Huxley H, Hanson J (1954) Changes in the cross-striations of muscle during contraction 17. Telley IA, Denoth J, Stüssi E, Pfitzer G, Stehle R (2006) Half-sarcomere dynamics in – BIOPHYSICS AND and stretch and their structural interpretation. Nature 173:973 976. myofibrils during activation and relaxation studied by tracking fluorescent markers. 3. Gilev VP (1962) A study of myofibril sarcomere structure during contraction. J Cell Biol Biophys J 90:514–530. COMPUTATIONAL BIOLOGY – 12:135 147. 18. Pavlov I, Novinger R, Rassier DE (2009) The mechanical behavior of individual sarco- 4. Maruyama K, Kimura S, Kuroda M, Handa S (1977) Connectin, an elastic protein of meres of myofibrils isolated from rabbit psoas muscle. Am J Physiol Cell Physiol 297: muscle. Its abundance in cardiac myofibrils. J Biochem 82:347–350. C1211–C1219. 5. Wang K, McClure J, Tu A (1979) Titin: Major myofibrillar components of striated 19. Maruyama K, Natori R, Nonomura Y (1976) New elastic protein from muscle. Nature muscle. Proc Natl Acad Sci USA 76:3698–3702. – 6. Agarkova I, Perriard JC (2005) The M-band: An elastic web that crosslinks thick fila- 262:58 60. ments in the center of the sarcomere. Trends Cell Biol 15:477–485. 20. Sosa H, Popp D, Ouyang G, Huxley HE (1994) Ultrastructure of skeletal muscle fibers 7. Gregorio CC, et al. (1998) The NH2 terminus of titin spans the Z-disc: Its interaction studied by a plunge quick freezing method: Myofilament lengths. Biophys J 67: with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity. J Cell Biol 143: 283–292. 1013–1027. 21. Linke WA, Ivemeyer M, Mundel P, Stockmeier MR, Kolmerer B (1998) Nature of PEVK- 8. Telley IA, et al. (2006) Dynamic behaviour of half-sarcomeres during and after stretch titin elasticity in skeletal muscle. Proc Natl Acad Sci USA 95:8052–8057. in activated rabbit psoas myofibrils: Sarcomere asymmetry but no ‘sarcomere pop- 22. Gordon AM, Huxley AF, Julian FJ (1966) The variation in isometric tension with sar- ping’. J Physiol 573:173–185. comere length in vertebrate muscle fibres. J Physiol 184:170–192. 9. Shimamoto Y, Suzuki M, Mikhailenko SV, Yasuda K, Ishiwata S (2009) Inter-sarcomere 23. Linari M, et al. (2015) Force generation by skeletal muscle is controlled by mecha- coordination in muscle revealed through individual sarcomere response to quick nosensing in myosin filaments. Nature 528:276–279. – stretch. Proc Natl Acad Sci USA 106:11954 11959. 24. Fusi L, Brunello E, Yan Z, Irving M (2016) Thick filament mechano-sensing is a calcium- 10. Rassier DE, Herzog W, Pollack GH (2003) Dynamics of individual sarcomeres during independent regulatory mechanism in skeletal muscle. Nat Commun 7:13281. – and after stretch in activated single myofibrils. Proc Biol Sci 270:1735 1740. 25. Rivas-Pardo JA, et al. (2016) Work done by titin protein folding assists muscle con- 11. Telley IA, Denoth J, Ranatunga KW (2003) Inter-sarcomere dynamics in muscle fibres. traction. Cell Reports 14:1339–1347. A neglected subject? Adv Exp Med Biol 538:481–500, discussion 500. 26. ter Keurs HE, Iwazumi T, Pollack GH (1978) The sarcomere length-tension relation in 12. Rassier DE, Pavlov I (2012) Force produced by isolated sarcomeres and half-sarcomeres skeletal muscle. J Gen Physiol 72:565–592. after an imposed stretch. Am J Physiol Cell Physiol 302:C240–C248. 27. Granzier HL, Pollack GH (1990) The descending limb of the force-sarcomere length 13. Horowits R (1992) Passive force generation and titin isoforms in mammalian skeletal – muscle. Biophys J 61:392–398. relation of the frog revisited. J Physiol 421:595 615. 14. Wang K, Ramirez-Mitchell R (1983) A network of transverse and longitudinal in- 28. Leite FS, et al. (2016) Reduced passive force in skeletal muscles lacking protein argi- – termediate filaments is associated with sarcomeres of adult vertebrate skeletal nylation. Am J Physiol Cell Physiol 310:C127 C135. muscle. J Cell Biol 96:562–570. 29. Campbell KS (2009) Interactions between connected half-sarcomeres produce emer- 15. Tskhovrebova L, Trinick J (2003) Titin: Properties and family relationships. Nat Rev Mol gent mechanical behavior in a mathematical model of muscle. PLOS Comput Biol 5: Cell Biol 4:679–689. e1000560.

de Souza Leite et al. PNAS | August 15, 2017 | vol. 114 | no. 33 | 8799 Downloaded by guest on September 24, 2021