Residual force depression in single sarcomeres is abolished by MgADP-induced activation

The mechanisms behind the shortening-induced force depression commonly observed in skeletal muscles remain unclear, but have been associated with sarcomere length non-uniformity and/or crossbridge inhibition. The purpose of this study was twofold: (i) to evaluate if force depression is present in isolated single sarcomeres, a preparation that eliminates sarcomere length non-uniformities and (ii) to evaluate if force depression is inhibited when single sarcomeres are activated with MgADP, which biases crossbridges into a strongly-bound state. Single sarcomeres (n = 16) were isolated from rabbit psoas myofibrils using two micro-needles (one compliant, one rigid), piercing the sarcomere externally adjacent to the Z-lines. The sarcomeres were contracted isometrically and subsequently shortened, in both Ca2+- and MgADP-activating solutions. Shortening in Ca2+-activated samples resulted in a 27.44 ± 9.04% force depression when compared to isometric contractions produced at similar final sarcomere lengths (P < 0.001). There was no force depression in MgADP-activated sarcomeres (force depression = −1.79 ± 9.69%, P =  0.435). These results suggest that force depression is a sarcomeric property, and that is associated with an inhibition of myosin-actin interactions.

inducing strong binding of crossbridges and actin 9 , decreasing the crossbridge inhibition that may have been caused by the strain of actin filaments.
In order to clearly elucidate the mechanisms of force depression, it is necessary to isolate the contribution of sarcomeric properties from between-sarcomere properties. According to the sarcomere length non-uniformity theory, at least two sarcomeres in series are necessary to produce residual force depression after shortening. Therefore, the goals of this study were: (i) to examine if force depression was present in single sarcomeres, and (ii) to examine if force depression could be prevented by increasing crossbridge binding during/after shortening. In order to reach these goals, we used a single sarcomere preparation developed in our laboratory 15 , that was activated either with Ca 2+ or MgADP -the latter biasing crossbridges into a strong binding state with actin 9 . Figure 1 shows a typical contraction produced by a sarcomere during Ca 2+ activation, with the corresponding SL and ramp traces. After ~5 s, Ca 2+ -activating solution was flushed into the experimental chamber and the sarcomere was activated. The force stabilized after ~1 s. In the shortening contraction, the imposed shortening occurred at ~7 s, resulting in a similar final SL in the isometric contraction. Relaxing solution was flushed once a steady-state force was re-established after the imposed shortening, at ~8.5 s. In the isometric contraction, relaxing solution was administered after ~5 s at a maximum steady-state force, at ~10.5 s. The black and grey arrows indicate the timing of solution administration in the isometric and shortening contraction, respectively. Figure 2 shows two superimposed graphs of force, length and ramp traces in MgADP-activated sarcomere. The force plateau of the isometric was time-matched with the force plateau upon force redevelopment in the shortening contraction in order to compare force at the same final SL. In Fig. 2A, in the isometric contraction, activation was induced at 12 s, and in the shortening contraction, activation was induced at 7 s -as shown by the black and grey arrows, respectively. The imposed shortening occurred at ~10 s after the force has maximized, and we compared the steady-state force generated in both corresponding contractions at ~14.5, which are approximately at the same final SL. Relaxing solution was flushed at 16.5 s, as indicated by the arrows, and the force stabilized to zero. Note that that the shortening is imposed slightly before a steady-state force is achieved. It has been shown that a steady-state force is not required prior to active shortening to produce force depression 8,12 . Due to the longer duration needed  to attain maximal force using MgADP 9 , the duration of experiments was increased prior to shortening. This procedure does not affect the comparisons made between contractions. The traces in Fig. 2B shows another experiment in which MgADP activation was held for 35 s prior to the imposed shortening, and the force depression was similar to the experiment depicted in Fig. 2A. Note that the force was still increasing over time, although it was slowly reaching a plateau towards the end of the contraction.

Results
The results from Figs. 1,2 were confirmed statistically as shown in Fig. 3, which illustrates the mean forces produced in all conditions. Post hoc analysis using the Holm-Sidak method indicated that the only significant difference in force existed between Ca 2+ -activated isometric (43.21 ± 6.08 nN/μ m 2 ) and shortening contractions (29.66 ± 6.19 nN/μ m 2 ) (t(15) = 3.939, p < 0.001). The final SL and the amount of shortening were not different between Ca 2+ -and MgADP-activation. The imposed shortening in Ca 2+ -activated samples resulted in a force depression of 27.44 ± 9.04% whreas force depression was virtually eliminated when the sarcomere was activated by MgADP.

Discussion
The current study is the first to show that the shortening-induced force depression commonly observed in skeletal muscles is present at the single sarcomere level with Ca 2+ activation. Furthermore, this study shows that force depression is abolished when samples are activated with MgADP, which biases crossbridges into a strongly-bound state. These findings suggest that force depression is caused partly by crossbridge binding site inhibition. Such a mechanism would be consistent with an actin distortion in the newly formed overlapped zone during shortening 9,14 .
When single sarcomeres were activated with Ca 2+ and subsequently shortened, force was noticeably depressed in comparison to the isometric contraction at the corresponding final SL. One suggested mechanism for force depression is sarcomere length non-uniformities 11,12 -a result of strength differences between sarcomeres in series. The heterogeneity in sarcomere length shortening results in some sarcomeres being longer, lying on the descending limb of the force-length relationship, and sarcomeres on the ascending limb matching the force produced by the other group of sarcomeres. The actual force produced is thus less than that predicted during isometric contractions. While the observation of force depression in Ca 2+ -activated samples in our study is consistent with previous studies using muscle fibres and myofibrils 2,7,9 , this study is the first to assess the magnitude of force depression when removing the contribution of non-uniformity between sarcomeres.
Previously, we observed that force depression was greatly reduced when myofibrils were activated with MgADP 9 . In the present study, we showed a full abolishment of force depression when single sarcomeres were activated in MgADP solution. The difference may reside in the fact that in myofibril preparations, some degree of sarcomere length non-uniformities may contribute to the overall force depression.
The absence of force depression in MgADP-activated samples supports a mechanism of force depression, intrinsic to the sarcomere, that has been suggested many years ago; a shortening-induced inhibition of myosin-actin attachment in the newly formed overlap zone during shortening 14 . Such an inhibition may be caused by a stress-deformation of actin, hindering its myosin-binding sites. Activation via MgADP happens when ADP enters the myosin nucleotide-binding pocket, inducing some crossbridges to transit into a strongly-bound state 16 . The strong binding of myosin-actin in turn may introduce conformational changes in the tropomyosin filaments, cooperatively exposing adjacent myosin-binding sites on actin, ultimately leading to further force-generating crossbridge attachments 17,18 . Furthermore, the strong binding from MgADP-activation has been suggested to compress the myofilament lattice 19 , pulling actin and myosin closer together.
It is tempting to speculate that MgADP activation allows the sarcomere to counteract and overcome the shortening-induced force depression by improving the alignment and resultant probability of actomyosin formation. MgADP induces strong binding between myosin crossbridges and actin in a cooperative fashion. Studies performed in solution with reconstituted filaments show that strong biding of myosin crossbridges to actin filament induces the binding of adjacent crossbridges 20,21 . Such strongly-bound crossbridges may cause conformational changes of the thin filaments that may in fact enhance P i release or other steps during isomerization of the actin-myosin-ADP-P i complex. Another effect of strong binding between crossbridges and actin is the compression of the myofilament lattice space upon muscle activation 19 , approximating thick and thin filaments. A decreased lattice space will likely increase the probability of crossbridge attachment to actin, which could counteract some of the shortening induced depression effects on force. Finally, the rigidity of the actin filament is reduced upon strong binding of myosin crossbridges 22 , which would release the stress caused by activation and shortening to the thin filaments. Altogether, these observations as well as our results indicate that the main difference between sarcomeres activated with Ca 2+ or MgADP is the myosin-actin binding probability. When myofibrils are activated and shortened with continuous flow of MgADP, the strong binding of crossbridges will be enhanced, increasing myosin cooperativity while decreasing the lattice spacing and the rigidity of the actin filaments. The newly formed overlap zone after shortening will be better aligned for myosin binding to actin.
In summary, this study was the first to provide direct support for a purely sarcomeric mechanism of force depression. While between-sarcomere mechanisms cannot be discounted in myofibril preparations, the observation that MgADP-activation abolishes force depression suggests that force depression is caused by an inhibition of myosin-actin interactions in the newly formed overlap zone during shortening.

Muscle Preparation and Single Sarcomere Isolation. Sections of rabbit psoas muscles (3-4 cm)
were dissected and subject to a standard permeabilization process 23  Small sections of a muscle sample were cut and placed in a 2 mL Eppendorf tube containing fresh rigor solution. The sample was thawed for one hour in the fridge (4 °C), and then transferred to approximately 5 mL of rigor solution in a 10 mL test tube. This sample was then homogenized using the following procedure: twice for 5 s at 18,000 rpm, twice for 5 s at 26,000 rpm and once for 3 s at 34,000 rpm. The homogenate, containing single myofibrils, was pipetted onto a glass cover-slip (thickness: 0.15 mm), which was vacuum grease-sealed to an experimental chamber positioned on the stage of an inverted microscope (NIKON Eclipse TE 2000U). Surrounding the chamber, a circulating cooling solution controlled the temperature at 10 °C. After 10 minutes, the homogenate was replaced with a relaxing solution (pH 7.0), which was subsequently used to fill the chamber, minimizing the appearance of floating debris. A myofibril was chosen for mechanical experimentation based on its striation pattern appearance, upon which a single sarcomere could be selected for isolation.

Micro-needle production and calibration. A vertical pipette puller (KOPF 720, David Kopf
Instruments) was used to make two glass micro-needles. Calibration of the micro-needles was done using a cross-bending method using a pair of micro-fabricated cantilevers of known stiffness (51.8 and 52.6 nN/μ m) 24 . For each experiment, a thin, compliant micro-needle (stiffness varied between 30 and 110 nN/μ m) and a thick, rigid micro-needle were used.
Mechanical isolation, visualization, and force measurement of single sarcomeres. Using the two pre-calibrated micro-needles controlled by micromanipulators (Narishige NT-88-V3, Tokyo, Japan), Scientific RepoRts | 5:10555 | DOi: 10.1038/srep10555 single sarcomeres were pierced externally adjacent to Z-lines. The sarcomeres were raised from the glass coverslip by 0.5-1.0 μ m. Under high magnification provided by an oil immersion phase-contrast lens (Nikon plan-fluor, X100, numerical aperture 1.30), the images of the single sarcomeres were further magnified X1.5 by an internal microscope function. A video was taken throughout sarcomere isolation and mechanical experimentation. After the mechanical isolation of the sarcomere, a contraction was initiated by a computer-controlled release of the activating solution through a double-barreled pipette connected to a multichannel perfusion system (VC-6M, Harvard Apparatus) 15,25 . To avoid contamination within the perfusion system, each side of the double-barrel was equipped to flush its own, independent activating solution contacting Ca 2+ or MgADP, as well as relaxing solution.
The contrast between the micro-needles produces a pattern of light intensity peaks that allow for tracking of their centroids frame-by-frame using a particle-tracker algorithm. The force produced during activation of the single sarcomere was obtained by measuring the displacement of the micro-needles, as previously described 15 . Experimental Protocol. Single sarcomeres (n = 16) were first activated isometrically and relaxed after force reached a steady-state for several seconds. Activation was induced in separate contractions using either Ca 2+ or MgADP solutions. Subsequently, the same sarcomere was passively stretched to a longer sarcomere length and activated. After force reached a steady-state (Ca 2+ activation) or a quasi-steady-state (MgADP activation), a computer-controlled shortening with an amplitude of 0.40 ± 0.03 μ m at a speed of 203.14 ± 14.45 nm.s −1 was imposed by the rigid micro-needle. In order to clearly identify force depression, the potential difference in forces was always investigated at the same final SL's. Relaxing solution was flushed into the experimental bath in order to stop the contractions. It is known that, during MgADP activation, the rate of force development is significantly slower than that produced during Ca 2+ activation 9 , and it may take up to a few minutes. Therefore, shortening was not always imposed during steady-state force in MgADP-activated sarcomeres, and in some cases force was still rising. However, it has been shown that force depression is present when shortening is imposed in the force rising phase, at levels that are qualitatively similar to those observe when shortening is imposed during the plateau of the force response in fully activated muscle fibres [8][9][10][11][12] .
The order of different activations (Ca 2+ and MgADP) and contraction types (isometric and shortening) was randomized to account for confounding effects. Figure 4 shows the needle movement through each phase of a typical MgADP-activated, shortening contraction. Data Analysis. The amount of force depression was measured by calculating the difference between the isometric force and the force produced after the imposed shortening in each sarcomere. The values taken for the calculation were averaged over 1 second in both contractions.
The results are presented as means ± standard error (SEM). Since the data were not normally distributed, a two-way ANOVA for repeated measures on ranks (n = 16) was used to identify potential differences in force and final SL between the four experimental conditions between groups. The Holm-Sidak post-hoc test was used to locate these differences when they were present. A level of significance (P) was set at 0.05. Since there is a relationship between the amount of shortening and active force depression, a student's t-test (n = 12) was used to compare the amount of shortening between the two activating solutions.