Preparation of the single and half-sarcomeres

Single and half sarcomeres were isolated from rabbit psoas muscle using a modified procedure explained previously by our group7. Shortly, small bundles of the muscles were tied to wooden sticks and chemically permeabilized using a standard protocol8. The muscles were incubated in rigor solution (pH = 7.0) for approximately 4 hours, after which they were transferred to a rigor:glycerol (50:50) solution for 20 hours. The samples were placed in a new rigor:glycerol (50:50) solution with the addition of a mixture of protease inhibitors (Roche Diagnostics, USA) and stored in a freezer (−20°C) for at least seven days. The protocol was approved by the McGill University Animal Care Committee and complied with the guidelines of the Canadian Council on Animal Care.

On the day of the experiment, a muscle sample was transferred to a fresh rigor solution and stored in the fridge for one hour before use. A small section of the sample was extracted (~1 mm3) and homogenized in a rigor solution (pH = 7.0) using the following sequence: twice for 5 s at 7,500 rpm and once for 3 s at 18,000 rpm. The homogenizing protocol produces a supernatant containing single myofibrils. This homogenate was transferred into an experimental chamber with the bottom made of a vacuum grease-sealed glass coverslip (thickness: 0.15 mm), placed on the stage of an inverted microscope (NIKON Eclipse TE 2000U). The chamber was filled with rigor solution and the temperature was controlled at ~10°C with a circulating cooling solution running through a channel surrounding the chamber. The sample was rinsed several times and after a rest period of 5 min, the rigor solution was slowly exchanged by a relaxing solution. A myofibril was chosen based on its striation appearance and either a single sarcomere or a half sarcomere was selected for mechanical experimentation.

Micro-needle production and calibration

The micro-needles were produced with a vertical pipette puller (KOPF 720, David Kopf Instruments) and calibrated by a cross-bending method7, using a pair of micro-fabricated cantilevers of known stiffness (489 and 592 nN/μm). The final stiffness of the micro-needles used during these experiments varied between 35 and 400 nN/μm.

Mechanical isolation, visualization and force measurement of single and half-sarcomeres

Using micromanipulators (Narishige NT-88-V3, Tokyo, Japan), single sarcomeres or half-sarcomeres were captured by two pre-calibrated micro-needles (Figures 3 and 4). The dimensions of the micro-needles were similar to those used in our previous study using single sarcomeres; the needles present a conical-shaped tip and only a small length (~1.5 μm, Figure 3 in red) is inserted into the myofibril. The diameters of the circular cross-sectional area of the portion of the cone inside the myofibril are normally bellow ~0.5–0.7 μm, while its tip is under 0.2 μm. The calibration of the micro-needles also followed a protocol established earlier in our laboratory7 and of others25. The needles were pierced externally adjacent to Z-lines in the case of sarcomeres (Figure 4D and Movie 1 suppl.), or between the Z-line and externally adjacent to the M-line (Figures 4B, 4C and Movie 2 suppl.). The samples were raised from the glass coverslip by ~0.5-1.0 μm. Under high magnification provided by an oil immersion phase-contrast lens (Nikon plan-fluor, ×100, numerical aperture 1.30), the images of the single and half-sarcomeres were further magnified 1.5× by an internal microscope function. The contrast between the micro-needles produces a pattern of light intensity peaks that allow for tracking of their centroids using a particle tracker algorithm26. The half-sarcomere length was obtained by interpolating the displacement of the micro-needles from the initial to the final distances measured from the Z-line to the center of the sarcomere. The force produced during activation of the single and half-sarcomeres was obtained by measuring the displacement of the micro-needles, as described elsewhere7.

Figure 3 Needle dimensions. Tip of a glass micro-needle piercing a myofibril (on the left) externally adjacent to the Z-line from one of its sarcomeres. The figure shows representative measurements (on the top-left, yellow arrows) of five arbitrary cross-sectional areas of the conical-shaped tip of the needle. The red arrow represents how much of this tip was inserted into the myofibril; this value was never higher than 1.5 μm. Magnification = 150X. Full size image

Figure 4 Half and single sarcomere isolation. A - Glass micro-needles approaching a single myofibril on the microscope coverslip (magnification = 90X). B - Half-sarcomere caught between the micro-needles. The myofibril is still on the coverslip (magnification = 90X). C - Half-sarcomere isolated and lifted (~2 μm) from the coverslip (magnification = 150X). D - Single sarcomere caught between two micro-needles (c). Full size image

Solutions

The rigor solution (pH 7.0) was composed of (in mM): 50 Tris, 100 NaCl, 2 KCl, 2 MgCl 2 and 10 EGTA. The activating (pCa2+ of 4.5) and relaxing (pCa2+ of 9.0) - pH 7.0- solutions contained (in mM): 20 imidazole, 14.5 creatine phosphate, 7 EGTA, 4 MgATP, 1 free Mg2+, free Ca2+ in two concentrations adjusted to obtain pCa2+ of 4.5 (32 μM) and 9.0 (1 nM); KCl was used to adjust the ionic strength to 180 mM in all solutions.

Protocol

Single sarcomeres (n = 18) and half-sarcomeres (n = 17) were immersed in relaxing solution for 1–2 s. The solution was rapidly replaced by an activating solution using a computer-controlled, multichannel perfusion system (VC-6M, Harvard Apparatus) and a double-barreled pipette7. When surrounded by the activating solution, the preparations contracted and produced force. Each experiment counted with 2–3 isometric contractions produced at different lengths and a contraction in which a stretch was imposed to the preparation. For the isometric contractions, the sarcomeres and half sarcomeres were passively adjusted to a desired length before activation; the nominal lengths were chosen to be 2.7 μm and 3.0 μm for the two contractions. However, differently from experiments performed with larger preparations, activation of sarcomeres and half-sarcomeres produced varying degrees of shortening, which made it difficult to have the experiments at exactly the same lengths.

For the stretch contraction, after full force development was obtained, single sarcomeres and half-sarcomeres were stretched by different magnitudes, ranging from 15–36% of half-sarcomere length (HSL), at speeds ranging from 1.35 to 3.15 μm·s−1·HSL−1. After the end of the stretch, the myofibrils were held isometric for at least 5 s before relaxation. Nominal length changes induced to the preparation resulted in varying levels of actual sarcomere/half-sarcomere stretching, which made a precise pre-determination of the final lengths difficult. For each preparation we performed a series of passive stretches, starting at ~2.4 μm and ending at ~4.0 μm, with intervals of at least 10 s between stretches.

Data analysis

The results of this study are reported as means ± standard deviations (SD), since all the data analyzed were normally distributed. To compare the mean isometric forces between sarcomeres and half-sarcomeres, we used T-tests for independent samples. A two-way, mixed model, analysis of covariance (ANCOVA) was used to compare averages of forces produced after stretch and during isometric contractions at similar conditions, as well as the two groups investigated in this study (sarcomeres and half-sarcomeres), after adjusting for the amount of stretch. The mixed model ANCOVA takes into account the possible correlation between repeated measures within each experiment because they were subjected to both conditions (isometric and after stretch) and enabled the amount of stretch, which may influence the levels of force enhancement, to be used as a covariate in the model, to more accurately assess the potential differences in forces. The assumption of homogeneity of slopes for the ANCOVA model was checked by introducing and testing interaction effect terms between both the groups (sarcomere and half-sarcomere) and the conditions (isometric, after stretch) and the covariate (amount of stretch). The assumptions of linearity, normality and equal variances of errors, as well as the presence of possible outliers, were explored with analysis of residuals. The statistical analyses were carried out with SAS software (version 9.2). All hypothesis tests were two-sided and performed at the 0.05 significance level.