Animals
The Actn3KO mouse line was previously created in this laboratory (17), and experiments were performed on male animals at 12-15 months of age. A total of 6KO and 6WT mice were used in the present study. Use of animals was approved by the Animal Care and Ethics Committees of the Children’s Medical Research Institute and the University of New South Wales.
Skeletal Muscle single fibre enzymatic isolation
Flexor digitorum brevis (FDB) and extensor digitorum longus (EDL) muscles were digested in Krebs solution composed of (in mM): 4.75 KCl, 118 NaCl, 1.18 KH2PO4, 1.18 MgSO4, 24.8 NaHCO3, 2.5 CaCl2 and 10 glucose containing 3 mg/ml collagenase type IV A (Sigma Aldrich, USA), gently bubbled with carbogen (95% O2, 5% CO2) and maintained at 37°C. After 25-30 minutes muscles were removed from the digest solution with a wide bore glass pipette and serially rinsed twice in Krebs solution containing 0.1% foetal calf serum. Single fibres were dispersed by gentle trituration. The FDB fibres were maintained in Krebs solution with 0.1% foetal calf solution at room temperature 21-23oC and continuously bubbled with carbogen. Using a pipette, 0.5 ml of solution was drawn and placed on a cleaned glass slide on an inverted microscope, each 0.5 ml contained between 10-50 fibres. FDB fibres attached firmly to the glass cover slip and were continually superfused with Krebs bubbled with carbogen at a rate of around 0.5 ml per minute. The FDB fibres were visualized at 200x magnification on a Nikon Eclipse Ti2-E Inverted Research Microscope. For fibre length and diameter measurements (Supplementary figure A), a grid was placed in the eye piece of the microscope so that it occupied ~50% of the field of view and all fibres in this view were recorded and processed using ImageJ open-source software, the microscope was calibrated using a stage micrometre, and a total of 200 WT FDB fibres were measured. Post-digest EDL muscles were rinsed first in Krebs with 0.1% foetal calf serum to stop the collagenase reaction and then rinsed for a second time in Krebs with no foetal calf serum and no added calcium before being placed in a relaxing solution with the following composition (mM): 117 K+, 36 Na+, 1 Mg2+, 60 HEPES, 8 ATP, 50 EGTA (Note: as the fibres are effectively chemically skinned by the high EGTA concentration, this is an intracellular solution). Transfers between solutions were made by sucking the digested muscle mass into a wide bored pipette. Finally, the muscle was gently agitated using a wide bore pipette to release individual fibres from the muscle. Fibres were maintained in the relaxing solution at four degrees centigrade for up to four hours before use.
High-speed acquisition of transillumination images
We selected FDB fibres with a width of 35 micrometres or greater (supplementary figure A), FDB is a fast-twitch muscle and we only used fibres which responded briskly and repeatedly to a 1msec activating pulse, over 90% of FDB fibres are fast-twitch, however, we occasionally came across fibres which were slower to contract and relax (visual inspection), these fibres were not used (18). Intact single FDB fibres were electrically field-stimulated with supramaximal voltage pulses of 1 ms duration, 10 V amplitude over a range of frequencies from 10 Hz to 100 Hz. The stimulator probe was bipolar, with two fine platinum wires isolated up to the ends, the wires were attached to a fine Perspex rod mounted on a micromanipulator to enable it to be placed close (~10µm) to the neuromuscular junction of the selected FDB fibre. A CMOS PCO1200hs high-speed camera (PCO AG, Kehlheim, Germany) was mounted to the camera side-port of the Nikon inverted microscope. The Peltier-cooled camera was connected to a computer for acquisition control and data storage. Single fibres approximately covered a 520×160 pixel area when visualised through a 20x objective which allowed frame rates for shortening sequences of 4,200 frames per second. Recordings were synchronised with the induction of a single twitch and image read-out and storage from the ring-buffer of the camera was performed offline. For offline analysis of each experiment, an image sequence of approximately 1,000 to 1,700 frames per fibre were analysed using a modification of a previously written processing algorithm in interactive data language environment (8).
EDL skinned fibre solutions
A single large (top 30% diameter of the fibres) intact EDL fibre was selected from the population of fibres using a fine bore pipette. We have previously shown that in mice there is a strong correlation between fibre size and type with fast fibres having nearly twice the cross-sectional area (CSA) compared to slow-twitch type 1 (9). The selected fibre was tied onto a sensitive force transducer of the MyoRobot biomechatronics system (19). After tying, it was placed for 10 min in solution A (see later) with 2% Triton X-100 added to chemically skin all remaining membranous cell elements. The fibre was then exposed to a series of solutions of different free Ca2+ concentrations. The strongly buffered Ca2+ solutions were prepared by mixing specific proportions of EGTA-containing solution (solution A) and Ca-EGTA–containing solution (solution B). Solution A contained 117 mM K+, 36 mM Na+, 8 mM adenosine triphosphate (ATP, total), 1 mM free Mg2+, 10 mM creatine phosphate, 50 mM EGTA (total), 60 mM N-[2- hydroxyethyl] piperazine-N’-[2-ethanesulfonic acid] (HEPES), and 1 mM NaN3 (pH 7.10). Solution B was similar to solution A, with the exception that the EGTA and Ca2+-EGTA concentrations of solution B were 0.3 and 49.7 mM, respectively. The free Ca2+ concentrations of the solutions were calculated using a Kapparent for EGTA of 4.78 x 106 M-1 (20). Maximal force was determined by exposure to solution B, containing a free Ca2+ concentration of 3.5 x 10-5 M. Force was returned to baseline after maximal activation by exposure to solution A. The plateaus of the force responses elicited by exposure to solutions of increasing free Ca2+ concentration are expressed as a percentage of maximum Ca2+-activated force and plotted as a function of pCa. The force–pCa data were fitted with Hill curves using GraphPad Prism8.
The MyoRobot, automated biomechatronics system
For full details of the MyoRobot see Haug (19). The following procedures were carried out on the EDL fibres using the MyoRobot. Force-pCa: the fibre was immersed in wells containing highly-EGTA buffered internal solutions with decreasing pCa values, made up by mixing solutions A&B (see above). Exposure to each pCa was for 20 seconds.
Slack test; speed of shortening:
The slack test assumes a constant shortening velocity of muscle fibres upon imposing a sudden small slack to the fibre when isometrically activated. Fibres were held at resting length L0, transferred to a maximally activating Ca2+ solution and maintained in this solution until the force produced by the fibre reached a steady-state plateau. The voice coil actuator (with fibre attached) was then linearly moved at maximum speed (250 mm/s) towards the transducer pin (other end of fibre attached) for a given slack length (5–40% L0). While force declined to zero, the force was continuously monitored at 2 kHz high sampling rate until force redeveloped through ongoing fibre shortening, re-establishing isometric force production. When the next steady-state force level was reached, the preparation was dipped in high EGTA relaxing solution where the voice coil pin was returned to L0 under relaxing conditions before the next slack test was imposed.
Passive axial elasticity, resting length-tension curves:
To assess axial fibre compliance through resting length-tension curves when the fibre was relaxed in low Ca2+ the voice coil was driven at very slow speed (quasi-static) to stretch the fibre while passive restoration force was sampled at 200 Hz. Since the skinned fibres possess viscous properties (e.g., presence of titin), the stretch velocity was optimized to values slow enough to be in a steady-state between instantaneous elastic restoration force and viscous relaxation.
Eccentric contractions:
The fibre was placed in a maximal Ca2+ activating solution and allowed to produce maximal isometric force; it was then stretched by 20% of L0 for two seconds, the stretch was released for a further two seconds before the fibre was relaxed in a low Ca2+, high EGTA solution. The procedure was carried out three times in total, and a final maximal Ca2+-activating force recorded.
Second harmonic generation imaging of single fibres:
Single EDL fibres were tied to thin glass rods and fixed in 0.1% glutaraldehyde solution for SHG microscopy. Glass rods with one EDL fibre each were mounted into a microscopy chamber immobilized between Vaseline® stripes for multiphoton imaging, for details see Friedrich (21).
Statistics
Data were presented as means ± SD. Differences occurring between genotypes were assessed by one-way ANOVA with respect to genotype. Post hoc analysis was performed using Holm-Sidak’s multiple comparisons test. The Logrank test was used to compare survival distributions of muscle fibres during contraction and the Mann Whitney test used for comparing angular variability of myofibres between groups. All tests were conducted at a significance level of 5%. All statistical tests and curve fitting were performed using a statistical software package Prism Version 8 (GraphPad, USA).