Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of Actn3KO mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics

Figures

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  Fig. 1. MyoRobot biomechatronics system. a Displays a schematics setup of the automated robotised system, whose functionality is centred around an optical force transducer, a voice-coil actuator and a 34-well rack housing bioactive solutions. b Diagram of the attachment points of force transducer and voice-coil between which the single muscle fibre is mounted; the fibre is shown in red. Upon immersion in different wells or by precisely elongating its extension, active and passive force recordings can be carried out in an automated fashion. c Shows a photograph of the MyoRobot with the 34-well rack removed to show the optics system used to record the muscle fibre diameter in a transmission illumination setting

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  Fig. 2. Unloaded speed of shortening during external field stimulation of single enzymatically dissociated intact FDB fibres. a Example images during a shortening sequence of a Actn3KO single fibre stimulated at 100 Hz for 250 ms and recorded at 4166 fps. Also shown (right panel, fibre image on a black background) are the automated analysis images from which the shortening parameters, fibre length and shortening velocity were recorded. b Analysed time traces of these parameters for the same fibre at indicated stimulation frequencies. c Minimum shortening length was not different between WT and Actn3KO mice; however, there was a significant reduction in both genotypes at higher frequencies 20–100 Hz as indicated *. d Absolute maximum shortening velocities were not different between genotypes apart from 30 Hz where Actn3KO were significantly faster as indicated by *. Significance WT vs. KO based on one-way ANOVA test indicated as follows: *p < 0.05

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  Fig. 3. Maximum myofibrillar force in single EDL fibres from Actn3KO mice is unaltered compared to WT. a Statistical analysis of maximum force and specific force (normalised to fibre diameter-derived cross-sectional area) values from EDL muscles of WT and Actn3KO mice. No significant differences based on one-way ANOVA tests were apparent. b Analysis of fibre diameter distributions shows a trend to smaller diameter in the Actn3KO fibres. c Calcium sensitivity shown as pCa-force relationship. Average data are displayed along with the reconstructed average fit

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  Fig. 4. Resting length-tension curves and steady-state compliance of EDL Actn3KO and wildtype single fibres. a Specific restoration force (stress) at 140% L0 analysed in WT and Actn3KO fibres. b Steady-state stiffness values vs. strain indicate a slight trend to lower mechanical stiffness in the Actn3KO background compared to the WT

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  Fig. 5. Single fibre visco-elasticity in EDL muscle from adult Actn3KO mice. a Example trace of ‘strain-jumps’ of increasing percentage of L0 amplitudes quickly applied to the fibre. b Each sudden stretch is answered by an instantaneous increase in restoration force FR to a new maximum Fabs (c) before exponentially relaxing to achieve a new steady-state level with relaxation amplitude Frelax (d) and a time constant trelax

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  Fig. 6. Contraction-induced breakages are more pronounced in EDL fibres from Actn3KO mice. a Schematic of the eccentric contractions protocol, maximally activating a single fibre and then imposing a 20% stretch before returning to resting length L0 and subsequent relaxation. Three eccentric contractions were carried out followed by a final assessment of maximum isometric force. b Actn3KO fibres showed much lower survival and higher rate of breakage during the sequence to a significance of p = 0.02 based on logrank analysis. In b, fibres that broke are shown in the segments of the pie chart with diagonal lines, while nonbreaking fibres are shown in solid fill

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  Fig. 7. Second harmonic imaging and quantitative morphometry imaging in single dissociated EDL fibres reveal misregistered myofibrillar ultrastructure in Actn3KO. a Representative example images from the middle plane of a single WT and Actn3KO EDL fibre (top) with the automatically detected verniers shown as red dots and short red lines the VD and CAS values derived from the morphometry analysis are given in text on the bottom right of each panel. A magnified rectangular section is given below the images to enable a clearer view of the automatically detected (in red) myofibrillar disruptions in the Actn3Ko compared with the absence of such deviations in the WT fibre. b Group analysis in a substantial number of single fibres from several animals reveals significantly higher VD values in Actn3KO fibres over fibres from WT littermates, indicative of a higher linear out-of-register disorder. As for the angular variability of myofibrils, the CAS values were similar in both groups. **p < 0.001, Mann–Whitney rank sum test. (n/m): n single fibres from m animals