Diltiazem improves contractile properties of skeletal muscle in dysferlin-deficient BLAJ mice, but does not reduce contraction-induced muscle damage

Figures

• 

  Figure 1. Setup for in vivo torque measurement and MSFEE. We measured contractile torque of the dorsiflexors of the left hindlimb and performed MSFEE on this muscle group with a custom‐built dynamometer (A and B). We elicited contractions of the dorsiflexors group of muscles, in which the prime mover is the TA muscle, by depolarizing the common peroneal nerve with a bipolar transcutaneous electrode (A). To perform forced eccentric contractions during MSFEE, we tetanically stimulated the dorsiflexors muscles, and forced the foot into plantarflexion (90–160°) with a stepper motor (B–F). We were able to trigger the electrical stimulator, control the motion of the stepper motor, and record torque data with custom‐written software. Representative torque traces for a single twitch, a maximal tetanic contraction, and a forced eccentric contraction, are shown in panels G, H, and I respectively. A single bout of MSFEE involved 40 eccentric contractions, performed in four sets of 10 repetitions, with 2 min rest between sets.

• 

  Figure 2. DTZ improves contractile torque before and immediately after MSFEE, but does not improve recovery at 3 days post‐MSFEE. DTZ increased baseline twitch torque (*), but did not increase baseline tetanic torque (A). Pre‐MSFEE torque‐frequency data suggest that, DTZ improves contractile torque at low frequencies (*, 1–30 Hz), but not at stimulation frequencies higher than 30 Hz (B). The frequency labeled reverse 1 Hz (Rev 1 Hz), is a twitch that is induced after the last tetanic contraction, in order to assess post‐tetanic changes in twitch torque (B–D). Torque data collected immediately after MSFEE suggest that, DTZ offers significant protection against acute torque loss from MSFEE (*, C). However, the protection against acute torque loss following MSFEE, is not seen at frequencies higher than 100 Hz (C). Torque data collected at 3 days post‐MSFEE suggest that, DTZ does not help improve torque recovery, except at the stimulation frequency of 10 Hz (D). Data are reported as mean ± SD. Asterisks denote a significant difference between DTZ and VEH.

• 

  Figure 3. DTZ does not reduce delayed‐onset muscle damage at 3 days post‐MSFEE. Our data suggest that, extensive myofiber damage can be detected at 3 days post‐MSFEE in BLAJ mice, irrespective of whether the animals are given DTZ or VEH (A–F). This damage can be detected by the presence of myofibers with pale and/or disrupted cytoplasm in H&E stained sections (A and B), and by the inclusion of endogenous IgG (C and D) and loss of desmin (E and F) within myofibers. Since H&E staining, and IgG and desmin colabeling, were performed on serial cross‐sections of the same TA muscle; the superimposed asterisks (*) in the image panels, denote the same subset of damaged fibers in panels A, C, E (DTZ) and panels B, D, F (VEH). Scale bar = 100 μm. Our quantitative analyses on images, such as, panels A–F, are shown in panels G (damaged fiber counts from H&E stained sections) and H (damaged fiber counts from IgG‐ and desmin‐labeled sections). Our data suggest that, at 3 days post‐MSFEE, DTZ does not reduce myofiber damage, as assessed by H&E staining (G) or IgG and desmin labeling (H). Data are reported as mean ± SD.