Characteristics of myogenic response and ankle torque recovery after lengthening contraction-induced rat gastrocnemius injury

Abstract

Background: Although muscle dysfunction caused by unfamiliar lengthening contraction is one of most important issues in sports medicine, there is little known about the molecular events on regeneration process. The purpose of this study was to investigate the temporal and spatial expression patterns of myogenin, myoD, pax7, and myostatin after acute lengthening contraction (LC)-induced injury in the rat hindlimb.

Methods: We employed our originally developed device with LC in rat gastrocnemius muscle (n = 24). Male Wistar rats were anesthetized with isoflurane (aspiration rate, 450 ml/min, concentration, 2.0%). The triceps surae muscle of the right hindlimb was then electrically stimulated with forced isokinetic dorsi-flexion (180°/sec and from 0 to 45°). Tissue contents of myoD, myogenin, pax7, myostatin were measured by western blotting and localizations of myoD and pax7 was measured by immunohistochemistry. After measuring isometric tetanic torque, a single bout of LC was performed in vivo.

Results: The torque was significantly decreased on days 2 and 5 as compared to the pre-treatment value, and recovered by day 7. The content of myoD and pax7 showed significant increases on day 2. Myogenin showed an increase from day 2 to 5. Myostatin on days 5 and 7 were significantly increased. Immunohistochemical analysis showed that myoD-positive/pax7-positive cells increased on day 2, suggesting that activated satellite cells play a role in the destruction and the early recovery phases.

Conclusion: We, thus, conclude that myogenic events associate with torque recovery after LC-induced injury.

Figure 1

Time course of changes in isometric torque after a lengthening contraction (LC). Isometric tetanic torque was examined before and after LC-induced muscle injury. **: p < 0.01, ***: p < 0.001, compared to pre-treatment values. Values are means and SDs.

Figure 2

Muscle contents of myogenin, myoD, pax7, and myostatin as determined by Western blotting. The expression of myogenin before and after LC-induced muscle injury was examined. Obtained bands and their quantifications are shown (A). The expression of myoD (B), pax7 (C), myostatin (D) were also examined. Details are described in the materials and methods section. **: p < 0.01, ***: p < 0.001, compared to pre-treatment levels. Values are means and SDs.

Figure 3

Typical photomicrographs showing the localizations of myoD and pax7 at day 2 and their quantifications. Triple immunostaining (A) of myoD (green), laminin (red), nuclei (blue), and merge for myoD-negative/pax7-positive, myoD-positive/pax7-negative, and myoD-positive/pax7-positive. Arrows indicate immunoreactive positive regions. Bar = 30 μm. The graph (B) shows the percentages of myoD-negative/pax7-positive cells (red bar), myoD-positive/pax7-negative (blue bar), and myoD-positive/pax7-positive (black bar). ***: p < 0.001, compared to pre-treatment and day 7 levels of the percentages of myoD-positive/pax7-positive cells.

Figure 4

Schematic of the 3 recovery phases following a LC-induced muscle injury. Based on our results on the isometric torque change, we approximated the regeneration process into 3 phases including destruction, recovery (days 0–5), and remodeling (days 5–7).