Myofiber apoptosis occurs in the inflammation and regeneration phase following eccentric contractions in rats

Abstract

Eccentric contractions (ECC) induce myofibrillar collapse, edema, and inflammation in muscle cells. Although apoptosis of myonuclei following ECC is activated during the inflammatory phase, the apoptosis response of the regenerative phase remains to be elucidated. The aim of the present study was to determine the inflammatory and regenerative phase of the apoptosis responses induced by ECC. In anesthetized rats, the tibialis anterior muscles were subjected to ECC repeated 40 times, evoked by surface electric stimulation (100 Hz, 10 V) with mechanical muscle stretch. Apoptosis was examined in the control group and in groups 1, 3, 7, and 14 days after ECC (each group, n = 4-6). Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive myonuclei were assessed by further labeling with dystrophin staining and DAPI. The expression of proteins related to apoptosis (Bcl-2 and Bax) was examined by Western blot assay. At 1 and 3 days, focal edema and necrotic myofibers invaded by mononuclear phagocytes were present, whereas regenerated myofibers with central nuclei were detected at 7 and 14 days. The occurrence of TUNEL-positive myonuclei increased significantly at 7 (7.0 +/- 1.5%) and 14 days (5.6 +/- 0.6%) compared with control (0.9 +/- 0.5%). Further we found that myonuclear apoptosis was restricted to the subsarcolemmal space at 7 and 14 days and markedly absent from the central nucleus. The Bax/Bcl-2 ratio was significantly higher at 3 (4.5 +/- 0.9) and 7 days (3.4 +/- 0.5) after ECC. In conclusion, myofiber apoptotic responses following ECC are present not only in the inflammatory phase but also persist during the regenerative phase.

Fig. 1

a Hematoxylin and eosin staining 3 days after ECC. Damaged myofibers were defined as those inflammatory cells with swollen (outlined arrows), swollen with infiltration (arrows), and infiltration (arrow-heads) appearance. b The multiple central nuclei can be detected (arrows) after 7 days of ECC. c TUNEL-positive nuclei were found both inside (a) and outside (b) myofibers. Muscle transverse sections were immunofluorescent-stained with dystrophin antibody to identify the sarcolemma (red), fluorescein-mediated TUNEL assay was performed to identify apoptotic nuclei (light blue), and all nuclei were labeled by DAPI staining (blue). TUNEL-positive nuclei positioned inside the dystrophin stain (a) were identified as myofiber nuclei, whereas other nuclei located outside (b) were counted as endothelial or interstitial cell nuclei. Bar 50 μm

Fig. 2

Serial transverse sections from control (a–c), 3 days (d–f), and 7 days (g–i) after ECC in TA muscle. H&E staining (a, d, g) was performed to examine the histological features of muscle damage, triple histochemical staining (b, e, h) for myofiber apoptotic nuclei was used as shown in Fig. 1, and AP-DPPIV staining was performed to identify endothelial cells (c, f, i). Bar 50 μm

Fig. 3

Percentage of myofiber and endothelial cell nuclei identified as apoptotic in control and post-ECC muscles at 1, 7, and 14 days. Values are mean ± SE. *p < 0.05, **p < 0.01, significant difference from control

Fig. 4

Longitudinal sections taken 7 and 14 days after ECC in TA muscle. All nuclei were stained with DAPI (blue), and nuclei with internal dystrophin stain (red) were defined as myofiber nuclei. TUNEL-positive myofiber nuclei (arrow) were located in the subsarcolemma, but central nuclei were not detected. Bar 50 μm

Fig. 5

Immunoblot analysis of the protein expression of Bcl-2 and Bax. Inset displays representative images for control and ECC muscles (a). Quantification by densitometry of Bcl-2 protein was significantly decreased 1, 3, and 7 days after ECC compared with control (b). Values are expressed as the percentage of the mean values of the control. Bax/Bcl-2 ratio was significantly higher at 3 and 7 days after ECC (c). *p < 0.05, significant difference from control