Cell death in denervated skeletal muscle is distinct from classical apoptosis

Denervation of skeletal muscle is followed by the progressive loss of tissue mass and impairment of its functional properties. The purpose of the present study was to investigate the occurrence of cell death and its mechanism in rat skeletal muscle undergoing post-denervation atrophy. We studied the expression of specific markers of apoptosis and necrosis in experimentally denervated tibialis anterior, extensor digitorum longus and soleus muscles of adult rats. Fluorescent staining of nuclear DNA with propidium iodide revealed the presence of nuclei with hypercondensed chromatin and fragmented nuclei typical of apoptotic cells in the muscle tissue 2, 4 and to a lesser extent 7 months after denervation. This finding was supported by electron microscopy of the denervated muscle. We found clear morphological manifestations of muscle cell death, with ultrastructural characteristics very similar if not identical to those considered as nuclear and cytoplasmic markers of apoptosis. With increasing time of denervation, progressive destabilization of the differentiated phenotype of muscle cells was observed. It included disalignment and spatial disorganization of myofibrils as well as their resorption and formation of myofibril-free zones. These changes initially appeared in subsarcolemmal areas around myonuclei, and by 4 months following nerve transection they were spread throughout the sarcoplasm. Despite an increased number of residual bodies and secondary lysosomes in denervated muscle, we did not find any evidence of involvement of autophagocytosis in the resorption of the contractile system. Dead muscle fibers were usually surrounded by a folded intact basal lamina; they had an intact sarcolemma and highly condensed chromatin and sarcoplasm. Folds of the basal lamina around the dead cells resulted from significant shrinkage of cell volume. Macrophages were occasionally found in close proximity to dead myocytes. We detected no manifestations of inflammation in the denervated tissue. Single myocytes expressing traits of the necrotic phenotype were very rare. A search for another marker of apoptosis, nuclear DNA fragmentation, using terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (the TUNEL method) in situ, revealed the presence of multiple DNA fragments in cell nuclei in only a very small number of cell nuclei in 2 and 4 month denervated muscle and to less extent in 7 month denervated muscle. Virtually no TUNEL reactivity was found in normal muscle. Double labeling of tissue denervated for 2 and 4 months for genome fragmentation with the TUNEL method and for total nuclear DNA with propidium iodide demonstrated co-localization of the TUNEL-positive fragmented DNA in some of the nuclei containing condensed chromatin and in fragmented nuclei. However, the numbers of nuclei of abnormal morphology containing condensed and/or irregular patterns of chromatin distribution, as revealed by DNA staining and electron microscopy, exceeded by 33-38 times the numbers of nuclei positive for the TUNEL reaction. Thus, we found a discrepancy between the frequences of expression of morphological markers of apoptosis and DNA fragmentation in denervated muscle. This provides evidence that fragmentation of the genomic DNA is not an obligatory event during atrophy and death of muscle cells, or, alternatively, it may occur only for a short period of time during this process. Unlike classical apoptosis described in mammalian thymocytes and lymphoid cells, non-inflammatory death of muscle fibers in denervated muscle occurs a long time after the removal of myotrophic influence of the nerve and is preceded by the progressive imbalance of the state of terminal differentiation. Our results indicate that apoptosis appears to be represented by a number of distinct isotypes in animals belonging to different taxonomic groups and in different cell lineages of the same organism.