Apoptosis and necrosis mediate skeletal muscle fiber loss in age-induced mitochondrial enzymatic abnormalities

Abstract

Sarcopenia, the age-induced loss of skeletal muscle mass and function, results from the contributions of both fiber atrophy and loss of myofibers. We have previously characterized sarcopenia in FBN rats, documenting age-dependent declines in muscle mass and fiber number along with increased fiber atrophy and fibrosis in vastus lateralis and rectus femoris muscles. Concomitant with these sarcopenic changes is an increased abundance of mitochondrial DNA deletion mutations and electron transport chain (ETC) abnormalities. In this study, we used immunohistological and histochemical approaches to define cell death pathways involved in sarcopenia. Activation of muscle cell death pathways was age-dependent with most apoptotic and necrotic muscle fibers exhibiting ETC abnormalities. Although activation of apoptosis was a prominent feature of electron transport abnormal muscle fibers, necrosis was predominant in atrophic and broken ETC-abnormal fibers. These data suggest that mitochondrial dysfunction is a major contributor to the activation of cell death processes in aged muscle fibers. The link between ETC abnormalities, apoptosis, fiber atrophy, and necrosis supports the hypothesis that mitochondrial DNA deletion mutations are causal in myofiber loss. These studies suggest a progression of events beginning with the generation and accumulation of a mtDNA deletion mutation, the concomitant development of ETC abnormalities, a subsequent triggering of apoptotic and, ultimately, necrotic events resulting in muscle fiber atrophy, breakage, and fiber loss.

Keywords: ETS abnormalities; aging; apoptosis; cell death; mitochondria; necrosis; sarcopenia; skeletal muscle.

Figure 1

Detection of myofiber cell death in the quadriceps of 12‐ and 36‐month‐old rats. (A) Representative micrographs containing a fiber positive for cell death. The tissue sections were stained with hematoxylin and eosin or immunostained using antibodies against truncated form of Bid, PUMA, cleaved caspase‐3, C5b‐9, and CD68. The scale bar = 25 μm. (B) Number of fibers per 1000 μm of tissue analyzed that stained positive for the cell death markers in 12‐ and 36‐month‐old rat quadriceps. *Significant, P < 0.05, increase in aged rats. Data presented as mean ± SEM.

Figure 2

Myofiber cell death in electron transport chain (ETC)‐abnormal fibers of 36‐month‐old rats. (A) Abundance of ETC‐abnormal fibers per 1000 micrometers analyzed in the quadriceps of 12‐ and 36‐month‐old rats. No ETC‐abnormal fibers were detected in 12‐month‐old rats. Data presented as mean ± SEM. (B) The percent of ETC enzymatic phenotype of myofibers positive for apoptotic and necrotic markers in the rectus femoris of aged rats. (C) Representative ETC‐abnormal fiber undergoing cell death. Micrographs of histochemical staining for COX and SDH activity and immunostaining with antibodies to cleaved caspase‐3, C5b‐9, and CD68 are presented. The scale bar = 25 μm. (D) Digital reconstruction of the fiber in panel C. The red region indicates the ETC‐abnormal region, and the yellow region depicts transition area within the fiber. Increased staining for cleaved caspase‐3, C5b‐9, and CD68 is depicted in blue, green, and brown, respectively.

Figure 3

Prevalence of cell death in electron transport chain (ETC)‐abnormal fibers in 36‐month‐old rats. The percent of ETC‐abnormal fibers from quadriceps of 36‐month‐old rats that were positive for one or more cell death marker in the different combinations detected.

Figure 4

Fibers with longer electron transport chain (ETC)‐abnormal regions are positive for cell death markers. (A) A significant increase is observed in ETC‐abnormal length for fibers positive in cell death markers, P value < 0.0001 (n = 68 fibers). (B) The length of ETC‐abnormal region was measured for fibers positive for only tBid and subsequent addition of PUMA, cl‐Cas3, C5b‐9, and CD68. Fibers with the longest ETC‐abnormal region are positive for all five cell death markers. A total of 32 fibers were analyzed.

Figure 5

Apoptosis and necrosis are prevalent in atrophic and broken electron transport chain (ETC)‐abnormal fibers. (A) Intrafiber atrophy is more prevalent in ETC‐abnormal fibers (n = 102 fibers) than in ETC‐normal fibers from either 36‐month‐old (n = 99 fibers) or 12‐month‐old (n = 30 fibers) rats. Significant variance (P value < 0.0001) in cross‐sectional area ratio (CSAR) values between ETC‐abnormal and ETC–normal fibers. (B) There is increased atrophy in ETC‐abnormal fibers positive for cell death. Cross‐sectional area ratios of COX−/SDH++ fibers and COX‐normal/SDH‐normal fibers were determined. Significant atrophy in ETC‐abnormal fibers (P value = 0.0016). (C) Atrophic and broken ETC‐abnormal fibers stain positive for cleaved caspase‐3, C5b‐9, and CD68. A total of 32 fibers were analyzed.

Figure 6

Model of myofiber loss. (A) Bundle of hypothetical fibers that contain wild‐type mtDNA with normal electron transport chain (ETC) function. (B) A mtDNA deletion mutation, presumably resulting from an mtDNA replication error, is generated. The deletion‐containing mtDNA genomes accumulate in a segment of fiber disrupting ETC enzymatic activity (gray). (C) The deficiency results in activation of Bid and PUMA (black). (D) As the ETC‐abnormal region expands, apoptosis is mediated by cl‐Cas3 leading to intrafiber atrophy. (E) Upon the activation of apoptosis and necrosis, fiber breakage occurs within the ETC‐abnormal region. (F) Apoptotic and necrotic region expands in the fiber. (G) Fiber loss occurs. (H) Another individual fiber, with accumulation of a deleted mtDNA genome, undergoes C–G again.