Latent mitochondrial DNA deletion mutations drive muscle fiber loss at old age

Abstract

With age, somatically derived mitochondrial DNA (mtDNA) deletion mutations arise in many tissues and species. In skeletal muscle, deletion mutations clonally accumulate along the length of individual fibers. At high intrafiber abundances, these mutations disrupt individual cell respiration and are linked to the activation of apoptosis, intrafiber atrophy, breakage, and necrosis, contributing to fiber loss. This sequence of molecular and cellular events suggests a putative mechanism for the permanent loss of muscle fibers with age. To test whether mtDNA deletion mutation accumulation is a significant contributor to the fiber loss observed in aging muscle, we pharmacologically induced deletion mutation accumulation. We observed a 1200% increase in mtDNA deletion mutation-containing electron transport chain-deficient muscle fibers, an 18% decrease in muscle fiber number and 22% worsening of muscle mass loss. These data affirm the hypothesized role for mtDNA deletion mutation in the etiology of muscle fiber loss at old age.

Keywords: aging; mitochondrial DNA deletion mutation; muscle; sarcopenia.

Figure 1

Morphometric data from aged control and GPA‐treated rats. (A) Representative muscle cross‐sectional images stained with hematoxylin and eosin. The bar denotes 5 mm. The encircled area delineates the rectus femoris muscle with cross‐sectional areas of 39.5 and 33.2 mm² for control and GPA treated, respectively. (B) Quadriceps muscle sections stained with Masson's trichrome to identify collagen (blue) deposition. The bar denotes 5 mm. (C) Higher magnification imaging of fibrotic tissue deposited in aged rats. The bar denotes 100 μm. (D) Quadriceps muscle mass, rectus femoris fiber number and cross‐sectional area, and quadriceps fibrotic tissue abundance in control and GPA‐treated aged rats.

Figure 2

GPA treatment of aged rats results in a 1200% increase in the abundance of segmental ETC abnormal fibers. (A) COX‐negative, SDH hyper‐ETC abnormal muscle fibers are prevalent in GPA‐treated rat muscle. The scale bar in each image is 0.25 mm. (B) ETC abnormality abundance estimates in rectus femoris and quadriceps muscles. (C) GPA treatment decreases ETC abnormal segment length. Frequency distributions are fitted with Gaussian curves using a least squares approach.

Figure 3

Increased fiber death in GPA‐induced ETC abnormal fibers. (A) Representative ETC abnormal fiber undergoing apoptosis and necrosis in a GPA‐treated rat. Dual and single histochemical staining for COX and SDH. Immunohistochemical staining for cl‐Cas3 and c5b‐9. The scale bar denotes 50 μm. (B) A greater fraction of ETC abnormal fibers are positive for apoptotic (cl‐Cas3) and necrotic (c5b‐9) cell death in GPA‐treated rat muscle as compared to control. (C) Cell death processes are activated in shorter ETC abnormal fiber segments of GPA‐treated rats than in controls.

Figure 4

Induction of mtDNA deletion mutation accumulation by GPA treatment in aged rats. (A) Elevated mtDNA deletion mutation frequency in GPA‐treated rat quadriceps muscles. (B) Mitochondrial DNA deletion mutations from GPA‐treated rats amplified by long extension PCR from laser capture microdissected COX‐fibers (C) Copy numbers of total mtDNA as compared to WT mtDNA in single, microdissected control or COX‐fibers showing degrees of heteroplasmy. (D) mtDNA deletion mutation breakpoint sequence from a single microdissected COX‐fiber shown in panels B and C—Fiber 1.