Figure 2.

Morphological and Functional Changes in Muscles of Atg7^−/− during Aging

(A) Kaplan-Meier curve of Atg7^f/f and Atg7^−/− mice. Atg7^−/− mice die earlier than their Atg7^f/f counterparts. Comparison of Atg7^f/f and Atg7^−/− survival curves was performed by both Mantel-Cox and Gehan-Breslow-Wilcoxon tests. Atg7^−/− mice show a significant reduction in the lifespan compared to controls (^∗p < 0.05).

(B) Morphological analyses of aged Atg7^f/f and Atg7^−/− tibialis anterior (TA) muscles. H&E staining shows that Atg7^−/− muscles are atrophic with interstitial inflammation and center-nucleated fibers.

(C) Higher magnification of atrophic fibers of aged Atg7^−/− mice showing angular, flat fibers and extremely small fibers (arrows).

(D) Quantification of the cross-sectional area (CSA) of glycolitic myofibers in TA muscles of adult and aged Atg7^f/f and Atg7^−/− mice. Values are mean ± SEM. At least four muscles of each group were analyzed; ^∗∗p < 0.01; ^∗p < 0.05.

(E) Quantification of CSA of oxidative myofibers of TA. Values are mean ± SEM; n > 4 for each group; ^∗p < 0.05.

(F and G) Force measurements performed in vivo. (F) Atg7^−/− mice show a profound decrease in maximal force generation in adult animals. Values are mean ± SEM (n = 5); ^∗∗p < 0.01. (G) Aging reduces force production in both Atg7^f/f and Atg7^−/− mice, therefore aggravating the already profound weakness of Atg7^−/− mice. Values are mean ± SEM; n = 4; ^∗p < 0.05.

(H) Quantification of center-nucleated myofibers in TA (n > 4 for each group; ^∗∗p < 0.01). Values are mean ± SEM.

(I) Representative images of immunostaining for NCAM expression in aged mice. Localization of NCAM along the entire fibers indicates the loss of muscle-nerve interaction.

(J) Immunoblotting for NCAM protein on muscle extracts from adult and aged gastrocnemius muscles. Atg7^−/− muscles express higher levels of NCAM than age-matched Atg7^f/f.

(K) Quantification of NCAM-positive fibers. Values were normalized for the total number of myofibers in muscle section (at least four muscles of each group were analyzed; ^∗∗p < 0.01). Atg7^−/− mice are characterized by much-higher age-dependent increase in NCAM-positive fibers than age-matched Atg7^f/f. Values are mean ± SEM.

(L) Expression levels of acetylcholine receptor AChR γ-subunit (CHRNG) (left) and MuSK (right). MuSK and AChR are upregulated in adult and aged Atg7^−/− muscles (n > 5; ^∗p < 0.05). Values are mean ± SEM.