Blunted angiogenesis and hypertrophy are associated with increased fatigue resistance and unchanged aerobic capacity in old overloaded mouse muscle

Abstract

We hypothesize that the attenuated hypertrophic response in old mouse muscle is (1) partly due to a reduced capillarization and angiogenesis, which is (2) accompanied by a reduced oxidative capacity and fatigue resistance in old control and overloaded muscles, that (3) can be rescued by the antioxidant resveratrol. To investigate this, the hypertrophic response, capillarization, oxidative capacity, and fatigue resistance of m. plantaris were compared in 9- and 25-month-old non-treated and 25-month-old resveratrol-treated mice. Overload increased the local capillary-to-fiber ratio less in old (15 %) than in adult (59 %) muscle (P < 0.05). Although muscles of old mice had a higher succinate dehydrogenase (SDH) activity (P < 0.05) and a slower fiber type profile (P < 0.05), the isometric fatigue resistance was similar in 9- and 25-month-old mice. In both age groups, the fatigue resistance was increased to the same extent after overload (P < 0.01), without a significant change in SDH activity, but an increased capillary density (P < 0.05). Attenuated angiogenesis during overload may contribute to the attenuated hypertrophic response in old age. Neither was rescued by resveratrol supplementation. Changes in fatigue resistance with overload and aging were dissociated from changes in SDH activity, but paralleled those in capillarization. This suggests that capillarization plays a more important role in fatigue resistance than oxidative capacity.

Keywords: Aerobic capacity; Angiogenesis; Hypertrophy; Muscle aging; Resveratrol.

Fig. 1

Effects of age, overload and resveratrol on in situ plantaris muscle fatigue resistance. Plantaris force (% of the force during the first contraction) is expressed as contraction number percentage of first contraction in the m. plantaris for adult (circle), old (triangle) and old-res (square) mice in both control (black) and overloaded (white) conditions

Fig. 2

Effects of age, overload and resveratrol on m. plantaris FCSA. FCSA was smaller in old compared to adult muscle (^a P < 0.05), bigger in old-res than old muscle (^o P < 0.05) and increased after overload in adult and old (*P < 0.01), but not in old-res muscle. Values are represented as percentage of the adult control muscle; mean ± SEM

Fig. 3

SDH activity and integrated SDH activity per fiber type for adult, old and old-res muscle. SDH activity for adult, old and old-res muscle in control and overloaded condition for type A, X, and B fibers in the oxidative (a) and glycolytic (b) region of the muscle. c Mean SDH for adult, old and old-res mice in both control (black) and overloaded (white) conditions. SDH activity in old muscle was higher than in adult muscle (^a P < 0.05). SDH activity was higher in old compared to adult muscle for all three fiber types (P < 0.05). Furthermore, the SDH activity in IIB of the glycolytic region of the muscle was lower than of type IIB fibers in the oxidative region (^g P < 0.05). Resveratrol did only increase SDH activity in type IIB fibers of the glycolytic region of the muscle (P < 0.05). SDH activity was lower in type IIB compared to IIX fibers (^χ P < 0.01) and lower in IIX compared to IIA fibers (^α P < 0.01). Integrated SDH activity for adult, old, and old-res muscle in control and overloaded condition for type A, X, and B fibers in the oxidative region (d) and glycolytic region (e) of the muscle. f Mean integrated SDH for adult, old, and old-res mice in both control (black) and overloaded (white) conditions. Integrated SDH activity increased after overload for all three fiber types (P < 0.01). Old muscle showed a higher integrated SDH activity in type IIA fibers, compared to adult (P < 0.01). Type IIX fibers had a lower integrated SDH activity in the glycolytic region, compared to the oxidative region (P < 0.01). Data shown are mean ± SEM

Fig. 4

Flk-1, SDH, and COX-4 protein expression in adult, old and old-res muscle. a SDH protein expression was similar in adult and old muscle, while COX-4 protein expression was decreased in old muscle (^a P < 0.05). The SDH/COX-4 ratio (b) was not significantly different in aged muscle. c Flk-1 protein expression in m. plantaris, an important receptor for VEGF, was decreased in old compared to adult muscle (^a P < 0.05), but not affected by overload or resveratrol. The results are presented as the ratio protein of interest/loading control (eEF2). Values are mean ± SEM

Fig. 5

Effects of age, overload, and resveratrol on CD, C/F, and Log_rSD. a CD was unaffected by age, however increased by overload (P < 0.01). Resveratrol blunted the overload-induced increase in CD, but only in the glycolytic region of the muscle (resveratrol × overload, P < 0.05). b C/F was increased by overload (P < 0.01), but unaffected by age or resveratrol. Both CD and C/F were higher in the oxidative region of the m. plantaris compared to the glycolytic region. c Log_rSD for adult, old and old-res control (black) and overloaded (white) muscle in the oxidative and glycolytic muscle regions. Log_rSD was higher in the glycolytic than in the oxidative region of the m. plantaris muscle (^g P < 0.01), but not affected by age. Overload reduced Log_rSD (*P < 0.01), while resveratrol did not affect the Log_rSD (P > 0.05). Values are mean ± SEM. Black bars control situation; white bars overload situation

Fig. 6

Illustration of the analyses of capillary data. a Typical example of capillaries identified with lectin staining in mice muscle. b In red, the capillaries, and in green, the muscle fiber outlines are shown, while in blue (c), the calculated capillary domains are depicted. d The overlap of domains and fibers, used to calculate fiber-specific capillary supply, as described in the methods section. Bar represents 30 μm (color figure online)

Fig. 7

Effects of age, overload, muscle area and resveratrol supplementation on local capillary to fiber ratio and capillary fiber density. LCFR for adult control (black), overloaded (white), old control (dashed), and overloaded (striped) muscle for type A, X, and B fibers in the oxidative (a) and glycolytic (b) region of the muscle. LCFR activity for old control (black), overloaded (white), old-res control (dashed) and overloaded (striped) muscle for type A, X, and B fibers in the oxidative (c) and glycolytic (d) region of the muscle. Overload increased the LCFR in fibers of all types in both regions (P < 0.05), except for oxidative type IIB fibers (P > 0.05). An interaction effect (age × overload) showed that the increase in LFCR was larger for adult than for old muscle (P < 0.05). Resveratrol did not affect the LCFR (P > 0.05). For both old and old-res muscle, the LCFR was higher in the oxidative, compared to the glycolytic part (P < 0.01). CFD (mm⁻²) for adult control (black), overloaded (white), old control (dashed), and overloaded (striped) muscle for type A, X, and B fibers in the oxidative (e) and glycolytic (f) region of the muscle. CFD (mm⁻²) for old control (black), overloaded (white), old-res control (dashed) and overloaded (striped) muscle for type A, X and B fibers in the oxidative (g) and glycolytic (h) region of the muscle. CFD showed an age × overload interaction for type IIAX and IIX fibers, only in the glycolytic region (P < 0.05). Resveratrol decreased the CFD for IIAX, X and B fibers in the glycolytic region of the muscle (P < 0.05). For type AX fibers, overload increased the CFD in both old and old-res muscle (P < 0.05). Furthermore, in IIX and IIB fibers CFD was lower in the glycolytic, compared to the oxidative region (age × overload interaction; P < 0.05). Data shown are mean ± SEM

Fig. 8

SDH activity and FCSA are inversely related. The figure shows the relationship between mean SDH activity per muscle and FCSA for adult and old muscles. Overload affects this relationship by inducing a rightward shift (R ² = 0.74; P < 0.01), compared to the control (R ² = 0.67; P < 0.01). The black lines represent the best fit hyperbola. Values are mean ± SEM