Muscle aging and oxidative stress in wild-caught shrews

Abstract

Red-toothed shrews (Soricidae, subfamily Soricinae) are an intriguing model system to examine the free-radical theory of aging in wild mammals, given their short (<18months) lifespan and high mass-specific metabolic rates. As muscle performance underlies both foraging ability and predator avoidance, any age-related decline should be detrimental to fitness and survival. Muscle samples of water shrews (Sorex palustris) and sympatrically distributed short-tailed shrews (Blarina brevicauda) were therefore assessed for oxidative stress markers, protective antioxidant enzymes and apoptosis. Activity levels of catalase and glutathione peroxidase increased with age in both species. Similarly, Cu,Zn-superoxide dismutase isoform content was elevated significantly in older animals of both species (increases of 60% in the water shrew, 25% in the short-tailed shrew). Only one oxidative stress marker (lipid peroxidation) was age-elevated; the others were stable or declined (4-hydroxynonenal adducts and dihydroethidium oxidation). Glutathione peroxidase activity was significantly higher in the short-tailed shrew, while catalase activity was 2x higher in water shrews. Oxidative stress indicators were on average higher in short-tailed shrews. Apoptosis occurred in <1% of myocytes examined, and did not increase with age. Within the constraints of the sample size we found evidence of protection against elevated oxidative stress in wild-caught shrews.

Fig. 1

Mean (± SE) activity of citrate synthase (U·μg protein⁻¹) in skeletal muscle homogenate from two shrew species. Closed bars represent samples from hindlimb (old: n=8 WS, n=7 STS; young: n=9 WS, n=7 STS), open bars from forelimb (old: n=8 WS, n=8 STS; young: n=6 WS, n=7 STS). LSD post-hoc tests reveal a species effect in adult forelimb (P=0.003, denoted `**') and in limb site (for adult STS only P=0.012, denoted `***').

Fig. 2

Mean (± SE) activities of catalase (CAT) and glutathione peroxidase (GPx) in skeletal muscle homogenate (U·μg protein⁻¹) from two species of shrew. Pooled data from hindlimb and forelimb are presented, with old animals (CAT: n=8 WS, n=8 STS; GPx: n=8 WS, n=8 STS) noted by black bars, and young animals (CAT: n=9 WS, n=7 STS; GPx: n=9 WS, n=7 STS) by gray bars. LSD post-hoc tests revealed a significant age effects (denoted `*') for CAT in WS (P=0.025) and GPx in STS (P=0.046), as well as species effect (denoted `**') for WS CAT (P=0.026) and for GPx in both cohorts (P=0.001 adults; P=0.014 juveniles).

Fig. 3

Protein contents of Mn-SOD and CuZn-SOD isoforms, expressed in arbitrary units, in skeletal muscle hindlimb homogenate from two species of shrew (means ± SE for pooled forelimb/hindlimb data). Black bars represent data from adult shrews (CuZn: n=5 WS, n=8 STS; Mn: n=6 WS, n=7 STS), and gray bars represent juvenile data (CuZn: n=9 WS, n=6 STS; Mn: n=4 WS, n=5 STS). LSD post-hoc tests revealed a significant age effect for CuZn SOD in WS (P=0.030), denoted `*'.

Fig. 4

Lipid peroxidation (μM t-butyl hydroperoxide Eq·μg protein⁻¹) in skeletal muscle homogenate from two species of shrew (means ± SE). Closed bars represent samples from hindlimb (old: n=9 WS, n=8 STS; young: n=10 WS, n=7 STS), open bars from forelimb (old: n=9 WS, n=8 STS; young: n=10 WS, n=7 STS). LSD post-hoc tests revealed an age effect in WS hindlimb (P=0.003, denoted `*') and in limb site (for juvenile WS only P=0.006, denoted `***').

Fig. 5

Dihydroethidium oxidation (DHE, xanthine oxidase Eq. U·μg protein⁻¹) and 4-hyroxynonenal adducts (4HNE, protein content arbitrary units) in skeletal muscle homogenate from two shrew species (pooled means ± SE from forelimb/hindlimb data). Black bars represent data from adult shrews (DHE: n=7 WS, n=9 STS; 4HNE: n=8 WS, n=8 STS), and gray bars represent juvenile data (DHE: n=10 WS, n=6 STS; 4HNE: n=9 WS, n=7 STS). LSD post-hoc tests revealed a significant species effects for DHE in adults (P=0.016) and 4HNE (P<0.001 for both cohorts), which are indicated with `**'.

Fig. 6

(COLOR FIGURE IN PRINT) Triply labeled gracilis muscle of a representative water shrew. TUNEL-positive areas are labeled with green florescence, laminin with red, and all nuclei (DAPI) with blue florescence. A: representative section of gracilis muscle with triple label; B: positive TUNEL control; C: negative TUNEL control.