β-Hydroxy-β-methylbutyrate reduces myonuclear apoptosis during recovery from hind limb suspension-induced muscle fiber atrophy in aged rats

Abstract

β-Hydroxy-β-methylbutyrate (HMB) is a leucine metabolite shown to reduce protein catabolism in disease states and promote skeletal muscle hypertrophy in response to loading exercise. In this study, we evaluated the efficacy of HMB to reduce muscle wasting and promote muscle recovery following disuse in aged animals. Fisher 344×Brown Norway rats, 34 mo of age, were randomly assigned to receive either Ca-HMB (340 mg/kg body wt) or the water vehicle by gavage (n = 32/group). The animals received either 14 days of hindlimb suspension (HS, n = 8/diet group) or 14 days of unloading followed by 14 days of reloading (R; n = 8/diet group). Nonsuspended control animals were compared with suspended animals after 14 days of HS (n = 8) or after R (n = 8). HMB treatment prevented the decline in maximal in vivo isometric force output after 2 wk of recovery from hindlimb unloading. The HMB-treated animals had significantly greater plantaris and soleus fiber cross-sectional area compared with the vehicle-treated animals. HMB decreased the amount of TUNEL-positive nuclei in reloaded plantaris muscles (5.1% vs. 1.6%, P < 0.05) and soleus muscles (3.9% vs. 1.8%, P < 0.05). Although HMB did not significantly alter Bcl-2 protein abundance compared with vehicle treatment, HMB decreased Bax protein abundance following R, by 40% and 14% (P < 0.05) in plantaris and soleus muscles, respectively. Cleaved caspase-3 was reduced by 12% and 9% (P < 0.05) in HMB-treated reloaded plantaris and soleus muscles, compared with vehicle-treated animals. HMB reduced cleaved caspase-9 by 14% and 30% (P < 0.05) in reloaded plantaris and soleus muscles, respectively, compared with vehicle-treated animals. Although, HMB was unable to prevent unloading-induced atrophy, it attenuated the decrease in fiber area in fast and slow muscles after HS and R. HMB's ability to protect against muscle loss may be due in part to putative inhibition of myonuclear apoptosis via regulation of mitochondrial-associated caspase signaling.

Figures

Fig. 1.

Body weight was determined longitudinally in animals at 4 different time points. The first was prior to giving the animals any dietary intervention (Start). The second (time 0), was after the animals were given either β-hydroxy-β-methylbutyrate (HMB) or the vehicle (water) by gavage for 7 days. Final points were after the animals had undergone 14 days of hindlimb suspension (HS) and after 14 days of HS followed by 14 days of reloading (R). Cage control-vehicle-treated (CC-Veh) and cage control-HMB (CC-HMB)-treated animals did not receive hindlimb suspension but were examined at the same time points as the other animals. Body weight was not significantly different between the HMB and vehicle control groups at any time point. An ANOVA followed by Bonferroni post hoc analyses was used to evaluate the differences between the group means. *Body weight was significantly lower (P < 0.05) than the time points before HS for the same treatment. **Body weight was significantly lower (P < 0.05) in vehicle and HMB-treated groups after both HS and R compared with CC-Veh or CC-HMB groups.

Fig. 2.

Isometric force. Maximal isometric force production was measured longitudinally in vivo in the plantar flexor muscles before experimental intervention (time 0), after 14 days of HS, and after 14 days of HS followed by 14 days of R. Animals received either HMB, or the vehicle (water) daily, for 7 days before time 0 and throughout the experimental period. An ANOVA followed by Bonferroni post hoc analyses was used to evaluate the differences between the group means. †P < 0.05, vehicle vs. HMB; *force/body weight was significantly lower (P < 0.05) compare to points before HS for the same treatment; **force/body weight was significantly lower (P < 0.05) in vehicle and HMB-treated groups after both HS and R compared with CC-Veh or CC-HMB groups.

Fig. 3.

Muscle wet weight was obtained in plantaris (A) and soleus (C) muscles of control animals for the HS group (HS-Con), the R group (R-Con), and in experimental animals after 14 days of HS or after 14 days of HS followed by 14 days of R. The ratio of muscle wet weight to body weight is presented for the plantaris (B) and the soleus (D) muscles of the aged animals after each condition. Animals received HMB or the vehicle (water) daily by gavage, for a total of 21 days (HS-Con and HS) or for 32 days (R-Con and R). An ANOVA followed by Bonferroni post hoc analyses was used to evaluate the differences between the group means. *P < 0.05, vehicle vs. HMB within the same condition; †P < 0.05 HS or R vs. control animals for that experimental condition.

Fig. 4.

Fiber cross-sectional area (CSA) was obtained by planimetry in the plantaris (A) and the soleus (C) muscles of control animals for the HS group (HS-Con), the recovery group (R-Con), and in experimental animals after 14 days of HS, or after 14 days of HS followed by 14 days of R. Animals received HMB or the vehicle (water) daily by gavage, for 7 days of pretreatment, followed by 14 days (HS-Con and HS) or for 28 days (R-Con and R) of the respective interventions. Fiber area-to-fiber frequency distribution is shown in 100-μm2 bin widths for the plantaris (B) and soleus (D) muscles of animals in the R group. An ANOVA followed by Bonferroni post hoc analyses was used to evaluate the differences between the group means. *P < 0.05, vehicle vs. HMB within the same condition; †P < 0.05. HS or R vs. control animals for that experimental condition.

Fig. 5.

A, top row: representative tissue sections from the plantaris muscle with fluorescent staining for TUNEL (green) to identify apoptotic nuclei in control and hindlimb suspended muscles. DAPI identified all nuclei (blue). Basal lamina (red) was identified to confirm that the TUNEL-positive nuclei were myonuclei. Conditions were vehicle control (Vehicle-Con), HMB control (HMB-Con), vehicle after 14 days of HS (Vehicle-HS), and HMB after 14 days of HS (HMB-HS). Arrows show TUNEL-positive nuclei lying below or immediately adjacent to the basal lamina of the muscle fibers. Bottom row: a higher magnification showing the individual markers for TUNEL, laminin, DAPI, and the combined images. Arrows show TUNEL-positive nuclei lying below or immediately adjacent to the basal lamina of the muscle fibers. B, top row: representative tissue sections from the plantaris muscle, with fluorescent staining for TUNEL (green) to identify apoptotic nuclei in control and reloaded muscles. DAPI identified all nuclei (blue). Basal lamina (red) was identified to confirm that the TUNEL-positive nuclei were myonuclei. Conditions were the same as in A. Arrows show TUNEL-positive nuclei lying below or immediately adjacent to the basal lamina of the muscle fibers. Bottom row: a higher magnification showing the individual markers for TUNEL, laminin, DAPI, and the combined images. Arrows show TUNEL-positive nuclei lying below or immediately adjacent to the basal lamina of the muscle fibers. C: apoptotic index was calculated from tissue cross sections of the plantaris muscle by determining the ratio of TUNEL-positive nuclei to total nuclei in plantaris muscles of control animals for the HS group (HS-Con), the recovery group (R-Con), and in experimental animals after 14 days of HS or after 14 days of HS followed by 14 days of reloading (R). Only nuclei that were directly below or touching the basal lamina were counted. Animals received HMB or the vehicle (water) daily by gavage, for a total of 21 days (HS-Con and HS) or for 32 days (R-Con and R). An ANOVA followed by Bonferroni post hoc analyses was used to evaluate the differences between the group means. *P < 0.05, Vehicle vs. HMB within the same condition; †P < 0.05, HS or R vs. control animals for that experimental condition.

Fig. 6.

A, top row: representative tissue sections from the soleus muscle with fluorescent staining for TUNEL (green) to identify apoptotic nuclei in control and hindlimb suspended muscles. DAPI identified all nuclei (blue). Basal lamina (red) was identified to confirm that the TUNEL-positive nuclei were myonuclei. Conditions were vehicle control (Vehicle-Con), HMB control (HMB-Con), vehicle after 14 days of HS (Vehicle-HS), and HMB after 14 days of HS (HMB-HS). Arrows show TUNEL-positive nuclei lying below or immediately adjacent to the basal lamina of the muscle fibers. Bottom row: a higher magnification showing the individual markers for TUNEL, laminin, DAPI, and the combined images. Arrows show TUNEL-positive nuclei lying below or immediately adjacent to the basal lamina of the muscle fibers. B, top row: representative tissue sections from the soleus muscle, with fluorescent staining for TUNEL (green) to identify apoptotic nuclei in control and reloaded muscles. DAPI identified all nuclei (blue). Basal lamina (red) was identified to confirm that the TUNEL-positive nuclei were myonuclei. Conditions are the same as in A. Arrows show TUNEL-positive nuclei lying below or immediately adjacent to the basal lamina of the muscle fibers. Bottom row: a higher magnification showing the individual markers for TUNEL, laminin, DAPI, and the combined images. Arrows show TUNEL-positive nuclei lying below or immediately adjacent to the basal lamina of the muscle fibers. C: apoptotic index was calculated from tissue cross sections of the soleus muscle by determining the number ratio of TUNEL-positive nuclei to total nuclei in soleus muscles of control animals for the HS group (HS-Con), the recovery group (R-Con), and in experimental animals after 14 days of HS or after 14 days of HS followed by 14 days of reloading (R). Only nuclei that were directly below or touching the basal lamina were counted. Animals received either HMB or the vehicle (water) daily by gavage, for a total of 21 days (HS-Con and HS) or for 32 days (R-Con and R). An ANOVA followed by Bonferroni post hoc analyses was used to evaluate the differences between the group means. *P < 0.05, vehicle vs. HMB within the same condition; †P < 0.05, HS or R vs. control animals for that experimental condition.

Fig. 7.

Bax protein abundance was determined by Western blot analysis in the plantaris (A) and soleus (B) muscles of rats under control, HS, or reloading conditions. Groups include: controls for the HS group (HS-Con), controls for the recovery group (R-Con), and in experimental animals after 14 days of HS or after 14 days of HS followed by 14 days of reloading (R). Animals received HMB or the vehicle (water) daily by gavage, for a total of 21 days (HS-Con and HS) or for 32 days (R-Con and R). Eight animals were in each diet and experimental group. α-Tubulin was used as a loading control. Data were normalized to α-tubulin and were expressed as means ± SD. An ANOVA followed by Bonferroni post hoc analyses was used to evaluate the differences between the group means. *P < 0.05, vehicle vs. HMB within the same condition; †P < 0.05, HS or R vs. control animals for that experimental condition.

Fig. 8.

Cleaved caspase-9 protein abundance was determined by Western blot analysis in the plantaris (A) and soleus (B) muscles of rats under control, HS, or reloading conditions. Groups include controls for the HS group (HS-Con), controls for the recovery group (R-Con), and in experimental animals after 14 days of HS or after 14 days of HS followed by 14 days of reloading (R). Animals received HMB or the vehicle (water) daily by gavage, for a total of 21 days (HS-Con and HS) or for 32 days (R-Con and R). Eight animals were in each diet and experimental group. α-Tubulin was used as a loading control. Data were normalized to α-tubulin and were expressed as means ± SD. An ANOVA followed by Bonferroni post hoc analyses was used to evaluate the differences between the group means. *P < 0.05, vehicle vs. HMB within the same condition; †P < 0.05, HS or R vs. control animals for that experimental condition.

Fig. 9.

Cleaved caspase-3 protein abundance was determined by Western blot analysis in the plantaris (A) and soleus (B) muscles of rats under control, HS, or reloading conditions. The groups include controls for the HS group (HS-Con), controls for the recovery group (R-Con), and in experimental animals after 14 days of HS or after 14 days of HS followed by 14 days of reloading (R). The animals received HMB or the vehicle (water) daily by gavage, for a total of 21 days (HS-Con and HS) or for 32 days (R-Con and R). Eight animals were in each diet and experimental group. α-Tubulin was used as a loading control. Data were normalized to α-tubulin and were expressed as means ± SD. An ANOVA followed by Bonferroni post hoc analyses was used to evaluate the differences between the group means. *P < 0.05, vehicle vs. HMB within the same condition; †P < 0.05, HS or R vs. control animals for that experimental condition.

Fig. 10.

Bcl-2 protein abundance was determined by Western blot analysis in the plantaris (A) and soleus (B) muscles of rats under control, HS, or reloading conditions. The groups include controls for the HS group (HS-Con), controls for the recovery group (R-Con), and in experimental animals after 14 days of HS or after 14 days of HS followed by 14 days of reloading (R). The animals received HMB or the vehicle (water) daily by gavage, for a total of 21 days (HS-Con and HS) or for 32 days (R-Con and R). Eight animals were in each diet and experimental group. α-Tubulin was used as a loading control. Data were normalized to α-tubulin and were expressed as means ± SD. An ANOVA followed by Bonferroni post hoc analyses was used to evaluate the differences between the group means. †P < 0.05, HS or R vs. control animals for that experimental condition.