Development aggravates the severity of skeletal muscle catabolism induced by endotoxemia in neonatal pigs

Abstract

Accretion rates of muscle protein are elevated in normal neonates, but this anabolic drive decreases with maturation. As this change occurs, it is not known whether development also influences muscle protein catabolism induced by sepsis. We hypothesize that protein degradation in skeletal muscle induced by endotoxemia becomes more severe as the neonate develops. Fasted 7- and 26-day-old pigs were infused for 8 h with LPS (0 and 10 μg·kg(-1)·h(-1)), while plasma amino acids (AA), 3-methylhistidine (3-MH), and α-actin concentrations and muscle protein degradation signal activation were determined (n = 5-7/group/age). Plasma full-length α-actin was greater in 7- than 26-day-old pigs, suggesting a higher baseline protein turnover in neonatal pigs. LPS increased plasma total AA, 3-MH, and full-length and cleaved α-actin in 26- than in 7-day-old pigs. In muscle of both age groups, LPS increased AMPK and NF-κB phosphorylation, the abundances of activated caspase 3 and E-3 ligases MuRF1 and atrogin1, as well as the abundance of cleaved α-actin, suggesting activation of muscle proteolysis by endotoxin in muscle. LPS decreased Forkhead box 01 (Fox01) and Fox04 phosphorylation and increased procaspase 3 abundance in muscle of 26-day-old pigs despite the lack of effect of LPS on PKB phosphorylation. The results suggest that skeletal muscle in healthy neonatal pigs maintains high baseline degradation signal activation that cannot be enhanced by endotoxin, but as maturation advances, the effect of LPS on muscle protein catabolism manifests its severity.

Fig. 1.

Plasma 3-methyl histidine (3-MH) concentration (A) and full-length (42 kDa) α-actin abundance (B) in 7- and 26-day-old pigs infused with LPS for 8 h. 3-MH was measured by HPLC, and α-actin abundance was estimated by Western blot analysis and was normalized to the total protein content in the aliquot. Values are means ± SE; n = 5–7/treatment/age. ^a,b,cValues with different superscript letters differ significantly (P < 0.05).

Fig. 2.

PKB and AMPK phosphorylation (A and C, respectively) and abundances (B and D, respectively) in skeletal muscle of 7- and 26-day-old pigs infused with LPS for 8 h. Amounts of the phosphorylated (p) forms of each protein were normalized to its abundance (t), which is the total protein content recovered from the immunoblot. Values are expressed as means ± SE; n = 5–7/treatment/age. ^a,bValues with different superscript letters differ significantly (P < 0.05).

Fig. 3.

Fox01 (A) and Fox04 (B) phosphorylation in skeletal muscle of 7- and 26-day-old pigs infused with LPS for 8 h. Amounts of the phosphorylated (p) forms of each protein were normalized to its abundance (t), which is the total protein content recovered from the immunoblot. Values are expressed as means ± SE; n = 5–7/treatment/age. ^a,b,cValues with different superscript letters differ significantly (P < 0.05).

Fig. 4.

NF-κB phosphorylation (A) and procaspase 3 (B) and activated (cleaved) caspase 3 abundances (C) in skeletal muscle of 7- and 26-day-old pigs infused with LPS for 8 h. Amounts of the phosphorylated (p) form of NF-κB were normalized to its abundance (t), which is the total protein content recovered from the immunoblot. Procaspase 3 and cleaved caspase 3 abundances were normalized to total β-actin abundance recovered from the immunoblot. Values are expressed as means ± SE; n = 5–7/treatment/age. ^a,b,c,dValues with different superscript letters differ significantly (P < 0.05).

Fig. 5.

Atrogin1 (A) and MuRF1 (B) abundances in skeletal muscle of 7- and 26-day-old pigs infused with LPS for 8 h. Abundances were normalized to total β-actin abundance recovered from the immunoblot. Values are expressed as means ± SE; n = 5–7/treatment/age. ^a,b,cValues with different superscript letters differ significantly (P < 0.05).

Fig. 6.

Cleaved (14 kDa) α-actin abundance in muscle (A) and plasma (B) in 7- and 26-day-old pigs infused with LPS for 8 h. α-Actin abundance was estimated by Western blot analysis and normalized to the total protein content in the aliquot. Values are expressed as means ± SE; n = 5–7/treatment/age. ^a,b,cValues with different superscript letters differ significantly (P < 0.05).