Heat stress causes oxidative stress but not inflammatory signaling in porcine skeletal muscle

Abstract

Heat stress is associated with death and other maladaptions including muscle dysfunction and impaired growth across species. Despite this common observation, the molecular effects leading to these pathologic changes remain unclear. The purpose of this study was to determine the extent to which heat stress disrupted redox balance and initiated an inflammatory response in oxidative and glycolytic skeletal muscle. Female pigs (5-6/group) were subjected to thermoneutral (20 °C) or heat stress (35 °C) conditions for 1 or 3 days and the semitendinosus removed and dissected into red (STR) and white (STW) portions. After 1 day of heat stress, relative abundance of proteins modified by malondialdehyde, a measure of oxidative damage, was increased 2.5-fold (P < 0.05) compared with thermoneutral in the STR but not the STW, before returning to thermoneutral conditions following 3 days of heat stress. This corresponded with increased catalase and superoxide dismutase-1 gene expression (P < 0.05) and superoxide dismutase-1 protein abundance (P < 0.05) in the STR but not the STW. In the STR catalase and total superoxide dismutase activity were increased by ~30% and ~130%, respectively (P < 0.05), after 1 day of heat stress and returned to thermoneutral levels by day 3. One or 3 days of heat stress did not increase inflammatory signaling through the NF-κB pathway in the STR or STW. These data suggest that oxidative muscle is more susceptible to heat stress-mediated changes in redox balance than glycolytic muscle during chronic heat stress.

Keywords: NF-kB; free radicals; inflammation; mitochondria; oxidative; pig.

Figure 1. Heat stress increased oxidative injury in oxidative, but not glycolytic muscle. Oxidative stress was measured by quantifying the relative abundance of MDA modified proteins, a marker of lipid peroxidation. (A) MDA modified proteins increased 2.5-fold compared with TN after 1 d of HS in the STR (TN, n = 8; 1 HS, n = 5; 3 HS, n = 5) (B) but remained unchanged in the STW (TN, n = 6; 1 HS, n = 5; 3 HS, n = 4). Representative blots are shown as well as corresponding Ponceau S staining to demonstrate equal loading. *indicates significantly different from TN; P < 0.05.

Figure 2. Gene expression of antioxidant enzymes following heat stress. (A) HS increased gene expression of both CAT (TN, n = 6; 1 HS, n = 6; 3 HS, n = 6) and SOD2 (TN, n = 6; 1 HS, n = 5; 3 HS, n = 6) following 1 d of HS, but not that of SOD1 (TN, n = 6; 1 HS, n = 5; 3 HS, n = 6) in the STR. Expression of all antioxidant enzymes was decreased after 3 d of HS compared with 1 d of HS in the STR. (B) HS increased SOD2 gene expression after 1 d of HS compared with TN, though expression of all antioxidant enzymes was decreased by day 3 in the STW (TN, n = 6; 1 HS, n = 6; 3 HS, n = 6). *indicates significantly different from TN; # indicates significantly different from 1-HS; P < 0.05.

Figure 3. Protein expression of catalase and SOD following heat stress. (A) Catalase protein content was unchanged with HS in the STR. However, MnSOD protein content was increased after 1 and 3 d of HS in the STR. (B) Catalase and MnSOD protein content remain unchanged with HS in the STW. Representative blots are shown as well as corresponding Ponceau S staining to demonstrate equal loading. * indicates significantly different from TN; P < 0.05.

Figure 4. Antioxidant enzyme activities following HS treatment in porcine skeletal muscle. (A) One day of HS increased catalase activity in STR (TN, n = 6; 1 HS, n = 5; 3 HS, n = 6) (B) but not STW (TN, n = 6; 1 HS, n = 6; 3 HS, n = 6) compared with TN. (C) Likewise, SOD activity was increased after 1 d of HS in the STR (TN, n = 10; 1 HS, n = 6; 3 HS, n = 6) (D) but remained similar in STW (TN, n = 10; 1 HS, n = 6; 3 HS, n = 6). *Indicates significantly different from TN; # indicates significantly different from 1-HS; P < 0.05.

Figure 5. Protein expression of inflammatory signaling molecules in the NF-κB pathway. (A) In STR, IkB-α, NF-κB, phospho-NF-κB and IL-6 protein abundance were similar between all treatment groups in the STR, however, TNF-α was increased 2.5-fold following 1 and 3 d of HS (n = 5–6/group). (B) In the STW, TNF-α, NF-κB and phospho-NF-κB protein content remain unchanged, however, HS decreased IkB-α and IL-6 protein content (n = 5–6/group). Representative blots are shown as well as corresponding Ponceau S staining to demonstrate equal loading. *indicates significantly different from TN; P < 0.05.

Figure 6. Gene expression of inflammatory cytokines in skeletal muscle following HS. (A) Gene expression of TNF, IL1B, and IL15 was similar between all treatment groups in STR (TNF and IL1B n = 6/group; IL15 TN n = 8, 1 HS = 3, 3 HS = 5). (B) Gene expression of TNF and IL1B, was similar between all treatment groups, but IL15 was increased after 3 d of HS in STW (TNF and IL1B n = 6/group; IL15 TN n = 8, 1 HS = 5, 3 HS = 4). *Indicates significantly different from TN; P < 0.05.