Effects of crowding on the three main proteolytic mechanisms of skeletal muscle in rainbow trout (Oncorhynchus mykiss)

Abstract

Background: Skeletal muscle is one of the tissues most affected by stress conditions. The protein degradation in this tissue is vital for the supply of energy mediated by different proteolytic pathways such as the ubiquitin-proteasome (UPS), autophagy-lysosome (ALS) and the calpain/calpastatin system (CCS). Nevertheless, the regulation of this proteolytic axis under stress conditions is not yet completely clear. Chile is the main producer of rainbow trout (Oncorhynchus mykiss) in the world. This intensive fish farming has resulted in growing problems as crowding and stress are one of the major problems in the freshwater stage. In this context, we evaluated the crowding effect in juvenile rainbow trout kept in high stocking density (30 kg/m³) for 15, 45 and 60 days, using a control group of fish (10 kg/m³).

Results: Plasmatic cortisol and glucose were evaluated by enzyme immunoassay. The mRNA levels of stress-related genes (gr1, gr2, mr, hsp70, klf15 and redd1), markers of the UPS (atrogin1 and murf1) and CCS (capn1, capn1, cast-l and cast-s) were evaluated using qPCR. ALS (LC3-I/II and P62/SQSTM1) and growth markers (4E-BP1 and ERK) were measured by Western blot analysis. The cortisol levels increased concomitantly with weight loss at 45 days of crowding. The UPS alone was upregulated at 15 days of high stocking density, while ALS activation was observed at 60 days. However, the CCS was inactivated during the entire trial.

Conclusion: All these data suggest that stress conditions, such as crowding, promote muscle degradation in a time-dependent manner through the upregulation of the UPS at early stages of chronic stress and activation of the ALS in long-term stress, while the CCS is strongly inhibited by stress conditions in the rainbow trout muscle farmed during freshwater stage. Our descriptive study will allow perform functional analysis to determine, in a more detailed way, the effect of stress on skeletal muscle physiology as well as in the animal welfare in rainbow trout. Moreover, it is the first step to elucidate the optimal crop density in the freshwater stage and improve the standards of Chilean aquaculture.

Keywords: Autophagy; Calpain/calpastatin; Fish; Stress response; Ubiquitin-proteasome.

Fig. 1

High density-induced stress on the cortisol signaling pathway in skeletal muscle. Relative expression (fold change) of (a) corticosteroid receptors gr1, gr2 and mr (b) GR target genes: redd1, klf15, and hsp70 normalized with fau and β-actin. Results are shown as the means ± SEM (n = 5 per group). LD, low stocking density; HD, high stocking density. Significant differences are indicated with a line (* = P < 0.05; ** = P < 0.01)

Fig. 2

Effect of high density-induced stress on the IGF-1 system. a Representative Western blots of key genes of IGF-1 system, ERK and p4E-BP1, in phosphorylated and total forms, with H2B as the loading control. b Fold change of pERK/ERK ratio (c) Fold change of p4E-BP1/4E-BP1 ratio. Results are shown as the means ± SEM (n = 5 per group). LD, low stocking density; HD, high stocking density. Significant differences are indicated with a line (* = P < 0.05)

Fig. 3

Effect of high density-induced stress on the ubiquitin-proteasome system. a Relative expression (fold change) of key genes of the ubiquitin proteasome system, atrogin1 and murf1, normalized with fau and β-actin. b Representative Western Blot of Ubiquitinated proteins. Coomassie blue-stained loading control proteins. Fold change of ubiquitinated proteins/free ubiquitin ratio. Results are shown as the means ± SEM (n = 5 per group). LD, low stocking density; HD, high stocking density. Significant differences are indicated with a line and an asterisk (* = P < 0.05; ** = P < 0.01; **** = P < 0.0001)

Fig. 4

Effect of high density-induced stress on the autophagy-lysosome system. Representative blots of the content of key proteins of autophagy system (a) LC3 II/I and (b) P62/SQSTM1 normalized with H2B. The results are shown as the means ± SEM (n = 5 per group). LD, low stocking density; HD, high stocking density. Significant differences are indicated with an asterisk (*** = P < 0.001)

Fig. 5

Effect of high density-induced stress on the Calpain/calpastatin system. Relative expression (fold change) of (a) calpain 1–2 and (b) the specific inhibitor calpastatin (short and long isoforms). Each gene was normalized using fau and β-actin. c Calpain enzymatic activity in relative fluorometric units (RFU). The results are shown as the means ± SEM (n = 5 per group). LD, low stocking density; HD, high stocking density. Significant differences are indicated with a line and an asterisk (* = P < 0.05; ** = P < 0.01)