Proteomic analysis of anoxia tolerance in the developing zebrafish embryo

Abstract

While some species and tissue types are injured by oxygen deprivation, anoxia tolerant organisms display a protective response that has not been fully elucidated and is well-suited to genomic and proteomic analysis. However, such methodologies have focused on transcriptional responses, prolonged anoxia, or have used cultured cells or isolated tissues. In this study of intact zebrafish embryos, a species capable of >24 h survival in anoxia, we have utilized 2D difference in gel electrophoresis to identify changes in the proteomic profile caused by near-lethal anoxic durations as well as acute anoxia (1 h), a timeframe relevant to ischemic events in human disease when response mechanisms are largely limited to post-transcriptional and post-translational processes. We observed a general stabilization of the proteome in anoxia. Proteins involved in oxidative phosphorylation, antioxidant defense, transcription, and translation changed over this time period. Among the largest proteomic alterations was that of muscle cofilin 2, implicating the regulation of the cytoskeleton and actin assembly in the adaptation to acute anoxia. These studies in an intact embryo highlight proteomic components of an adaptive response to anoxia in a model organism amenable to genetic analysis to permit further mechanistic insight into the phenomenon of anoxia tolerance.

Keywords: Anoxia; proteomic; zebrafish.

Fig. 1

Appearance of normoxic and anoxic zebrafish embryos. (A) Normoxic embryo at 24 hpf and (B) 24 hpf embryo after an additional 24 h of anoxia. (C) Two-dimensional gel of proteins labeled with cyanine dyes from zebrafish embryos at 24 hpf normoxic (Cy2), 48 hpf normoxic (Cy3) and 24 hpf + 24 h anoxia (Cy5) (Gel 3).

Fig. 2

2D-DIGE analysis of proteomic changes induced by acute anoxia using an internal standard. (A) Schematic of experimental design in which three 2D gels were run with lysates from 24 hpf embryos using the 24 hpf, normoxic sample as the internal standard (Cy2, Cy3, Cy3 in gels 1, 4, and 5 respectively), and comparing 24 hpf + 1 h normoxia (Cy3, Cy5, Cy2) with 24 hpf + 1 h anoxia (Cy5, Cy2, Cy5). (B) Heat map analysis of statistically significant (p<0.05) proteomic changes after 1 h of anoxia. The color in each panel represents the relative change compared to the internal standard (24 hpf, normoxic) in each individual gel. (C) Image of Gel 4 indicating the location of the spots comprising the heat map.

Fig. 3

Analysis of cofilin changes in acute and prolonged anoxia, and in CO₂ and KCN. (A–C) 3D quantitative representation of a spot determined to represent muscle cofilin 2 at 24 hpf, 24 + 1 h normoxia and 24 + 1 h anoxia. (D–F) Cofilin abundance in the same spot at 24 hpf, 24 + 24 h normoxia and 24 +24 h anoxia. (G–I) Cofilin abundance in 24 hpf embryos compared to 24 + 1 h in 5% CO₂ or 1 h in 500 μM KCN. Fold changes between each sample are indicated on the right.

Fig. 4

Pathways associated with proteins identified by 2D-DIGE as changing in acute or prolonged anoxia. Ingenuity Pathway Analysis software (Ingenuity Systems, Inc.) was utilized to map proteins identified onto existing pathways that are based on established interactions. Shading indicates proteins identified by 2D-DIGE/MS, while unshaded proteins represent components of these pathways not detected by 2D-DIGE/MS. The full name of each protein is given in Table S3.