Cloning and characterization of the executioner caspases 3, 6, 7 and Hsp70 in hyperthermic Atlantic salmon ( Salmo salar) embryos

Abstract

Hyperthermia during embryogenesis has been reported to induce deformities in Atlantic salmon ( Salmo salar). To examine the involvement of executioner caspases in hyperthermia-induced cell-death in a poikilotherm vertebrate species, five genes encoding caspase-3,-6, and -7 were cloned from Atlantic salmon, and the expression was studied in thermal stressed salmon embryos. The salmon genome contained two genetically distinct variants of both salmon caspase-3 and caspase-6 that is likely the result of two independent chromosome or genome duplications. Whereas only partial caspase-3A encoding sequences were isolated, the full-length caspase-3B cDNA encodes the inactive proenzyme of 279 amino acids (aa) consisting of an N-terminal prodomain and the large and the small subunit. The salmon caspase-6A and caspase-6B proenzymes include an additional linker region between the two subunits. The deduced salmon caspase-7 consists of only 245 aa and lacks the prodomain and part of the large subunit similar to the predicted caspase-7 of the puffer fish Tetraodon sp.. Increased apoptotic activity as evidenced by cleavage of nuclear DNA was demonstrated in salmon embryos incubated at 18–20 °C for 84 h after acclimatization at 8 °C. Hyperthermia-induced activation of the executioner caspases was indicated by the increased mRNA levels of caspase-3B, caspase-6A/B and caspase-7 after 54 h heat exposure as quantified by real-time RT-PCR. The 2–2.5 fold increase in the mRNA expression of the heat shock protein Hsp70 gene coincided with the peak mRNA values of the executioner caspases. Whole-mount in situ hybridization of the salmon embryo identified caspase-7 mRNA in the lens exclusively, while caspase-3B and caspase-6A/B were expressed in multiple tissues of exposed and control embryos. Interestingly, cardiac expression of caspase-6A/B was only identified in heat stressed embryos . Altogether, these results shed light on evolutionary aspects of the executioner caspases in vertebrates and their expression in salmon embryos exposed to hyperthermia. In particular, the heat sensitive caspase-6 expression in the embryonic heart is of interest since cardiac malformations are an emergent problem in salmon aquaculture.