Calsequestrin (CASQ) is the main Ca(2+) binding protein within the sarcoplasmic reticulum (SR) of the vertebrate heart. The contribution of SR Ca(2+) stores to contractile activation is larger in atrial than ventricular muscle, and in ectothermic fish hearts acclimation to low temperatures increases the use of SR Ca(2+) in excitation-contraction coupling. The hypotheses that chamber-specific and temperature-induced differences in SR function are due to the increased SR CASQ content were tested in rainbow trout (Oncorhynchus mykiss) acclimated at either 4 degrees C (cold acclimation, CA) or 18 degrees C (warm acclimation, WA). To this end, the trout cardiac CASQ (omCASQ2) was cloned and sequenced. The omCASQ2 consists of 1275 nucleotides encoding a predicted protein of 425 amino acids (54 kDa in molecular mass, MM) with a high (75-87%) sequence similarity to other vertebrate cardiac CASQs. The transcript levels of the omCASQ2 were 1.5-2 times higher in CA than WA fish and about 2.5 times higher in the atrium than ventricle (P<0.001). The omCASQ2 protein was measured from western blots using a polyclonal antibody against the amino acid sequence 174-315 of the omCASQ2. Unlike the omCASQ2 transcripts, no differences were found in the abundance of the omCASQ2 protein between CA and WA fish, nor between the atrium and ventricle (P>0.05). However, a prominent qualitative difference appeared between the acclimation groups: two CASQ isoforms with apparent MMs of 54 and 59 kDa, respectively, were present in atrial and ventricular muscle of the WA trout whereas only the 54 kDa protein was clearly expressed in the CA heart. The 59 kDA isoform was a minor CASQ component representing 22% and 13% of the total CASQ proteins in the atrium and ventricle of the WA fish, respectively. In CA hearts, the 59 kDa protein was present in trace amounts (1.5-2.4%). Collectively, these findings indicate that temperature-related and chamber-specific differences in trout cardiac SR function are not related to the abundance of luminal Ca(2+) buffering by cardiac CASQ.