2+uptake in cardiac homogenates of the cold-acclimated (CA) trout (Aho and Vornanen, 1998;Aho and Vornanen, 1999). These findings strongly suggest that temperature-induced changes in cardiac contractility in the CA trout are partly due to increases in the activity of the SR Ca 2+ pump. However, the molecular basis of these changes is still poorly elucidated.The interplay between cardiac SERCA2 and phospholamban (PLN) is crucial for Ca 2+ cycling through the SR and therefore for Accepted 5 December 2011 SUMMARY In the heart of rainbow trout (Oncorhynchus mykiss), the rate of contraction and Ca 2+ uptake into the sarcoplasmic reticulum (SR) are faster in atrial than ventricular muscle, and contraction force relies more on SR Ca 2+ stores after acclimation to cold. This study tested the hypothesis that differences in contractile properties and Ca 2+ regulation between atrial and ventricular muscle, and between warm-(WA) and cold-acclimated (CA) trout hearts, are associated with differences in expression of sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) and/or phospholamban (PLN), an inhibitor of the cardiac SERCA. Quantitative PCR (SERCA only) and antibodies raised against SERCA and PLN were used to determine abundances of SERCA2 transcripts and SERCA and PLN proteins, respectively, in atrium and ventricle of trout acclimated to cold (+4°C, CA) and warm (+18°C, WA) temperatures. Expression of SERCA2 transcripts was 1.6 and 2.1 times higher in atrium than ventricle of WA and CA trout, respectively (P<0.05). At the protein level, differences in SERCA expression between atrium and ventricle were 6.1-and 23-fold for WA and CA trout, respectively (P<0.001). Acclimation to cold increased SERCA2 transcripts 2.6-and 2.0-fold in atrial and ventricular muscle, respectively (P<0.05). At the protein level, cold-induced elevation of SERCA (4.6-fold) was noted only in atrial (P<0.05) but not in ventricular tissue (P>0.05). The expression pattern of PLN was similar to that of the SERCA protein, but chamber-specific and temperature-induced differences were much smaller than in the case of SERCA. In the ventricle, PLN/SERCA ratio was 2.1 and 7.0 times higher than in the atrium for WA and CA fish, respectively. These findings are consistent with the hypothesis that low PLN/SERCA ratio in atrial tissue enables faster SR Ca 2+ reuptake and thus contributes to faster kinetics of contraction in comparison with ventricular muscle. Similarly, cold-induced decrease in PLN/SERCA ratio may be associated with faster contraction kinetics of the CA trout heart, in particular in the atrial muscle.

SUMMARY Calsequestrin (CASQ) is the main Ca2+ 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 chamberspecific 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°C (cold acclimation, CA) or 18°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.

Cardiac function in fish acclimates to long-term temperature shifts by generating compensatory changes in structure and function of sarcoplasmic reticulum (SR) including the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2). The current study compares temperature responses of the cardiac SERCA in two fish species, burbot (Lota lota) and crucian carp (Carassius carassius), which differ in regard to thermal tolerance and activity pattern. Burbot are cold stenothermal and cold-active, while crucian carp are eurythermal and cold-dormant. The fish were acclimated at 4 °C (cold-acclimation, CA) or 18 °C (warm-acclimation, WA) and expression of SERCA proteins and transcript was measured from atrium and ventricle. Burbot heart expresses one major isoform of SERCA (110 kDa), while crucian carp heart expresses two isoforms (110 and 93 kDa). Expression of SERCA proteins was about four times higher (P < 0.05) in the heart of CA burbot than WA burbot, in both cardiac chambers. In the heart of crucian carp, thermal acclimation did not affect SERCA proteins, in either chamber (P > 0.05). The expression of SERCA transcripts did not follow the expression pattern of SERCA protein in either species, suggesting that SERCA expression is mainly regulated posttranscriptionally. These findings show that the stenothermal and cold-active burbot compensates for the decrease in ambient temperature by increasing the expression of SERCA. In the eurythermal and cold-dormant crucian carp SERCA expression is independent of temperature, while the presence of two SERCA isoforms may provide some thermal independence in SR Ca(2+) pumping.

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