Expression of calsequestrin in atrial and ventricular muscle of thermally acclimated rainbow trout.

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.

Knocking down type 2 but not type 1 calsequestrin reduces calcium sequestration and release in C2C12 skeletal muscle myotubes.

We examined the roles of type 1 and type 2 calsequestrins (CSQ1 and CSQ2) in stored Ca2+ release of C2C12 skeletal muscle myotubes. Transduction of C2C12 myoblasts with CSQ1 or CSQ2 small interfering RNAs effectively reduced the expression of targeted CSQ protein to near undetectable levels. As compared with control infected or CSQ1 knockdown myotubes, CSQ2 and CSQ1/CSQ2 knockdown myotubes had significantly reduced stored Ca2+ release evoked by activators of intracellular Ca2+ release channel/ryanodine receptor (10 mM caffeine, 200 microM 4-chloro-m-cresol, or 10 mM KCl). Thus, CSQ1 is not essential for effective stored Ca2+ release in C2C12 myotubes despite our in vitro studies suggesting that CSQ1 may enhance ryanodine receptor channel activity. To determine the basis of the reduced stored Ca2+ release in CSQ2 knockdown myotubes, we performed immunoblot analyses and found a significant reduction in both sarco/endoplasmic reticulum Ca2+-ATPase and skeletal muscle ryanodine receptor proteins in CSQ2 and CSQ1/CSQ2 knockdown myotubes. Moreover, these knockdown myotubes exhibited reduced Ca2+ uptake and reduced stored Ca2+ release by UTP (400 microM) that activates a different family of intracellular Ca2+ release channels (inositol 1,4,5-trisphosphate receptors). Taken together, our data suggest that knocking down CSQ2, but not CSQ1, leads to reduced Ca2+ storage and release in C2C12 myotubes.