Abstract
Calumenin is a multiple EF-hand Ca2+-binding protein localized in the sarcoplasmic reticulum (SR) lumen. Evidence of the interaction between calumenin and SERCA2 in rat cardiac SR was shown recently (Mol. Cells, 26:265-269, 2008). To elucidate the possible role of calumenin in cardiac excitation-contraction (E-C) coupling, calumenin was knocked down by transfection of mouse cardiac cell line (HL-1 cells) with calumenin specific siRNA oligonucleotides. After 72 hrs of transfection, calumenin protein level was reduced by 75% without any obvious changes in the expression levels of other E-C coupling proteins such as RyR2, SERCA2, NCX, CSQ and PLB. A field stimulation (1Hz) of KD cells (n = 58) led to significantly increased Ca2+ transient peak height (1.02 ± 0.02 vs. 0.82 ± 0.03 fura-2 ratio at 340 and 380 nm, p < 0.05), decreased time to peak (0.093 ± 0.001 vs. 0.107 ± 0.003 s, p < 0.05) and time to 50% baseline (0.172 ± 0.005 vs. 0.235 ± 0.006 s, p < 0.05) as compared to control cells (n = 44). On the other hand, the SR Ca2+ load remained unchanged in KD cells. Pull-down experiments with GST fusion proteins showed that calumenin interacts with both RyR2 and SERCA2 in a Ca2+ dependent manner. Taken together, the present results suggest that calumenin is related to SR Ca2+ homeostasis. Currently, the molecular interactions between calumenin and SERCA2 or RyR2 are being examined by using various deletion mutants.
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