Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca2+-binding protein changes in CSQ2 knockout mice.
Calsequestrin 2 (CSQ2) is generally regarded as the primary Ca2+-buffering molecule present inside the sarcoplasmic reticulum (SR) in cardiac cells, but findings from CSQ2 knockout experiments raise major questions about its role and necessity. This study determined the absolute amount of CSQ2 present in cardiac ventricular muscle to gauge its likely influence on SR free Ca2+ concentration ([Ca2+]) and maximal Ca2+ capacity. Ventricular tissue from hearts of freshly killed sheep was examined by SDS-PAGE without any fractionation, and CSQ2 was detected by Western blotting; this method avoided the >90% loss of CSQ2 occurring with usual fractionation procedures. Band intensities were compared against those for purified CSQ2 run on the same blots. Fidelity of quantification was verified by demonstrating that CSQ2 added to homogenates was detected with equal efficacy as purified CSQ2 alone. Ventricular tissue from sheep (n=8) contained 24±2 μmol CSQ2/kg wet wt. Total Ca2+ content of the ventricular tissue, measured by atomic absorption spectroscopy, was 430±20 μmol/kg (with SR Ca2+ likely<250 μmol/kg) and displayed a linear correlation with CSQ2 content, with gradient of ∼10 Ca2+ per CSQ2. The large amount of CSQ2 bestows the SR with a high theoretical maximal Ca2+-binding capacity (∼1 mmol Ca2+/kg ventricular tissue, assuming a maximum of ∼40 Ca2+ per CSQ2) and would keep free [Ca2+] within the SR relatively low, energetically favoring Ca2+ uptake and reducing SR leak. In mice with CSQ2 ablated, histidine-rich Ca2+-binding protein was upregulated ∼35% in ventricular tissue, possibly in compensation.
Department of Zoology, La Trobe University, Melbourne, Victoria, 3086, Australia.
Robyn M
RM
Murphy
Janelle P
JP
Mollica
Nicole A
NA
Beard
Bjorn C
BC
Knollmann
Graham D
GD
Lamb
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002155:Calsequestrin / Q000235:genetics / Q000378:metabolism*; D006352:Heart Ventricles; D051379:Mice; D018345:Mice, Knockout; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012756:Sheep; D016622:Silver Staining; D013054:Spectrophotometry, Atomic
D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D002118:Calcium