New method for determining total calcium content in tissue applied to skeletal muscle with and without calsequestrin.
Bottom Line: In both fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscles from mice, [CaT]WM increased approximately linearly with decreasing muscle weight, increasing approximately twofold with a twofold decrease in muscle weight.Knocking out the high capacity Ca-binding protein calsequestrin (CSQ) did not significantly reduce [CaT]WM in mouse EDL or soleus muscle.Because greater reductions in [CaT]WM would be predicted in both muscle types, we hypothesize that there is a substantial increase in Ca bound to other sites in the CSQ knockout muscles.
Affiliation: Institute of Sport, Exercise and Active Living, Victoria University, Melbourne, Victoria 8001, Australia.Show MeSH
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Mentions: Inverse relationship between [CaT]WM and muscle weight in muscles from mice. (A–D) The open circles in A and C plot [CaT]WM versus muscle weight for EDL and soleus muscles, respectively, from a set of 4–6-mo-old C57BL/6 mice. B and D plot these same results in A and C, respectively, along with several other sets of muscles. Each set of muscles is plotted with a different symbol, and all muscles in a set were processed together on the same day or, in one case, a 2-d period. As for the set plotted with open circles from A and C, the sets plotted with open squares and open diamonds were also obtained with 4–6-mo-old C57BL/6 mice. All of the other four sets were genetically modified to allow for selective deletion of normally expressed genes. Because the genes function normally and because the mice were crossbred on background C57BL/6 or C57BL/6J mice, results for these other four sets would not be expected to differ from those with the C57BL/6 mice. For the two sets plotted with + and × symbols, the mice (RANKfloxed/floxed) were genetically modified to allow for deletion of the gene for the receptor activator of nuclear factor κB (RANK), an enzyme involved in regulating bone remodeling. These mice also underwent mock surgical experiments involving exposing the sciatic nerve without cutting it, and then closing the wound 2 wk before the measurements here. Because this would not be expected to alter [CaT]WM, it seems reasonable to include these sets. For the two sets plotted with open triangles and open-inverted triangles, the mice were modified to allow for deletion of CSQ (results given in Fig. 7). The RANKfloxed/floxed mice were 3–4 mo old, somewhat younger than the range of 4 to 6 mo for the mice in the other sets. The lines in each panel were obtained with least-squares best fits to the data. The best-fit slopes in units of millimoles/kilogram per milligram of muscle weight (y intercepts in millimoles/kilogram; p-values) for the lines in A–D are, respectively, −0.2866 (5.13; P = 0.0072), −0.2347 (4.92; P < 0.0001), −0.2489 (4.48; P = 0.0539), and −0.3102 (5.46; P < 0.0001). The average values (and n values) of [CaT]WM in A–D are, respectively, 2.30 (16), 2.71 (54), 2.02 (16), and 2.62 (54). For EDL muscles in A and B, the horizontal scales on top were calculated with the linear least-squares best-fit line of R versus muscle weight in Fig. S5 E given by R = −0.187 × muscle weight + 4.59. The corresponding relationship for soleus muscles in C and D is given by R = −0.239 × muscle weight + 5.14.
Affiliation: Institute of Sport, Exercise and Active Living, Victoria University, Melbourne, Victoria 8001, Australia.