CAPUT MORTUM

Ca2+ release assay conditions in myotubes were chosen to be the same as for B-lymphoblastoid cell assays. They were carried out for cells derived from patient A and for control patients C1 and C2 only since no cells were available from any other patients carrying the R2452W mutation. Myoblasts were seeded into collagen-coated 96-well plates and grown to about 70 % confluence before the medium was changed to differentiation medium containing 2 % horse serum and 1 % penicillin/streptomycin. Cells were left to differentiate for 2 to 3 weeks depending on the cell line.

For Ca2+ release assays, individual cells were selected under the microscope and Ca2+ release was measured in response to different concentrations of 4CmC. Results from individual cells were combined and the average of Ca2+ released in response to 4CmC was calculated. Ca2+ release was calculated as the % of Ca2+ released normalised to 1 mM 4CmC. This is shown in figure 3.11A while an example of raw data for Ca2+ _{release of patient C1 in}

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Figure 3.11 Ca2+ release data and concentration-response curve for myotubes upon 4CmC stimulation.

A) Ca2+ release data normalised to 1 mM 4CmC. Values are given as the average of all measurements ± SEM (n= 14-30) depending on myotubes availability. B) shows an example of raw data from Ca2+ release assays in myotubes when stimulated with 1 mM 4CmC. As previously shown for B-lymphoblastoid cells, the baseline ratio of 340/380 nm was determined before the addition of the agonists. The peak Ca2+ release was determined and subtracting the ratio for the peak Ca2+ release from the baseline ratio yields the Δ Ca2+ release.

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Because of high variability between Ca2+ releases of individual cells, which could be at different stages of differentiation, results of all individual cells were averaged and one sigmoid fitted curve was obtained and the EC50 read

from the graph. The resulting EC50 values are shown in table 3.6.

patients EC50 values (μM 4CmC) ±SEM MH status

A 309±45 * MHS

C1 612±54 MHN

C2 638±11 MHN

Table 3.6 EC50 values for 4CmC stimulation calculated from concentration-response curves

using myotubes.

EC50 values were calculated from a sigmoid fitted curve and expressed as the average EC50

value ± SEM (n=14-30). Significantly different EC50 values compared to MHS controls C1

and C2 are marked with an asterisk.

The EC50 value for patient A was significantly lower using One-Way ANOVA

with Bonferroni post-hoc analysis when compared to both control individuals (p< 0.005) whereas there was no significant difference between samples C1 and C2 (p>0.05). Ca2+ release for myotubes shows activation at lower 4CmC concentrations indicating higher Ca2+ release compared to both control patients. Concentration-response curves for C1 and C2 result in different EC50 values but are not statistically different. Since these two patients are not

related the differences could be due to the genetic background or within experimental error.

Ca2+ _{release assays for the MHE samples were carried out as described}

above but cells of patient U1 were differentiated for 2 weeks whereas cells of patient U2 took almost 5 weeks to differentiate into myotubes.

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Figure 3.12 Concentration-response curve using myotubes of MHE patients after 4CmC stimulation.

Values are presented as the average of all measurements ± SEM (n= 6-30) and are normalised to 1 mM 4CmC.

Figure 3.12 shows concentration-response curves fitted using a sigmoid curve function. Myoblasts for patient U1 showed similar Ca2+ release as MHS patient A however Ca2+ release in response to 100 µM 4CmC was lower. Ca2+ release for patient U1 was high for all 4CmC concentrations above 100 µM which would class this patient as MHS (compared to MHS individual A). The sigmoid curves for both patients were similar suggesting patient U1 has a MHS phenotype in muscle cells. On the other hand patient U2 showed similar Ca2+ release for 400 and 600 µM 4CmC as the MHN control whereas Ca2+ release is much higher for 200 and 800 µM 4CmC where values are similar to MHS patient A. Therefore patient U2 cannot be classified as either MHN or MHS using this assay. EC50 values for all samples were calculated

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patients EC50 values (μM 4CmC) ±SEM MH status

A 309±45 * MHS

C1 612±54 MHN

U1 334±87 * MHEh

U2 473±11 MHEh

Table 3.7 EC50 values for myotubes of MHE patients upon 4CmC stimulation.

Values are presented as the average of all measurements ± SEM (n= 6-30) and are normalised to 1 mM 4CmC. Significantly different EC50 values compared to MHN control C1

are marked with an asterisk.

Results were subject to One-Way ANOVA for each individual 4CmC concentration measured using a Bonferroni post-hoc analysis to determine significant differences between either MHN and MHE or MHS and MHE samples. A significant difference was found between samples from patient A and C1 (p<0.05) for all 4CmC concentrations used. U1 and C1 showed significant differences for all concentrations apart from 100 µM 4CmC at a confidence interval of 95 %. U1 compared to C1 showed significant differences for most 4CmC concentrations measured, apart from 100 and 600 µM 4CmC which could be due to the smaller sample size used (n=6), therefore patient C1 could be classed as MHS.