Myotubes

3.4.2.1 Myoblast extraction optimisation

In order to obtain high quality and quantity myoblast cultures extracted from muscle biopsies the method was first optimised comparing two different methods. Cells were extracted using the modified method described by Wehner et al. [143]. Myoblasts were enriched but the population of fibroblasts was too high so cells could not be readily differentiated as fibroblasts tended to overgrow the myoblasts. Therefore the explant method was used to prepare myoblasts. This method was optimised to support rapid myoblast proliferation and generally pure myoblast cultures were obtained. The literature described growing myoblasts in DMEM supplemented with 20 % FCS and 1% penicillin/streptomycin but this seemed to improve fibroblast proliferation. The use of Ham’s F10 medium supplemented with 20% FCS, 1% P/S and 40 ng/µL rhFGF seemed to be an optimal growth medium for myoblasts (adapted from [175]) and has been described for primary myoblast cultures previously [181, 182]. Separation of myoblasts and fibroblasts by the addition of PBS [175] was trialled and found to be suitable because myoblasts would detach and could be removed for separate cultures while fibroblast-like cells remained attached to the plate. This optimised protocol led to relatively pure myoblast cultures which could be used for differentiation into myotubes and subsequent use in Ca2+ release assays. Immunostaining, using the myocyte-specific antibody desmin, was carried out to confirm success of the myoblast extraction protocol.

3.4.2.2 Ca2+ release assays

For Ca2+ release assays collagen-coated 96-well plates were used. Differentiation took, depending on the cell line, between 1 and 5 weeks and progress was monitored regularly. Multiple differentiated myotubes were present in a single well and were observed under the microscope simultaneously and stimulated with 4CmC. Cells, depending on their

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differentiation state and also the size and the number of nuclei, reacted with different intensities to the agonist. Therefore averaged results for one given 4CmC concentration were calculated in an attempt to obtain robust data. The SEM (between 14 and 30 myotubes for each 4CmC concentration) was also calculated. Data showed that R2452W positive cells responded to much lower 4CmC concentrations compared to MHN cells C1 and C2. The EC50

value for patient A was approximately half that of control patients C1 and C2 (table 3.6).

Ca2+ release assays for patients carrying the adjacent I2453T mutation [110], estimated the EC50 value at 93.9 µM 4CmC compared to EC50 values for

MHN patients ranging between 192.5 and 352.3 µM 4CmC. While these values are different to those observed in the current study the functional assays for myotubes carrying the I2453T mutation were carried out in buffer containing 2 mM free Ca2+. This could influence the amount of Ca2+ released from the stores as demonstrated previously for B-lymphoblastoid cells. Nevertheless the difference in EC50 values between MHN and MHS cell lines

were relatively similar, being approximately threefold.

Ca2+ release in myotubes measured using Krebs-Ringer solution containing 0.5 mM EGTA was studied by Ducreux et al. [151]. EC50 values of 10 µM

4CmC for cells carrying an MH-associated mutation V2168M and 50 µM for the CCD-associated mutation I4898T have been found as well as EC50 value

of 120 µM 4CmC for a MHN control individual. Wehner et al., using an imaging buffer containing 2 mM Ca2+, obtained EC50 values of 118 µM 4CmC

for MHS and 210 µM 4CmC for MHN individuals [183]. Discrepancies between different studies cannot be easily explained but indicate the importance of standard assay conditions to accurately compare MHN and MHS samples.

Ca2+ release assays using myotubes have also been studied by Kaufmann et al. [184] using 4CmC as well as caffeine and halothane as RYR1 agonists. Their study showed EC50 values of 100 µM 4CmC for MHN control

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show a hypersensitive ryanodine receptor 1 for myotubes carrying the R2458H mutation when using 4CmC as agonist. Nevertheless the R2458H mutation has already been classed as MH causative by the EMHG according to Yang et al. [141].

These examples indicate that even though relative EC50 values differ

between different assays and conditions, in general a two- to threefold decrease in EC50 for MH or CCD causative mutations is observed compared

to WT RYR1. This conclusion is supported by the present study where EC50

values were halved, compared to MHN cells, in patient A carrying the R2452W mutation (table 3.6). Ca2+ _{release in both myotubes and}

lymphocytes from patient A was altered. Since B-lymphoblastoid cells from two unrelated families with the R2452W mutation gave a MHS phenotype in Ca2+ _{release assays it is likely that the R2452W mutation is responsible for}

this phenotype since lymphocytes do not express any other proteins associated with muscle contracture or the muscle triad. This leads to the conclusion that R2452W can be classed as an MH causative mutation.

Ca2+ release assays for myotubes from two unrelated MHE patients U1 and U2 gave less clear results. EC50 values were determined and showed that

values for patient U1 were in the same range as for MHS patient A. The error bars obtained for these measurements are small and not overlapping with the control patient and statistical analysis of the results show a significant difference between U1 and C1, classing this patient as MHS using myotubes, indicating a hyperactive RYR1 channel. Therefore the MHE classification from the IVCT may have been incorrect. Taking the results for the B- lymphoblastoid cell Ca2+ release assays into account, RYR1 can be excluded as candidate gene for MH in patient U1. Ca2+ release data for patient U2 are equivocal. Sigmoid curve fitting for myotubes Ca2+ release data resulted in a curve intermediate between those of MHN and MHS samples (figure 3.12). Considering the individual data points, only Ca2+ release in response to 800 µM is significantly increased to the level observed for MHS samples. For other 4CmC concentrations the measured Ca2+ release appeared similar to MHN patients. Taking into account the B-lymphoblastoid Ca2+ release data, it

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is unclear whether this patient had a false positive IVCT result, a mutation in a gene other than RYR1, involved in Ca2+ release in muscle cells or this patient is correctly classified as MHE and may or may not be susceptible to triggering agents.

There are several reports showing that 4CmC is not able to discriminate between MHE and MHS cells [185, 186]. Weigl et al. [186] failed to demonstrate altered Ca2+ release in MH equivocal myotubes with caffeine leading to the assumption that neither 4CmC nor caffeine can be used as agonist to demonstrate functional effects in MHE cells. If both agonists failed to show altered Ca2+ _{release it is possible that the IVCT gave a false positive}

result. Kaufman et al. [184] showed that R2458H positive myotubes failed to show a hypersensitive Ca2+ release with 4CmC, but altered Ca2+ was shown when stimulated with caffeine. This suggests that some patients might carry mutations which can only be detected with Ca2+ release assays using caffeine as agonist which might be the case for patient U2. To obtain further information about Ca2+ release in myotubes of patient U2, further assays could be performed using caffeine as the agonist. Caffeine also activates RYR3 and therefore a more non-specific reaction may be detected [100, 101] which may confound results.

In summary, patient B-lymphoblastoid cells and/or myoblasts can be used in Ca2+ release assays to determine whether a RYR1 mutation alters Ca2+ release from the SR. This study shows that patients from two unrelated families carrying the R2452W mutation show altered Ca2+ release resulting in a hypersensitive receptor phenotype. These results confirm that the R2452W is causative of MH and this study can be used to support the addition of the R2452W mutation to the list of MH causative mutations. MHE samples used in this study did not show altered Ca2+ release in B-lymphoblastoid cells and therefore may not carry a mutation in RYR1. Myotube Ca2+ release assays did not yield a clear result for patient U2 while patient U1 can be classed as MHS according to myotube assays. Since this patient showed a negative phenotype in B-lymphoblastoid cells this patient is likely to carry a mutation in a different, as yet unidentified, gene involved in Ca2+ release in muscle cells.

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Chapter 4 Functional characterisation of the