Dantrolene is a skeletal muscle relaxant and the only clinically available drug for treatment of MH.21 We examined the effects of dantrolene on WT-RyR1–transfected cells, Ala4894Thr-RyR1–transfected cells, and Ala4894Ser-RyR1–transfected cells. Cells were perfused with 10 μM dantrolene in HBSS for 15 minutes, then loaded with increasing concentrations of caffeine solutions containing 10 μM dantrolene for 2 minutes. We plotted the dose-response curves of WT-RyR1–transfected cells, Ala4894Thr-RyR1–transfected cells, and Ala4894Ser-RyR1–transfected cells to caffeine with dantrolene and found that the curves were shifted to the right as compared with those without dantrolene (Fig. 3). The 50% effective concentration values of WT-RyR1–transfected cells, Ala4894Thr-RyR1–transfected cells, and Ala4894Ser-RyR1–transfected cells for caffeine with dantrolene were 3.18 ± 0.31, 2.03 ± 0.54, and 1.21 ± 0.03 mM, respectively (Table 1).
The present study revealed that MH-associated, Ala4894Thr-RyR1–transfected cells are more sensitive to caffeine and 4CmC than WT-RyR1 cells, whereas those responses were inhibited by dantrolene. In addition, CNMDU1-associated, Ala4894Pro-RyR1–transfected cells were found to be insensitive to caffeine and 4CmC, whereas cells transfected with Ala4894Ser, a new artificial variant, were also more sensitive to caffeine than WT cells. Our findings also showed that cells transfected with Ala4894Gly, another new artificial variant, were less sensitive to caffeine than WT cells. Together, these results indicate that different RyR1 mutations at Ala4894 lead to different physiological responses, which may represent clinically relevant findings.
Most mutations reported in studies of MH patients are located in the N-terminal or central domains. Ala4894Thr, found in a Japanese MH patient, is a mutation located in the C-terminal domain. That patient was clinically diagnosed with MH and examined with the calcium-induced calcium release (CICR) rate test.2 In Japan, a CICR rate test using skinned muscle fibers was performed to diagnose susceptibility to MH24–26 and the patient had a significantly accelerated CICR rate (Table 4). The present results indicate that the Ala4894Thr-RyR1 mutation is consistent with known responses of the MH-causal RyR1 mutation.
We found that dantrolene suppressed the response of Ala4894Thr-transfected cells to caffeine. Dantrolene shows clinical efficacy in many MH cases; however, the scientific evidence that dantrolene is effective for any mutations of RyR1 that cause MH has not been obtained, and it is difficult to solve the problem. Therefore, it is important to show the effect of dantrolene for each variant reported from MH. Our results indicate that dantrolene may be effective for MH associated with Ala4894Thr.
We also found that Ala4894Pro-transfected cells, as a homozygous model, were completely insensitive to caffeine and 4CmC. Because humans are heterozygous for nearly all RyR1 mutations, and because such patients survive, albeit with modified skeletal muscle function, these patients are presumably heterozygous as well. We therefore hypothesize that this mutation would bestow a relative insensitivity of RyR1 to normal agonist stimuli during normal physiological function in these individuals, resulting in a decrement of excitation-coupled Ca2+ release and decreased contractile function. This is in line with the recent classification of mutated RyR1 into 3 types: leaky channels, EC uncoupling, and overactive channels. Leaky channels, which remain in an open state and leak Ca2+ from the SR, cause depletion of Ca2+ in the SR and increase of intracellular Ca2+ in a resting state. As a result, there is a decrease in Ca2+ release in an excited state, which causes muscle weakness.27 The Tyr523Ser CCD mutant was reported as leaky channels.28 EC uncoupling is a dysfunction of the timing of the open states of 2 receptors, dihydropyridine receptor (DHPR) and RYR1, disabling their coupling. DHPR is an L-type Ca2+ channel and known as a voltage sensor of skeletal muscle. The interaction of DHPR with RYR1 regulates the release of Ca2+ from SR. Consequently, EC uncoupling leads to lack of Ca2+ release from the SR.29 The Ile4897Thr CCD mutant was reported as EC uncoupling.30 Overactive channels are hypersensitive channels associated with MH. In our experiments, there was no difference in intracellular Ca2+ between WT- and Ala4894Pro-transfected cells in a resting state and maximal response. These results indicate that the amount of Ca2+ in the ER of Ala4894Pro-transfected cells is not different from that of WT-transfected cells, which leads us to conclude that the Ala4894Pro mutant is not the leaky channels type. EC uncoupling is insensitive to caffeine loading.27 Therefore, our results may indicate that Ala4894Pro-RyR1 is the EC uncoupling type. However, it is necessary to evaluate the coupling functions of DHPR and RyR1 before making a final conclusion. There are no data yet to link this RyR1 mutation and perturbations in Ca2+ release fluxes to the lack of development of type 2 fibers in CNMDU1, which also must contribute to the myopathy seen in these patients.
We also noted that Ala4894Ser-RyR1–transfected cells were more sensitive to caffeine. Because both serine and threonine contain a hydroxyl group, the hydroxyl group of amino acids at Ala4894 may enhance RyR1 function. Amino acids 4895 to 4901 correspond to pore-forming regions,31–33 and Ala4894 is the amino acid adjacent to the beginning of this region. Mutations in the pore-forming region have been reported to change channel conductance and ion selectivity.31 Similarly, replacement of Ala4894 with an amino acid that contains a hydroxyl group may change channel gating and/or Ca2+ conductance in such a manner as to enhance RyR1 function and make it more sensitive to trigger MH.
Ala4894Gly-transfected cells were less sensitive to caffeine. The characteristics of glycine are similar to alanine. However, the function of Ala4894Gly was found to be different from that of the WT. Most mutations in the C-terminal domain have been reported in patients with congenital myopathy,34–37 and various artificial variations in the pore-forming region are reported to lead to RyR1 dysfunction.30 Additional studies of variants in the pore-forming region may provide useful knowledge regarding RyR1 function.
The present study was a cell culture–based, functional evaluation of transfected transgene-RyR1, both WT and specifically mutated. Although it is preferable to use tissue-specific biological models, such as murine models, with a specific knock-in transgene in appropriate tissue, i.e., skeletal muscle, our results in cell culture correspond well with patient clinical episodes. We suggest, therefore, that Ala4894Thr and Ala4894Pro are associated with MH and CNMDU1, respectively.
In conclusion, we evaluated RyR1 function after transfection of Ala4894-mutated RyR1 into HEK-293 cells. The Ala4894Thr-transfected cells were more sensitive to caffeine and 4CmC than the WT. In contrast, Ala4894Pro-transfected cells had no response to caffeine or 4CmC. Our results indicate that hypersensitive Ala4894Thr-RyR1 is associated with MH, whereas the poorly functional Ala4894Pro-RyR1 is associated with CNMDU1.
We thank Professor David H. MacLennan for the generous gift of Rabbit-RyR1/pcDNA. We also thank Professor Ichizo Nishino (Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan) for his kind advice. This study was performed at the Analysis Center of Life Science, Hiroshima University, and was supported in part by the Tsuchiya Foundation.
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