TRPV1 variants impair intracellular Ca2+ signaling and may confer susceptibility to malignant hyperthermia

Figures

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  Fig. 1. Volatile anesthetics activate TRPV1 channels heterologously expressed in HEK-293 cells and in flexor digitorum brevis (FDB) isolated fibers. (a,b) Original recordings of the baseline TRPV1 current (ITRPV1) (acquired at point 1 of the time course in (a)) and isoflurane-activated (0.125 mM) (acquired at point 2 of the time course) in response to pulse protocol with voltage-ramp portion shown above the recordings. Isoflurane activated a membrane current with biophysical properties, such as a prominent outward rectification and close to 0 mV reversal potential, which is similar to the current activated by the established TRPV1 stimulus, capsaicin (data not shown) (n = 7). (c) Examples of the baseline and halothane-activated ITRPV1 in response to the depicted voltage-clamp protocol, which were used to measure voltage dependence of TRPV1 channel open probability (Po in (d); experiments were performed at 20 °C); arrows in (c) point to the ITRPV1 tail currents at + 60 mV. Tail currents were measured during the first millisecond of the final step +60 mV and normalized to the maximal tail current. Normalized amplitude as a function of conditioning depolarizing pulse (ranging from –120 to…

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  Fig. 2. TRPV1 variations from patients who suffer from malignant hyperthermia. (a) Segregation ofTRPV1 variations. Individuals tested MHEh are depicted with hatched symbols. Black symbols refer to patients diagnosed with a congenital myopathy and MH sensitivity. Arrows indicate probands referred to the laboratory for genetic studies according to initial diagnosis: malignant hyperthermia crisis for family 1 and myopathy for family 2. Family 1: the proband was referred to genetic investigation for postoperative hyperthermia after anesthesia. No significant variant was found in the RYR1 gene sequence. The c.1836C>T; pThr612Met variation was found in targeted analysis of the gene. Family 2: the proband was referred to genetic investigation for muscle weakness. He responded positively to the in vitro contracture test (MHS) and further familial studies were undertaken for MH sensitivity. Two mutations in the RYR1 gene (c.1205T>C;p.Met402Thr and c.11653C>T;p.Arg3885X) were found inherited from each of the parents in the proband and his symptomatic sister’s DNA. The haplo insufficiency that was revealed during sequencing of the father RYR1 messenger RNA (mRNA) cannot account for its MH…

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  Fig. 3. Decrease in capsaicin sensitivity and increase in isoflurane sensitivity in mutants TRPV1 (T612M; N394del) transfected in FDB muscles fibers of trpv1−/− mice and effect of Trpv1 inhibition by capsazepine on heat-induced sudden death in Y524S mice heat challenged at 37 °C. Changes in fluorescence ratio F/F0 (peak-resting) induced by (a,b) (100 μM) capsaicin or (c,d) isoflurane (6 mM) in C57Bl6J (black), TRPV1−/− (red), humanTRPV1-mcherry (gray), TRPV1 T612M-mcherry (blue), or TRPV1 N394del-mcherry (green). Corresponding scatterplots of max value expressed as median. For the capsaicin response, data are from 42 cells (WT), Eleven cells (hTRPV1), 12 cells (T612M), 10 cells (N394del), and 9 cells (TRPV1−/−) from at least 4 independent fiber preparations. For the isoflurane response, data are from 29 cells (WT), 18 cells (hTRPV1), 11 cells (T612M), 12 cells (N394del), and 13 cells (TRPV1−/−) from at least 4 independent fiber preparations. Kruskal–Wallis tests with Dunn's post hoc tests were performed: *p < 0.05, **p < 0.001, ***p < 0.0001, ****p < 0.0001. (e) Oxygen consumption (VO2) during a 15 min exposure of mice at 37 °C in Y524S RYR1 mouse model (YS) or in WT mouse (WT). n = 8…