RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice

Abstract

Mice with a malignant hyperthermia mutation (Y522S) in the ryanodine receptor (RyR1) display muscle contractures, rhabdomyolysis, and death in response to elevated environmental temperatures. We demonstrate that this mutation in RyR1 causes Ca(2+) leak, which drives increased generation of reactive nitrogen species (RNS). Subsequent S-nitrosylation of the mutant RyR1 increases its temperature sensitivity for activation, producing muscle contractures upon exposure to elevated temperatures. The Y522S mutation in humans is associated with central core disease. Many mitochondria in the muscle of heterozygous Y522S mice are swollen and misshapen. The mutant muscle displays decreased force production and increased mitochondrial lipid peroxidation with aging. Chronic treatment with N-acetylcysteine protects against mitochondrial oxidative damage and the decline in force generation. We propose a feed-forward cyclic mechanism that increases the temperature sensitivity of RyR1 activation and underlies heat stroke and sudden death. The cycle eventually produces a myopathy with damaged mitochondria.

Figure 1. Effects of warming on RyR1^Y522S/wt mice

A. Rectal temperatures in RyR1^wt/wt mice. Mice were anesthetized with etomidate (i.p.) and placed in an environmental chamber (41°C) and rectal temperatures were measured as a function of time in RyR1^wt/wt mice (●, n=10) and in mice treated for 3–5 days with NAC (○, n=6), L-NAME (gray circles, n=4) or combined NAC and L-NAME (◇, n=4). B. Rectal temperatures in RyR1^Y522S/wt mice. Similar measurements were made with RyR1^Y522S/wt mice (■, n=7) and mice treated with NAC (□, n=4), L-NAME (gray squares, n=4), and combined NAC and L-NAME (◇, n=4). The dashed line represents the WT curve from Figure 1A for comparison. When the curves were compared by F-tests, NAC (p<0.05), L-NAME (p<0.001) and NAC + L-NAME (p<0.001) significantly attenuated the response versus no treatment in the RyR1^Y522S/wt mice. C. VO₂ is increased at thermoneutral conditions in RyR1^Y522S/wt mice. VO₂ was measured at 32°C using 4–5 mice in each group *p<0.05 vs. RyR1^wt/wt. D. Temperature sensitivity of basal stress in RyR1^wt/wtsolei. Solei from RyR1^wt/wt (with and without prior treatment with NAC or L-NAME) were isolated, attached to force transducers, and heated progressively from an initial temperature of 25°C to 41°C. n=3–8 mice/group. E. Temperature sensitivity of basal stress in RyR1^Y522S/wt solei. Group, temperature, and interaction effects were significant (p<0.0001) when data were analyzed by 2-way ANOVA. *p<0.05 to p<0.001 RyR1^Y522S/wt vs RyR1^Y522S/wt NAC, #p<0.05 to p<0.01 RyR1^Y522S/wt vs. RyR1^Y522S/wt L-NAME. For clarity, significant differences between L-NAME and NAC treated groups at 39°C and 41°C are not indicated on the graph. F. GSH/GSSG ratios. The data (n=3–4) were compared between untreated mice of each genotype and the mice receiving the indicated treatment. *p<0.05. G. Fluorescence imaging of myotubes loaded with DCF. All imaging data were obtained from multiple cells across at least three independent myotube preparations. Representative images obtained with DCF-loaded RyR1^wt/wt and RyR1^Y522S/wt myotubes at 25ºC and 37ºC in the presence and absence of ryanodine (20 μM). H. Changes in DCF fluorescence with temperature. ROS production was detected with DCF in the presence absence of ryanodine (20μM), L-NNA (50 μM) and GSHEE (5mM). I. Fluorescence imaging of myotubes loaded with DAF-AM. Representative images obtained with DAF-AM loaded RyR1^wt/wt and RyR1^Y522S/wt myotubes at 25ºC and 37ºC in the presence and absence of ryanodine (20 μM). J. Changes in DAF fluorescence with temperature. RNS production was detected with DAF in the presence or absence of ryanodine (20μM), L-NNA (50 μM) and GSHEE (5mM). All data in this figure are shown as mean ± S.E.M.

Figure 2. Temperature dependent increases cytosolic Ca²⁺ levels in RyR1^Y522S/wt myotubes and solei

A. Temperature dependent increases in cytosolic Ca²⁺ levels measured with fura-2. Myotubes loaded with fura-2AM were warmed to the indicated temperatures in the presence and absence of 5mM GSHEE. Values are mean ± SEM for 3 independent cultures for each group: RyR1^wt/wt (●, n=27), RyR1^wt/wt + GSHEE (○, n=31), RyR1^Y522S/wt (■, n=29), RyR1^Y522S/wt + GSHEE (□, n=32) (*p < 0.001, one-way ANOVA followed by Scheffe's comparison). B. Effect of L-NNA on temperature dependent increase in resting Ca²⁺. Myotubes loaded with fura-2AM were warmed to the indicated temperatures in the presence or absence of 50μM L-NNA. RyR1^Y522S/wt (■, n= 27) RyR1^Y522S/wt + L-NNA (□, n= 31), RyR1^wt/wt (●, n= 28) and RyR1^wt/wt + L-NNA (○, n= 33). *p<0.05, one way ANOVA followed by Scheffe’s comparison. C. Temperature dependent increases in cytosolic free Ca²⁺ concentration in RyR1^Y522S/wt myotubes. Indo-1-loaded myotubes were warmed to the indicated temperatures and indo-1 ratios were calibrated as described in Methods. *p<0.05 compared to RyR1^wt/wt at 23°C. D. Temperature dependent increases in cytosolic Ca²⁺ in solei fibers. Solei fibers of RyR1^Y522S/wt mice were loaded with fura-2 and resting Ca²⁺ was measured in the presence or absence of ryanodine (20μM). RyR1^Y522S/wt (■, n= 17), RyR1^Y522S/wt + 20μM ryanodine (□, n=10), RyR1^wt/wt (●, n= 12) and RyR1^wt/wt + 20μM ryanodine (○, n= 4). *p<0.05, one-way ANOVA. E. Effects of GSHEE on the voltage dependence of L-type Ca²⁺ currents. Voltage dependence of average (± SEM) peak L-currents at room temperature in RyR1^wt/wt myotubes (●), RyR1^Y522S/wt myotubes (■), and RyR1^Y522S/wt myotubes preincubated with 5 mM GSHEE (□). F. The effects of GSHEE on the voltage dependence of intracellular Ca²⁺ release. Ca²⁺ transients at room temperature were measured in RyR1^wt/wt myotubes (●), RyR1^Y522S/wt myotubes (■), and RyR1^Y522S/wt myotubes preincubated with 5 mM GSHEE (□). G. Temperature dependence of L-type Ca²⁺ currents in RyR1^wt/wt and RyR1^Y522S/wt myotubes. Voltage dependence of average (± SEM) peak L-currents at 23°C (closed symbols) and 37°C (open symbols) in RyR1^wt/wt myotubes (circles) and RyR1^Y522S/wt (squares) myotubes. Each dataset was fit (smooth solid lines) using equations described previously (Chelu, 2006) in order to determine G_max, V_G1/2, k_G, and V_rev at both 23°C (167 nS/nF, 7.8 mV, 8.6 mV, and 72.6 mV for RyR1^wt/wt and 242 nS/nF, 17.3 mV, 7.1 mV, and 73.0 mV for RyR1^Y522S/wt, respectively) and 37°C (309 nS/nF, −3.0 mV, 7.0 mV, and 72.5 mV for RyR1^wt/wt and 442 nS/nF, −0.6 mV, 4.5 mV, and 78.2 mV for RyR1^Y522S/wt, respectively). H. Temperature dependence of intracellular Ca²⁺ transients in RyR1^wt/wt and RyR1^Y522S/wt myotubes. Voltage dependence of average (± SEM) peak Ca²⁺ transient amplitude at 23°C (closed symbols) and 37°C (open symbols) in RyR1^wt/wt myotubes (circles) and RyR1^Y522S/wt (squares) myotubes Each dataset was fit (smooth solid lines) using equations described previously (Chelu, 2006) in order to determine F_max, V_F1/2, and k_F at both 23°C (3.1, −16.8 mV, and 6.2 mV for RyR1^wt/wt and 2.7, −23.1 mV, and 7.5 mV for RyR1^Y522S/wt, respectively) and 37°C (1.4, −31.8 mV, and 5.7 mV for RyR1^wt/wt and 1.0, −35.6 mV, and 8.2 mV for RyR1^Y522S/wt, respectively for RyR1^Y522S/wt, respectively). All data in this figure are shown as mean ± S.E.M.

Figure 3. Redox modifications of RyR1 and functional consequences

A. Redox modifications of RyR1. Representative blots obtained with 3 independent microsomal preparations were obtained from RyR1^wt/wt (top) and RyR1^Y522S/wt (bottom) mice. Density of the bands corresponding to the redox modifications of RyR1 were obtained under control conditions (middle) or in the presence of either ascorbic acid (left) or DTT (right). B. Fluorescence signals for S-nitrosylation normalized to the Coomassie stain of each band. Data (mean ± SD, n=3–5) are presented as the ratio to untreated microsomes. *p<0.05 compared to control. C. Fluorescence signals for S-glutathionylation normalized to the Coomassie stain of each band. Data (mean ± SD, n=3–5) are presented as the ratio to untreated microsomes. *p<0.05 compared to control. D–F. Equilibrium [³H]ryanodine binding. Scatchard plot analysis (see Supplemental Figure 4) determination of K_D values for [³H]ryanodine binding to microsomes from RyR1^wt/wt and RyR1^Y522S/wt muscle (D). [³H]ryanodine binding was titrated at different Ca²⁺concentrations to calculate EC₅₀ (E) and IC₅₀ (F) values from traces as those shown in Supplemental Figure 5. *p<0.05 compared to RyR1^wt/wt or untreated controls. G. Temperature dependence of the association kinetics of [³H]ryanodine binding. Microsomes from untreated mice were pre-incubated in vitro with buffer (untreated), AA or DTT as in (A). [³H]ryanodine binding was assessed at different time points (1–90min) and k_obs values (mean ± SD) were determined from 3–4 independent experiments. Statistical significance for all panels was obtained by two-way ANOVA. *p<0.05 compared to RyR1^wt/wt or untreated controls. H. Rate of Ca²⁺ efflux from SR vesicles. Ca²⁺-induced Ca²⁺ release in the presence of 1mM free ATP and 9–10 μM free Ca²⁺ was measured using stopped-flow spectrofluorometry. Ca²⁺ release was measured in RyR1^wt/wt and RyR1^Y522S/wt vesicles using extravesicular Calcium Green-5N under control conditions or following treatment with AA. Release rate constant values (k) were obtained by peak differential analysis of fluorescence data (representative traces shown in Supplemental Figure 7). All data in this figure are shown as mean ± S.E.M.

Figure 4. Effects of the Y522S mutation on mitochondrial structure and muscle function

A–F. Mitochondrial ultrastructure is altered in muscle of RyR1^Y522S/wt mice. A. Mitochondria of RyR1^wt/wt fibers at 2–3 months of age are usually regularly shaped, appearing either round (panels 1 and 2) or slightly elongated (panel 3), with a dark/dense internal matrix. Abnormal mitochondria (panel 4) are rare in RyR1^wt/wt fibers. B. In RyR1^Y522S/wt fibers, some normal mitochondria are present (panel 1), but abnormal mitochondria are frequent (panels 2–4). C–F. At 1 year, a much larger percentage of mitochondria are severely swollen/disrupted than at 2–3 months of age in both in FDB and soleus muscles from RyR1^Y522S/wt mice (D and F, stars). In wt FDB (C) and soleus (E) muscles, on the other hand, mitochondria appear similar to those at 2–3 month of age. G. Mitochondrial lipid peroxidation in young and aged mice. Mitochondrial enriched fractions from 2 or 12 month-old mice with or without chronic NAC-treatment (≥ 2 months) were isolated and TBARS were measured in acid supernatants after protein precipitation, as detailed in Methods. Shown are the mean of 3 independent determinations ± SD. H–J. Effect of chronic NAC treatment on skeletal muscle function of aged mice. One year old male RyR1^wt/wt and RyR1^Y522S/wt mice were treated with NAC (1% w/v) in their drinking water for at least 2 months prior to sacrifice. Solei were removed and stimulated in vitro, as described in Experimental Procedures. Shown are data for stress developed at increasing frequency of electrical stimulation (H) and the maximal tension per cross-sectional area obtained by maximal electrical stimulation (I) or application of 20mM caffeine (J). All data in this figure are shown as mean ± S.E.M.

Figure 5. Proposed model of exertional/environmental heat stress and myopathy in RyR1^Y522S/wt mice

➀ SR Ca²⁺ release channels in RyR1^Y522S/wt mice are more sensitive to voltage, ligand, and Ca²⁺ activation and open more readily, producing small, possibly local, increases in resting Ca²⁺. ➁ Increased cytosolic Ca²⁺ levels enhance ROS/RNS production. Increases in RNS are likely to be produced by nitric oxide synthases (NOS). Possible sources of ROS include mitochondria, xanthine oxidase (XO) and NAD(P)H oxidases (NOX). ➂ Although both S-glutathionylation and S-nitrosylation of RyR1^Y522S/wt occurs, our data suggest that S-nitrosylation alone enhances the temperature sensitivity of RyR1. ➃ S-nitrosylation of RyR1^Y522S/wt increases its sensitivity to temperature and decreases its sensitivity to Ca²⁺ inhibition further promoting SR Ca²⁺ leak. ➄ Ca²⁺ increases further enhance ROS/RNS production. ➅ In response to heat stress, Ca²⁺ release from the modified RyR1^Y522S/wt is greatly and persistently augmented, leading to heat stroke. ➆ and ➇ Chronically-elevated levels of Ca²⁺ and ROS/RNS damage mitochondria and contribute to the development of myopathy.