Strenuous exercise triggers a life-threatening response in mice susceptible to malignant hyperthermia

Abstract

In humans, hyperthermic episodes can be triggered by halogenated anesthetics [malignant hyperthermia (MH) susceptibility] and by high temperature [environmental heat stroke (HS)]. Correlation between MH susceptibility and HS is supported by extensive work in mouse models that carry a mutation in ryanodine receptor type-1 (RYR1^Y522S/WT) and calsequestrin-1 knockout (CASQ1-null), 2 proteins that control Ca²⁺ release in skeletal muscle. As overheating episodes in humans have also been described during exertion, here we subjected RYR1^Y522S/WT and CASQ1-null mice to an exertional-stress protocol (incremental running on a treadmill at 34°C and 40% humidity). The mortality rate was 80 and 78.6% in RYR1^Y522S/WT and CASQ1-null mice, respectively, vs. 0% in wild-type mice. Lethal crises were characterized by hyperthermia and rhabdomyolysis, classic features of MH episodes. Of importance, pretreatment with azumolene, an analog of the drug used in humans to treat MH crises, reduced mortality to 0 and 12.5% in RYR1^Y522S/WT and CASQ1-null mice, respectively, thanks to a striking reduction of hyperthermia and rhabdomyolysis. At the molecular level, azumolene strongly prevented Ca²⁺-dependent activation of calpains and NF-κB by lowering myoplasmic Ca²⁺ concentration and nitro-oxidative stress, parameters that were elevated in RYR1^Y522S/WT and CASQ1-null mice. These results suggest that common molecular mechanisms underlie MH crises and exertional HS in mice.-Michelucci, A., Paolini, C., Boncompagni, S., Canato, M., Reggiani, C., Protasi, F. Strenuous exercise triggers a life-threatening response in mice susceptible to malignant hyperthermia.

Keywords: calsequestrin-1; excitation-contraction coupling; ryanodine receptor; sarcoplasmic reticulum; skeletal muscle.

Figure 1.

Mortality rate and core temperature of mice during the ES protocol. A) Incidence of sudden (black) and delayed (gray) deaths during ES protocol (see also Supplemental Table S1). B) Time to onset of lethal crises in MH-susceptible mice (measured from the beginning of the ES protocol). C, D) Changes in absolute (C) and relative (ΔT) (D) core temperature during ES protocol, measured at the beginning (t₀) and at the end (t_f) of the experiment (see also Supplemental Table S2). Data in B–D are given as means ± sem. Azu, azumolene; Y522S, RYR1^Y522S/WT. *P < 0.05 (compared with azumolene-treated mice; C, D).

Figure 2.

Immunofluorescence of EDL fibers and blood levels of CK, K⁺, and Ca²⁺ after the ES protocol. A–E) Double immunolabeling of EDL muscle fibers with Abs against RYR1 (red) and TOM20 (green) in samples from WT (A), RYR1^Y522S/WT (Y522S; B), and CASQ1-null (C) mice and azumolene (Azu)-treated Y522S (D) and CASQ1-null (E) mice. Asterisks (B) and dashed oval mark (C) indicate contractures and a fiber losing cross-striation, respectively. F–H) Blood levels of CK in serum (F) and K⁺ (G) and Ca²⁺ (H) in plasma, collected from the 5 groups of animals after ES protocol (see also Supplemental Fig. S1 for immunofluorescence and blood analysis in control conditions). Data are given as means ± sem. Scale bars, 10 µm (insets, 5 µm). *P < 0.05.

Figure 3.

Quantitative analysis in histologic sections of EDL fibers presenting structural damage following the ES protocol. A–C) Histologic examination of EDL muscles after ES protocol allowed classification of fibers in 3 main classes: fibers with no apparent sign of damage (A); fibers losing striation (dashed oval; B); and fibers with contractures (asterisks; C). D) Quantitative analysis showing the percentage of EDL fibers presenting the different features classified in A–C. White, fibers with no apparent damage; gray, fibers losing striation; black, fibers with contractures (also see Supplemental Table S3). Azu, azumolene; Y522S, RYR1^Y522S/WT. Scale bar, 10 µm. *P < 0.05.

Figure 4.

Responsiveness of EDL muscles and FDB fibers to electrical stimulation and caffeine. A, B) Average basal tension during high-frequency (80 Hz) electrical stimulation in intact EDL muscles from RYR1^Y522S/WT (Y522S; A) and CASQ1-null (B) mice in the absence or presence of 50 µM azumolene (Azu). C) Specific basal tension (mN/mm²) recorded at the end of the experiment (i.e., 20 min). D, E) Average basal tension during an IVCT performed exposing EDL muscles from Y522S (D) and CASQ1-null (E) mice to increasing caffeine concentrations in the absence or presence of 50 µM Azu. F) Specific basal tension (mN/mm²) recorded at the end of the experiments (i.e., 22 mM of caffeine). G, H) Average Fura-2 fluorescence ratio curves, normalized to 0 mM of caffeine, recorded in single FDB fibers from Y522S (G) and CASQ1-null (H) mice in the absence or presence of 50 µM Azu. I) Myoplasmic Ca²⁺ levels recorded at the end of the experiment (i.e., 10 mM of caffeine). Data are given as means ± sem. *P < 0.05 vs. WT and/or Azu-treated samples, as inidcated.

Figure 5.

Total calpain activity in EDL muscle homogenates in basal condition and after the ES protocol. Calpain activity, expressed as relative luminescence units normalized to the total protein content (µg), measured in muscle homogenates from mice in basal conditions (A) and from mice subjected to the ES protocol untreated or pretreated with azumolene (B). Azu, azumolene; Y522S, RYR1^Y522S/WT. Data are given as means ± sem. *P < 0.05.

Figure 6.

3-NT levels in EDL muscles in basal condition and after ES protocol. A) Representative immunoblot showing expression levels of 3-NT in EDL muscles from control mice (Ctrl, basal conditions), mice that were subjected to the ES protocol (ES), and MH-susceptible mice that were pretreated with azumolene (Azu) before exposure to the ES protocol. B, C) Relative band densities expressed as 3-NT/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio in basal conditions (B) and after exposure to the ES protocol in mice untreated or pretreated with Azu (C). Y522S, RYR1^Y522S/WT. Data are given as means ± sem. *P < 0.05.

Figure 7.

NF-κB p65 subunit (p65) activation in EDL muscles in basal condition and after ES protocol. A) Representative immunoblot showing expression levels of phospho-p65 (p-p65) and total p65 in EDL muscles from control mice (Ctrl, basal conditions), mice that were subjected to the ES protocol (ES), and MH-susceptible mice that were pretreated with azumolene (Azu) before exposure to the ES protocol (samples for these experiments were from mice also used in Fig. 6B, C). B, C) Relative band densities, expressed as p-p65/p65 ratio in basal conditions (B) and after exposure to the ES protocol in mice untreated or pretreated with Azu (C). Y522S, RYR1^Y522S/WT. Data are given as means ± sem. *P < 0.05.

Figure 8.

Schematic model illustrating the possible molecular pathways underlying muscle fiber rhabdomyolysis during ES. During exposure to strenuous exercise, excessive SR Ca²⁺ release (Fig. 4) and overproduction of oxidative species (Fig. 6) likely promote a feed-forward mechanism (31), which causes the generation of abnormal muscle tension (Fig. 4) and damage/rhabdomyolysis of muscle fibers (Figs. 2 and 3), possibly as the consequence of enhanced activation of calpains and NF-κB pathways (Figs. 5 and 7).