Anesthetic- and heat-induced sudden death in calsequestrin-1-knockout mice

Abstract

Calsequestrin-1 (CASQ1) is a moderate-affinity, high-capacity Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) terminal cisternae of skeletal muscle. CASQ1 functions as both a Ca(2+)-binding protein and a luminal regulator of ryanodine receptor (RYR1)-mediated Ca(2+) release. Mice lacking skeletal CASQ1 are viable but exhibit reduced levels of releasable Ca(2+) and altered contractile properties. Here we report that CASQ1-null mice exhibit increased spontaneous mortality and susceptibility to heat- and anesthetic-induced sudden death. Exposure of CASQ1-null mice to either 2% halothane or heat stress triggers lethal episodes characterized by whole-body contractures, elevated core temperature, and severe rhabdomyolysis, which are prevented by prior dantrolene administration. The characteristics of these events are remarkably similar to analogous episodes observed in humans with malignant hyperthermia (MH) and animal models of MH and environmental heat stroke (EHS). In vitro studies indicate that CASQ1-null muscle exhibits increased contractile sensitivity to temperature and caffeine, temperature-dependent increases in resting Ca(2+), and an increase in the magnitude of depolarization-induced Ca(2+) release. These results demonstrate that CASQ1 deficiency alters proper control of RYR1 function and suggest CASQ1 as a potential candidate gene for linkage analysis in families with MH/EHS where mutations in the RYR1 gene are excluded.

Figure 1.

CASQ1-null male mice exhibit a high incidence of spontaneous mortality and enhanced sensitivity to heat stress (41°C) and halothane exposure (2%), which are prevented by dantrolene pretreatment. A) Age-dependent survival analysis of male and female WT and CASQ1-null mice housed under standard conditions evaluated using the Kaplan-Meier method. B) Incidence of sudden death in WT and CASQ1-null mice as a result of halothane exposure (2% for 1 h). C) Incidence of sudden and delayed (within 24 h after challenge) death in WT and CASQ1-null mice as a result of heat challenge (41°C for 30 min). D) Dantrolene administration (4 mg/kg) protects male CASQ1-null mice from heat- and halothane-induced sudden death (compare & and # in B–D). *P < 0.01 vs. WT (see Supplemental Table 1).

Figure 2.

Time-dependent increase in core temperature during heat challenge is greater in CASQ1-null mice. A) Increase in internal temperature during heat stress (41°C, 30 min) in CASQ1-null and WT male and female mice. B) Semilog plot showing results of Student’s t test performed during 30 min of exposure to heat (calculated every 2 min). C) Table summarizing absolute and relative changes in internal core temperatures in male and female WT and CASQ1-null mice at beginning (t₀) and end (t₃₀) of heat stress protocol.

Figure 3.

Rhabdomyolysis in CASQ1-null mice after heat stress. A, B) Damaged EDL fibers are frequent (B, asterisks) in male CASQ1-null mice after heat stress-induced sudden death but very rare in male WT mice exposed to the same treatment (A) (see Supplemental Table 2). C) Areas of excessive contracture (solid arrows) and overstretched sarcomeres (open arrows) shown in a representative damaged fiber (inset, asterisk) from a post-trigger CASQ1-null male mouse. Scale bars= : 10 μm (A, B); 2 μm (C).

Figure 4.

Temperature and caffeine dependence of basal muscle tension and resting cytosolic Ca²⁺ concentration in EDL muscles, single FDB fibers, and cultured primary myotubes. A) Temperature dependence of basal tension in EDL muscles isolated from male WT (○) and CASQ1-null (•) mice; n = 10; *P < 0.05. B) Percentage of EDL muscles from male WT (left) and CASQ1-null (right) mice exhibiting a contracture at caffeine concentration below 5 mM during exposure to increasing concentrations of caffeine (0.5, 1, 2, 4, 6, 8, 10, 12, and 15 mM). C, D) Temperature dependence of resting cytosolic Ca²⁺ concentration in single FDB muscle fibers (C) and cultured myotubes (D) from WT (○) and CASQ1-null (•) mice; n = 10; *P < 0.05; **P < 0.01.

Figure 5.

Maximal voltage-gated Ca²⁺ calcium release is enhanced in myotubes from CASQ1-null mice. A) Representative L-type Ca²⁺ currents (bottom traces) and intracellular Ca²⁺ transients (top traces) elicited by 200 ms depolarization to the indicated test potential in WT (left) and CASQ1-null (right) myotubes. B, C) Average voltage dependence of peak L-type Ca²⁺ current density (B) and intracellular Ca²⁺ transients (C) in WT (•, n=21) and CASQ1-null (○, n=20) myotubes (see also Supplemental Table 3 and Supplemental Fig. 2).