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.

No abstract

Amplitude of Ca(2+) transients, ultrastructure of Ca(2+) release units, and molecular composition of sarcoplasmic reticulum (SR) are altered in fast-twitch skeletal muscles of calsequestrin-1 (CASQ1)-null mice. To determine whether such changes are directly caused by CASQ1 ablation or are instead the result of adaptive mechanisms, here we assessed ability of CASQ1 in rescuing the null phenotype. In vivo reintroduction of CASQ1 was carried out by cDNA electro transfer in flexor digitorum brevis muscle of the mouse. Exogenous CASQ1 was found to be correctly targeted to the junctional SR (jSR), as judged by immunofluorescence and confocal microscopy; terminal cisternae (TC) lumen was filled with electron dense material and its width was significantly increased, as judged by electron microscopy; peak amplitude of Ca(2+) transients was significantly increased compared with null muscle fibers transfected only with green fluorescent protein (control); and finally, transfected fibers were able to sustain cytosolic Ca(2+) concentration during prolonged tetanic stimulation. Only the expression of TC proteins, such as calsequestrin 2, sarcalumenin, and triadin, was not rescued as judged by Western blot. Thus our results support the view that CASQ1 plays a key role in both Ca(2+) homeostasis and TC structure.

the procedure. The other electrode was used to dialyze the fiber with a solution containing either 6-15 mM BAPTA, or up to 60 mM EGTA (pCa¼7.0), and adjusted to pH¼7.0 with 20 or 60 mM MOPS, respectively. Free myoplasmic Ca 2þ concentration ([Ca 2þ ]) changes were measured with the low affinity indicator OGB-5N (200 mM). SR Ca 2þ release was elicited by either 20 mM caffeine, or 1 mM 4-chloro-methy-phenol (4CmC), added to the external solution. The maximal fluorescence change of the Ca 2þ indicator was assessed at the end of the protocol by exposing the fibers to saponin (0.1 mg/ml in isotonic CaCl 2 , pH¼7.0 with 20 mM MOPS). To prevent changes in shape of the fibers under these conditions, they were pretreated for 1 min with 1% formaldehyde (in Tyrode). The experiments were conducted at 20 C. A single model compartment was used to estimate (from the [Ca 2þ ] changes) the total Ca 2þ released in response to caffeine/4CmC application. We obtained values of SR Ca 2þ content in the range of 15-27 mM for normal FDB fibers. Interestingly, comparable values were obtained in fibers from mdx mice.

Lack of both CASQ isoforms was confirmed by western blot. The doublenull mice are viable and breed normally, however the rate of spontaneous mortality of male animals is higher than CASQ1-null animals. Whereas the overall phenotype of mice is similar to that of CASQ1-null mice, significant differences are found in Soleus. From the structural point of view, in Soleus muscle we find many fibers (about 30%) with severe structural damage that were not found in CASQ1-null animals. Functional studies indicate significant prolongation in twitch time parameters, increased twitch tension and impaired tension generation during prolonged tetani both in EDL and Soleus, likely related to abnormal calcium release kinetics. These findings suggest that: a) expression of CASQ2 is essential for the maintenance of a subpopulation of Soleus fibers; and b) lack of both CASQ1 and 2 exacerbates the overall phenotype of CASQ1null mice.