Inhibition of sarcoplasmic Ca²⁺-ATPase increases caffeine- and halothane-induced contractures in muscle bundles of malignant hyperthermia susceptible and healthy individuals

In skeletal muscle, the action potential passes along the surface membrane of the muscle fibre into the transverse tubular system. Depolarisation of the voltage sensitive dihydropyridine receptor leads to an opening of the ryanodine receptor in the nearby sarcoplasmic reticulum (SR). Sarcoplasmic calcium (Ca²⁺) release via the ryanodine receptor raises cytosolic Ca²⁺ and activates muscle contraction. Energy-dependent Ca²⁺ reuptake into the SR is caused by the SR Ca²⁺-ATPase (SERCA) and enables skeletal muscle relaxation [1]. In individuals susceptible to the autosomal dominant skeletal muscle disorder malignant hyperthermia (MH), electro-mechanical coupling is disturbed. Due to MH-associated mutations in the ryanodine receptor, triggering agents such as halogenated anaesthetics cause an excessive Ca²⁺ release from the SR resulting in intracellular hypermetabolism, increased mitochondrial energy-turnover and metabolic failure with a deficiency of adenosine-triphosphate [2]. This may also lead to energetic exhaustion of the SERCA, the main transporter for Ca²⁺ ions across the sarcoplasmic membrane. Cyctosolic Ca²⁺ concentration is determined by sarcoplasmic Ca²⁺ release and it's reuptake via the SERCA [3]. The mycotoxin cyclopiazonic acid (CPA) is a selective inhibitor of SR Ca²⁺ reuptake [4] that has been used previously to study SERCA in different tissues [5, 6].

We hypothesized that in skeletal muscle, preincubation with CPA enhances halothane- and caffeine-induced contractures in MH susceptible (MHS) more than in non-susceptible (MHN) skeletal muscle.

Muscle bundles of 30 patients undergoing a diagnostic in-vitro contracture test (IVCT) were investigated to detect MH susceptibility. With informed consent, surplus muscle bundles were studied by the same IVCT protocol following SERCA inhibition by CPA.

Thirty patients, 9 female and 21 male, with a mean age of 28 (15 – 32) years and a mean weight of 74 (62 – 87) kg were studied. 7 patients were classified as MHS, 7 as MHEh (susceptible only for halothane) and 16 as MHN according to the criteria of the European Malignant Hyperthermia group diagnostic protocol. In every patient, an additional IVCT with CPA was performed. Muscle bundles used for the IVCT and CPA-IVCT did not differ regarding to length, weight, maximum twitch height, predrug resting tension and predrug twitch height (Table 1).

In the caffeine contracture test, prior incubation with CPA resulted in significant higher contractures compared to the diagnostic IVCT in the MHS and MHEh group (Table 2). At the diagnostic threshold dose of caffeine 2 mM, MHS muscles developed significantly higher contractures with 32 (25 – 38) mN following preincubation with CPA vs. 8 (4 – 12) mN without CPA. In the MHEh group CPA preincubation lead to significantly higher contractures with 12 (11 – 27) mN vs. 1 (0 – 1) mN without CPA, while the contractures of MHN muscle bundles did not differ with or without CPA.

At halothane 0.44 mM, CPA preincubation increased contractures of MHS and MHEh muscle bundles significantly to 59 (33 – 73) mN respectively 45 (24 – 55) mN compared to standard IVCT conditions with 20 (16 – 26) mN respectively 4 (2 – 4) mN. In addition, in the MHN group at halothane 0.44 mM contractures were significantly increased by CPA preincubation to 16 (4 – 34) mN vs. 1 (1 – 1) mN without CPA (Table 3).

In MH uncontrolled SR Ca²⁺ release, caused by MH associated mutations mainly in the ryanodine receptor gene, is widely accepted as the underlying pathophysiological mechanism of hypermetabolism [8]. However, the detection of a mutation in the alpha 1-subunit of the voltage sensitive dihydropyridine receptor in a French MH family suggests a more complex pathogenesis of MH [9]. According to the unique mechanism of intracellular Ca²⁺ cycling that induces contraction and relaxation in vertebrate skeletal muscle, sarcoplasmic Ca²⁺ release and sarcoplasmic Ca²⁺ reuptake determine the mainstays of Ca²⁺ regulation. Undoubtly, an altered SR Ca²⁺ release plays a crucial role in the development of MH. However, it is completely unclear why many MHS individuals may suffer from MH only after several uneventful exposures to trigger agents during anaesthesia. Several modulating factors have been postulated to modulate cytosolic Ca²⁺ concentrations, e.g. magnesium [10], sympathetic activity [11], temperature [12], volatile anesthetics [13] or channel's redox state [14]. While SR Ca²⁺ release was extensively studied in MH [15], the impact of an altered SR Ca²⁺ reuptake on the pathogenesis of MH by intrinsic or extrinsic factors is poorly understood. Theoretically, a reduced activity of the skeletal muscular SERCA type 1 may result in an elevated cytosolic Ca²⁺ level due to a persistent slow Ca²⁺ efflux out of the SR that is otherwise balanced by reuptake [16]. A critical threshold of cytosolic Ca²⁺ may then be exceeded and may lead to contracture development in vitro and to the MH syndrome in susceptible patients. Interestingly, CPA alone did not induce skeletal muscle contractures at 25 μM [17]. We assume that in our study SERCA was inhibited almost completely, since CPA 10 μM reduced the SERCA activity approximately by 70% in frog skinned fibres [16] and nearly by 100% in rat skinned fibres [18].

In the presented study, CPA preincubation lead to a high variability of halothane- respectively caffeine-induced contractures especially in the MHS and MHEh group, despite SERCA distribution does not differ between MHS and MHN muscle [19]. Interestingly, the response of MHEh muscle bundles to caffeine was enhanced by CPA preincubation. However, at this stage, our results do not suggest CPA as an alternative approach to improve differentiation of MHE from MHN respectively MHS individuals.

Ca²⁺ uptake capacity and SERCA activity was found to be significantly increased in MHS pigs [20] and in HEK-293 cells transfected by MH mutants [21] but was described to be lower in MHS muscle compared to normal human skeletal muscle [22]. Since a leaky ryanodine receptor in MHS individuals may lead to increased cytosolic calcium, it looks feasible that SR-Ca-ATPase may be upregulated by a compensatory mechanism.

Another option is that CPA itself modulates directly the effect of the trigger agent. This is less likely since halothane and caffeine do have different binding sites at the sarcoplasmic membrane [23].

The role of a reduced SERCA activity in the pathogenesis of Brody's disease, a skeletal muscular myopathy, is well known and characterized by painless muscle cramping and exercise-induced muscle stiffness linked to a mutation in the gene encoding SERCA [24, 25]. The left-shift of the dose-response curve for halothane- and caffeine-induced contractures following inhibition of the sarcoplasmic Ca²⁺ reuptake by CPA points out the essential part of SERCAs in the regulation of cytoplasmic Ca²⁺. We believe this may be an explanation why some MH susceptible patients develop a MH crisis while others never or only after several trigger exposures suffer from MH despite a proven in vitro susceptibility. In this context, an altered activity of SERCA due to intrinsic or extrinsic factors may play a crucial role in the evolution of MH.

The present study demonstrates that CPA preincubation enhances halothane- and caffeine-induced muscle contractures in the IVCT of MHS, MHEh more than in MHN patients.

Modulation of SERCA may play a significant role in the development of malignant hyperthermia. Patients with a high activity may compensate an increased Ca²⁺ release or leakage from the SR while patients with a low activity of the SERCA do not. Further investigations with focus on extrinsic and intrinsic factors that modulate SERCA activity may be helpful to understand why MH patients may have had several anaesthesias including trigger agents without a significant reaction while developing a fulminate MH crisis at another occasion.