1. IntroductionMalignant hyperthermia (MH) is a potentially life-threatening pharmacogenetic reaction to volatile anesthetics and/or depolarizing muscle relaxants, characterized by hyperpyrexia, hypercapnia, muscle breakdown, metabolic decompensation and, if untreated, death [[1]Rosenberg H Pollock N Schiemann A Bulger T Stowell K. Malignant hyperthermia: a review.]. MH was initially recognized as an autosomal-dominant trait and subsequently attributed to mutations in the RYR1 gene [[2]Fujii J Otsu K Zorzato F de Leon S Khanna VK Weiler JE et al.Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia.], encoding the skeletal muscle ryanodine receptor-1 (RyR1), the sarcoplasmic reticulum (SR) calcium release channel playing a crucial role in excitation-contraction coupling (ECC). RYR1-related MH occurs in humans and other mammals, notably in swine with the “porcine stress syndrome” (PSS) [[3]Gillard EF Otsu K Fujii J Duff C de Leon S Khanna VK et al.A substitution of cysteine for arginine 614 in the ryanodine receptor is potentially causative of human malignant hyperthermia.], and in several horse breeds [[4]Aleman M Nieto JE Magdesian KG. Malignant hyperthermia associated with ryanodine receptor 1 (C7360G) mutation in Quarter Horses.]. Whilst RYR1 mutations remain the most common genetic cause of MH, other genes implicated include CACNA1S [[5]Monnier N Krivosic-Horber R Payen JF Kozak-Ribbens G Nivoche Y Adnet P et al.Presence of two different genetic traits in malignant hyperthermia families: implication for genetic analysis, diagnosis, and incidence of malignant hyperthermia susceptibility.], encoding the alpha 1-subunit of the dihydropyridine receptor (DHPR), the voltage sensing calcium channel activating RyR1, and STAC3 [[6]Horstick EJ Linsley JW Dowling JJ Hauser MA McDonald KK Ashley-Koch A et al.Stac3 is a component of the excitation-contraction coupling machinery and mutated in Native American myopathy.], encoding a regulatory protein with a putative role in stabilizing the DHPR complex. In about 65% of MH cases, an RYR1 variant has been identified [[1]Rosenberg H Pollock N Schiemann A Bulger T Stowell K. Malignant hyperthermia: a review.].There is considerable overlap between MH-associated RYR1 mutations and neuromuscular disorders, particularly Central Core Disease (CCD) and other RYR1-related disorders [[7]Jungbluth H Treves S Zorzato F Sarkozy A Ochala J Sewry C et al.Congenital myopathies: disorders of excitation-contraction coupling and muscle contraction.]. RYR1 mutations have also been identified as a common cause of rhabdomyolysis triggered by exercise, intercurrent viral illness, heat, drugs, or a combination of the above [[8]Dlamini N Voermans NC Lillis S Stewart K Kamsteeg E-J Drost G et al.Mutations in RYR1 are a common cause of exertional myalgia and rhabdomyolysis.,[9]Molenaar JP Voermans NC van Hoeve BJ Kamsteeg EJ Kluijtmans LA Kusters B et al.Fever-induced recurrent rhabdomyolysis due to a novel mutation in the ryanodine receptor type 1 gene.]. Indeed, the finding of MH-associated RYR1 mutations in a large cohort of individuals presenting with exertional myalgia/rhabdomyolysis (ERM) but without a MH history suggests that ERM may be a more common manifestation of MH-associated RYR1 mutations than MH itself [[8]Dlamini N Voermans NC Lillis S Stewart K Kamsteeg E-J Drost G et al.Mutations in RYR1 are a common cause of exertional myalgia and rhabdomyolysis.]. Exertional heatstroke (EHS) is another presentation expanding the spectrum of MH-associated RYR1 mutations [10Roux-Buisson N Monnier N Sagui E Abriat A Brosset C Bendahan D et al.Identification of variants of the ryanodine receptor type 1 in patients with exertional heat stroke and positive response to the malignant hyperthermia in vitro contracture test., 11Fiszer D Shaw MA Fisher NA Carr IM Gupta PK Watkins EJ et al.Next-generation Sequencing of RYR1 and CACNA1S in Malignant Hyperthermia and Exertional Heat Illness., 12Gardner L Miller DM Daly C Gupta PK House C Roiz de Sa D et al.Investigating the genetic susceptibility to exertional heat illness.]. Interestingly, a substantial proportion of individuals referred to MH centers following a presentation with ERM and EHS rather than MH are subsequently found to be MH susceptible on In Vitro Contracture Testing (IVCT) [[13]Miller DM Daly C Aboelsaod EM Gardner L Hobson SJ Riasat K et al.Genetic epidemiology of malignant hyperthermia in the UK.], further confirming the close association between these RYR1-related manifestations.Despite the discoveries in genetics and principal pathophysiology of MH [[14]Riazi S Kraeva N Hopkins PM. Malignant hyperthermia in the post-genomics era: new perspectives on an old concept.], some aspects remain poorly understood even 50 years after its original description. One question concerns the unexplained discrepancy between the low prevalence of MH reactions (1 in 15-75000 general anesthetics) [[15]Prevalence of malignant hyperthermia diagnosis in hospital discharge records in California, Florida, New York, and Wisconsin.,[16]Incidence of malignant hyperthermia in Denmark.], and the relatively high combined prevalence of MH-causative RYR1 variants (1:2800 in the general population, according to the exome data from 60000 individuals (https://gnomad.broadinstitute.org/gene/ENSG00000196218?dataset=gnomad_r2_1). This discrepancy may be partly explained by lack of exposure of genetically predisposed individuals to MH-triggering agents. However, there are a substantial number of MH-susceptible individuals with uneventful repeated exposure to MH-triggering agents before suffering an MH reaction [[17]Riazi S Larach MG Hu C Wijeysundera D Massey C Kraeva N. Malignant hyperthermia in Canada: characteristics of index anesthetics in 129 malignant hyperthermia susceptible probands.], suggesting the presence of additional modifying factors as a possible explanation for the observed variable penetrance. The presence of such factors is supported by reports of emotional stress contributing to MH [[18]Letter: Malignant hyperthermia: a human stress syndrome?.,[19]Mogensen JV Misfeldt BB Hanel HK. Letter: Preoperative excitement and malignant hyperthermia.], and the observation of a fatal MH reaction in a patient exposed to MH-triggering agents following intense exercise [[20]Forrest KM Foulds N Millar JS Sutherland PD Pappachan VJ Holden S et al.RYR1-related malignant hyperthermia with marked cerebellar involvement - a paradigm of heat-induced CNS injury?.]. Post-mortem genetic analysis in this patient identified the RYR1 p.Val2168Met variant, a common European MH mutation [[21]Girard T Cavagna D Padovan E Spagnoli G Urwyler A Zorzato F et al.B-lymphocytes from malignant hyperthermia-susceptible patients have an increased sensitivity to skeletal muscle ryanodine receptor activators.]; the mutation was subsequently also identified in his relatives with previous repeated uneventful exposures to MH-triggering agents.

Based on these observations, we hypothesized that MH is a multifactorial event, determined not only by genotype but also non-genetic factors, in particular exercise and pyrexia at the time of general anesthesia (GA) with MH-triggering agents, all of which have also been implicated in RYR1-related rhabdomyolysis and EHS. To investigate a potential association between MH, exercise and pyrexia, we undertook a retrospective case study of MH cases seen at five MH referral centers.

2. Patients and methods

Patients were included for this retrospective case study from three European (Antwerp, Lund, and Nijmegen), one Australian (Melbourne) and one North American (Toronto) MH center. This retrospective case study was based on a collaboration agreed at a meeting of the European Malignant Hyperthermia Group (EMHG) in Antwerp in 2017, following presentation of a case who had developed MH when anesthetized in timely association with strenuous exercise. The aim of this collaboration was to retrospectively identify similar cases through a collaborative international effort. Research Ethics Board approvals were obtained from each participating centers. Data collection, governance and analysis were performed in accordance with the proposal after approval by the institutional review board.

We reviewed case notes from patients presenting with an MH reaction to any of the participating centers between 1995–2015. Patients were included if they fulfilled the following inclusion criteria: 1) clinical features suggestive of an MH reaction based on a Clinical Grading Score (CGS) of at least 20 or above [[22]Larach MG Localio AR Allen GC Denborough MA Ellis FR Gronert GA et al.A clinical grading scale to predict malignant hyperthermia susceptibility.], 2) confirmation of MH susceptibility on Contracture Testing (IVCT or CHCT) and/or RYR1 genetic testing and a history of 3) strenuous exercise within 72 h and/or pyrexia >37.5 oC prior to the GA. Strenuous exercise was defined according to the US Physical Activity Guidelines for adults 2008 (https://health.gov/sites/default/files/2019-09/paguide.pdf). Body temperature had been measured as tympanic measurement on admission, and as core temperature (typically on the mid-oesophageal or nasopharyngeal level) in the operating room in all patient groups [[23]Perioperative temperature monitoring.]. Inclusion criteria for this study are summarized in Fig. 1.

Fig. 1Inclusion criteria for this study. GA = General anesthesia, IVCT = In Vitro Contracture Test, MH = Malignant Hyperthermia.

We compared the characteristics of GAs associated with an MH event (“GA+MH”) and of GAs without an MH event (“GA-MH”) in the patients and/or their similarly mutated relatives; comparison with similarly mutated relatives (rather than unrelated control individuals) was elected to reduce genetic background variability. We recorded a history of intense exercise and/or body temperature >37.5 ℃ within 72 h prior to the GA, the indications for the GA (categorized as “trauma”, “acute abdomen” and “other”); if (where applicable) the surgery was elective or emergency; the specific anesthetic agents used, including use of succinylcholine. We compared the frequency of these parameters between GA+MH, and GA-MH

We also recorded the sex of each patient, and for all GA+MH, the age of the patient at the time of the MH event.

Data collected for this study are summarized in Fig. 1. Missing data has been indicated where applicable.2.1 Statistical analysis

Data were presented descriptively as means, medians, or proportions. Fisher exact test was used to compare categorical predictor variables, with the dependent variables being the MH reaction. Logistic regression analysis, with an adjustment for clustering on patient visit, was used to assess risk factors for the development of MH via a backwards elimination process with p < 0.05.

Since the outcomes of interest (pyrexia and exercise) were rare or non-existent in the control group, sample size was not calculated.

All of the statistical analyses were performed using Stata, release 16.0 (Stata Corp., USA).

3. Results

We identified 41 cases from 40 families who had a GA resulting in an MH event (GA+MH, n = 41) and fulfilled the inclusion criteria of strenuous exercise within 72 h and/or pyrexia >37.5 oC prior to the GA, amounting to 8.6% out of a total of 476 cases who were referred under the suspicion of MH to one of the participating MH centers during the study period and in whom MH susceptibility was subsequently confirmed. MH susceptibility was confirmed based on diagnostic RYR1 variants (n = 35) and/or positive contracture test (n = 6). In one patient (Patient 7.1) who had died because of the MH event (with an CGS of 70), the MH-associated RYR1 genotype was deducted based on genetic testing of both parents.

In addition to GAs with an MH event (GA+MH, n = 41), we also identified additional GAs without an MH event (GA-MH, n = 51) in the index cases and their relatives (n = 20) in whom MH susceptibility had been confirmed based on the identification of the familial RYR1 mutation(s) (n = 19) or a positive contracture test (n = 1). Detailed data from index patients and their relatives are summarized in Supplemental Table ST 1.

We then compared the characteristics of GA+MH and GA-MH (summarized in Table 1 and illustrated in Fig. 2). In the GA+MH group, there were 32 male and 9 females, with a median age of 21.5 years (range 5-68 years). There was a history of intense exercise within 72 h and/or pyrexia prior to the MH-triggering GA in 24 and 21 patients, respectively. Out of the 21 patients who presented with pyrexia, 3 had an additional history of intense exercise within 72 h and/or pyrexia prior to the MH-triggering GA. The majority of GAs in the GA+MH group was given on an emergency basis (n = 34), whilst a smaller proportion was administered electively (n = 7). All these patients received volatile anesthetic agents and 18 patients received succinylcholine (no data available for 1 patient). The most common surgery indications were trauma (n = 17), typically suffered during intense exercise, or acute abdomen (n = 10), most often attributed to appendicitis (n = 7), and in four cases with the latter diagnosis associated with pyrexia. In the GA-MH group, there were 31 male and 20 females. There was one individual with a history of pyrexia; no one had exercised within 72 h of the administration of a potentially MH-triggering agent. Majority of the GAs were elective (n = 42), whilst a smaller proportion was performed as emergency (n = 9). All patients received volatile anesthetic agents. Twelve patients received succinylcholine (no data available for 6 patients). The most common surgery indication was “other” (n = 44), whilst “trauma” (n = 4) and “acute abdomen” (n = 3) were much less common in this group.

Table 1Comparison of general anesthesias with MH (GA+MH) (i.e. anesthesias resulting in an MH event) and general anesthesia without MH (GA-MH).

Fig. 2Comparison of general anesthesia with MH (GA+MH) (i.e. anesthesia resulting in an MH event) and general anesthesia without MH (GA-MH). Bar charts illustrating percentage distribution of A) sex (males = black; females = grey), B) emergency (black) vs elective (grey) surgery and C) use of succinylcholine use (yes = black; no = grey; no data = light grey) in eventful (i.e. GA+MH, indicated as “+”) and uneventful general anesthesia (i.e. GA+MH, indicated as “-”). D) The surgery indications in the two groups are also indicated (trauma = horizontal stripes; acute abdomen = diagonal stripes; other = dots).

Comparing the two groups of GA+MH and GA-MH, as expected based on the selection criteria there were significant differences in history of intense exercise and/or pyrexia within 72 h prior to the triggering GA (P = 0.000), but also the use of succinylcholine (P = 0.000), frequency of emergency surgery (P = 0.000), and the combined incidence of trauma/acute abdomen as surgical indications (P = 0.000). Male predominance was seen in both groups, with no significant difference (P = 0.113).

Furthermore, multivariate analysis suggested that both the urgency of the surgery and the use of succinylcholine were associated with higher likelihood of an MH reaction.

4. Discussion

In this retrospective case study, we investigated strenuous exercise within 72 h and/or pyrexia prior to a triggering anesthetic leading to an MH reaction (GA+MH) in susceptible individuals. We compared GA+MH events with unselected uncomplicated GA (GA-MH) events in the same individuals (n = 17) and/or their MH susceptible relatives (n = 34); comparison with similarly mutated relatives (rather than unrelated control individuals) was elected to reduce genetic background variability. As expected, based on our selection criteria strenuous exercise within 72 h and pyrexia prior to the triggering anesthetic were common in the GA+MH group (n = 24 and n = 21, respectively) but not in the GA-MH group (n = 0 and n = 1, respectively). Three out of 41 individuals in the GA+MH group had a history of both pyrexia and of pre-operative exercise, and we cannot exclude that the elevated body temperature in these individuals may have been secondary to strenuous exercise rather than representing an independent factor. Furthermore, the use of succinylcholine, frequency of emergency surgery and the combined incidence of trauma/acute abdomen as surgical indications were higher in the GA+MH group.

These observations correspond to findings in RYR1-related ERM and EHS, where genetically predisposed individuals may suffer a single ERM/EHS episode after having exercised intensely, often in the presence of intercurrent viral illness with pyrexia, increased environmental temperatures, or intake of medical or recreational drugs, including alcohol [[8]Dlamini N Voermans NC Lillis S Stewart K Kamsteeg E-J Drost G et al.Mutations in RYR1 are a common cause of exertional myalgia and rhabdomyolysis.,[9]Molenaar JP Voermans NC van Hoeve BJ Kamsteeg EJ Kluijtmans LA Kusters B et al.Fever-induced recurrent rhabdomyolysis due to a novel mutation in the ryanodine receptor type 1 gene.].Our observations provide a possible, at least partial explanation for the currently unexplained discrepancy between the prevalence of MH-related RYR1 variants and the incidence of actual MH events [[1]Rosenberg H Pollock N Schiemann A Bulger T Stowell K. Malignant hyperthermia: a review.], suggesting an increase of the likelihood of an MH reaction when the trigger is given in the context of a pre-primed muscle (e.g. after intense exercise, already increased body temperature, or a combination of the above). We illustrated that when the index case or their MH susceptible relatives did not have any of the above-listed additional factors, they did not trigger despite receiving MH triggering agents. This is also supported by the finding that the ryr1Y522S MH-mouse model triggers an MH reaction when exposed to high ambient temperature, even in the absence of an MH triggering agent [[24]Durham WJ Aracena-Parks P Long C Rossi AE Goonasekera SA Boncompagni S et al.RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice.]. We identified strenuous exercise within 72 h and/or pyrexia > 37.5 oC prior to the GA in 8.6% of all MH cases confirmed on IVCT seen at the participating centers during the observation period, however, this is likely an underestimate as these features are not routinely captured in the anesthetic history and may thus not have specifically been asked for.The time interval of 72 h for pre-operative exercise was chosen based on the empirical characteristics of RYR1-related (exertional) myalgia and rhabdomyolysis, which (in contrast to exertional myalgia and rhabdomyolysis associated with, for example, glycogen or fatty acid oxidation defects) does not usually manifest during or immediately after exercise but after an interval of typically 24–72 h, occasionally even longer [[25]Scalco RS Snoeck M Quinlivan R Treves S Laforet P Jungbluth H et al.Exertional rhabdomyolysis: physiological response or manifestation of an underlying myopathy?.]. Muscle damage in RYR1-related (exertional) myalgia and rhabdomyolysis is thus not a direct effect of metabolic deficiency but of increased calcium levels and cellular damage; in this sense it is more like “pure overload RM” in healthy individuals in response to extreme triggers (but with a lower trigger threshold), where CK elevations usually peak at 24–36 h after exertion and then decreases back to baseline at a rate of about 40% per day [[26]Giannoglou GD Chatzizisis YS Misirli G. The syndrome of rhabdomyolysis: Pathophysiology and diagnosis.].Identification of several recurrent MH-associated RYR1 variants mirrored their relative high frequency in MH populations [[13]Miller DM Daly C Aboelsaod EM Gardner L Hobson SJ Riasat K et al.Genetic epidemiology of malignant hyperthermia in the UK.]. The impact of genotype on developing an MH event may be variable depending on the mutation and the distribution of mutated and wildtype proteins within the RyR1 channel. It is conceivable that some RYR1 variants have stronger MH-inducing effects: On the cellular level, RYR1 variant-dependent differences in calcium homeostasis have been documented [[27]Monnier N Kozak-Ribbens G Krivosic-Horber R Nivoche Y Qi D Kraev N et al.Correlations between genotype and pharmacological, histological, functional, and clinical phenotypes in malignant hyperthermia susceptibility.], however, our study was not designed to investigate this question.The observation of many asymptomatic relatives in our series confirms the recognized low and variable penetrance of MH, and further supports our hypothesis of pre-operative muscle priming as a potential modifying factor in MH. The presumption of MH and ERM as multifactorial events is supported by observations in animal models. The Y522S MH mouse model can exercise uneventfully under normal environmental temperatures, however, an increase in environmental temperature whilst exercising will result in ERM, rapid metabolic decompensation and death, due to a vicious and self-sustaining cycle of decompensated calcium leak, increased reactive nitrogen species, enhanced S-nitrosylation of the RyR1 receptor resulting in increased temperature sensitivity of the receptor and a further increase of calcium leak [[24]Durham WJ Aracena-Parks P Long C Rossi AE Goonasekera SA Boncompagni S et al.RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice.,[28]Chelu MG Goonasekera SA Durham WJ Tang W Lueck JD Riehl J et al.Heat- and anesthesia-induced malignant hyperthermia in an RyR1 knock-in mouse.]. This cycle can be fed into by several external factors, in addition to exercise and increased environmental temperatures also including potentially triggering anesthetic agents. A study in rats further demonstrated that acute exercise results in increased pRyRSer2840 levels and dissociation of stabilizers from the RyR1 receptor [[29]Suhr F Braun K Vanmunster M Bloch W. Acute skeletal muscle contractions orchestrate signaling mechanisms to trigger nuclear nfatc1 shuttling and epigenetic histone modifications.]. Intense exercise can also result in extensive fragmentation of the RyR1 in human muscle biopsies and isolated mouse fibers, and in lower levels of RyR1-bound FKBP (FK506-binding protein), leading to unstable RyR1 tetramers and calcium leak [[29]Suhr F Braun K Vanmunster M Bloch W. Acute skeletal muscle contractions orchestrate signaling mechanisms to trigger nuclear nfatc1 shuttling and epigenetic histone modifications.]. If MH triggering agents are given to a genetically susceptible patient on the background of a muscle already primed through the mechanisms outlined above, this may lower lower the threshold, resulting in an MH reaction as observed in our study. This hypothesis is also in line with current hypotheses concerning rhabdomyolysis, also considered a composite outcome of genetic predisposition and additional environmental factors [[30]Kruijt N den Bersselaar LV Snoeck M Kramers K Riazi S Bongers C et al.RYR1-related rhabdomyolysis: a spectrum of hypermetabolic states due to ryanodine receptor dysfunction.,[31]Kruijt N van den Bersselaar LR Kamsteeg EJ Verbeeck W Snoeck MMJ Everaerd DS et al.The etiology of rhabdomyolysis: an interaction between genetic susceptibility and external triggers.]. Fig. 3 illustrates suggested pathophysiological mechanisms underlying the relationship between exercise, pyrexia and MH.

Fig. 3Proposed mechanisms in Malignant Hyperthermia (MH). Based on the findings in our cohort, we propose A) that MH is the combined outcome of both genetic and non-genetic factors. B) Non-genetic factors include exposure to triggering agents, intense exercise, pyrexia and possibly others. Depending on the “strength” of the genetic background, the relative contribution of genetic and non-genetic factors may be variable.

In addition, increased sweating during exercise and pyrexia may lead to decreased body water and electrolyte disturbances, processes that play a major role in the etiology of ERM and EHS [[32]Westwood CS Fallowfield JL Delves SK Nunns M Ogden HB Layden JD. Individual risk factors associated with exertional heat illness: A systematic review.] and, based on our observation, may also be an important confounder for MH. Because of the retrospective nature of our study, we were not able to collect data comparing the hydration status of patients during GA+MH and GA-MH. In addition to a disturbed body water and electrolyte disturbance, hypermetabolic states induced by exercise or pyrexia may lower the threshold for the decompensated hypermetabolism characteristic of MH.Another important observation in our cohort is the predominance of trauma and acute abdomen as surgical indications in the GA+MH group. Most fractures requiring surgical intervention were not only suffered in the course of intense physical exercise, an independent risk factor for MH identified in this study, but often in the context of physical exercise involving heavy gear (such as skiing, playing ice hockey, etc.), a factor that may have had an additional priming effect through increasing body temperature before receiving the trigger. In particular, whilst exercise-induced increases in body temperature are usually controlled by thermoregulation and brought down with decreasing metabolic rates post-exercise, it is conceivable that whilst not its primary cause, the use of such gear may well have interfered with above above compensatory mechanisms. Moreover, the number of appendectomies in the GA+MH group was much higher than expected based on the number of appendectomies in a randomly taken survey of surgery indications [[33]

Statistiek CBv, Operaties in het ziekenhuis; soort opname, leeftijd en geslacht 1995-2010, in 1995-2010.

]. While succinylcholine, a known trigger was used in all the acute abdomen cases, our multivariate analysis showed the emergency nature of the surgery in addition to acute abdomen and trauma occurred significantly higher in the GA+MH group. It is of particular interest in the context of our findings that acute rhabdomyolysis may mimic an acute abdomen [[34]Exercise-induced abdominal wall muscle injury resulting in rhabdomyolysis and mimicking an acute abdomen.,[35]Newman J Case Kandikattu B. 4: Recurrent orange urine and abdominal pain in a 13-year-old boy.], and thus tempting to speculate that the patients in our series anesthetized for emergency surgical exploration of an acute abdomen (with “acute appendicitis” the most common surgical indication within this subgroup) may in fact have been in established rhabdomyolysis, resulting in a lower threshold to develop an MH event with administration of triggering agents on the background of a “primed” muscle. This hypothesis could not be further verified because of the lack of pre-operative CK samples in our series, but is supported by the previous observation of a fatal MH reaction during an emergency appendectomy in timely association with intense and unaccustomed exercise [[20]