Malignant Hyperthermia (MH) and EHS Share Genotype-Associated Susceptibility

Among individuals with genetic susceptibility to MH, there are those who appear to be at risk also for what is termed “awake” or nonanesthetic malignant hyperthermia that was defined early on as porcine stress syndrome in the 1960s and linked to human conditions [66, 67]. More than 50 years later, EHS links to MH have been more specifically defined with terminology such as “MH-like syndrome” to address possible variations of the syndromes [59]. Novel variants or presence of two mutations on different alleles [68] may decrease the thermal tolerance of an individual to environmental conditions, exercise, infection, and any drugs that affect heat balance. Data confirm the likelihood of relationships between MH-related genotypes and exertional heat stroke or sensitivity to exercise-heat stress. Strenuous treadmill running induces hyperthermia and rhabdomyolysis in mice with altered RYR1 genes [65]. Researchers have performed diagnostic contracture tests on individuals who have collapsed with EHS [69], observing that up to 45.6% of EHS survivors were positive for an in vitro contracture test, indicating MH susceptibility [63]. Multiple case reports have described cases in which individuals who suffer from an exertional heat stroke respond positively to an IVCT [10, 11]. Wappler et al. identified 10 out of 12 patients presenting with EHS symptoms to also be MH-susceptible. Three of these ten had candidate ryr1 variants [70]]. Bendahan et al. diagnosed 26 with EHS and all of them were positive for an IVCT test [71].

A dysfunctional RYR1 variant confirms a diagnosis of MH in conjunction to an IVCT, but MHS (malignant hyperthermia susceptibility) is genetically heterogeneous [69]. Genetic variants other than those associated with RYR1 are associated with different forms of MH and thus perhaps with EHI/EHS susceptibility. Among MH cases not associated with RYR1 variants, ~1% [60] (MH5) are associated with defects in the CACNA1S gene. CACNA1S encodes the alpha-1 subunit of an L-type voltage-gated calcium channel in skeletal muscle that responds to a depolarizing stimulus [61, 72]. Another calcium channel gene associated with MH, CACNA2D1 [73, 74], encodes the subunit of a calcium channel in the skeletal muscle that is associated with RYR1 at triadic junctions. These and other genetic variants associated with less common types of malignant hyperthermia (MH2–6) exhibit clinical manifestations that may be related to thermal intolerance and risk for exertional heat complications, but no consistent and direct link has been found between many of these variants and risk for EHS or thermal intolerance in exercise-heat stress settings. Studies of MH and EHI/EHS patients have revealed isolated cases of individuals positive for mutations in CACNA1S and CACNA2D1 [75, 76].

Other proteins such as calsequestrin-1 (CASQ1) have also been explored as candidate genes for MH and EHS susceptibility [77, 78]. CASQ1 encodes a protein that regulates RYR1-mediated calcium release. In 2009, researchers [77] created a CASQ1 knockout model in mice and observed that these mice had increased susceptibility to and spontaneous mortality during halothane anesthetic administration and heat stress. CASQ1 null mice experienced hyperthermia and rhabdomyolysis during heat stress and were successfully treated if administered with dantrolene, a drug that is known to reduce RyR1 calcium release and is used to treat malignant hyperthermia. In a study of 75 MH-susceptible (MHS) individuals diagnosed by positive IVCT, clinicians and researchers determined that a CASQ1 variant (c.260 T > C) was present in 6 heterozygous individuals also carrying an MH-causative mutation in RYR1; further analysis of 130 additional patients with MH susceptibility (positive IVCT and causative RYR1 mutation) revealed 9 additional heterozygous individuals for the CASQ1 variant. However, based on analysis of additional MHS and MHS-negative samples, researchers determined that the CASQ1 variant they observed was not likely to be a major MHS locus in the North American population [79]. Other investigators [78] have identified additional CASQ1 variants that appear to be linked to heat stroke patients in populations outside North America and further identified other possible genetic links to heat intolerance and heat stroke susceptibility and pathophysiology.

Gene variants associated with additional MHS classifications have not been as well-studied with respect to EHS susceptibility, but they are considered candidate genes to explore in the future. MHS2 is a form of malignant hyperthermia that stems from a mutation on the gene coding for the voltage-gated sodium channel SCNA4 on chromosome 17q [80, 81]. Problems with this channel typically result in hyperkalemic periodic paralysis, but Olckers et al. [81] felt there was enough evidence to link mutations in the SCNA4 gene to malignant hyperthermia susceptibility. MHS4 was determined to exist in a single German family by Sudbrak et al. (1995). They linked markers defining a 1-cM interval on chromosome 3q13.1. They confirmed the MH phenotype via an in vitro contracture test [82]. MHS6 was discovered in 1997 by Robinson et al. A genome-wide search of a Belgian family with MH revealed linkage to a region on chromosome 5p. Robinson et al. concluded their study by suggesting that there is at least one more MHS locus [72].

Novel genes not yet associated with specific MH subtypes also are interesting candidates to consider in EHS susceptibility. One recent target is the TRPV1 (transient receptor potential vanilloid 1) nonselective cation channel which is responsible for sensing noxious stimuli in the nervous system. This receptor which is capable of detecting temperature, pain, and other stimuli can vary among individuals and appears to be affected by the hypermetabolic state in MH. One comprehensive study in 2018 [83] identified that TRPV1 is activated by anesthetics in cell culture models of MH. More compelling are their findings that of 28 MH patients, 2 unrelated patients possessed rare genetic TRPV1 variants; transfection of these variants into cell culture affected calcium signaling and response to anesthetic-induced events in vitro/in situ. Treatment of animal models of heat stroke with GSK2193874, a TRPV4 channel, was not prophylactically effective but was effective in reducing heat stroke-related injury when administered at the onset of heat stroke [84]. Physiological, tissue-level, biomarker, and lethality outcomes were improved with the administration of this drug [84], contributing to the evidence that this may also be a novel MHS genotype that can be further explored in future EHS research. There are likely other heritable factors that contribute to different classifications of MH and may be associated with EHS but much remains unknown in this area.

Non-MH Related Heritability Is Associated with Exercise-Heat Intolerance

Data linking MH and EHI/EHS are compelling. Less understood is how genotypes associated with other clinical conditions are related to high incidence of intolerance to exercise-heat stress. For example, mutations in gene coding for RYR2, a ryanodine receptor found in cardiac muscle, can lead to sudden, exercise-induced death (catecholaminergic ventricular tachycardia). There are two proposed mechanisms. The first is that a decreased concentration of channel stabilizing protein calstabin-2, which binds and stabilizes the “closed” state of RYR2, causes a calcium leak into the cardiac tissue, triggering cardiac arrhythmias. The second mechanism purports that during exercise, RYR2 is phosphorylated by PKA, causing dissociation of the binding protein FKBP12.6. In patients with stress-induced polymorphic ventricular tachycardia, mutations in the genes coding for RYR2 resulted in a decrease in affinity for FKBP12.6. Similar to the first mechanism, this dissociation causes an increase in intercellular calcium, resulting in cardiac dysfunction [85].

Another example of a heritable condition increasing susceptibility to exercise-heat stress is sickle cell trait/disease. Individuals with sickle cell trait (SCT) are already at a greater risk of exercise-induced death illness given the nature of the disease. Exertional sickling can be compounded by high heat, humidity, and dehydration, coupled with intense bouts of exercise in which blood oxygen is reduced [85].

SCT is no longer considered a result of one genetic mutation but rather a combination of multiple genotypes that determine the severity of an individual’s disease [86, 87]. Hemoglobin, the oxygen-carrying protein in red blood cells, comprises two α– and two β-subunits. Fetal hemoglobin contains γ-subunits instead of β-subunits. Sickle cell hemoglobin (HbS) is monogenetically inherited and is common across all phenotypes. A single nucleotide substitution occurs on chromosome 11p15.5 and results in hydrophobic valine replacing hydrophilic glutamic acid in the β-subunit of hemoglobin (Hb), denoted as β^S [88]. This mutation can interact with other β-subunit mutations to produce varying severities of sickle cell disease. Other genotypes can influence sickle cell aggregation to the endothelial cells, the degree of sickling, and the concentration of fetal hemoglobin. Homozygotes for β^S, which lead to a condition known as sickle cell anemia (HbSS) , demonstrate the most severe phenotype, which manifests almost identically to another genotype, heterozygous HbS/β⁰ thalassemia. β⁰ thalassemia is a condition in which no β-subunits are produced. HbS/β⁺ thalassemia is a heterozygotic disease in which β-subunits are produced, but in lower amounts, and phenotypically is much less severe [89]. Individuals with HbS combined with a hereditary persistence of fetal Hb demonstrate a mild phenotype or are symptom-free [87, 88].

Healthy erythrocytes have a high degree of deformability, allowing them to traverse small arterioles and capillaries. In individuals with sickle cell trait, stress (e.g., low oxygen, dehydrated, high temperature, low pH) induces HbS polymerization and erythrocyte sickling [87]. Sickled red blood cells [26, 27] become trapped resulting in vaso-occlusion and reduced blood flow to tissues including skeletal muscle. Other events compounding this include adhesion of erythrocytes to the epithelium and the binding of leukocytes to the sickled cells [27]. The presence of epinephrine can also exacerbate the adhesion of sickled cells [88]. Local inflammatory responses continue to aggravate the adhesion and the binding of immune cells, and the local hypoxic environment caused by the vaso-occlusion contributes to further sickling of cells. This causes a positive feedback loop that ultimately results in the “metabolic failure” of exercising muscle [87].

The risk of death from exertional sickling is greatly elevated in both athletes and military personnel [90, 91]. Most reported cases of exertional collapse associated with sickle cell trait (ECAST) occur during preseason training, or after return from injury. Other musculoskeletal stresses, especially those which have been shown to lead to exertional rhabdomyolysis (ER) , can decrease the “threshold” required for a severe ECAST [92].

Of note, while many case studies examine exertional sickling, rhabdomyolysis, and exertional heat stroke during the football preseason when athletes are exposed to hot and humid environmental conditions in an unacclimatized state, high temperatures are not a prerequisite for exertional sickling. Individuals will often have an ECAST episode early enough during the exercise without core body temperature reaching pathologically high levels. Thus, patients lack the elevated core temperature found in EHS patients and do not have impaired CNS function [92, 93]. It is thought that leading cause of exertional sickling is exercise intensity [93]. The link between SCT and EHS genetic susceptibilities is thus not entirely clear, though data linking SCT and EHI/EHS is suggestive, both in military and athlete populations [94–96].

Metabolic Disturbances Likely Reduce Tolerance to Exercise-Heat Stress

Rhabdomyolysis is a condition during which striated muscle, typically skeletal muscle, breaks down due to high mechanical and metabolic stress. The consequent release of the muscle contents can lead to myoglobinuria, acute kidney injuries, acute compartment syndrome, and, in extreme cases, arrhythmias and death [97]. Exertional rhabdomyolysis (ER) is a pathological condition where muscle breakdown occurs after a bout of exercise. In most cases, ER occurs as a result of excessive, prolonged, or repeated eccentric exercises [97, 98]. Individuals with a low baseline fitness level or low heat tolerance are also at risk for ER [29]. There are several inherited skeletal muscle metabolic disorders that increase the risk of developing ER as a result of intense exercise.

Carnitine palmitoyltransferase (CPT) II is an enzyme located on the inner mitochondrial membrane that catalyzes the formation of fatty acyl co-A. The CPT system facilitates long-chain fatty acid transport into the mitochondria. Mutations in the gene coding for CPT II can cause deficiency of the enzyme, though the degree of disease caused by CPT II deficiency is variable [32]. Nonfatal forms of the disease are characterized by exercise-induced muscle pain, rhabdomyolysis, and myoglobinuria [99]. A Japanese study [100] of 24 heat stroke (non-exertional) patients revealed a common CPTII variant (F352C) in 11 patients that was associated with greater JAAM DIC diagnostic criteria score and SOFA indicating greater coagulopathy and organ dysfunction that was associated with double mean hospital stays (21.5 ± 23.0 vs. 9.5 ± 13.2 days). All other aspects of acute illness including body temperature, albumin, and creatine kinase, were similar between those with F352C variant and control patients.

Very long-chain acetyl-CoA dehydrogenase deficiency (VLCAD) is an inherited metabolic disease of fatty acid beta-oxidation. Clinically, VLCAD deficiency manifests in one of three ways: (1) early onset, which is very severe and has a high mortality rate, (2) childhood onset with intermediate severity and a more favorable outcome, and (3) adult onset, which is usually characterized by rhabdomyolysis and myoglobinuria after exercise [101]. The disease is the result of a homozygous mutation in the gene coding for very long-chain acyl-CoA dehydrogenase on chromosome 17p13 [101]. Other common inherited fatty acid metabolic disorders leading to rhabdomyolysis are medium-chain acyl-CoA dehydrogenase (MCAD) and trifunctional enzyme deficiency [102, 103].

McArdle disease , or glycogen storage deficiency V is another heritable metabolic disease that increases susceptibility to rhabdomyolysis. GSD5 is caused by a homozygous mutation in the genes coding for muscle glycogen phosphorylase, at chromosome 11q13 [104]. Affected individuals are unable to mobilize muscle glycogen stores [105]. During exercise, patients experience muscle cramps, weakness, and myalgia [35]. Transient episodes of myoglobinuria can occur as a result of rhabdomyolysis, and severe myoglobinuria can lead to kidney failure [104, 106].

Other forms of glycogen storage deficiencies can lead to an increased risk of exertional myopathies. GSD7 is an autosomal recessive disorder and comes from a mutation on chromosome 12q13, the gene coding for muscle phosphofructokinase (PFK) [106]. Symptoms include muscle cramping, exercise intolerance, and exertional myopathies such as a rhabdomyolysis. In severe cases, myoglobinuria occurs as a result of muscle damage. Unique to GSD7 are a partial loss of muscle PFK and partial loss of red cell PFK activity [107]. GSD11 is caused by a mutation in the gene coding for muscle lactate dehydrogenase (LDHM), on chromosome 11p15. As with other glycogen storage deficiencies, patients complain of exercise intolerance and myoglobinuria. GSD11 has been documented only in the Japanese populations, thus far, and is relatively rare [108]. In many cases, glycogen storage deficiencies are mild, and many patients do not seek medical attention [107].

The link between rhabdomyolysis and exertional heat stroke is complex though the conditions often occur concurrently due to MOD and specifically muscle tissue damage often seen in EHS. However, individuals with genetic predisposition to rhabdomyolysis and other muscle disorders often present exercise intolerance as a symptom, due to factors like ineffective fatty acid metabolism and issues related to glycogen storage and use. This could limit the ability of an athlete to exercise effectively in the hot conditions.