1. Malignant Hyperthermia Joseph Blommesteyn Leanne Kong Ryan Marko Dario Moscoso PHM142 Fall 2014 Instructor: Dr. Jeffrey Henderson

2. Overview Genetic Basis Biochemical Mechanisms Diagnosis and Treatment Susceptibility Testing and Prevention

3. Malignant Hyperthermia Inherited disorder that is usually triggered by exposure to certain general anesthetics, specifically volatile anesthetic agents and succinylcholine Susceptible individuals may experience excessive production of heat and lactic acid, which can lead to acidosis and death if not treated immediately

4. Muscle Contractions – EC Coupling 1.Contractile signal received at NMJ 2.ACh release and sarcolemma depolarization 3.DHPRs undergo conformational change 4.RYR1s undergo conformational change 5.Ca 2+ released from SR 6.Myofilament contraction 7.Ca 2+ re-sequestering by ATP-dependent pumps on the SR 8.Process is ready to start again Anaesthesia and Intensive Care Medicine, 12(6), 263-265

5. Genetic Basis of MH Pharmacogenetic disorder Autosomal dominant inheritance Epidemiology: Incidence: – ~ 1/15,000 children – ~ 1/50,000 adults Genetic susceptibility: – Between 1/3,000 and 1/10,000 ~ 2X more common in males than in females Orphanet Journal of Rare Diseases, 2, 1-14. Pflugers Arch. 460(2), 467-480.

6. Genetic Basis of MH Heterogeneous genetic disorder: 6 different loci containing MH-associated mutations LocusChromosomal LocationGene MHS119q13.1RYR1 MHS217q11.3-q24Unidentified MHS37q21-q22Unidentified MHS43q13.1Unidentified MHS51q32CACNA1S (DHPR) MHS65pUnidentified Loci and genes involved in MH: Isr. Med. Assoc. J., 9(1), 39-41.

7. RYR1 (~ 50-70%) DHPR (~ 1%) Anaesthesia and Intensive Care Medicine, 12(6), 263-265

8. Ryanodine Receptor 1 RYR1: Skeletal muscle isoform ~ 2 MDa homotetramer 300 known genetic variants 151 MH-associated point mutations 34 causative mutations (so far) (Top-down view) 12 34 Nature, 468(7323), 585-588

9. Mutation site: RyR1 AA sequence: RYR1 Mutation Hot-Spots T-tubule SR DHPR RYR1 Mutations cluster in “hot-spots”: DomainAmino Acids N-terminalC35 – R614 CentralA2129 – R2458 C-terminalI3916 – G4942 These clusters correspond to regions at the interface between the individual subunits Cold Spring Harbor Perspectives in Biology, 2(11), a003996. Biochem. Biophys. Res. Comm., 322(4), 1280-1285.

10. RYR1s and MH Mutations make the RYR1 more hypersensitive to channel-opening stimuli

11. Triggering Agents of MH Depolarizing Muscle Relaxants Succinylcholine is more potent than ACh Longer duration of effect, does not allow the muscle cells to repolarize Ca 2+ normally removed independent of repolarization With leaky RYR1s Ca 2+ conc. remains high in cytoplasm Inhalant Anesthetics Direct effect on DHPR-RYR1 complex that occurs irrespective depolarization Mg 2+ binds to RYR1s and inhibit their opening Mutant RYR1s have less affinity for Mg 2+ Inhalant anesthetics can overcome the inhibiting effects of the weakly associated Mg 2+ People susceptible to MH should avoid strenuous activity in hotter conditions as well since Ca 2+ reuptake cannot keep up with the leaky RYR1’s

12. Biochemical Consequences of MH ATP depletion from an increase in compensatory Ca 2+ reuptake processes and sustained muscle contractions ADP stimulates metabolic processes, and thus increases oxygen consumption, CO 2 production Heat generated from sustained muscle contractions Increase in permeability of cell membrane due to hyperthermia and muscle activity Leakage of cellular constituents Anaesthesia and Intensive Care Medicine, 12(6), 263-265

13. Diagnosis and Treatment of Malignant Hyperthermia

14. Differential Diagnosis Certain other disorders can imitate Common symptoms: masseter spasm hypercapnia (ETCO 2 >55mmHg), hyperthermia, tachycardia, arrhythmia, ECG changes Patient history: thyroid storm, infection/sepsis, pheochromocytoma

15. MH Crisis Confirmed Primary treatment centered on 3 pillars: Increasing inspired O 2 Discontinuing triggering agents Administering dantrolene: dose dependent on anesthetic dose used

16. Reasoning Behind Treatment High CO 2 needs to be brought to equilibrium with O 2 Triggering agents will continue to cause pathophysiology Dantrolene inhibits calcium release into the SR by binding ryanodine receptors

17. Treatment Options Charcoal filter prevents residual anesthetic from reaching patient Dantrolene sodium to be reconstituted: 2 formulations, new is hyperconcentrated

18. Secondary Treatment Treatment of hyperkalemia: CaCl 2, insulin + dextrose, furosemide Treatment of metabolic acidosis: HCO 3 Cooling of internal/external body surfaces/cavities: IV saline, ice (body temperature>39 C)

19. Susceptibility Testing In vitro caffeine-halothane contracture test (IVCT) – Involves a muscle biopsy in which the biopsy is bathed in solutions of halothane receptor agonists, caffeine and halothane, and tested for contraction – European Malignant Hyperthermia Group – North American Malignant Hyperthermia Group DNA Testing – RYR1 receptor mutation http://bestpractice.bmj.com/best-practice/monograph/1053/diagnosis/step-by-step.html

20. Prevention Patients should be screened before any procedure with anesthesia is being performed If patient is MH susceptible, trigger (potent volatile) anesthetics should be avoided Avoid use of depolarizing muscle relaxants Anesthesia machines should also be flushed to remove remnants of trigger anesthetics Ensure that dantrolene is available Performing regular MH drills to be prepared

21. Summary Malignant hyperthermia is a heterogeneous pharmacogenetic disorder that's typically inherited in an autosomal dominant fashion. 6 MH susceptibility genes have been mapped, but only two have been unambiguously identified: ryanodine receptor 1 (RYR1) and dihydropyridine receptor (DHPR). RYR1 mutations are associated with 50-70% of MH cases. RYR1 is a large homotetrameric calcium channel in the sarcoplasmic reticulum. MH-associated mutations tend to cluster in "hot-spots" at the interfaces between the four subunits. Mutant RYR1s are hypersensitive to Ca 2+ releasing stimuli. High cytoplasmic Ca 2+ concentration leads to increased activity of compensatory reuptake mechanisms (ATP-dependent Ca 2+ pumps). Depletion of ATP from these compensatory mechanisms as well as muscle contractions leads to increased metabolic processes. Sustained muscle contractions produce excess heat, increased oxygen consumption, increased CO 2 production, and lactate buildup. There is also leakage of cellular constituents (hyperkalemia and rhabdomyolysis). First one must establish patient is actually experiencing MH crisis; consider susceptibility, history, and administered agent. MH patients exhibit signs such as elevated body temperature, tachycardia, arrhythmia, muscle spasm and ECG changes. Primary treatment include ventilation with O 2, IV dantrolene, and removing triggers. Secondary treatment involves insulin/glucose, bicarbonate, diuretics and ice/cool saline. The gold standard for susceptibility testing of MH is the in vitro caffeine-halothane contracture test (IVCT), which involves taking a skeletal muscle biopsy and testing the contracture with soaking the biopsy in ryanodine receptor agonists (caffeine and halothane). High contracture denotes susceptibility to MH. Preventive measures: Avoid triggering anesthetics, avoid depolarizing muscle relaxants, and be prepared for the possibility of an episode occurring upon anesthesia by stocking dantrolene and doing drills

22. References Benkusky, N. A., Farrell, E. F., & Valdivia, H. H. (2004). Ryanodine receptor channelopathies. Biochemical and Biophysical Research Communications, 322(4), 1280-1285. Betzenhauser, M. J., & Marks, A. R. (2010). Ryanodine receptor channelopathies. Pflugers Archiv: European Journal of Physiology, 460(2), 467-480. Brandom, B.W., & Lehman, E.B. (2010). Clinical presentation, treatment, and complications of malignant hyperthermia in North America from 1987 to 2006. Anesthesia & Analgesia, 110, 498-507. Denborough, M. (1998). Malignant hyperthermia. The Lancet, 352, 1131-1136. Greenbaum, I., Weigl, Y., & Pras, E. (2007). The genetic basis of malignant hyperthermia. The Israel Medical Association Journal, 9(1), 39-41. Hopkins, P. (2011) Malignant Hyperthermia. Anaesthesia and Intensive Care Medicine, 12(6), 263-265 Kobayashi, S., Bannister, M. L., Gangopadhyay, J. P., Hamada, T., Parness, J., & Ikemoto, N. (2005). Dantrolene stabilizes domain interactions within the ryanodine receptor. The Journal of Biological Chemistry, 280(8), 6580-6587. Lanner, J. T., Georgiou, D. K., Joshi, A. D., & Hamilton, S. L. (2010). Ryanodine receptors: structure, expression, molecular details, and function in calcium release. Cold Spring Harbor Perspectives in Biology, 2(11), a003996. Larach, M.G., Gronert, G.A., Allen, G.C., MacLennan, D.H., & Phillips, M.S. (1992) Malignant hyperthermia. Science, 256, 789- 194. Litman, R., & Rosenberg, H. (2005) Malignant Hyperthermia: Update on Susceptibility Testing. Journal of the American Medical Association, 293, 2918-2924. Rosenberg, H., Davis, M., James, D., Pollock, N., & Stowell, K. (2007) Review: Malignant Hyperthermia. Orphanet Journal of Rare Diseases, 2, 1-14. Stowell, K. M. (2008). Malignant hyperthermia: a pharmacogenetic disorder. Pharmacogenomics, 9(11), 1657-1672. Tung, C. C., Lobo, P. A., Kimlicka, L., & Van Petegem, F. (2010). The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule. Nature, 468(7323), 585-588.