Malignant Hyperthermia and Pickled Pigs PHM142 Fall 2015 Instructor: Dr. Jeffrey Henderson Malignant Hyperthermia and Pickled Pigs Tony Huang, James Yan, Shelley Shen, Jia Nan (Cathy) Xu

Malignant hyperthermia in humans Triggered after administration of certain anaesthetics hypermetabolism and buildup of lactic acid autosomal dominant mutation of calcium channel RYR1 Alternative point mutations in skeletal muscle sodium channels and DHPR Malignant Hyperthermia is a life threatening condition that affects individuals with an autosomal dominant mutation in the RYR1 calcium channel. It occurs after administration of both non-halogenated anaesthetics such as diethyl ether and halogenated anaesthetics such as Halothane. If left untreated, the patient’s condition can quickly deteriorate as a result from uncontrolled temperature elevation and buildup of lactic acid. Malignant Hyperthermia primarily results from an autosomal dominant mutation in the RYR1 Calcium Channel. However, additional point mutations in skeletal muscle sodium channels and skeletal muscle Dihydropyridine receptors have also been found to be linked to Malignant hyperthermia. http://www.amc.seoul.kr/asan/healthinfo/disease/diseaseDetail.do?contentId=32326 (1) Pessah, I. N., and Allen, P. D. (2001) Malignant hyperthermia. Best Pract. Res. Clin. Anaesthesiol. 15, 277–288. (2) Treves, S., Anderson AA., Ducreux, S., Divet, A., Bleunven, C., Grasso, C., Paesate, S., Zorzato, F. Ryanodine receptor 1 mutations, dysregulation of calcium homeostasis and neuromuscular disorders.Neuromuscul Discord. 15(9-10), 577-87.

Pickled Pigs and Malignant Hyperthermia Porcine Stress Syndrome Mutation found on porcine chromosome 6q12 R615C in RYR1 Observed frequently due to founder effect Malignant Hyperthermia Mutation found on human chromosome 19q R614C in RYR1 prevalence ~ 1 in 15 000 to 100 000 patients A similar condition exists in pigs and its symptoms mirror the effects of Malignant Hyperthermia. Pigs with Porcine Stress Syndrome experience a similar state of elevated body temperature, acidosis, and muscle rigidity. Both diseases can be linked to mutations in the Type 1 Ryanodine Receptor in skeletal muscle. In pigs, the genes coding for this receptor can be found on chromosome 6q12 and 19q in pigs and humans respectively. Arginine to cysteine substitution mutations at the 615th position in pigs and 614th position in humans create mutant proteins with similar irregular functions. However, there are other known mutations that trigger MH in humans. This mutation is observed more frequently in pigs relative to humans because of a founder effect. As a result, the use of pigs suffering from porcine stress syndrome as an animal model for human malignant hyperthermia is beneficial simply due to the ability to draw data from a larger sample. Homologous mutations in the RYR1 calcium channel disrupt calcium homeostasis and result in hypermetabolism and lactic acidosis Jurkat-Rott, K. Genetics and pathogenesis of malignant hyperthermia. Muscle & Nerve 23, 4–17.

Tachycardia and Tachypnoea Anaesthetics Skeletal Muscles Masseter spasm Calcium build up Muscle contraction hypermetabolism Heat Fever Tachycardia and Tachypnoea Early Clinical Signs of Malignant Hyperthermia It is almost impossible to identify a patient who is susceptible to malignant hyperthermia without a detailed family medical history. Therefore, it is crucial to recognize the early symptoms to prevent long-term damage or even death to MH-susceptible patients. Shortly after the administration of certain anaesthetics, various symptoms of malignant hyperthermia can occur. TRigidity of the masseter muscle is one of the earliest signs of MH. As an abundance of calcium build up in the skeletal muscle, it forces the muscle to contract involuntarily. In addition, an overabundance of intracellular calcium stimulates metabolism by activating phosphrylase and increasing glycosis. This results in an overproduction of heat, which may display as a high fever. Tachycardia, which is an abnormally high pulse rate and tachypnoea, which is abnormally rapid breathing, will also occur due to hypermetabolism. Hopkins, P. M. (2000) Malignant hyperthermia: advances in clinical management and diagnosis. Br. J. Anaesth. 85, 118–128.

Late Clinical Signs of Malignant Hyperthermia Generalized skeletal muscle rigidity Ability to generate ATP is nearly exhausted. Lactic acidosis Cyanosis Low oxygen saturation Skin appear to be purple or blue Dark urine increased creatine kinase Temperature rise above 40ºC Death This can occur in as quickly as 10 minutes after the first display of symptoms. Hopkins, P. M. (2000) Malignant hyperthermia: advances in clinical management and diagnosis. Br. J. Anaesth. 85, 118–128.c

Mechanism of Calcium Overabundance Motor neuron T-tubule Ryanodine Receptor I will now review the process of a muscle contraction and the effects of malignant hyperthermia. The action potential from the motor end plate travels down the T-tubules where it is detected by the DHP receptor. The DHP receptor opens the Ryandoine/Ca receptor causing facilitated diffusion down the concentration gradient. Ca binds Troponin which pushes tropomyosin off the myosin binding head, and with ATP will cause the power stroke for contraction. In patients with malignant hyperthermia, the Ryanodine receptor is non functional causing Ca to flow in continuously causing sustained contraction and ATP use. With that, muscle will begin using anerobic resepiration and produce lactic acid. DHP Receptor Sarcoplasmic reticulum Ca2+ actin © Pearson Education, 2009 myosin

Possible Mechanism of Hyper-metabolism High intracellular [Ca2+] activates phosphorylase kinase activity Active Phosphorylase Kinase High Ca2+ ATP ADP Active Glycogen phosphorylase Glycogen phosphorylase Pi Glycogen Glucose-1-phosphate explain how everything downstream of glycogenolysis is triggered and how high ATP production coupled with high ATP consumption cause overheating In order to maintain the supply of ATP, the body will require more glucose from glycogen stores. This is a potential High intracellular Ca levels help activate phosphorylase kinase which phosphorylates the protein glycogen phosphorylase. The active Glycogen phosphorylase This enzyme promotes the cleavage of glycogen via the alpha 1,4 glycolytic bond and phosphorylation for preparation for glycolysis to generate more ATP. As more ATP are consumed by the muscles, it releases lots of heat causing the rise in body temperature. Additionally, other catabolic enzymes in glycolysis and TCA cycle are activated by Ca2+ Glycolysis Fletterick RJ, Sprang SR. Glycogen phosphorylase Structures and function. Accounts of Chemical Research. 1982 Nov; 15(11):361-369.

Effects of MH Mutations in RYR1 T-tubule Potential difference Repolarized Depolarized DHPR voltage sensor T-tubule associated Inactivated Activated RYR1 Ca2+ channel SR associated Closed, insensitive Closed, sensitive Open Inhibitors Agonists Physiological & pharmacological agonists: Ca2+, calmodulin, ATP, caffeine, 4-chloro-m-cresol, halothane Mg2+, ruthenium red and dantrolene. Treves, S. (2005) Ryanodine receptor 1 mutations, dysregulation of calcium homeostasis and neuromuscular disorders. Neuromuscular Disorders 15, 577–587.

Effects of MH Mutations in RYR1 T-tubule Potential difference Repolarized Depolarized DHPR voltage sensor T-tubule associated Inactivated Activated RYR1 Ca2+ channel SR associated Closed, insensitive Closed, sensitive Open Inhibitors 1 Agonists Lower potential for depolarization state: -30 mV in normal vs -50 mV in MH (1) Treves, S. (2005) Ryanodine receptor 1 mutations, dysregulation of calcium homeostasis and neuromuscular disorders. Neuromuscular Disorders 15, 577–587. (2) Estève, E., Eltit, J. M., Bannister, R. A., Liu, K., Pessah, I. N., Beam, K. G., Allen, P. D., and López, J. R. (2010) A malignant hyperthermia–inducing mutation in RYR1 (R163C): alterations in Ca2+ entry, release, and retrograde signaling to the DHPR. J Gen Physiol 135, 619–628. (3) Fruen, B. R., Mickelson, J. R., and Louis, C. F. (1997) Dantrolene Inhibition of Sarcoplasmic Reticulum Ca2+Release by Direct and Specific Action at Skeletal Muscle Ryanodine Receptors. J. Biol. Chem. 272, 26965–26971.

Effects of MH Mutations in RYR1 T-tubule Potential difference Repolarized Depolarized DHPR voltage sensor T-tubule associated Inactivated Activated RYR1 Ca2+ channel SR associated Closed, insensitive Closed, sensitive Open Inhibitors 1 2 Agonists 2) Longer active conformation of DHPR, stabilizes RYR1 at sensitive state (ie. Sensitive to agonists) (1) Treves, S. (2005) Ryanodine receptor 1 mutations, dysregulation of calcium homeostasis and neuromuscular disorders. Neuromuscular Disorders 15, 577–587. (2) Estève, E., Eltit, J. M., Bannister, R. A., Liu, K., Pessah, I. N., Beam, K. G., Allen, P. D., and López, J. R. (2010) A malignant hyperthermia–inducing mutation in RYR1 (R163C): alterations in Ca2+ entry, release, and retrograde signaling to the DHPR. J Gen Physiol 135, 619–628. (3) Fruen, B. R., Mickelson, J. R., and Louis, C. F. (1997) Dantrolene Inhibition of Sarcoplasmic Reticulum Ca2+Release by Direct and Specific Action at Skeletal Muscle Ryanodine Receptors. J. Biol. Chem. 272, 26965–26971.

Effects of MH Mutations in RYR1 T-tubule Potential difference Repolarized Depolarized DHPR voltage sensor T-tubule associated Inactivated Activated RYR1 Ca2+ channel SR associated Closed, insensitive Closed, sensitive Open Inhibitors 1 2 Agonists 3 3 3) Mutations in agonist binding and auto-inhibitory regions of RYR1 cause hypersensitivity RYR1 open with Lower [agonists] (Ca2+, halothane) (1) Treves, S. (2005) Ryanodine receptor 1 mutations, dysregulation of calcium homeostasis and neuromuscular disorders. Neuromuscular Disorders 15, 577–587. (2) Yamamoto, T., El-Hayek, R., and Ikemoto, N. (2000) Postulated Role of Interdomain Interaction within the Ryanodine Receptor in Ca2+ Channel Regulation. J. Biol. Chem. 275, 11618–11625.

Effects of MH Mutations in RYR1 T-tubule Potential difference Repolarized Depolarized DHPR voltage sensor T-tubule associated Inactivated Activated RYR1 Ca2+ channel SR associated Closed, insensitive Closed, sensitive Open Inhibitors 4 1 2 Agonists 3 3 4) Desensitize response to Mg2+, an antagonist. (1) Duke, A. M., Hopkins, P. M., Halsal, J. P., and Steele, D. S. (2004) Mg2+ dependence of halothane-induced Ca2+ release from the sarcoplasmic reticulum in skeletal muscle from humans susceptible to malignant hyperthermia. Anesthesiology 101, 1339–1346.

Effects of MH Mutations in RYR1 T-tubule Potential difference Repolarized Depolarized DHPR voltage sensor T-tubule associated Inactivated Activated RYR1 Ca2+ channel SR associated Closed, insensitive Closed, sensitive Open Inhibitors 4 1 2 5 Agonists 3 3 5) Retrograde effect: mutated RYR1 conformation stabilizes activated DHPR conformation more (1) Estève, E., Eltit, J. M., Bannister, R. A., Liu, K., Pessah, I. N., Beam, K. G., Allen, P. D., and López, J. R. (2010) A malignant hyperthermia–inducing mutation in RYR1 (R163C): alterations in Ca2+ entry, release, and retrograde signaling to the DHPR. J Gen Physiol 135, 619–628. (2) Bannister, R. A., Estève, E., Eltit, J. M., Pessah, I. N., Allen, P. D., López, J. R., and Beam, K. G. (2010) A malignant hyperthermia–inducing mutation in RYR1 (R163C): consequent alterations in the functional properties of DHPR channels. The Journal of general physiology 135, 629–640.

Effects of MH Mutations in RYR1 T-tubule Potential difference Repolarized Depolarized DHPR voltage sensor T-tubule associated Inactivated Activated RYR1 Ca2+ channel SR associated Closed, insensitive Closed, sensitive Open Inhibitors 4 1 2 5 Agonists 3 3 Increase RYR1 channel open probability, Increase Ca2+ flux, sustained muscle contraction (1) Jurkat-Rott, K. Genetics and pathogenesis of malignant hyperthermia. Muscle & Nerve 23, 4–17. (2) Treves, S. (2005) Ryanodine receptor 1 mutations, dysregulation of calcium homeostasis and neuromuscular disorders. Neuromuscular Disorders 15, 577–587.  

Summary: Treatment Dantrolene T-tubule DHPR RYR1 Inhibitors Agonists Potential difference Repolarized Depolarized DHPR voltage sensor T-tubule associated Inactivated Activated RYR1 Ca2+ channel SR associated Closed, insensitive Closed, sensitive Open Inhibitors Agonists Muscle relaxant Decrease Ca2+ efflux from SR to sarcoplasm Direct or indirect closure of RYR1 Similar effects in MH susceptible as in normal tissues Azumolene (alanlogue) of dantrolene shows similar effects (1) Krause, T. Dantrolene – A review of its pharmacology, therapeutic use and new developments. Anaesthesia 59, 364–373. (2) Fruen, B. R., Mickelson, J. R., and Louis, C. F. (1997) Dantrolene Inhibition of Sarcoplasmic Reticulum Ca2+Release by Direct and Specific Action at Skeletal Muscle Ryanodine Receptors. J. Biol. Chem. 272, 26965–26971. (3) Do Carmo, P. L., Zapata-Sudo, G., Trachez, M. M., Antunes, F., Guimarães, S. E. F., Debom, R., Rizzi, M. D. R., and Sudo, R. T. (2010) Intravenous Administration of Azumolene to Reverse Malignant Hyperthermia in Swine. Journal of Veterinary Internal Medicine 24, 1224–1228.

Summary Malignant Hyperthermia is a deadly disease triggered by certain anaesthetics in individuals with mutations in skeletal muscle proteins Pigs experience similar symptoms with Porcine Stress Syndrome and thus are good animal models Calcium overabundance induces uncontrollable muscle contractions which leads to hypermetabolism and excess heat. Caused by a mutation of the ryanodine receptor 1 (RYR1) which is responsible for calcium homeostasis Early clinical features include hyperthermia and lactic acidosis Later clinical signs include rhabdomyolysis, cyanosis, and death Drugs such as dantrolene and azumolene can reverse the effects of Malignant Hyperthermia