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

Tony Huang, James Yan, Shelley Shen, Jia Nan (Cathy) Xu

Malignant Hyperthermia and Pickled Pigs

PHM142 Fall 2015 Instructor: Dr. Jeffrey Henderson

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

(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.

http://www.amc.seoul.kr/asan/healthinfo/disease/diseaseDetail.do?contentId=32326

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

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.

Anae

sthe

tics

Skeletal Muscles

Masseter spasm

Calcium build up

Muscle contraction

hypermetabolism

Heat

Fever

Tachycardia and Tachypnoea

Early Clinical Signs of Malignant Hyperthermia

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

Hopkins, P. M. (2000) Malignant hyperthermia: advances in clinical management and diagnosis. Br. J. Anaesth. 85, 118–128.c

Mechanism of Calcium Overabundance

Sarcoplasmic reticulum

Motor neuron

T-tubule

Ca2+

Ryanodine Receptor

DHP Receptor

myosin

actin

© Pearson Education, 2009

Possible Mechanism of Hyper-metabolism

High intracellular [Ca2+] activates phosphorylase kinase activityActive Phosphorylase

Kinase

Glycogen phosphorylase

Glycogen phosphorylase

ATP ADP

GlycogenGlucose-1-phosphate

Glycolysis

High Ca2+

Fletterick RJ, Sprang SR. Glycogen phosphorylase Structures and function. Accounts of Chemical Research. 1982 Nov; 15(11):361-369.

Active

Pi

Additionally, other catabolic enzymes in glycolysis and TCA cycle are activated by Ca2+

T-tubulePotential difference

Repolarized

Depolarized

Agonists

Inhibitors

Effects of MH Mutations in RYR1

• Physiological & pharmacological agonists:

Ca2+, calmodulin, ATP, caffeine, 4-chloro-m-cresol, halothane

• Physiological & pharmacological agonists:

Mg2+, ruthenium red and dantrolene.

Treves, S. (2005) Ryanodine receptor 1 mutations, dysregulation of calcium homeostasis and neuromuscular disorders. Neuromuscular Disorders 15, 577–587.

T-tubulePotential difference

Repolarized

Depolarized

Agonists

Inhibitors

Effects of MH Mutations in RYR1

1) Lower potential for depolarization state:-30 mV in normal vs -50 mV in MH

1

(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.

T-tubulePotential difference

Repolarized

Depolarized

Agonists

Inhibitors

Effects of MH Mutations in RYR1

2) Longer active conformation of DHPR, stabilizes RYR1 at sensitive state (ie. Sensitive to agonists)

1 2

(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.

T-tubulePotential difference

Repolarized

Depolarized

Agonists

Inhibitors

Effects of MH Mutations in RYR1

3) Mutations in agonist binding and auto-inhibitory regions of RYR1 cause hypersensitivityRYR1 open with Lower [agonists] (Ca2+, halothane)

1 2

3 3

(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.

T-tubulePotential difference

Repolarized

Depolarized

Agonists

Inhibitors

Effects of MH Mutations in RYR1

4) Desensitize response to Mg2+, an antagonist.

1 2

3

4

3

(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.

T-tubulePotential difference

Repolarized

Depolarized

Agonists

Inhibitors

Effects of MH Mutations in RYR1

5) Retrograde effect: mutated RYR1 conformation stabilizes activated DHPR conformation more

1 2

3

4

5 3

(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.

Increase RYR1 channel open probability, Increase Ca2+ flux, sustained muscle contraction

T-tubulePotential difference

Repolarized

Depolarized

DHPRvoltage sensor

T-tubule associated

Inactivated

Activated

RYR1Ca2+ channel

SR associated

Closed, insensitive

Closed, sensitive

OpenAgonists

Inhibitors1 2

3

4

5 3

Effects of MH Mutations in RYR1

(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.

T-tubulePotential difference

Repolarized

Depolarized

DHPRvoltage sensor

T-tubule associated

Inactivated

Activated

RYR1Ca2+ channel

SR associated

Closed, insensitive

Closed, sensitive

OpenAgonists

Inhibitors

Dantrolene

• 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: Treatment

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