Pickled Pigs and Malignant Hyperthermia

1. PHM142 Fall 2013 Instructor: Dr. Jeffrey Henderson Pickled Pigs and Malignant Hyperthermia Augustina Esedebe, Colin Hesp, Gerick Abaca, Yan Zhuo October 9, 2013

2. Overview • Malignant hyperthermia • Pickled pigs • Genetic Basis • Mechanism • Treatment & Prevention • Summary

3. What is Malignant Hyperthermia?

4. What is Malignant Hyperthermia? Hyperthermia vs. Fever • Hyperthermia: body absorbs more heat than it can lose • Fever: increase in set point for thermoregulation; fight infection

5. What is Malignant Hyperthermia? Hyperthermia vs. Fever • Hyperthermia: body absorbs more heat than it can lose • Fever: increase in set point for thermoregulation; fight infection Malignant Hyperthermia • Rare, life-threatening disease • Inherited; autosomal dominant

6. What is Malignant Hyperthermia? Symptoms • Dramatic rise in body temperature (>38ºC) • Sweating

7. What is Malignant Hyperthermia? Symptoms • Dramatic rise in body temperature (>38ºC) • Sweating • Acidosis: ↓ pH • Metabolic – body produces excessive acid; not enough time to remove in kidney • Respiratory – increased [CO2] in the blood • hypoventilation

8. What is Malignant Hyperthermia? Symptoms • Dramatic rise in body temperature (>38ºC) • Sweating • Acidosis: ↓ pH • Metabolic – body produces excessive acid; not enough time to remove in kidney • Respiratory – increased [CO2] in the blood • hypoventilation • Skeletal muscle rigidity & stiffness • dark brown urine

9. What is Malignant Hyperthermia? Onset • Life-threatening side effect occurs within minutes of exposure to anesthetics: • Inhaled Anesthetics: Halothane (most common), enflurane, isoflurane, sevoflurane • Other suspected drugs: monoamine oxidase inhibitors, catecholamines, and phenothiazines.

10. How are pickled pigs and malignant hyperthermia related? • Human syndrome: Malignant hyperthermia • Pig syndrome: porcine stress syndrome • Pigs exposed to halothane (anaesthesia): • Rise in body temperature • Rigidity in muscles • Pig death by MH  pickled pigs • Flesh turn watery • Pale • Very low pH; anaerobic fermentation = ?

11. Why are pickled pigs important? HEALTH: Pig used as a model for humans • Human syndrome is rare; use pigs as proxy for research • Porcine stress syndrome is similar to human malignant syndrome • Study: Harrison and colleagues showed three pigs dying from hyperthermia through inhaled anaesthesia, halothane. • Eradication Importance: Human syndrome > Porcine syndromes

12. Genetic Basis of Malignant Hyperthermia • Autosomal dominant mode of inheritance in humans. In swine, it is an autosomal recessive disorder • Single amino acid substitution on the RYR1 gene from cysteine to arginine at position 615 in swine susceptible to porcine stress syndrome • In humans, mutations in two genes leading to malignant hyperthermia susceptibility have been identified: • RYR1 • Chromosomal locus 19q13.2 • Encodes the type 1 ryanodine receptor of skeletal muscle • Mutations in RYR1 account for 70-80% of individuals with confirmed malignant hyperthermia susceptibility

13. Genetic Basis of Malignant Hyperthermia • CACNA1S • Chromosomal locus 1q32.1 • Encodes the α1-subunit of the voltage-dependent L-type calcium channel found in skeletal muscle • Mutations in CACNA1S account for 1% of individuals with confirmed malignant hyperthermia susceptibility • Four additional loci have been mapped to specific chromosome intervals; however the genes have not been identified: • MHS2 (linked to chromosomal locus 17q11.2-q24) • MHS4 (3q13) • MHS6 (5p) • MHS3 (7q21-q22)

14. Mechanism of muscle contraction

15. Mechanism of Malignant Hyperthermia http://www.biozentrum.uni-wuerzburg.de/fileadmin/REPORT/HUMGE/humge006.htm

16. Mechanism • Electric signal from nerve reaches sarcoplasmic membrane • Depolarization travels down T tubule • Activate voltage-gated DHPR receptor in terminal cisternae of sarcoplasmic reticulum (SR) • Activate RYR1, Ca2+ is released • Ca2+ binds with troponin → muscle contraction • Ca2+ binds with phosphorylase kinase  activate glycolysis  pyruvate •  lactic acid •  aerobic respiration in mitochondria  ATP • Normally, Ca2+ is actively transported back to SR through SERCA (sarcoplasmic endoplasmic reticulum calcium-ATPase)

17. Mechanism • In MH, opening of RYR1 is prolonged, the reuptake process is overwhelmed  excess accumulation of Ca2+ in cells • Muscle contraction without relaxation  spasm / contracture  increase the extra vascular resistance to muscle perfusion  ischemia • Incessant glycolysis & glucogenolysis • Remember: muscle mass constitute 30-40% of body mass, so this hypermetabolism leads to excess generation of heat / lactic acid / CO2 metabolic exhaustion, muscle edema, muscle breakdown

18. RYR1 • Homotetrameric from 565kD subunit http://www.bcm.edu/physio/hamilton/

19. Preventing MH Episodes • best way to prevent MH is to detect those at risk PRIOR to administering anaesthesia triggering agents Screening for MH susceptibility • caffeine halothane contracture test (CHCT), which is performed on a small sample of biopsied muscle and measures the strength of contraction in response to caffeine or halothane • Intramuscular halothane injection of 5-6 % followed by pCO2 measurement. pCO2 in MH > pCO2 in non-MH • They can still have surgery, but healthcare workers should take necessary precautions, including the use of either a local anesthetic or a non-triggering general anesthetic

20. Dantrolene – a skeletal muscle relaxant Influx of Ca2+ into the muscle cells due to leaky channels Solution is Dantrolene (since 1979) The only accepted drug to treat MH Exact action of dantrolene is not clear Direct or indirect inhibition of the ryanodine receptor Ca2+ channels resulting in normal Ca2+ intracellular concentrationlevels to be restored After its introduction, mortality of MH episodes decreased from 80% in the 1960s to <10% today Main side effect is muscle weakness

21. Dantrolene – a skeletal muscle relaxant Total paralysis cannot be obtained due to the poor water solubility Dantrolene is metabolized to 5-hydroxydantrolene, which also acts as a skeletal muscle relaxant

22. Treatment Immediate Therapy (in OR) 1) Volatile agent vaporisers removed from the anaesthetic machine 2) Patients’ lungs ventilated with 100% oxygen 3) Dantrolene preparation and administration intravenously (2.5 mg/kg) and repeated as necessary MH progresses very rapidly, steps 1-3 are time sensitive Supportive Therapy - Body cooling down to 38 oC - Sodium bicarbonate - to treat acidosis - Beta-blockers - in case of cardiac arrhythmias - Glucose-insulin infusions – in case of hyperkalemia, hypercalcaemia or myoglobinuria

23. Summary • Malignant hyperthermia is the dramatic rise in body temperature triggered by inhaled anaesthetic, most commonly by halothane. • In humans, malignant hyperthermia is inherited in an autosomal dominant pattern. • 70-80% of all cases of malignant hyperthermia are associated with mutations In RYR1 which encodes ryanodine receptor type 1 on chromosome 19 in humans. • The anesthetic triggers inappropriate release of Ca2+ from the RYR1 transporter on the sarcoplasmic reticulum and uncontrolled stimulation of heat producing processes, including myosin ATPase, glycogenolysis, glycolysis, and cyclic uptake and release of Ca2+ by mitochondria and sarcoplasmic reticulum. • Pickled pigs are formed after susceptible pigs die from inhaled anaesthesia, halothane, causing malignant hyperthermia (called porcine stress syndrome in pigs) and thus, can be used as a model for the human syndrome and its eradication. • Dantrolene is an important drug used to restore intracellular calcium concentrations to normal during MH emergencies by inhibiting the ryanodine receptor Ca+2 channels

24. References • Devlin, T.M. (2011). Textbook of biochemistry: with clinical correlations. Hoboken, NJ. John Wiley & Sons, 7th ed. Pg. 612. • MacLennan, D.H., Phillips, M.S. (1992). Malignant Hyperthermia. Science. 256 (5058): 789-794 • Martini, F.H., Timmons, M.J., Tallitsch, R.B. (2009) Human Anatomy, 6E. Chapter 9, Figure 11. • Mitchell, G., Heffron, J. (1982). Porcine stress syndromes. Advances in Food Research. 28:167-178 • T. Krause, M. U. Gerbershagen, M. Fiege, R. Weihorn and F. Wappler. (2004) Dantrolene – A review of its pharmacology, therapeutic use and new developments. Anaesthesia. 59, 364–373. • K. O. Ellis and F. L. Wessels. (1978) Muscle Relaxant Properties of the Identified Metabolites of Dantrolene. Naunyn-Schmiedeberg's Archives of Pharmacology. 301, 237-240. • Rosenberg, H., Sambuughin, N., Riazi, S., Dirksen, R. (2003). Malignant Hyperthermia Susceptibililty. GeneReviews. Seattle (WA): University of Washington, Seattle; 1993-2013. • Schuster F, Gardill A, Metterlein T, Kranke P, Roewer N, Anetseder M (2007). A minimally invasive metabolic test with intramuscular injection of halothane 5 and 6 vol% to detect probands at risk for malignant hyperthermia. Anaesthesia62 (9): 882–887