Electron Micrograph of RyR1 Ryanodine Receptor in Skeletal Muscle “Catecholamine Induced RyR1 Ca2+ Release in Malignant Hyperthermia Sensitive Human B-Lymphocytes .   Lt. Colonel Susan M. Perry, PhD, CRNA, USAF, NC

Disclaimer The view expressed in this presentation are those of the authors and do not reflect the official policy or position of the Department of the Air Force, the Department of Defense, or the Uniformed Services University, or the United States Government. Funding for this research was provided by a grant from the American Association of Nurse Anesthetist Foundation

BACKGROUND Malignant Hyperthermia (MH) is an autosomal inherited disorder associated with the RyR1 (Ryanodine) receptor in skeletal muscle that pre-disposes the susceptible individual to a life threatening hypermetabolic syndrome. Although it is predominantly exhibited during general anesthesia, exertional heat and emotional stress also have been shown to trigger MH. Without the definitive treatment of dantrolene sodium and discontinuation of all triggering agents, mortality is in excess of 70%. MH is an autosomal inherited disorder or skeletal muscle that pre-disposes to a life threatening hypermetabolic syndrome predominantly exhibited during general anesthesia. However, there are MHS humans and animals that trigger in the absence of any pharmacologic trigger as a result of heat or emotional stress.

Graphic Representation: Clinical Signs of MH Ca2+ Release Melzer & Dietz Acta Physiol Scan, 2001

Unresolved Issues..what else acts as trigger? MH in the absence of anesthetic triggers 2 year old girl Family History of MH Machine was “clean” flushed overnight/10L/min No triggering agent used/IV Propofol and morphine/glycopyrolate Triggered 15 minutes into case 57% NAMH/More than 2 uneventful anesthetics Larach et al. 2010

MH and Stress in Humans In the 1970s and 1980s there were many studies and case reports implicating emotional and exercise induced stress or pain as contributing to the development of MH in humans. Moulds, 1975; Katz, et al. 1976; Huckell, et al., 1978; Gronert, et al., 1980; Wingard, 1981; Grinberg, 1983; Britt, et al., 1988; Montegi, et al., 1996; Muldoon, et al., 2004).

These questions continue today Researchers Muldoon, Deuster, Brandom and Bunger examined the relationship between MH and exertional heat stress. In their paper they discuss variability as a “striking characteristic” of the disease MH. These researchers suggested that MH “stems from an interaction between genes and environmental factors” and estimated that between “5-8%” of MHS individuals develop symptoms with exercise, emotional stress and or environmental heat exposure. “Is There a Link Between Malignant Hyperthermia and Exertional Heat Injury?” Exercise and Sports Science reviews, 2004

Research Question Is the variability seen in MHS individuals related to an abnormal adrenergic response related to an increased sensitivity to catecholamines? I needed a human model.

Human Lymphocyte Model Human B Cells express RyR1 Sei, et al. (1999): RYR1 expressed in human B-lymphocyte cell lines was identical to the skeletal muscle type (RYR1). Ca2+ release in B cells was significantly altered by 4-chloro-m-cresol and ryanodine (J Biol Chem, 1999) The Ca2+ responses to caffeine or 4-chloro-m-cresol in B lymphocytes showed significant differences between MHS and MHN (or control) individuals.(Anesthesiology,2002)

Human Lymphocyte Model McKinney, et al. (2006): RyR1-mediated Ca2+ signals could be distinguished from other intracellular sources of Ca2+ in human B cells using fluorescent measurements of the response to the RyR1 agonist 4-chloro-m-cresol (4-CmC). Lymphocytes from MHS pigs displayed increased sensitivity to 4-CmC when compared to cells from normal pigs. (EC(50) = 0.47 vs. 0.81 mm for normal cells) (Anesthesiology, 2006)

Hypothesis Activation of the adrenergic stress response in malignant hyperthermia susceptible (MHS) B- lymphocytes will result in an augmentation of intra-cellular Ca2+ release in response to the RyR1 agonist 4 Chloro-m-cresol (4-CmC) in a way that is different from MHN cells.

Norepinephrine Protocol 50 Seconds 250 Seconds 0 1 2 3 4 0 = Cells at room temperature/ 40 minutes minimum 1 = Cells spun/ Fresh Buffer added/ Cells re-suspended 2 =Norepinephrine 1μM added to Cells 3 = Baseline measurement taken (50 seconds) and 4 CmC added to cells 4 = Area Under Curve measurement taken at 250 seconds/ Peak Fura 2 Emissions noted

4-CmC Dose Response in MHN Cell Line Yellow: 4-CmC alone Red: 4 CmC with NE

4-CmC Dose Response in MHS Cell Line Yellow: 4-CmC alone Red: 4-CmC with NE

Norepinephrine 4-CmC Response Results MHS vs MHN groups demonstrated statistically significant differences in response to norepinephrine augmentation of 4-CmC Ca2+ emissions when comparing Base Area (BA), Peak Emissions (PE) and Area Under Curve (AUC). P < .05 Effect Size = 0.96

MHS vs MHN Baseline Area Ca 2+Emissions Differences in Baseline Ca2+ emissions in the presence of Norepinephrine MHS = 65 MHN = 61 P < 0.05 Baseline responses were removed from analysis of 4-CmC response during analysis.      

AUC Comparison for Fura 2 Ca2+ Emissions in MHS vs MHN Human B AUC Comparison for Fura 2 Ca2+ Emissions in MHS vs MHN Human B. Lymphocytes There was no statistically significant difference in MHN AUC response to 4-CmC in the presence or absence of norepinephrine (P > .05). There was a statistically significant difference in MHS cell lines response to 4-CmC in the presence of norepinephrine (P < .05). There was a statistically significant difference in MHS vs MHN B-cell lines in AUC response to 4-CmC both in the absence and presence of norepinephrine (P < .05). This was true for all doses of 4 CmC (paired student t- tests) * 2.0m M Response may be non-specific for RyR1 Calcium Release alone AUC ANOVA Results

Fura 2 Ca2+ PE Comparison between MHS vs MHN Human B- Lymphocytes ANOVA Results There was no statistically significant difference in MHN PE Ca2+ response to 4- CmC in the presence or absence of norepinephrine (P > .05). There was a statistically significant difference in MHS cell lines PE Ca2+ response to 4-CmC in the presence of norepinephrine (P < .05). There was a statistically significant difference in MHS vs MHN B-cell lines in PE response to 4- CmC both in the absence and presence of norepinephrine (P < .05). This was true for all doses of 4 CmC (paired student t-tests) * 2.0 Response may be non-specific for RyR1 Calcium Release alone

Norepinephrine/Propranolol Study Yellow: 4 CmC alone Red: 4 CmC with NE Teal: Propranolol Effect 4 Separate MHS Cell lines

Norepinephrine/Phentolamine Study Yellow: 4- CmC Alone Red: 4- CmC with NE Teal: Phentolamine Effect 4 Separate MHS Cell Lines

Isoproterenol/Propranolol Study Yellow: 4- CmC alone Red: 4-CmC with Isoproterenol Teal: Propranolol Effect 4 MHS Separate Cell Lines

Conclusions Human B-Lymphocytes from Malignant Hyperthermia susceptible (MHS) individuals display a significantly increased sensitivity to norepinephrine induced adrenergic augmentation of intra-cellular Ca2+ release from the Ryanodine (RyR1) receptor when compared to MHN controls and this response is effectively blocked by the αadrenergic blocker phentolamine.

Conclusions EBV Immortalized human B-lymphocytes provided a useful cell line model to examine intracellular Ca2+ handling under various hormonal and pathological conditions.

Conclusions Because the Ca2+ release response was enhanced in the presence of a CONSTANT concentration of 1μM of norepinephrine, the data suggest that the MHS cell lines exhibit an increased sensitivity to adrenergic αagonists, compared to MHN cell lines.

Conclusions The statistical evidence shows that only about 65% of the variability in the measured Ca2+ response in both the MHN and MHS cell lines can be attributed to RyR1 agonism due to 4-CmC in combination with norepinephrine. This means that 35% of the observed variability remains unexplained. I conclude from this that there should be other cellular mediators that contribute to the response. The β adrenergic response requires further study.

Future Research In Vivo study Immune response in MHS and EHI individuals Use in development of new testing/diagnosis in MH and EHI individuals Recent studies on MHS B-Lymphocytes have implicated a possible role for cytokines in the pathologic MH response. Girard et al., in a study published in The Journal of Biological Chemistry (2001) report that EBV immortalized B-Cells” produced more interleukin (IL)-1b after treatment with the RyR activators caffeine and 4-chloro-m-cresol”. They go on to suggest that perhaps the symptoms seen during an MH episode may be related to the effects of the production of IL-1b. Other possibilities to explain the variance include downstream effects from increased levels of adenylate cyclase and cAMP; effects from aberrant protein kinase (PKA) activity; or alterations in phosphorylation processes. In recent investigations, researchers have used skeletal muscle, knock in mice, and B-Lymphocyte models to investigate genetic differences in the function of the FK506 binding protein FKB12, Calsequestirin, and ATP levels in MHS groups (Gaburjakova, et al, 2001; Marx, et al, 2001, Chelu, et al, 2004; Lanner, et al, 2010; Dainese, M, et al. 2009; Dulhunty, A., et al, 2001). Many of these studies suggest that differences in phosphorylation processes involving multiple areas related to the stabilization of the RyR1 receptor, may be involved in the pathogenesis of MH and other RyR1 related disorders

Future Studies and Limitations Variability in numbers of adrenergic receptors on MHS B-Lymphocytes. Intracellular stores of Calcium (Not only RyR1) Immune cells vs myofibrils: What effect does stress have on immune response in muscle and immunology. Limitations Immune cells vs myofibrils: need to examine effect on myofibrils.

Acknowledgements Christine Kasper, PhD, RN, FAAN, FACSM, Professor, PhD Program, GSN, USU Sheila Muldoon, M.D. Professor of Anesthesiology/Director of MH Diagnostic Center, USU Rolf Bunger, M.D., PhD, Professor of Physiology, School Of Medicine, USU Colonel Michaela Shafer, PhD, RN, Chief Nurse Executive, Assistant Professor, GSN, USU

Questions?