Electron Micrograph of RyR1 Ryanodine Receptor in Skeletal Muscle “ Catecholamine Induced RyR1 Ca 2+ Release in Malignant Hyperthermia Sensitive Human B- Lymphocytes . Lt. Colonel Susan M. Perry, PhD, CRNA, USAF, NC
Jan 15, 2016
Electron Micrograph of RyR1Ryanodine 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%.
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.
0 1 2 3
0 = Cells at room temperature/ 40 minutes minimum 1 = Cells spun/ Fresh Buffer added/ Cells re-suspended2 =Norepinephrine 1μM added to Cells 3 = Baseline measurement taken (50 seconds) and 4 CmC added to cells4 = Area Under Curve measurement taken at 250 seconds/ Peak Fura 2 Emissions noted
Norepinephrine Protocol
50 Seconds 250 Seconds
4
4-CmC Dose Response in MHN Cell Line
Yellow: 4-CmC aloneRed: 4 CmC with NE
4-CmC Dose Response in MHS Cell Line
Yellow: 4-CmC aloneRed: 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.
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
AUCANOVA Results
Fura 2 Ca2+ PE Comparison between MHS vs MHN Human B- Lymphocytes
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
PE ANOVA Results
Norepinephrine/Propranolol Study
Yellow: 4 CmC aloneRed: 4 CmC with NETeal: Propranolol Effect
4 Separate MHS Cell lines
Norepinephrine/Phentolamine StudyYellow: 4- CmC AloneRed: 4- CmC with NETeal: Phentolamine Effect
4 Separate MHS Cell Lines
Isoproterenol/Propranolol StudyYellow: 4- CmC aloneRed: 4-CmC with IsoproterenolTeal: 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
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.
LimitationsImmune 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?