A Stressful Question ‐ How Does 3′ 5′ cyclic adenosine monophosphate Signaling Affect Oxygen Metabolism Within Malignant Hyperthermia Susceptible (MHS) B‐lymphocytes?
Authors: Michaelson Luke , Oates Paige 
Published: May 2022

Background

Even though the MHS trait is inherited by an autosomal dominant pattern, the variable penetrance and symptom presentation suggest involvement of oligogenic or post‐translational modifications. Since malignant hyperthermia (MH) includes hypermetabolic pathologies as its defining characteristics, the metabolic pathways offer biomarker targets which may segregate with the MHS phenotype.

Problem

Identifying the malignant hyperthermia susceptible (MHS) trait using pathogenic mutations has limitations. Causative mutations within the ryanodine receptor type I (RYR1),the SH3 and cysteine rich domain 3 (STAC3),or the (CACNA1S), voltage dependent calcium channel, alpha‐1s subunit genes account for only 50‐86% of subjects diagnosed with MH.

Hypothesis

The adrenergic agonist norepinephrine (NOR) and the membrane permeable 3′,5′ cyclic adenosine monophosphate (cAMP) analog, adenosine 3ʹ,5ʹ‐cyclic monophosphate, 8‐bromo‐, sodium salt, (8Br‐cAMP‐Na) will significantly alter oxygen metabolism between human MHS and MH non‐susceptible (MHN) b‐lymphocyte groups.

Methods

MHN and MHS cells (8.5x104 and 3.7 x104 cells/well, respectively), were treated with NOR 1μM, or 10μM 8Br‐cAMP‐Na, for 1 hr. Electron transport chain inhibitors were sequentially added to form a bioenergetic profile. The Seahorse Xfe96 AnalyzerTM analyzed the oxygen consumption rate (OCR, pmol/min/cells) to calculate the basal and maximal OCR and the spare respiratory capacity (SRC). This value quantifies the metabolic ability of the cell to respond to an energetic demand.

Results

When compared to the naive MHN group, 8Br‐cAMP‐Na decreased the MHN SRC by 48%, p <.001. In contrast, the SRC increased 67%, p< .01, within the 8Br‐cAMP‐Na MHS group vs the MHS naive group. 1μM NOR produced similar SRC changes within each group. The SRC decreased by 22% ,p <.05, and increased by 35%, p >.05, within the NOR MHN and MHS groups, respectively (n = 4‐12 wells/group).

Conclusion

The intracellular cAMP signaling affect the oxygen metabolism differently within MHN and MHS groups during adrenergic stress. The cAMP pathways contributing to a subclinical hypermetabolic response need further research. The adrenergic signaling may “prime” the conditions for an MH crisis until a specific metabolic threshold is obtained. The MHN group compensates for the increased metabolism and does not reach a thresh hold.

Disclaimers

The opinions and assertions expressed herein are those of the author(s) and do not necessarily reflect official policy or position of the Uniformed Services University or the Department of Defense.

This work was prepared by a military or civilian employee of the US Government as part of the individual’s official duties and therefore is in the public domain and does not possess copyright protection (public domain information may be freely distributed and copied; however, as a courtesy it is requested that the Uniformed Services University and the authors be given an appropriate acknowledgement).

The protocol DBS.2019.008, was reviewed and approved by the USU Institutional Review Board, in accordance with all applicable Federal regulations governing the protection of humans in research.