Investigating the fundamental mechanisms underlying the phenotypic diversity observed in RYR1-associated Malignant Hyperthermia.

Calcium is a crucial regulator of normal function within all cells but in skeletal muscle it has additional roles in allowing the muscle to contract. Therefore, muscle cells (myocytes) have evolved unique structures and pathways to regulate calcium levels within them. The normal functioning of these systems are incompletely understood, as is the detail of how they fail in disease. One of the most dramatic examples of skeletal muscle calcium dysregulation is a potentially lethal reaction to general anaesthetics (GA) called malignant hyperthermia (MH). People with a genetic risk for MH are apparently healthy until exposed to the strong anaesthetic gases which then results in a rapidly progressive and life-threatening (malignant) increase in heat generation (hyperthermia). The MH reaction is due to muscle over-activity from calcium dysregulation. These genetic changes in MH can lead to ongoing calcium dysregulation even without anaesthetic exposure. This then causes long-term adaptations within the myocytes, resulting in muscle pain and damage especially when exposed to stress such as exercise and statin medication. Exercise can also lead to excess heat production and the development of heat illness of which the most severe form known as heat stroke can be fatal. As a result, research into MH has benefits for various people; it can directly help patients at risk of MH (up to 1 in 2,000 people), but also patients with these other disease processes.

The studies on MH have provided important discoveries on the mechanisms of normal skeletal muscle function, as well as revealed how such calcium dysregulation can cause problems in other tissues including the heart and brain. A problem with previous research has been the lack of availability of the correct samples to allow a suitable understanding of the mechanisms in humans. I am fortunate to be able to undertake this research in the only MH unit in the UK which has human tissue samples donated by patients undergoing investigations for MH.

Recent studies have shown that calcium levels in skeletal myocytes are higher in people who have had MH compared to those who have not. Consequently, this research will address the fundamental need to understand how the genetic differences seen in patients with MH affect the entry and regulation systems of calcium in skeletal myocytes. To understand these mechanisms I will examine how calcium entry into myocytes from patients with MH differs to those without. The initial focus will be to study the most common and serious mutations using various molecular research techniques. One of the techniques I will use is highly specialised, but allows me to directly measure the amount of calcium in myocytes. To learn and import this technique to the UK, I will travel to America for six months. Once I have measured the calcium levels in the different myocyte samples, I can then use several cutting-edge research tools to try and identify the mechanisms of calcium dysregulation and how this can be controlled. After completing these sets of experiments, my research will then examine how the number of mutations affects the amount of calcium that enters and stays in cells. The reason for this is that there are some families with MH where the condition appears to result from the effects of more than one gene interacting with each other. This provides an additional genetic mechanism placing patients at risk of MH. The knowledge gained will allow doctors to better recognize how the genetic changes in patients at risk of MH are likely to affect the patient on exposure to GA, as a result allow patients to receive a safer GA.

To summarise, by identifying the structures involved in short and long-term skeletal muscle calcium dysregulation and how they interact, this innovative research could detect potential targets for the development of new drugs. These could be used to prevent and treat MH and various long-term conditions associated with calcium dysregulation.

Malignant hyperthermia (MH) is a pharmacogenetic condition resulting in a potentially lethal reaction triggered during general anaesthesia. In susceptible individuals, this rapid hypermetabolic response results from skeletal muscle calcium (Ca2+) dysregulation. The majority of MH predisposing mutations relate to the RYR1 gene encoding the skeletal muscle Ca2+ release channel, ryanodine receptor 1 (RyR1). Gain of function mutations in RYR1 are increasingly associated with a greater risk of several skeletal muscle pathologies including exertional heatstroke, statin-induced myopathy and premature muscle ageing. The mechanisms of the phenotypic diversity associated with such RYR1 variants and hence potential therapeutic approaches, have yet to be established. However, recently multiple pathways have been implicated in the skeletal muscle Ca2+ dysregulation in MH tissue: store operated Ca2+ entry (SOCE), excitation-coupled Ca2+ entry, and a raised resting Ca2+ entry (RCaE).

Our hypothesis is that RYR1 variant-specific differences in the influx of extracellular Ca2+ result in a chronic elevation of resting skeletal muscle Ca2+ concentration ([Ca2+]i), but also dictates the magnitude of response to acute stimulation. This elevation in [Ca2+]i is in turn responsible for the observed MH reaction and the non-anaesthetic manifestations of MH susceptibility. I will use several approaches to alter the Ca2+ handling of immortalized myoblasts derived from genotyped MH susceptible and normal patients to determine the following objectives: (i). the molecular mechanisms underlying SOCE in MH, (ii). the contribution of RCaE to resting [Ca2+]i in MH, (iii). the influence of RYR1 genotype on SOCE and RCaE. Identifying the effects of these pathways on Ca2+ regulation in normal and MH tissue will provide a better mechanistic understanding of calcium homeostasis in health, MH and related diseases. This may reveal novel targets for the development new therapies for such conditions.

This fellowship will help improve the scientific command of two major topics, the first is an increased understanding of skeletal muscle calcium homeostasis during normal and pathological states, and the other is the mechanisms that underlie the phenotypic translation of genetic mutations in MH. The benefits can be described according to the different communities that would benefit.

Scientific Community
The scientific remunerations will extend well beyond those interested in MH, as the regulation of skeletal muscle Ca2+ homeostasis is fundamental to the normal functioning of muscle, with Ca2+ dysfunction is directly or indirectly implicated in a wide range of myopathies and premature muscle ageing. Hence, the findings would directly benefit physiologists seeking to understand the process of Ca2+ entry in skeletal muscle cells in health and disease. Furthermore, there has been a recent interest in investigating the effects of insulin on RyR mediated intracellular calcium homeostasis, as skeletal muscle plays an important role in glucose homeostasis and insulin resistance in Type 2 diabetes. Interestingly, recent literature is implicating mutations in RyR isotypes in conditions in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. My research could thus have an impact on these areas of science and medicine. Further descriptions of the impact on the immediate and distant scientific community is explained in detail in the 'Academic Beneficiaries' section.

Patients and Public
Although considerable progress has been made in identifying the genes associated with MH, a major gap exists with regards to the significance of how these manifest clinically. Such deficiencies are pertinent to address, particularly as MH can result in significant anaesthesia induced morbidity and mortality. Thus, an enhanced mechanistic understanding of MH can augment patient safety in anaesthesia through the development of better genomic tests. Such tests will also reduce delays or cancellations of operations, specialised investigations and the requirements for costly higher levels of hospital care.

The recent discoveries that mutations associated with MH result in accelerated muscle ageing and increased risk of several myopathies, also renders this fundamental research of benefit to society. This is particularly important as reduced muscle function in the aged is a major determinant of well-being, dependency on support services, together with morbidity and mortality in the longer term. Furthermore, a greater understanding of the RyR1 and intracellular Ca2+ homeostasis provides for the translation of this research into therapies in other medical fields (see below). This would improve the lives of suffers of these diseases and hence the population as a whole.

Clinical Community
During the medium-term the improved knowledge on the genotypic-phenotypic correlations in MH that this study provides would alleviate the clinical angst that occurs when in vitro contracture and genetic tests do not correlate. This will become pertinent as the data from the 100 000 genomes project becomes available in a few years. The clinical utility will intensify in the longer-term, as individualised care with point of access genomic testing becomes a fundamental part of anaesthesia. Furthermore, this fellowship also maintains the longevity of MH research in this country as I would develop within the only MH clinical unit in the UK. The funding would also highlight support for research in anaesthesia which has been declining over the last two decades.

MRC
This study will address the recent MRC strategic review highlighting the importance of understanding the fundamental biology in human health and disease, by studying the core mechanisms underlying the phenotypic diversity in MH. This will be achieved through an international collaboration which also aims to support me through a higher degree, hence encourage future UK research.