Progressive Muscular Weakness in Periodic Paralysis and Normal Ageing

The Periodic Paralyses (PP) are a rare group of inherited muscle disorders that result from changes in proteins called ion channels. Muscle ion channels are essential for the accurate communication of electrical signals. Changes in patients' ion channels mean muscle becomes paralysed when exposed to high or low potassium levels in the blood that can be triggered by something as simple as eating a pizza. Paralysis lasts from 1 hour to several days. In between attacks muscle strength is fully restored in the early stages of the condition. However, with age, although the frequency of attacks diminishes, patients report taking longer to recover from each attack and many develop permanent weakness that gets worse with age. By 40 the majority of patients are affected. The weakness can range in severity from mild limitation to having a major effect on independence and quality of life. There are treatments to prevent attacks of PP but there is no treatment for the permanent muscle weakness.

Loss of muscle mass and strength also occurs in normal ageing and is linked to falls, disability and death. There is evidence to suggest that with age, the proteins linking electrical signals with muscle contraction are less effective resulting in weakness. In addition, when looked at under a microscope, some of the same changes seen in aged muscle are also seen in younger people with PP, indicating that similar problems may occur at different times.

The cause of an acute attack of PP is now well understood. However, neither the short to medium-term effects of an attack of PP on muscle nor the reason for the development of permanent weakness is known.

Our overall aim is to understand if normal age-related changes in muscle contribute to an earlier onset of the disabling permanent weakness that occurs in people with periodic paralysis (PP).
Specific aims and objectives are to investigate:

1. What constitutes normal age-related change in the proteins linking electrical signals with muscle contraction?
In order to answer this we will use normal mouse and human muscle from young adult, early middle-aged, late middle-aged and old subjects. We will examine changes:
a in the strength of muscle of different ages and whether electrical signals are being converted to muscle contraction correctly
b in the code (RNA) of these proteins with age. Changes in RNA can affect the amount or function of a protein
c in muscle structure and define at what time point these start to occur
d in the proteins themselves including total levels and whether the protein is modified with age
This will provide an invaluable resource to facilitate further research in other muscle disease. It also has potential to identify new mechanisms underlying age-related weakness.

2. How do changes in PP compare to normal age-related changes?
We will use muscle from mice and humans with PP (ages as for aim 1) and examine them in exactly the same way as for aim 1. This will enable us to clarify the time line of changes in PP helping to identify an optimum time point for treatment to prevent permanent weakness. It may also identify new mechanisms and tests of disease progression.

3. What effect does a paralytic attack have on muscle?
To answer this we will either use chemicals added to normal muscle to mimic the effect of changes in patient's ion channels or PP mouse muscle and then expose it to low or high potassium. This will help us understand if:
a the linking of electrical signals to muscle contraction is impaired after a paralytic attack and the effect of age on this
b Novel or established age related changes occur in younger muscle after an attack of paralysis
c structural changes associated with PP can occur after a single paralytic attack indicating that attack prevention may prevent permanent weakness.
d We can minimise or prevent changes using drugs that target these proteins thus identifying potential treatments

1. Normal age-related changes in the skeletal muscle 'channelome', muscle structure and excitation-contraction (EC) coupling

EDL and soleus of wild-type male mice (3,12,18,24 months) and quadriceps muscle from normal males (18-33, 34-49, 50-64, 65+) will be used to examine age related changes in:
a) Muscle strength and EC coupling using muscle tension testing on explanted mouse EDL and soleus. Supramaximal electrical stimulation will be compared with supra maximal activation with caffeine.
b) The skeletal muscle channelome using exon level RNA sequencing (mouse & human muscle)
c) Muscle structure and ultrastructure will be examined using light and electron microscopy (mouse & human muscle)
d) Expression and post translational modification of proteins involved in excitation and EC coupling (i.e. voltage-gated sodium channel, calcium channel and ryanodine receptor) using immunohistochemistry (mouse & human muscle)

2. PP age-related changes in skeletal muscle 'channelome', muscle structure and EC coupling
HyperPP mouse muscle and muscle from male patients with HypoPP (Cav1.1 R528H or R1239H) and HyperPP (Nav1.4 T704M or M1592V) will be used. Muscle will be processed and examined as for Aim 1.

3. The effect of prolonged depolarisation on muscle and how it varies with age
To model an acute attack of PP we will induce prolonged depolarisation by exposing normal mouse muscle of different ages to either barium or gramicidin and low potassium solution, or Hyper PP mouse muscle of different ages to high potassium solution. We will then examine:
a. if EC uncoupling or established histological features of PP (i.e. vacuoles) occur after prolonged depolarisation and how age effects this
b. if age related changes occur in younger muscle after an episode of prolonged depolarisation
c. If there is a final common pathogenic pathway following prolonged depolarisation in vitro
d. If pathogenic changes can be minimised with certain pharmacological agents.

1. What are the benefits for patients and patient organisations?

a. In the short term, establishing whether acute attacks of paralysis cause muscle damage and whether this accumulates over time will help to answer whether early and aggressive attack prevention is important for longer term prevention of permanent weakness.
b. In the medium term clearer delineation of age-dependent changes in skeletal muscle ion channels and accessory proteins may help identify a key time point at which intervention is optimal to prevent longer term disability.
c. In the longer term the identification of novel pathways could lead to novel therapeutic or preventative treatment options. This could significantly improve quality of life for patients and their families as well as extending economic and creative life and prolonging functional independence.

2. What are the benefits for the wider public?
a. Investigating changes associated with normal aging may result in the identification of muscle protective pathways relevant to sarcopenia and possibly even other muscle conditions. In the longer term, this could lead to novel therapeutic and preventative strategies. As sarcopenia is highly predictive of increased falls, loss of functional independence and mortality this would be of great benefit.

3. What are the benefits of this work relevant to academic colleagues?
a. Addressing the gap in knowledge in terms of the acute, sub-acute and chronic effects of periodic paralysis on muscle as a whole.
b. Identification of normal age-related changes in the skeletal muscle ion channel and accessory protein transcriptome and comparison to that observed in periodic paralysis. This will improve understanding and awareness of the age-dependent phenotypic changes observed in periodic paralysis and possibly other muscle conditions. It may also identify novel muscle-protective and disease-specific pathways that would be of interest to other researchers in associated fields.
c. Identification of potential biomarkers for sarcopenia or periodic paralysis could help facilitate future therapeutic trials.
d. This project provides a replicable example of combining the investigation of normal aging with a genetic disorder with clear age-associated phenotypic changes in order to identify potentially important and novel pathways that may be of relevance for both.
e. It also will provide other academics with resources that will faciliate further research in ageing muscle via the generation of high depth RNA sequencing data for patient and control muscle and by depositing surplus aged tissue in a biorepository such as ShARM that can be accessed by other researchers wishing to investigate the effects of ageing on other tissues.

4. What are the benefits for commercial and private sector beneficiaries?
a. Identification of novel pathways involved in the development of age-related loss of muscle mass and function would be of significant interest for pharmaceutical companies.
b. The identification of biomarkers that might predict or monitor age related loss of muscle mass and function with age would be of significant interest for two reasons. Firstly, it could facilitate effective therapeutic trials for other compounds in the development pipeline; secondly it may be an assay worthy of developing and manufacturing for clinical use independently.

5. What are the benefits for policy makers?
a. In the longer term improved understanding of the biology of normal muscle ageing may help develop lifestyle and other preventative measures aimed at reducing age dependent loss of muscle mass and function thus prolonging economic activity, prolonging functional independence, reducing mortality and improving quality of life.