Periodic paralysis results from mutations in the skeletal muscle voltage gated sodium channel (NaV1.4) or calcium channel. (CaV1.1). While the direct effects of ...
Muscle Degeneration due to Skeletal Ion Channel Dysfunction Neta Amior with Professor Michael Duchen and Professor Michael Hanna MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology, Queen Square
BACKGROUND
Figure 2: PP mutations in CaV1.1
Figure 1: PP mutations in NaV1.4
Periodic paralysis results from mutations in the skeletal muscle voltage gated sodium channel (NaV1.4) or calcium channel (CaV1.1). While the direct effects of these mutations are known, the cause for long term muscle degeneration is not.
AIM The aim is to identify the link between ion channel dysfunction and development of muscle degeneration in PP. Initially this will involve developing and characterising a suitable model.
Francis et al., 2011
MODEL ONE: CELL CULTURE
MODEL TWO: MOUSE
Although PP affects muscles, human fibroblasts are more easily acquired. Fibroblasts were thus transfected with MyoD, causing them to develop a myoblast phenotype. These were then differentiated into mature myotube-like cells and were tested for myotube characteristics.
Two PP animal models with known human mutations exist, one hyperkalaemic (M1592V; Hayward et al., 2008) and one Hypokalaemic (R669H; Wu et al., 2011). Although isolated muscle from these present with sustained weakness upon perfusion with high or low potassium concentration respectively they don’t present with attacks of weakness in vivo.
Live Imaging Harwell* developed a mouse (I582V, known as Draggon) that does exhibit paralysis in vivo. The mutation does not correlate with a known human mutation. It is therefore important to test if muscle from these shows other characteristics of the disease.
Live imaging showing that classic mitochondrial networks are formed
Tetanic contraction produced by 1-ms pulse trains at 40, 80 and 100Hz, as well as initial single stimulation (twitch) (Tetanic/Twitch) Force Ratio
Immunofluorescence
6
5
5
4 3
WT Draggon
2 1
40 Hz
Control fibroblasts LC3 (antibody; red)
express
Figure 6: Desmin
Figure 7: DHPR
Control fibroblasts express desmin (antibody; green)
Control fibroblasts express DHPR (antibody; red)
As for figure 10
6
0
Figure 5: LC3
Figure 11: Draggon EDL Tension
Figure 10: Draggon TA Tension
0
80 Hz
Tetanic/Twitch) Force Ratio
Fluorescence intensity
4000
4 3
WT Draggon
2 1 0
100 Hz
40 Hz
80 Hz
100 Hz
Figure 12: TA Tensions
Figure 13: EDL Tensions
The difference between Draggon and WT TA tetanic force is not significant using 4 limbs, 3 animals
The difference between Draggon and WT EDL tetanic force is not significant using 4 limbs, 3 animals
Fatigue Tests Figure 14: WT fatigue Fatigue induced by repeated (once per second) 40 Hz stimulation of EDL
Fatigue Index
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Figure 15: Draggon fatigue
Figure 8: SERCA Control fibroblasts express SERCA (antibody; green)
As in figure 14
Figure 9: Actin and Myosin Control fibroblasts express actin filaments (stained with phalloidin; red) and myosins (antibodies; green)
WT
Draggon
Figure 16: Fatigue WT EDL tends to fatigue faster than Draggon EDL (p=0.06, 2 tailed students ttest). Fatigue index is taken as initial force divided by force after 180 stimulations
CONCLUSION The cell culture results suggest that fibroblasts can be used to produce functional myotubes – they release calcium and express skeletal muscle proteins. The mouse model fatigue results suggest that the Draggon mutation does indeed show the long term PP phenotype as well as the previously observed attacks of paralysis. The tetanic tension results are not conclusive.
FURTHER WORK Establish fibroblasts cultures from patient and control skin biopsies. Compare the characteristics of patient and control cells.
REFERENCES Francis D. G., et al. (2011) Neurology 76, 1635-1641. Hayward L. J., et al, (2008) J Clin Invest 118, 1437-1449. Wu F., et al. (2011) J Clin Invest 121, 4082-4094.
