BACKGROUND AIM MODEL ONE: CELL CULTURE ...

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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.

3500

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Cell 1 2500

Cell 2 2000

Cell 3 1500

Tetanic Tension Tests

Cell 4 1000

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1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127 133 139 145 151

Time (seconds)

Figure 3: Calcium release

Figure 4: Mitochondria and calcium

Calcium is released in response to 5mM caffeine

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

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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

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80 Hz

Tetanic/Twitch) Force Ratio

Fluorescence intensity

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4 3

WT Draggon

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40 Hz

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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.

* Harwell Oxford Research Centre, Oxfordshire