Animal Models for Autoimmune Myocarditis and Autoimmune Thyroiditis(1)

Myosin purification

Myosin can be purchased from many suppliers. However, preparation of myosin may be better to control the quality of myosin for better reproducibility of EAM. Prepare 160 mouse hearts. Ideally the hearts must be collected from a susceptible mouse strain. Wash with cold PBS and store at -80C until use.

Day 1

1. To 250 mL of modified Hasselbach-Schneider solution (1L for 22.35 g 0.30 M KCl, 26.1 g 0.15 M K2HPO7, 2.66 g, 0.01 M Na4P2O7, 0.095 g 1 mM MgCl2), add 0.08 g dithiothreitol (DTT), 250 μL leupeptin (1.0 mg/mL, aliquoted), and 350 μL PMSF (phenylmethyl-sulfonyl- fluoride; 50 mg/mL) (the final solution should contain 2 mM DTT, 1 μg/mL leupeptin, and 1 mM PMSF). The final pH should be 6.8 (original pH 8.7; use HCl).

2. Thaw hearts, rinse with cold PBS. Weigh all mice hearts (160 hearts should be approx 22 g wet weight).

3. Mince the hearts on ice with a razor blade. Add 100 mL of modified Hasselbach-Schneider solution with DDT, leupeptin, and PMSF.

4. Homogenize with motor-driven homogenizer, and add another 100 mL modified Hasselbach-Schneider solution.

5. Homogenize the whole volume for 1 minutes, let it rest for 1 min, and homogenize for another 5 minutes.

6. Extract the homogenate with gentle stirring for 90 minutes at 4°C.

7. Centrifuge at 12,000g for 12 minutes at 4°C and collect supernatant.

8. Centrifuge supernatant at 140,000g for 4 hours at 4°C.

9. Collect the supernatant.

10. Dilute supernatant (around 195 mL) with 20 times the volume of ddH2O (4°C) (about 3900 mL) by pouring myosin supernatant carefully into water. Let myosin precipitate settle overnight at 4°C.

Day 2

1. To 200 mL of Extraction buffer (1L for 22.35 g 0.30 M KCl, 0.681 g 0.01 M imidazole, 0.475 g 5 mM MgCl2), add 0.552 g ATP disodium salt (Na2ATP), 0.062 g DTT, 200 μL leupeptin (1.0 mg/mL, aliquoted), and 300 μL PMSF (50 mg/mL). The final solution should contain 5 mM Na2ATP, 2 mM DTT, 1 μg/mL leupeptin, and 1 mM PMSF. The final pH should be 6.8 (original pH 6.4–6.5; use 6N NaOH).

2. Very carefully pour the supernatant from the precipitated myosin. Do not pour all the supernatant so the myosin is not lost.

3. Centrifuge at 12,000g for 14 minutes at 4°C.

4. Dissolve the pellet in a 100 mL Extraction buffer and add an additional 100 mL Extraction buffer to reach a 200 mL final volume.

5. Homogenize with a Teflon-glass homogenizer.

6. Centrifuge the solution at 43,000g for 30 minutes at 4°C to remove actin.

7. Collect supernatant.

8. Myosin is precipitated from the supernatant (200 mL) by adding eight times the volume of ddH20 (1600 mL) and allowing it to stand overnight at 4°C. The precipitation is seen very soon after water is added.

Day 3

To 150 mL of extraction buffer 2 (1L for 22.35 g 0.30 M KCl, 0.681 g 0.01 M imidazole), add 0.048 g DDT, 150 μL leupeptin (1.0 mg/mL, aliquoted), and 225 μL PMSF (50mg/mL). The final solution should contain 2 mM DTT, 1 μg/mL leupeptin, and 1 mM PMSF at pH 6.8 (original pH 8.0–8.5; use 6N HCl).

2. Stir gently to mix precipitation.

3. Centrifuge at 14,077g for 14 minutes. Collect pellets. Centrifuge supernatant again to increase yield. Collect pellets.

4. Dissolve pellet with 150 mL extraction buffer 2 and homogenize it with a Teflon-glass homogenizer.

5. Centrifuge the solution at 43,000g for 30 min to remove actomyosin.

6. Collect supernatant (a sample of solution can be taken at this point to run on gel).

7. Myosin precipitate is collected by centrifugation at 14,077g for 18 minutes. Collect pellets and the supernatant should be clear.

8. Redissolve pellet with 15 mL extraction buffer 2. Homogenize it with a Teflon-glass homogenizer. Add 3mLextraction buffer 3, aliquot myosin (0.2–0.5 mL), and freeze aliquots immediately. Myosin can be stored up to 1 year at –80°C, or up to 1 month in 4°C.

9. Myosin concentration can be established by spectrophotometer or by a BCA protein assay.

1. D. Čiháková, R. B. Sharma, D. Fairweather, M. Afanasyeva, N. R. Rose, in Autoimmunity: Methods and Protocols, A. Perl, Ed. (Humana Press, Totowa, NJ, 2004), pp. 175–193.