Effect of removal and reconstitution of troponin and tropomyosin on Ca2+- activated tension generation in isolated myofibrils

Replacement of native troponin (Tn) and tropomyosin (Tm) with exogenous engineered proteins into striated muscle sarcomere can be a powerful tool to investigate the regulation of contraction. Here we described an effective Tm-Tn replacement method in isolated myofibrils. Myofibril suspensions were centrifuged several times in a low ionic strength solution at pH 8 to remove native Tm and Tn.The reconstitution of thin filament regulatory complex was made in a rigor solution in two steps, first adding Tm and then Tn. To measure the effects of this procedure, samples of myofibrils were analyzed at different stages of the protocol (control, extracted, reconstituted) both functionally (by loss and regain of Ca2+-dependent regulation) and by SDS-PAGE. The success of the exchange was determined by mechanical measurements of calcium dependent force activation on the reconstituted myofibrils. Small bundles of myofibrils were mounted in a force recording apparatus and activated by rapidly switching between low and high [Ca2+] solutions. Maximal isometric tension was 30-35% lower in the reconstituted myofibrils than in control myofibrils but the rate of force activation (kACT) and that of force redevelopment (kTR) was the same in the two myofibril groups. The effectiveness of Tm replacement in human cardiac myofibrils is under investigation. This approach can be used to test the functional impact of Tm mutations responsible for human cardiomyopathies. Supported by Telethon-Italy GGP07133