Tropomyosin is an essential component in the regulation of muscle contraction. A method has been described to remove endogenous tropomyosin (Tm) and troponin (Tn) and replace them with exogenous proteins into rabbit psoas myofibrils (Siththanandan et al., Biophys. J., 2007, suppl. 626a). In the present study the endogenous Tm and Tn are replaced into cardiac mouse myofibrils by a modification of that method. Endogenous Tm and Tn were extracted by washing cardiac mouse myofibrils in a low ionic strength solution at pH 8.0. The wash was repeated several times to increase the amount of extracted Tm. The reconstitution of thin filament regulatory proteins 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 Ca2þ -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 cardiac myofibrils were mounted in a force recording apparatus and activated by rapidly switching between low and high Ca2þ solutions (Tesi et al., Biophys. J., 2002, 83, 2142- 2151). 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.

Effects of removal and reconstitution of troponin and tropomyosin on Ca2+-activated tension generation in cardiac myofibrils / Scellini B.; Piroddi N.; Poggesi C.; Tesi C.. - In: BIOPHYSICAL JOURNAL. - ISSN 0006-3495. - STAMPA. - 96(3) suppl:(2009), pp. 228a-228a.

Effects of removal and reconstitution of troponin and tropomyosin on Ca2+-activated tension generation in cardiac myofibrils

SCELLINI, BEATRICE;PIRODDI, NICOLETTA;POGGESI, CORRADO;TESI, CHIARA
2009

Abstract

Tropomyosin is an essential component in the regulation of muscle contraction. A method has been described to remove endogenous tropomyosin (Tm) and troponin (Tn) and replace them with exogenous proteins into rabbit psoas myofibrils (Siththanandan et al., Biophys. J., 2007, suppl. 626a). In the present study the endogenous Tm and Tn are replaced into cardiac mouse myofibrils by a modification of that method. Endogenous Tm and Tn were extracted by washing cardiac mouse myofibrils in a low ionic strength solution at pH 8.0. The wash was repeated several times to increase the amount of extracted Tm. The reconstitution of thin filament regulatory proteins 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 Ca2þ -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 cardiac myofibrils were mounted in a force recording apparatus and activated by rapidly switching between low and high Ca2þ solutions (Tesi et al., Biophys. J., 2002, 83, 2142- 2151). 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.
2009
Scellini B.; Piroddi N.; Poggesi C.; Tesi C.
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/969000
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