Reconstitution of skinned cardiac fibres with human recombinant cardiac troponin‐I mutants and troponin‐C

Troponin C (TnC) could be extracted from skinned porcine cardiac muscle fibres and their Ca2+ sensitivity restored by reconstitution with recombinant human cardiac TnC. After extraction of troponin I (TnI) and TnC using the vanadate treatment method of Strauss et al. Strauss, J. D., Zeugner, C., Van Eyk, J.E., Bletz, C., Troschka, M. and Rüegg, J.C. (1992) FEBS Lett. 310, 229-234, skinned porcine cardiac muscle fibres were reconstituted with wild-type recombinant human cardiac TnC and either wild-type cardiac TnI or several mutant isoforms of human TnI. Reconstitution with wild-type proteins restored the Ca2+ sensitivity of the tissue and phosphorylation of the TnI with the catalytic subunit of protein kinase A reduced the Ca2+ sensitivity (i.e.-logCa2+ for 50% of maximal force) as has been shown by others. However, reconstitution with the TnI mutant Ser-23Asp/Ser-24Asp mimicking the phosphorylated form of cardiac TnI, led to a reduced Ca2+ sensitivity compared with reconstitution with wild-type TnI, whereas the mutant Ser-23Ala/Ser-24Ala behaved as the dephosphorylated form of TnI. These data confirm the importance of negative charge in this region of the TnI molecule in altering the Ca2+ responsiveness in this system.