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Biochemistry. 2018 Apr 17;57(15):2256-2265. doi: 10.1021/acs.biochem.8b00109. Epub 2018 Mar 30.

Reversible Covalent Reaction of Levosimendan with Cardiac Troponin C in Vitro and in Situ.

Author information

1
Department of Biochemistry, Faculty of Medicine and Dentistry , University of Alberta , Edmonton , Alberta T6G 2H7 , Canada.
2
Department of Chemistry, Faculty of Science , University of Alberta , Edmonton , Alberta T6H 2H7 , Canada.
3
Pharmaceutical and Health Benefits Branch, Ministry of Health , Government of Alberta , Edmonton , Alberta T5J 3Z5 , Canada.
4
Randall Centre for Cell and Molecular Biophysics and British Heart Foundation Centre of Research Excellence , King's College London , London SE1 1UL , U.K.

Abstract

The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties, including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target because cardiac troponin C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan. We have recently shown that when a covalent cTnC-levosimendan analogue is exchanged into cardiac muscle cells, they become constitutively active, demonstrating the potency of a covalent complex. We have also demonstrated that levosimendan reacts in vitro to form a reversible covalent thioimidate bond specifically with cysteine 84, unique to cTnC. In this study, we use mass spectrometry to show that the in vitro mechanism of action of levosimendan is consistent with an allosteric, reversible covalent inhibitor; to determine whether the presence of the cTnI switch peptide or changes in either Ca2+ concentration or pH modify the reaction kinetics; and to determine whether the reaction can occur with cTnC in situ in cardiac myofibrils. Using the derived kinetic rate constants, we predict the degree of covalently modified cTnC in vivo under the conditions studied. We observe that covalent bond formation would be highest under the acidotic conditions resulting from ischemia and discuss whether the predicted level could be sufficient to have therapeutic value. Irrespective of the in vivo mechanism of action for levosimendan, our results provide a rationale and basis for the development of reversible covalent drugs to target the failing heart.

PMID:
29558109
DOI:
10.1021/acs.biochem.8b00109
[Indexed for MEDLINE]

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