In vitro motility analysis of thin filaments from failing and non-failing human heart:: Troponin from failing human hearts induces slower filament sliding and higher Ca2+ sensitivity

A. KNOTT. I. PURCELL AND S. MARSTON. In vitro Motility Analysis of Thin Filaments from Failing and Non-failing Human Heart: Troponin from Failing Human Hearts Induces Slower Filament Sliding and Higher Ca2+ Sensitivity. Journal of Molecular and Cellular Cardiology (2002) 34, 469-482. Contractility of the myocardium is altered ill end-stage heart failure. We investigated whether this was related to functional changes in troponin. We isolated troponin from 1 g samples of end-stage failing, non-failing and foetal human heart and studied its regulation of actin-tropomyosin movement over immobilised HMM by in vitro motility assay. At pCa5.4 the sliding velocity of thin filaments reconstituted with non-failing heart troponin was 52+/-4%, more than actin-tropomyosin. with failing heart troponin velocity increased by 35+/-2% and with foetal heart troponin velocity increased by 11+/-4%. Thin filaments containing troponin from failing hearts were more Ca2+-sensitive than non-failing heart troponin. EC50 for the fraction of filaments motile and filament velocity decreased 1.76+/-0.20 and 1.89+/-0.62-fold respectively relative to non-failing heart troponin. With foetal heart troponin the EC50 dececased 2.16+/-0.23 and 3.50+/-1.73-fold for fraction and velocity respectively. Western blots revealed no difference in troponin T or troponin I isoform expression in troponin from failing and non-failing adult hearts but foctal isoforms of troponin I and T were observed in troponin from foctal heart. The level of PKA phosphorylation of troponin from failing and non-failing heart was not significantly different, however, complete non-specific dephosphorylation of troponin abolished most of the difference between failing and non-failing heart troponin. These findings show functional alterations in troponin in failing hearts which could account for the reduced contractile function but there is no change in troponin isoform expression or PKA phosphorylation. Differential phosphorylation by other kinases may account for altered troponin function. (C) 2002 Elsevier Science Ltd. All rights reserved.