Published online before print October 5, 2006, doi: 10.1161/01.RES.0000248753.30340.af
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Submitted on July 24, 2006
Revised on September 19, 2006
Accepted on September 25, 2006
Impaired Diastolic Function After Exchange of Endogenous Troponin I with C-Terminal Truncated Troponin I in Human Cardiac Muscle
Nadiya A. Narolska ;
From the Laboratory for Physiology (N.A.N., N.M.B., R.Z., R.J.M., J.v.d.V., G.J.M.S.), Institute for Cardiovascular Research, VU Medical Center, Amsterdam, the Netherlands; Dipartimento di Scienze Fisiologiche (N.P., A.B., B.S., C.T., C.P.), Università di Firenze, Italy; Research Laboratory of Molecular Cardiology (S.D., K.J.), Bergmannsheil/St. Josef-Hospital, Medical School of the Ruhr-University of Bochum, Germany; Muscle Research Unit (C.d.R.), Institute for Biomedical Research, The University of Sydney, Australia; and Department of Medicine (D.B.F., A.M.M., J.E.v.E.), Johns Hopkins University, Baltimore, Md.
* To whom correspondence should be addressed. E-mail: g.stienen{at}vumc.nl.
The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI1-192) and full-length cTnI were expressed in Escherichia coli, purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 µm, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic 
-adrenergic stimulation. One-dimensional gel electrophoresis, Western immunoblotting, and 3D imaging revealed that approximately 50% of endogenous cTnI had been homogeneously replaced by cTnI1-192 in both myofibrils and cardiomyocytes. Maximal tension was not affected, whereas the rates of force activation and redevelopment as well as relaxation kinetics were slowed down. Ca2+ sensitivity of the contractile apparatus was increased in preparations containing cTnI1-192 (pCa50: 5.73±0.03 versus 5.52±0.03 for cTnI1-192 and full-length cTnI, respectively). The sarcomere length dependency of force development and the desensitizing effect of PKA were preserved in cTnI1-192-exchanged cardiomyocytes. These results indicate that degradation of cTnI in human myocardium may impair diastolic function, whereas systolic function is largely preserved.
Key words: cardiac function • contractility • cardiomyocytes • cardiomyopathy • ischemia
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