Impaired diastolic function after exchange of endogenous troponin I with C-terminal truncated troponin I in human cardiac muscle.
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 (cTnI(1-192)) 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 mum, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic beta-adrenergic stimulation. One-dimensional gel electrophoresis, Western immunoblotting, and 3D imaging revealed that approximately 50% of endogenous cTnI had been homogeneously replaced by cTnI(1-192) 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. Ca(2+) sensitivity of the contractile apparatus was increased in preparations containing cTnI(1-192) (pCa(50): 5.73+/-0.03 versus 5.52+/-0.03 for cTnI(1-192) and full-length cTnI, respectively). The sarcomere length dependency of force development and the desensitizing effect of PKA were preserved in cTnI(1-192)-exchanged cardiomyocytes. These results indicate that degradation of cTnI in human myocardium may impair diastolic function, whereas systolic function is largely preserved.
Narolska
Nadiya A
NA
Laboratory for Physiology, Institute for Cardiovascular Research, VU Medical Center, Amsterdam, the Netherlands.
Piroddi
Nicoletta
N
Belus
Alexandra
A
Boontje
Nicky M
NM
Scellini
Beatrice
B
Deppermann
Sascha
S
Zaremba
Ruud
R
Musters
Rene J
RJ
dos Remedios
Cris
C
Jaquet
Kornelia
K
Foster
D Brian
DB
Murphy
Anne M
AM
van Eyk
Jennifer E
JE
Tesi
Chiara
C
Poggesi
Corrado
C
van der Velden
Jolanda
J
Stienen
Ger J M
GJ
D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003971:Diastole; D006801:Humans; D007537:Isometric Contraction; D007700:Kinetics; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000502:physiology / Q000648:ultrastructure; D009210:Myofibrils / Q000502:physiology / Q000648:ultrastructure; D012518:Sarcomeres / Q000648:ultrastructure; D017384:Sequence Deletion; D014336:Troponin / Q000032:analysis / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000378:metabolism*
D014336:Troponin; D019210:Troponin I; D017868:Cyclic AMP-Dependent Protein Kinases