© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
Troponin I protein kinase C phosphorylation sites and ventricular function
aCardiovascular Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA
bDepartment of Bioengineering, University of Pittsburgh, 749 Benedum, Hall Pittsburgh, PA 15261, USA
cPittsburgh NMR Center for Biomedical Research, Carnegie Mellon University, Pittsburgh, PA 15213, USA
dLaboratory of Functional and Molecular Imaging, NINDS, NIH, Bethesda, MD 20817, USA
* Corresponding author. Tel.: +1-412-624-2095; fax: +1-412-383-8788. Email address: sshroff{at}pitt.edu
Objective:Cardiac Troponin I (cTnI) phosphorylation by protein kinase C (PKC) results in a reduction of maximal actomyosin ATPase activity, an effect that is more marked at higher levels of calcium (Ca2+) and is likely to reduce active force development. We postulated that there would be greater Ca2+-dependent changes in ventricular function in hearts of cTnI transgenic (TG) mice expressing mutant troponin I lacking PKC sites compared to wild-type (WT). Methods:We studied left ventricular function in isolated perfused hearts over a wide range of left ventricular volumes (Frank-Starling relationships) and mechanical restitution at three levels of perfusate Ca2+ (1.5, 2.5, and 3.5 mM). Manganese-enhanced magnetic resonance imaging (MRI) was used to study in-vivo sarcolemmal Ca2+ influx. The phosphorylation status of cTnI was examined by western blot analysis. Results:Systolic contractile function in TG mice was altered in a calcium-dependent manner such that ventricular contractility was significantly greater in TG mice only at 3.5 mM perfusate Ca2+. The relaxation process and passive mechanical properties were unaltered in TG mice. Mechanical restitution parameters were abnormal in TG mice only at 1.5 mM perfusate Ca2+. In-vivo MRI data demonstrated up to 48% reduction in Mn2+-induced contrast enhancement, indicating reduced sarcolemmal Ca2+ influx. Western blot analysis indicated increased cTnI phosphorylation in TG mice. Conclusions:(1) TG mice exhibit calcium-dependent positive inotropy without slowed relaxation and this phenotype is mitigated by concomitant (compensatory) changes of reduced intracellular Ca2+ and increased phosphorylation of remaining cTnI sites. (2) The contractile phenotype in TG mice can be interpreted as an amplification of the normal response to changes in cellular Ca2+ observed in WT mice. Thus, PKC phosphorylation sites on cTnI play a role in attenuating contractile responses to changes in intracellular Ca2+.
KEYWORDS Protein kinase C; Ventricular function; Transgenic animal models; Calcium (cellular); Phosphorylation; Contractile function