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Effects of Protein Kinase C Phosphorylation of Cardiac Troponin I:An Experimental and Model-Based Study

Kirk, Jonathan Alder (2010) Effects of Protein Kinase C Phosphorylation of Cardiac Troponin I:An Experimental and Model-Based Study. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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This project was aimed at further elucidating the role of protein kinase C (PKC)-induced phosphorylation of troponin-I (cTnI) in cardiac contraction. We created a new transgenic mouse model (TG-E), expressing a mutant cardiac TnI constitutively pseudo-phosphorylated at the three PKC phosphorylation sites (S43, S45, T144 mutated to glutamate). 2D-DIGE (Difference in Gel Electrophoresis) gels indicated 7.2 ± 0.5% replacement, with no change in baseline level of actual phosphorylation of cTnI or other myofilamental proteins. Experiments were conducted in perfused isolated mouse hearts, isolated papillary muscles, and skinned fiber preparations. The mechanical measurements were complemented by biochemical and molecular biological measurements, and a mathematical model-based analysis for integrative interpretation. Compared to wild-type mice, TG-E mice exhibited negative inotropy in in vivo echocardiographic studies (9% decrease in fractional shortening), isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, Fmax). Additionally, TG-E mice exhibited slowed relaxation in echocardiographic studies, isolated hearts and intact papillary muscles. The TG-E mice showed no differences in calcium sensitivity, cooperativity, steady-state force-ATPase relationship, and calcium transient (amplitude and relaxation). The four-state model of cardiac contraction was used for a model-based analysis of the data. The model was verified as a priori globally identifiable using a differential algebraic approach. The model-based analysis revealed that experimental observations in TG-E mice could be reproduced by two simultaneous perturbations: a decrease in the rate of crossbridge formation and an increase in calcium-independent persistence of the myofilament active state. In summary, a modest increase in PKC-induced cTnI phosphorylation can significantly regulate cardiac muscle contraction: (1) negative inotropy via decreased crossbridge formation and (2) negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that the effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 vs. T144).


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Kirk, Jonathan
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee MemberLondon,
Committee MemberFuchs,
Committee MemberSalama, Guygsalama@pitt.eduGSALAMA
Committee MemberBorovetz, Harvey Sborovetzhs@upmc.eduBOROVETZ
Committee MemberCampbell, Kenneth
Committee MemberShroff, Sanjeev Gsshroff@pitt.eduSSHROFF
Date: 26 January 2010
Date Type: Completion
Defense Date: 19 August 2009
Approval Date: 26 January 2010
Submission Date: 28 July 2009
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Swanson School of Engineering > Bioengineering
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: calcium; identifiability; isolated hearts; isolated papillary muscles; skinned fibers
Other ID:, etd-07282009-133103
Date Deposited: 10 Nov 2011 19:54
Last Modified: 19 Dec 2016 14:36


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