Abstract
Protein kinase C (PKC) phosphorylates 3 clusters of residues within cardiac troponin I (cTnI) and yet, it is unclear whether phosphorylation at multiple sites produces additive and/or divergent functional modifications. Our goal was to evaluate the influence of cTnI with phosphomimetic substitutions on contractile performance under basal conditions and in response to PKC activation by endothelin. Endogenous cTnI was replaced with phosphomimetic substitutions using adenoviral-mediated gene transfer into adult rat cardiac myocytes. Phosphomimetics of Ser43/45, Ser43/45 plus Thr144, and Ser23/24 plus Ser43/45 were substituted with Asp to form AdcTnISer43/45Asp, cTnIAspTriple (e.g. cTnISer43/45AspThrT144Asp), and cTnIAspQuad (cTnISer23/24/43/45Asp). Isolated myocytes were electronically paced and studied 4 days after gene transfer. Gene transfer of epitope-tagged versions of each construct resulted in 30-40% replacement after 2 days and >65% replacement of endogenous cTnI 4 days after gene transfer without significant alterations in the expression of other myofilament proteins. In functional studies, peak shortening amplitude was significantly decreased in myocytes expressing cTnISer43/45Asp or cTnIAspQuad, while peak shortening in myocytes expressing cTnIAspTriple was not significantly different from controls. Relaxation was accelerated in myocytes expressing cTnIAspQuad, but was not different from controls in myocytes expressing cTnIAspTriple or cTnISer43/45Asp. Together, these results suggest the Ser23/24 and Ser43/45 sites have an additive influence on shortening, while substitution at Thr144 attenuates the influence of Ser43/45 on peak shortening. To further determine whether multiple phosphomimetic substitutions within cTnI influence myocyte shortening, we studied the change in peak shortening and relaxation produced by the PKC agonist, endothelin (10 nM). In preliminary studies, the increased peak amplitude and accelerated relaxation observed in control myocytes is not significantly different in myocytes expressing cTnISer43/45Asp. However, there is a trend for myocytes expressing cTnIAspQuad to show an attenuated amplitude and relaxation response to ET.
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