The long-range goal of this work is to determine the molecular basis of enhanced cardiac contractile performance resulting from myofilament protein phosphorylation. The overall working hypothesis is that phosphorylation causes intramolecular domain modifications within troponin I (TnI) that directly affect the interactions of TnI with other thin filament regulatory proteins to then cause the observed alterations in function. Two consecutive serines are present at positions 23 and 24 in TnI and each can be phosphorylated by protein kinase A (PKA) in species from mouse to humans. There are four possible phosphorylation states of TnI: un-phosphorylated, Ser23/Ser24 mono-phosphorylated, or di-phosphory-lated. The physiological significance of the four phosphorylation states of TnI has not been determined. A hypothesis of this proposal is that phosphorylation causes a modification in the interaction between acidic and basic amino acid clusters within the TnI N-terminus. This model requires flexibility in the N-terminus proposed to be provided by a proline cluster critically positioned between the charged residues. The testable outcome of this model is that neutralization of charge in the acidic amino acid or basic clusters, or substitution of the prolines, should mimic the effects of serine phosphorylation.
The specific aims are: 1) to determine the contribution of TnI phosphorylation, relative to C-protein phosphorylation, to cause alterations in contractile function in the adult single cardiac myocyte; 2) to establish the physiological significance of the four phosphorylation states of TnI in adult cardiac myocytes; and 3) to determine the molecular domain interactions within TnI and between TnI and TnC that are altered by phosphorylation of TnI in adult cardiac myocytes. The experimental strategy involves genetic dissection of TnI in adult cardiac myocytes in primary culture using recombinant adenovirus vectors.
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