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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL059301-04
Application #
6330137
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Reinlib, Leslie
Project Start
1997-12-08
Project End
2002-08-31
Budget Start
2000-12-01
Budget End
2002-08-31
Support Year
4
Fiscal Year
2001
Total Cost
$269,511
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Physiology
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Heinis, Frazer I; Vermillion, Katie L; Andrews, Matthew T et al. (2015) Myocardial performance and adaptive energy pathways in a torpid mammalian hibernator. Am J Physiol Regul Integr Comp Physiol 309:R368-77
Thompson, Brian R; Metzger, Joseph M (2014) Cell biology of sarcomeric protein engineering: disease modeling and therapeutic potential. Anat Rec (Hoboken) 297:1663-9
Bedada, Fikru B; Chan, Sunny S-K; Metzger, Stefania K et al. (2014) Acquisition of a quantitative, stoichiometrically conserved ratiometric marker of maturation status in stem cell-derived cardiac myocytes. Stem Cell Reports 3:594-605
Wang, Wang; Asp, Michelle L; Guerrero-Serna, Guadalupe et al. (2014) Differential effects of S100 proteins A2 and A6 on cardiac Ca(2+) cycling and contractile performance. J Mol Cell Cardiol 72:117-25
Martindale, Joshua J; Metzger, Joseph M (2014) Uncoupling of increased cellular oxidative stress and myocardial ischemia reperfusion injury by directed sarcolemma stabilization. J Mol Cell Cardiol 67:26-37
Thompson, Brian R; Houang, Evelyne M; Sham, Yuk Y et al. (2014) Molecular determinants of cardiac myocyte performance as conferred by isoform-specific TnI residues. Biophys J 106:2105-14
Heinis, Frazer I; Andersson, Kristin B; Christensen, Geir et al. (2013) Prominent heart organ-level performance deficits in a genetic model of targeted severe and progressive SERCA2 deficiency. PLoS One 8:e79609
Asp, Michelle L; Martindale, Joshua J; Heinis, Frazer I et al. (2013) Calcium mishandling in diastolic dysfunction: mechanisms and potential therapies. Biochim Biophys Acta 1833:895-900
Asp, Michelle L; Martindale, Joshua J; Metzger, Joseph M (2013) Direct, differential effects of tamoxifen, 4-hydroxytamoxifen, and raloxifene on cardiac myocyte contractility and calcium handling. PLoS One 8:e78768
Wang, Wang; Barnabei, Matthew S; Asp, Michelle L et al. (2013) Noncanonical EF-hand motif strategically delays Ca2+ buffering to enhance cardiac performance. Nat Med 19:305-12

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