Phosphorylation of the cardiac isoform of troponin I (cTnI) in vitro is associated with enhanced contractile function during protein kinase C (PKC) activation in intact myocytes. However, the specific role of cTnI phosphorylation in the acute contractile response to PKC is controversial. The overall objective of this proposal is to understand the contribution of cTnI, as well as regions within cTnI, to the PKC-mediated contractile response in intact cardiac myocytes. The working hypothesis is that cTnI phosphorylation plays a critical role in the relaxation phase of the contractile function response to PKC. This hypothesis will be tested using the potent PKC agonist endothelin-1 (ET), which reproducibly enhances myocyte contractile function in a largely PKC-dependent manner, and is released during several physiological/pathophysiological conditions.
The first aim of this proposal is to characterize cTnI phosphorylation and determine its role in the adult rat cardiac myocyte contractile response to acute PKC activation. Initial experiments will evaluate temporal and dose-dependent associations between TnI phosphorylation and the myocyte contractile shortening response to PKC activation by ET. Results from these experiments will lay an essential foundation for subsequently determining the contribution of phosphorylated cTnI to the myocyte shortening response to PKC. This later goal will be achieved using viral-based gene transfer to express unique TnI proteins in adult cardiac myocytes and then comparing PKC-dependent TnI phosphorylation and shortening responses in myocytes. Rapid, specific, and efficient gene transfer, protein expression and myofilament incorporation of delivered TnI genes is achieved in fully differentiated adult myocytes using this powerful approach. The first unique TnI selected for determining the contribution of cTnI phosphorylation to the PKC-mediated shortening response will be a TnI isoform expressed during fetal cardiac development, which lacks at least two putative sites phosphorylated by PKC. Information from these studies will provide a direct understanding of the role phosphorylated cTnI plays in the PKC-mediated myocyte shortening response. Insight also will be gained into differential developmental responses to PKC activation.
The second aim will be to identify site(s) within cTnI that are phosphorylated by PKC and determine the importance of each site in the myocyte contractile response to PKC. The relative importance of cTnI site(s) phosphorylated by PKC in the shortening response will be investigated in myocytes expressing mutant TnI containing substitutions in putative phosphorylation sites.
A third aim of this proposal is to determine the effect of isoform-specific TnI region(s) on the ability of TnI to be phosphorylated and/or influence the contractile response to PKC. Ultimately, insights gained from the proposed studies may aid in the design and delivery of unique TnI proteins to failing hearts experiencing altered or pathophysiological responses to PKC.
|Lang, Sarah E; Stevenson, Tamara K; Schatz, Tabea M et al. (2017) Functional communication between PKC-targeted cardiac troponin I phosphorylation sites. Arch Biochem Biophys 627:1-9|
|Kim, Evelyn H; Galchev, Vladimir I; Kim, Jin Young et al. (2016) Differential protein expression and basal lamina remodeling in human heart failure. Proteomics Clin Appl 10:585-96|
|Lang, Sarah E; Stevenson, Tamara K; Xu, Dongyang et al. (2016) Functionally conservative substitutions at cardiac troponin I S43/45. Arch Biochem Biophys 601:42-7|
|Lang, Sarah E; Schwank, Jennifer; Stevenson, Tamara K et al. (2015) Independent modulation of contractile performance by cardiac troponin I Ser43 and Ser45 in the dynamic sarcomere. J Mol Cell Cardiol 79:264-74|
|Lang, Sarah E; Westfall, Margaret V (2015) Gene transfer into cardiac myocytes. Methods Mol Biol 1299:177-90|
|Westfall, Margaret V (2014) The art of the deal in myofilament modulation of function. J Mol Cell Cardiol 72:238-40|
|Hwang, Hyosook; Robinson, Dustin; Rogers, Julie B et al. (2013) Agonist activated PKC?II translocation and modulation of cardiac myocyte contractile function. Sci Rep 3:1971|
|Lang, Sarah E; Robinson, Dustin A; Wu, Helen C et al. (2013) Myofilament incorporation and contractile function after gene transfer of cardiac troponin I Ser43/45Ala. Arch Biochem Biophys 535:49-55|
|Nichols, Ruthann; Bass, Chloe; Demers, Leslie et al. (2012) Structure-activity studies of RFamide-related peptide-1 identify a functional receptor antagonist and novel cardiac myocyte signaling pathway involved in contractile performance. J Med Chem 55:7736-45|
|Hwang, Hyosook; Robinson, Dustin A; Stevenson, Tamara K et al. (2012) PKC?II modulation of myocyte contractile performance. J Mol Cell Cardiol 53:176-86|
Showing the most recent 10 out of 14 publications