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Margaret V. Westfall, Ph.D.

Margaret V. Westfall, Ph.D. - Research

Structural and Functional Effects of Protein Kinase C-mediated Troponin I Phosphorylation in Adult Rat Cardiac Myocytes

Investigators: Margaret V. Westfall, PhD, Surgery

Funding: American Heart Association

Activation of protein kinase C (PKC) by several agonists has previously been demonstrated to cause phosphorylation of cardiac troponin I (cTnI), as well as other myofilament proteins. A primary goal of this research is to characterize acute and long-term PKC phosphorylation of myofilament proteins in the intact cardiac myocyte. A second goal is to determine the relative contribution of acute PKC-mediated cTnI phosphorylation to myofilament force generation in permeabilized adult cardiac myocytes. These studies are being accomplished by expressing slow skeletal TnI in adult myocytes using viral-mediated gene transfer, and then comparing TnI phosphorylation and myofilament function to myocytes expressing the endogenous cTnI. Experiments are also being carried out to determine whether long term activation of PKC continues to phosphorylate cTnI, and whether these changes in cTnI phosphorylation correlate with the onset of myofibrillar degradation.

Protein Kinase C. Signaling Through Troponin I in Adult Cardiac Myocytes

Investigators: Margaret V. Westfall, PhD, Surgery

Funding: Rackham Grant and Fellowship Program, University of Michigan

The overall aim of this group of experiments is to ascertain the direct influence of phosphorylated cTnI on adult myocyte contractile function in response to PKC activation by physiological agonists. Phosphorylation of myofilament proteins and myocyte sarcomere shortening responses to the PKC agonist endothelin are being examined for these studies. The time-dependence myofilament protein phosphorylation and myocyte shortening in response to different doses of ET are currently under investigation. Ongoing studies have determined that cTnI phosphorylation by PKC causes accelerated relaxation in intact cardiac myocytes. This enhancement in the rate of relaxation is removed in myocytes expressing the slow skeletal isoform of TnI, which is not phosphorylated by protein kinase C. Information gained from these studies can now be used to determine whether TnI phosphorylation and/or the TnI-mediated enhancement of relaxation produced in response to PKC is altered under physiological and pathophysiological conditions.

Influence of Troponin I Phosphorylation by Protein Kinase C on Cardiac Myocyte Contractile Function

Investigator: Margaret V. Westfall, PhD, Surgery

Funding: UM Cardiovascular Center McKay Grant

The overall objective of this proposal is to understand the contribution of individual phosphorylation sites within cTnI to the PKC_mediated enhancement of relaxation in intact cardiac myocytes. The working hypothesis is that phosphorylation sites Ser43/45 and Thr144 within TnI each play 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. Phosphorylation of TnI and myocyte shortening will be followed in parallel experiments using myocytes expressing mutant cTnI proteins with Ala substitutions at Ser43, Ser45, and/or Thr144. These mutant cTnI proteins will be expressed in adult myocytes using viral_based gene transfer to express the unique TnI proteins in adult cardiac myocytes. Rapid, specific, and efficient gene transfer, protein expression and myofilament incorporation of delivered TnI genes previously has been achieved in fully differentiated adult myocytes with this powerful approach. Information from these studies will provide a direct understanding of the role specific phosphorylation sites within cTnI play in the PKC_mediated myocyte shortening response. Ultimately, knowledge gained from the proposed studies may be used to design and deliver unique TnI proteins to failing hearts experiencing altered responses to PKC.

Influence of Troponin I Phosphorylation by PKC on Contractile Function

Investigators: Margaret V. Westfall, PhD, Surgery

Funding: National Institutes of Health

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