Sheri W. Faught
phone: 585-4314
pager: 339-3073
sheri.faught at m.cc.utah.edu
Fellowship Proposal
Undergraduate Research Opportunities Program
Summer Quarter 1998
Background Information
Protein phosphorylation (the attachement of a phosphate group to a protein) plays an important role in regulating contractility in cardiac myocytes. The activity of any protein regulated by phosphorylation depends on the balance between the activities of the kinases that phosphorylate it and the phosphatases that dephosphorylate it.1 Cyclic AMP produces its effects by activating cAMP-dependent protein kinases (A-kinases). The cumulative effects of agents that raise cyclic AMP concentrations and thus activate cAMP-dependent protein kinase is the phosphorylation of a large number of proteins in cardiac myocytes and an increase in contractility. Drugs that raise cAMP concentrations in cardiac myocytes improve contractility but unfortunately have deleterious effects on long-term survival.
The association of identified subunits of phosphatases to proteins in human myocardium is pertinent to the pharmacological investigation of heart failure and the long-term efficacy of certain therapeutic drugs. Selectivity of positive responses in the phosphorylation substrates of cAMP-dependent protein kinases may elicit responses that will improve contractility in the failing human heart. Subsequently, more selective increases in protein phosphorylation might allow the benefits of treatment without long-term harm. One way this might be achieved is by inhibition of the protein phosphatases that dephosphorylate the substrates of cAMP-dependent protein kinase. There are many families of phosphatases comprised of many isoforms, and these isoforms may be selectively coupled to different substrates. If so, selective inhibition of these phosphatases could result in more selective increases in protein phosphorylation and thus allow the increase in contractility without the deleterious long-term effects.
Signal transduction refers to the processes through which extracellular agents bind to receptors in the plasma membranes of targeted cells and thus cause intracellular events that alter the functions of those cells. Adenylate cyclase is a membrane-bound enzyme that catalyzes the production of cAMP from ATP. Cyclic AMP-mediated signal transduction specifically denotes the pathways that involve the stimulation of adenylate cyclase and the consequent increase of the second messenger cAMP. Also, adrenaline in the body acts by binding to b-adrenergic receptors on the muscle cell surface, thereby causing an increase in the level of cyclic AMP in the cytosol.1 When activated by cAMP, A-kinase causes a variety of intracellular effects. In this way, the activation of a receptor on the surface of a cell can generate a single intracellular signal that can regulate a variety of different processes within the cell. The complicated molecular pathways involved in signal transduction are abundant. Thus it is important to understand the complex actions and reactions possible with respect to the cellular pathophysiology of heart failure and its affects on other processes mediated by the same pathways.
Heart failure is the result of an impairment in the ability of the heart muscle, or myocardium, to generate force during contraction. As a response to drugs relating to the force of muscular contractions (intotropic drugs), the heart is still capable of an increase in contractility. If an associated reduced contractility of the heart muscle could be returned to normal, the remaining symptoms would be alleviated. Commonly used glycosides (which increase cardiac output) have a narrow margin of safety, and less toxic compounds with positive actions relating to the force of muscular contractions are needed. The administration of some drugs tend to improve contractility by increasing intracellular calcium concentrations and may be accompanied by symptoms such as excess fluid accumulation. These symptoms may be reduced by drugs such as antidiuretics which enhance the loss of sodium and water by the kidneys. These examples show that the utilization of drugs that affect processes other than calcium concentrations but that also improve contractility will be useful.Specific Aims
Our goal is to determine whether there is evidence that selective phosphatase inhibition could result in selective protein phosphorylation. To assess the plausibility of this hypothesis, we plan to determine whether different phosphatases have different substrates and/or binding proteins in cardiac myocytes.
Several families of protein phosphatases have been identified, cloned and sequenced. Dr. Movsesian’s lab has identified several specific isoforms of protein phosphatase 2A (PP2A), B56a and B56g, that are expressed at high levels in human cardiac myoctes. They have also expressed his-tagged recombinant B56a and B56g subunits of human PP2A in Sf9 cells an purified them via nickel-affinity chromatography. Proteins that bind to recombinant B56 subunits, whether they are substrates of PP2A or PP2A targeting proteins, will adhere to the nickel resins together with the his tagged recombinant subunits. Proteins will be eluted from these resins with imidazole and subjected to SDS-polacrylamide gel electrophoresis. We will determine whether the different B56 subunits bind to different proteins in human myocardium. A difference in the pattern of proteins that bind to different PP2A B subunits will be evidence in support of the notion that selective PP2A inhibition may lead to selective changes in protein phosphorylation. If our hypothesis is correct, a different set of proteins will co-purify with B56a and B56g, and this will be evident on SDS-polyacrylamide gel eletrophoresis. This will lead to further experiments, that are not part of this project, in which B56a and B56g-binding proteins will be identified by two-hybrid screening.
Significance of the Study
The processes that bring about the cellular responses associated with increases in intracellular cAMP concentrations are important in the pathogenesis and treatment of heart failure. The enhancement of myocardial contractility that results from exposure to b-adrenergic receptor agonists correlate specifically with increases in membrane-bound rather than cytosolic cAMP content. These findings indicate that the possibility of a selective increase in membrane-bound cAMP content would be beneficial to therapeutic practices. What purpose is served by the differential regulation of cytosolic and membrane-bound cAMP content in cardiac myocytes? Selective activation of cAMP-dependent protein kinases may permit control over the intracellular distribution of cAMP and the compartmentation of cAMP-mediated signal transduction. Will the administration of such appropriately selective activators or inhibitors result in a pattern of protein phosphorylation that allows inotropic or lusitropic responses to be elicited without the undesirable accompanying consequences of increases in cAMP content?2 Answers to the aforementioned questions will provide a better understanding of the molecular pathophysiology of the heart and allow for pharmacological advancements to improve the treatment of heart failure.
References
Movsesian MA. Cyclic AMP-mediated signal transduction in heart failure: molecular pathophysiology and therapeutic implications. J Investig Med 45 (8):432-440, 1997.Rhoades, Rodney, Ph.D. and Pflanzer, Richard, Ph.D., ed. 1996. Human Physiology. New York: College Publishing.
Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. 1994. Molecular Biology of the Cell. New York & London: Garland Publishing, Inc.
MA Movsesian, M.D., Associate Professor of Medicine (Cardiology) and Adjunct Associate Professor of Pharmacology & Toxicology. May 1998. Interview.
Hohl CM, Li Q. Compartmentation of cAMP in adult canine ventricular myocytes. Relation to single-cell free Ca2+ transients. Circulation. 69: 1369-1379, 1991.
Please contact sheri.faught at m.cc.utah.edu if you have any questions/comments.