Researchers Devise New Tools To Help Pinpoint Treatments For Heart Failure

In a paper to appear in the Aug. 7 online edition of Proceedings of the National Academy of Sciences, the researchers at the University of California, San Diego describe the use of an engineered protein partly derived from a jelly fish that fluoresces within heart cells in tandem with activation of the key enzyme called PKA (protein kinase A). By combining computer modeling with the novel fluorescence-imaging technique in living cells, the researchers were able to uncover new details in the molecular control of PKA.

PKA is an intensely studied regulatory enzyme whose activity in heart cells rises sharply in response to exercise or various stresses, priming the heart to beat faster and with more power, and to increase its metabolic rate to meet the increased energy demands.

"For the first time, this innovative visualization technique allowed us to refine our computational models get a better understanding of the interacting biochemical pathways in heart cells that involve PKA," said Andrew McCulloch, a professor and chair of the Department of Bioengineering at UCSD's Jacobs School of Engineering. "Now we're in a good position to do similar experiments with mutant strains of mice that experience heart failure in ways that mimic human disease."

McCulloch is an expert at mathematical modeling the interactions of hundreds of enzymes and other molecules in heart cells. McCulloch and a team of Ph.D. candidates in bioengineering, including recent graduate Jeffrey Saucerman, collaborated with another group at UCSD led by Roger Y. Tsien, a professor of medicine, pharmacology, and chemistry and biochemistry and a Howard Hughes Medical Institute investigator. Tsien and his team have developed a variety of molecular sensors that have revolutionized the optical monitoring of neurons and other cell types. Their tailor-made fluorescence-tagged proteins have permited scientists to visualize signaling processes in nerve cells in culture dishes and in the brains of living animals.

Fluorescence-tagged proteins created by Tsien's group have been used before to probe heart cells; however, the results reported in PNAS were the first in which the proteins have been used to visualize PKA activity in those cells. The most recent visualizations, combined with mathematical models, provide more detailed and quantitative measurements of PKA activation.

PKA affects the heart rhythm in ways that are readily detectable with an electrocardiogram. However, a better understanding of how the PKA-dependent regulatory system works in healthy heart cells, and how it is altered in diseased cells, may reveal underlying causes of heart failure.

"In order to validate and refine our computer models we must be able to measure the localized activity of PKA dynamically a living heart cell, and that's what we've been able to accomplish for the first time," said McCulloch. "We've done it by essentially lashing molecular flashlights to the backs of 'sensor' proteins that tell us what's going on inside heart cells."

By stimulating one end of the cell, the researchers were also able to watch a fluorescent wave of PKA activity travel along heart cells. They reproduced the behavior in their computer model. Making such a model that is capable of reproducing the spatial localization and movement of signaling events in heart cells should allow the researchers to gain a better understanding of the complexities of cardiac cell signaling. For example, it may help to explain why stimulation of the beta-adrenergic receptors increases the mechanical performance of the failing heart in the short term yet is detrimental in the long term.

The key enzyme in heart muscle signaling, PKA, is a member of a huge class of regulatory proteins called kinases. Each kinase is specialized to attach a phosphate molecule to a specific set of target proteins. These phosphorylation reactions switch targeted proteins from an inactive state to an active state. PKA actually activates other kinases, which in effect amplify the effect of PKA through a signaling cascade.

The activity of kinases is delicately balance by a group of enzymes called protein phosphatases, which simply remove phosphate groups from specific proteins, inactivating them. About 30 percent of all human proteins are regulated by kinases and phosphatases.

Activation of PKA is actually initiated at the exterior surface of heart cells where neurotransmitters and hormones bind to beta-adrenergic receptors. However, while drugs that boost PKA activity temporarily increased cardiac contractions, they also led to higher patient mortality in the long term.

A widely used class of drugs is called beta-blockers. Drugs in that class, including Atenolol, Bisoprolol, and Metoprolol are designed to take the opposite approach: they block the beta-adrenergic receptors, thereby reducing PKA activity and lowering cardiac output. Beta blockers are now taken daily by about 5 million U.S. patients suffering from heart failure, high blood pressure and other cardiovascular diseases. The effectiveness of beta blockers has highlighted the need to better understand the system of biochemical signaling within heart cells.

For example, the clinical observations and experimental findings of many scientists suggest that increasing the strength of heart cells contraction may be less beneficial to patients than restoring the normal PKA-dependent control system. During heart failure, the heart muscle contracts weakly, which causes the body to compensate by releasing more hormones and neurotransmitters to try to make the heart cell contractions return to normal strength. However, like an exhausted athlete being exhorted by a screaming coach to run faster, the beta-adrenergic receptor control system becomes unresponsive and the heart loses its ability to respond to changes in the body's demands. Paradoxically, blocking the beta-receptors actually reduces that desensitization and helps to slow the vicious cycle of heart failure.

To dissect the dynamics of signaling in individual living heart cells, Saucerman and his colleagues measured the expected PKA activation at the periphery of stimulated heart cells, but to their surprise, they measured a delay of PKA activation at the center of the cell. The McCulloch group's computer model suggests that microstructures near the cell membrane may retard the movement of molecules that activate PKA. Indeed, proteins directly or indirectly involved in activation of PKA are typically clumped to the cell membrane rather than distributed as a homogeneous soup throughout the cell. The researchers think this ability of different regions of the cell to respond differently to external stimuli may explain the ability of heart cells to produce appropriate responses to the myriad different stimuli they receive.

While the series of PKA signaling behaviors reported in the PNAS paper confirmed parts of a sophisticated computer model of myocytes regulation, there were surprises as well. "The devil is in the details of a biochemical system so complex, but by pursuing those details we may be able to help develop therapies designed to treat patients with inherited or acquired defects in this important system," said McCulloch. "The improvement of our computer models goes hand in hand with the ability to test them in living heart muscle cells with these novel visualization tools. This combined approach takes advantage of what we already know, but also opens up new opportunities to find missing pieces of the puzzle, any of which could end up being the target for new, more effective heart drugs."