Steps To Block Key Enzyme Action In Heart Failure Mapped

The Johns Hopkins team reports how the enzyme, phosphodiesterase 5, or PDE5A, slows down the breakdown of another, more vital compound in the body, cyclic guanosine monophosphate (cyclic GMP) which influences a variety of biological activities, including cell growth and muscle contraction. A buildup of cyclic GMP limits stress and overgrowth of heart tissue.

PDE5A is the same enzyme that earlier Johns Hopkins work in mice showed is slowed down by the drug sildenafil (Viagra), leading to a reverse of tissue damage from heart enlargement, or hypertrophy, and potentially heart failure. What the scientists are seeing more broadly in this new work is most likely the same braking mechanism, but through a natural chemical reaction in the cell instead of through a synthetic chemical.

In the latest study, to be presented Nov. 11 at the American Heart Association's annual Scientific Sessions in New Orleans, the Johns Hopkins team of protein biochemists confirmed precisely where a sulfur- and nitrogen-containing molecule, or S-nitrosyl group, chemically alters the enzyme's amino-acid building blocks. And they showed that so-called S-nitrosylation of amino acid cysteine 181 results in a 25 percent decrease in PDE5A activity, pinpointing how the enzyme's action is suppressed.

"Knowing the molecular make-up and activity of a protein is critical to understanding heart failure because these problem-specific biochemical reactions are magnified in the disease," says senior study investigator Jennifer Van Eyk, Ph.D., a professor at the Johns Hopkins University School of Medicine and its Heart and Vascular Institute.

"Targeted drug therapies can now be developed and tested to work specifically on cysteine 181, to block the PDE5A enzyme, lower the breakdown of cyclic GMP, and potentially stall progression of heart failure and hypertrophy," adds Van Eyk, director of the Johns Hopkins NHLBI Proteomics Group and the Proteomics Center at Johns Hopkins Bayview Medical Center.

Van Eyk says that previous research by co-investigator Hunter Champion, M.D., Ph.D., had shown that other chemical pathways in cyclic GMP were controlled by the placement of the sulfur-nitrosyl combo, prompting her team to investigate if PDE5A was similarly influenced.

In the first of a series of experiments led by biochemist Christopher Murray, M.Sc., a doctoral candidate at Johns Hopkins, test-tube samples of the enzyme and cyclic GMP were exposed to nitric oxide, which stimulates S-nitrosylation, alone and in the presence of sildenafil. Results showed that PDE5A activity, as measured by the breakdown of cyclic GMP, was at a maximum 100 percent if left untreated with no nitric oxide, but enzyme activity dropped by 25 percent once it was added.

"This was the first solid evidence of S-nitrosylation in PDE5A," says Murray, who, after having confirmed what was happening to the enyzme, proceeded to map out just where on the enzyme S-nitrosylation was taking place.

As cysteine is the only amino acid to which an S-nitrosyl group can attach, Murray's next step was to narrow the search from among the 20-plus possible cysteine locations found in PDE5A.

Two potential candidates emerged for having undergone S-nitrosylation, a cysteine located at the 181st position in the amino acid chain, and another cysteine at site 210. After exposing all PDE5A cysteines to possible S-nitrosylation and then substituting any attached nitrosyl groups with a chemical tag to extract any chemically modified parts, researchers used a mass spectrometry device to tell one amino acid from another.

In a separate experiment, Murray then determined which one of the two cysteines, or both, was being altered. Enzyme pools specific to each cysteine were created. One pool had cysteine 181 blocked, leaving only cysteine 210 available for possible chemical modification, while the other pool had cysteine 210 blocked, leaving open cysteine 181.

Separate chemical attempts at S-nitrosylation of each pool gradually showed that if cysteine 181 was unavailable to react, then enzymatic activity proceeded at the same rate as in a control group with all cysteines free to react.

Researchers say their next steps are to investigate how PDE5A affects where cyclic GMP is broken down or made, with the goal of determining if the enzyme also controls the known cell pooling of the compound. Future studies will also examine other potential effects of PDE5A production, and how alterations to its structure affect its function.

Study co-investigator and cardiologist David Kass, M.D., in whose lab in 2005 much of the original work on sildenafil and PDE5A was performed, also plans to develop a mouse model with blocked cysteine 181 to see what happens when these mice develop heart failure.

Funding for this study, which took three years to complete, was provided by the National Heart, Lung and Blood Institute (NHLBI), a member of the National Institutes of Health, and the AHA.

Besides Van Eyk, Champion, Murray and Kass, other Johns Hopkins researchers who took part in this study were Milena Gebska, M.D., Ph.D.; Azeb Haile, M.Sc.; and Manling Zhang, M.D. Kass is also the Abraham and Virginia Weiss Professor of Cardiology at Johns Hopkins. (Presentation title: cGMP Specific Phosphodiesterase Type 5A Activity is Regulated by S-nitrosylation at Cys 181)