Fickle enzyme helps protect, but also can promote heart failure

Apr 26 2005

Enzymes that make the gas nitric oxide (NO) not only protect the heart from damage due to high blood pressure or a heart attack, but also promote heart failure through overgrowth and enlargement of the muscle tissue, say animal researchers at Johns Hopkins.

The Hopkins study, to be published in the May 2 edition of the Journal of Clinical Investigation, is believed to be the first to suggest future therapies for heart failure using chemical cofactors that control the enzymes' action.

Nitric oxide's extensive portfolio of natural effects includes the ability to expand coronary arteries, which improves blood flow, and to help regulate the strength of the heart's contraction, notes cardiologist David Kass, M.D., a specialist in enlarged hearts, or hypertrophy, and a professor at The Johns Hopkins University School of Medicine and its Heart Institute.

But there is clearly a dark side, a biological cost, to this activity in some situations when the enzyme changes form, Kass added.

In several experiments, the researchers simulated hypertrophy for up to nine weeks in groups of 10 to 40 male mice, some bred with and some bred without the gene for the most prominent of the NO-making enzymes, nitric-oxide synthase-3 (NOS3).

NOS3 stops functioning normally when levels of its cofactor, called tetrahydrobiopterin (BH4), decrease.

Results not only showed that BH4 levels drop in hypertrophied hearts, but also that NOS3 uncouples, or splits apart, in the absence of its cofactor. Less NO is produced, and instead, the enzyme produces factors that contribute to oxidative stress in the heart. When the researchers restored levels of BH4, it reversed these harmful effects.

In the first experiment, mice without NOS3 better compensated for the damaging stress of hypertrophy, showing less muscle growth, and fibrosis (scar tissue) and better heart function than mice with the enzyme.

Normal mice with the gene for NOS3 could not adapt to the stress, leading the researchers to conclude that the enzymes had lost their protective value to the heart during hypertrophy.

Biochemical analysis revealed that mice with NOS3 had a mix of two chemical forms of the enzyme. The form of NOS3 that works best with the BH4 cofactor dominated in the non-enlarged hearts but uncoupled when levels of its cofactor declined. The scientists believe this enzymatic uncoupling is key to explaining what happens to cause heart enlargement and pumping failure.

"In these animals, it was better for the heart not to have NOS3 than to have the enzyme in its uncoupled state," says Kass.

In a second experiment to see if the effects of hypertrophy could be reversed, the researchers attempted to preserve normal NOS3 enzymatic function and fed supplements of cofactor BH4 to the group of mice with the enzyme. After three weeks of therapy, results showed that hypertrophy was markedly reduced and heart function improved.

For all mice with hypertrophy, the condition was surgically produced by constricting the main artery carrying blood from the heart to create pressure and oxidative stress. Hearts of untreated mice with NOS3 doubled in size after three weeks and almost tripled in size after nine weeks. Those treated with BH4 or lacking in NOS3 developed milder hypertrophy.

BH4 and other cofactors are "vitamin-like" chemicals required by enzymes to function properly.

"This study shows that nitric-oxide-making enzymes can have both beneficial and detrimental effects on the heart," says Kass. "However, the harmful effects can, at least in mice, be treated with its naturally occurring cofactor, BH4, suggesting a possible therapy in the future."

The researchers plan further experiments to evaluate the therapeutic effects of BH4 in hypertrophy and how it, together with NOS3, compensates for the damage that leads to heart failure.

Funding for the two-year study came from the National Institutes of Health (NIH), the American Heart Association, the Peter Belfer Laboratory Foundation, the American Physiological Society and the Bernard Family Foundation.

The lead Hopkins researchers who took part in this study were Eiki Takimoto, M.D., Ph.D.; Hunter Champion, M.D., Ph.D.; and Maxiang Li, M.D., Ph.D. Other Hopkins researchers who took part were Shuxun Ren, M.D.; E. Rene Rodriguez, M.D.; Nazareno Paolocci, Ph.D.; Kathleen Gabrielson, D.V.M., Ph.D.; and Yibin Wang, Ph.D. Other researchers included Barbara Tavazzi, Ph.D., University of Rome, Italy; and Giuseppe Lazzarino, Ph.D., University of Catania, Italy. Senior study author Kass is also the Abraham and Virginia Weiss Professor of Cardiology at Hopkins.

Research to date is limited on BH4, but the cofactor is being used as a treatment for phenylketonuria, a rare genetic disorder in children that results from a deficiency in the enzyme phenylalanine hydroxylase. Loss of this enzyme can lead to mental retardation, organ damage and unusual posture.

Cardiac hypertrophy commonly develops from high blood pressure, which forces the heart to pump harder to circulate blood throughout the body. According to the latest statistics from the American Heart Association, in 2002, 65 million Americans have high blood pressure (defined as systolic pressure of 140 millimeters of mercury or greater, and/or a diastolic pressure of 90 millimeters of mercury or greater, taking antihypertensive medication or being told at least twice by a physician or other health professional that they have high blood pressure). Patients who develop hypertrophy have two to three times the risk of suffering cardiovascular disease, including heart failure and sudden cardiac death.