By David N. Leff

It's a well-worn truism that coming events cast their shadows before. One such shadow is angina pectoris, the chest pain that foreshadows a heart attack.

Usually, that sharp twinge radiates down the left arm, across the shoulders, abdomen and mid-backbone. Angina is nature's heads-up alert that one's heart is gasping for breath - more specifically, for oxygen. This arises from a blockage in the arteries that deliver oxygen-laden red blood to the myocardial muscles. That early warning of ischemia, or oxygen starvation, means that myocardial infarction - the death of heart muscle cells - may be imminent.

To relieve angina's pain, the sufferer tucks a tablet of nitroglycerin under the tongue, to dissolve. It acts as a vasodilator, which widens the lumen of the occluded blood vessel. Incidentally, nitroglycerin is a starting material for the manufacture of dynamite and other high explosives. What those products have in common with the angina pill is the chemically volatile gas, nitric oxide.

"Clinical studies," observed cardiologist Roberto Bolli at the University of Louisville in Kentucky, "have looked at patients in whom episodes of angina - which prefaces ischemia - occurred 24 to 48 hours prior to the heart attack. They found that those who had angina episodes prior to the heart failure did better than those who did not. The severity of the attack was mitigated; there was less damage to the heart, and the patient had fewer complications, such as heart failure, heart rupture, arrythmias and shock.

Bolli went on to cite "many studies that this preconditioning effect, as it's called, decreases deaths of heart patients in the hospital, and the sustained reduction of mortality persisted five years later." This seemingly counterintuitive effect of incipient ischemia has generated a lively spate of research and theory in the cardiovascular community. So far, what's known is that preconditioning happens in two waves: a brief initial phase that lasts two to three hours, followed 12 to 24 hours later by a second window of cardiac protection that may persist for at least 72 hours.

Nitric Oxide - Friend And Foe

Bolli and his colleagues in the Experimental Research Laboratory of the university's Division of Cardiology set out to seek the basis for this phenomenon. They found it in the trendy molecule, nitric oxide, as reported in the current Proceedings of the National Academy of Sciences (PNAS), dated Sept. 28, 1999. Their paper, of which Bolli is senior author, bears the title: "The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO [nitric oxide] synthase gene." (See BioWorld Today, Sept. 22, 1995, p. 1.)

"This is the first direct evidence," Bolli told BioWorld Today, "that a protein - inducible NO synthase - has been shown to be responsible for preconditioning. It has been sought for at least 10 years, and many hypotheses have been put forth." NO synthase is the enzyme that gives rise to the nitrogen radical, nitric oxide.

"Our hypothesis," Bolli explained, "which is now substantiated by considerable evidence from animal experiments, shows that NO triggers the whole process of preconditioning during the initial exposure to ischemia, It's the stimulus that starts the preconditioning response. This response," he went on, "involves a lot of steps. The bottom line is that the synthesis of a new protein - the inducible NO synthase, or iNOS - makes NO 24 hours later, which then protects the heart against a heart attack."

Those experiments involved a total of 321 mice. "We exposed the mice to brief episodes of ischemia," Bolli recounted, "which is deprivation of blood to the heart, and let them recover. Then the next day, 24 hours later, we exposed these animals to a severe episode of ischemia, which caused a massive heart attack."

To mimic the preconditioning phase, the co-authors alternately tightened and loosened sutures around the animals' coronary arteries - four minutes of occlusion, then four of reperfusion, for six cycles. To create the heart-attack infarctions, they occluded the arteries for half an hour, then released the blockage to restore blood flow.

"First of all," Bolli said, "we found that normal wild-type mice - which carry the iNOS genes - are protected, or preconditioned, by the initial brief episodes of ischemia. So the severity of the heart attacks was much less than in mice that were not preconditioned. Infarct size was reduced by two-thirds. But in knockout mice that did not have the gene for iNOS, this protection was completely abolished. So we conclude that iNOS is necessary for this protection to occur."

Focus On New Drugs; Doubt On Gene Therapy

Any future clinical payoffs from these and related findings, Bolli deems "very speculative." He speculated, "To protect the heart, one could mimic the same increase in iNOS as preconditioning. We don't know how to do that way of increasing iNOS activity yet. What's important here is that it has to be a mild or modest increase, such as is associated with ischemic preconditioning, because a high increase is toxic to the heart."

He made the point, "iNOS is thought to be detrimental - which I'm sure it is - when it's produced at very high levels. For example, when you transplant a heart, the rejection is mediated in part by iNOS. Inflammation of the lungs also seems to involve the NO enzyme, and septic shock is another example where iNOS plays a very important role. So in humans it suggests that iNOS is a double-edged sword, with a dose-dependent profile. Low levels of induction can be very beneficial."

As for developing new drugs along these lines, Bolli said, "We are working on that but so far we don't have anything to report. That's certainly going to be our focus for the near future." He is less bullish on the outlook in this context for gene therapy.

"That's one of the possibilities we are exploring," he allowed. "The problem is delivering this iNOS gene to the heart. Nobody so far," Bolli pointed out, "has succeeded in a reproducible way to transfer genes to the heart muscle, which limits all kinds of gene therapy in that organ."