Mysteries of the Heart

Like all cardiologists, Roberto Bolli, M.D., has made a career out of listening to the human heart.

But Bolli, chief of the Division of Cardiology in the UofL School of Medicine, hears more than rhythmic beats and pulsing blood. He also hears answers that explain some of the heart's most fundamental mysteries.

Take the phenomenon of late preconditioning, in which a heart that suffers a brief loss of blood flow, or ischemia, actually becomes resistant to the damage that might be caused by a major heart attack any time in the next four days.

While most scientists were pondering the implications of this phenomenon, first discovered in 1993, Bolli sensed its significance immediately and began laying the groundwork for a research agenda that eventually would establish UofL as a world authority in cardiac ischemia.

"It seemed intuitive to me that the heart would know better than anyone how to protect itself from injury," Bolli says of his decision to pursue late preconditioning as a topic of study. "It's the heart's innate cardioprotective mechanism, developed over millions of years of evolution, to protect itself from injury. And when things evolve over millions of years, usually they are very effective."

Today, Bolli and his 44-member research team have, by and large, decoded the complex chain of molecular events necessary for the heart to establish preconditioning -- a landmark achievement that could lead to new drug or gene therapies for patients at risk of heart attack.

"The bottom line is that we have written a new chapter in pathophysiology," Bolli says. "Ten years ago, this was unknown, and we have systematically deciphered the steps whereby the heart becomes resistant to ischemia after preconditioning."

As remarkable as these discoveries are, Bolli's most impressive feat may be the research program behind them. Recruited to UofL from Houston's Baylor College of Medicine in late 1994 with a $2.3 million grant from Jewish Hospital, Bolli arrived to find a cardiology division with no federal research funding and just one research scientist -- himself.

Since then, the division has hired 11 faculty researchers, 19 research associates or fellows and 14 support staff. Collectively, those researchers have brought in an astounding $43.9 million in national competitive grants since 1995, including 21 from the National Institutes of Health and 11 from the American Heart Association.

Bolli alone has four NIH grants, including a 10-year, $2.7 million MERIT Award in recognition of his outstanding scientific discoveries.

The UofL team's resulting scholarship has produced an astonishing number of papers -- more than 210 highly regarded, peer-reviewed articles for top medical publications like the Journal of Clinical Investigation and Circulation Research. In fact, Bolli was the most-cited author in Circulation Research in 2001, and five of his papers have been referenced more than 300 times in the medical literature.

"Dr. Bolli's progress since coming to UofL has been quite incredible," says Richard Redinger, M.D., chairman of the university's Department of Medicine. "I think we now have the best basic-science, research-oriented cardiology program in the country, and we certainly can compete at the highest possible level against the best scientists anywhere in the world."

One Bolli colleague, Eduardo Marbán, M.D., Ph.D., isn't surprised by the cardiologist's remarkable progress here.

"Dr. Bolli has always been an extremely bright, industrious scientist of the highest order," says Marbán, chief of cardiology at Johns Hopkins University and an occasional collaborator of Bolli's. "His visionary research is truly groundbreaking."

Cardioprotection

A native of Perugia, Italy, Bolli spent a good portion of his childhood engrossed in study. Even his playtime activities -- chemistry sets and reagents -- foreshadowed a life of science.

He says he had always dreamed of being a doctor, in part because physicians "have the ability to give people the most precious gift, which is life itself."

By his senior year in high school, Bolli's future was set. He enrolled in medical school at the University of Perugia and began classes in the fall of 1970, quickly developing an interest in cardiology.

"I was just stunned by the marvelous logic of the cardiovascular system -- how you can understand the functioning of the heart in hydraulic terms, and how everything is so well designed," Bolli says. "The miraculous perfection of the cardiovascular system simply enthralled me."

Bolli graduated magna cum laude in 1976 and, following a brief period of private practice in Italy, accepted a fellowship in cardiovascular research at the National Institutes of Health two years later.

It was there, under the direction of Stephen Epstein, M.D., chief of the cardiology branch of the National Heart, Lung and Blood Institute, that Bolli received his introduction to the concepts of "cardioprotection" -- phenomena like preconditioning that reduce the damage caused to heart muscle during ischemia.

Bolli and other researchers working for Epstein began testing various anti-inflammatory agents like naproxen and ibuprofen to determine if they offered any protection to heart tissue during ischemia and reperfusion -- the final phase of some ischemic events, when blood flow is restored to ischemic heart tissue.

While this restoration of blood flow is necessary to prevent the death of heart muscle, it also can have negative consequences.

For example, reperfused heart tissue sometimes beats abnormally. These arrhythmias can evolve into ventricular tachycardia -- extremely fast heart rates -- and eventually degenerate into ventricular fibrillation, when the heart's muscle activity becomes so chaotic that it can no longer pump blood.

The NIH research failed to find any protective benefit to the anti-inflammatory agents, but it did pique Bolli's inherent curiosity: What, he wanted to know, was the root cause of these arrhythmias?

Working solo

Bolli left the NIH in 1980 to accept a 2 1/2-year clinical fellowship in cardiology at Baylor College of Medicine, and it was there, between clinical rotations, that Bolli began to pursue this question independently.

"Independently" is an apt description. Bolli was the only person -- faculty or fellow -- doing basic cardiology research at Baylor in the early '80s.

"The other fellows thought I was nuts spending my time doing basic research when everyone else was doing clinical training," Bolli recalls, chuckling at the memory. "But that was a very productive time for me. I was able to get seven papers published during my seven months in the lab."

Bolli's most significant finding was his discovery of evidence which disproved a then-common assumption: that so-called alpha-adrenergic receptors played a significant role in reperfusion arrhythmias.

"Whenever we get scared or excited, our bodies release a hormone called norepinephrine," which is closely related to adrenaline, Bolli explains.

"One of the things norepinephrine does is combine with these alpha receptors, which causes a variety of actions. There was some evidence at the time that one of those actions would precipitate reperfusion arrythmias."

But Bolli was able to show in experimental studies at Baylor that reperfusion arrhythimas continued, even if he blocked the ability of alpha receptors to bind with norepinephrine. Thus, Bolli concluded that alpha-adrenergic receptors played no role in causing reperfusion arrhythimas.

"That was quite controversial at the time," Bolli says, "because everybody thought they were involved."

When Bolli's fellowship ended, he carefully considered his career options and decided to stay put, beginning an 11-year tenure at Baylor that would culminate with his being named a full professor of medicine in 1994.

But Bolli's impetus for remaining was not the opportunity it provided to teach. Instead, he was enticed by the chance to build a first-rate basic research program as director of the Experimental Research Laboratory -- the very same, solitary lab in which he worked as a fellow, cranking out one study after another.

Bolli's resources at Baylor were modest -- he had limited physical space and, eventually, a staff of just two technicians and two or three fellows. But the lab's scientific contributions were world-class.

"I essentially created that lab from scratch and developed it to the point where, after a few years, we were one of the leading labs in myocardial ischemia in the world," Bolli says.

Perhaps their most significant discovery was the identification of the cause of myocardial stunning, a phenomenon in which reperfused hearts -- that is, ischemic hearts in which blood flow has been re-established -- fail to beat at all.

"When you reperfuse the heart, two things can happen: arrhythmias, in which the muscle contraction is rapid and may become chaotic; and stunning, when the heart does not contract at all even though the tissue is still alive because you were able to reperfuse it very early and prevent an infarction, or heart attack.

"It is what we call 'stunned' myocardium, and it may take days for the function to come back to the tissue after you reestablish blood flow."

Stunning is an important phenomenon because it can be a source of morbidity and mortality. For example, in patients who undergo cardiac surgery, heart tissue becomes ischemic when a surgeon clamps the aorta to stop the heart. Then, after surgery, the surgeon will declamp the aorta to re-establish normal blood flow.

"Now you have reperfusion, and, in some cases, a severely stunned heart that is unable to support circulation," Bolli says. "So the patient will need a lot of drugs -- inotropic agents -- to keep the heart beating and also maybe balloon pumps to assist the heart. Sometimes they can even die from shock."

Stunning also affects patients who suffer heart attacks and then undergo procedures like balloon angioplasty to remove any blockages from their coronary arteries.

Many hypotheses had been advanced to explain the causes of myocardial stunning, Bolli says, and none of them turned out to be correct. Bolli, meanwhile, had been thinking about free radicals, unstable molecules with a well-known ability to cause tissue damage.

"Our idea was that when you reperfuse the heart, you re-introduce oxygen," Bolli says. "And this tissue, which was ischemic, suddenly is confronted with an abundance of oxygen. It is unable to handle this, and so oxygen is converted into reactive oxygen species, or oxygen free radicals. These are highly reactive molecules that can cause all sorts of damage to the heart -- maybe even stunning."

It turned out that Bolli's initial hunch was correct, and he spent the next 10 years elucidating the processes by which stunning takes place, producing what is now known as the Oxyradical Hypothesis of Myocardial Stunning. His work has been reproduced by multiple labs and is now regarded as a proven hypothesis -- something in which Bolli takes great pride.

"In research, a lot of people put forth a lot of hypotheses, and it's very rare for one to be confirmed by several labs and finally become accepted," he notes. "Only a tiny fraction of initial hypotheses survive this test over time."

With another major discovery under his belt, Bolli began looking for new challenges -- and he found one in the mysteries surrounding cardiac preconditioning.

Discovered by a team of researchers at Duke University in 1986, preconditioning initially was described as a phenomenon that gave briefly ischemic hearts -- those whose blood flow was stopped for less than 10 minutes -- a window of protection from damage in the event of a heart attack. Because this window lasted only two hours, it wasn't really something researchers could exploit for therapeutic benefit.

Then, in 1993, it was discovered that a second window of protection -- called late preconditioning -- occurs 24 hours after the end of ischemia and lasts for up to four days. Bolli was especially intrigued by the discovery because it signaled the involvement of genetic reprogramming at the molecular level.

"It's hard to imagine something that lasts for four days that does not involve some kind of genetic reprogramming of the heart," Bolli notes.

To pursue this line of research, Bolli would need to incorporate molecular biology into his regimen of biochemistry and physiology -- a task that simply
wasn't possible given his limited resources at Baylor.

In the meantime, UofL was looking to recruit a new chief for its cardiology division, and it wanted someone with a strong research background to develop a leading program here.

Building from scratch

"We conducted a national search, and Dr. Bolli was by far the best candidate," recalls Redinger, chairman of the university's Department of Medicine. "His academic credentials were impeccable, and he had this incredible intensity about him. You could tell he was the kind of person who would work hard to build a top-notch program."

Bolli was offered $500,000 in start-up funds to supplement the $2.3 million Jewish Hospital grant, a newly renovated laboratory in the Medical-Dental Research Building and the promise of more lab space in the future. He also was told he could develop his research program any way he saw fit.

The offer gave Bolli just about everything he was seeking, and so he signed on in 1994 to begin building yet another research program from the ground up. He found much the same state of affairs in Louisville as he did in Houston: no basic cardiology research.

But the absence of any existing program gave Bolli a blank sheet of paper on which to draft a multi-disciplinary program that would integrate biochemistry, physiology and molecular biology.

Bolli wasted little time executing his plan, and a rapidly growing staff of scientists quickly began zeroing in on the mechanisms of late preconditioning.

"We wanted to understand how the heart becomes preconditioned, because if you understand the mechanism, then you can exploit it, therapeutically, to precondition patients," Bolli says. "That has always been my goal: to exploit our knowledge of the mechanism in order to come up with a treatment that would precondition the heart of patients at risk for heart attacks."

In the process, Bolli's team made several key discoveries. Among these, that late preconditioning protects against stunning as well as tissue death.

The team also identified the role of nitric oxide in preconditioning -- a landmark discovery that has opened the door for treatments that may substantially reduce the damage caused by heart attacks, which strike 1.2 million people annually in the United States.

"Nitric oxide, or NO, is a hugely important biological mediator," Bolli says, explaining how his team decided to focus its research. "It is involved in essentially every biological process. We also knew that brief ischemia, which preconditions, causes the release of nitric oxide."

Armed with this knowledge, Bolli hypothesized that nitric oxide acted as a trigger, setting in motion a cascade of molecular events that eventually results in late preconditioning.

Remarkably, once again, Bolli's hypothesis proved correct. When researchers shut down the production of nitric oxide in experimental animals, their hearts could no longer precondition following ischemia.

Taking this concept to the next level, Bolli and his collaborators then demonstrated that heart tissue becomes preconditioned simply by exposure to nitric oxide. In other words, an ischemic event wasn't necessary to produce preconditioning. Nitric oxide alone would do the trick.

"We were the first to show a new property of nitrates, which is the ability they have to put the heart in a preconditioned state -- a defensive or protected state that makes the heart resistant to ischemia," Bolli says.

The UofL team then extended its research to humans, giving intravenous nitroglycerin to patients who were scheduled to undergo balloon angioplasty the next day.

"As you know, when you inflate the balloon in a coronary artery to remove a blockage, you stop the blood flow to heart tissue," Bolli explains. "So that region of the heart is ischemic. The patient experiences chest pain and you see big changes on the electrocardiogram, just like at the beginning of a heart attack. You also see that this part of the heart is not moving very well -- we call that wall motion abnormality."

Amazingly, the patients in Bolli's study experienced a marked reduction in all these symptoms -- in some cases by up to 70 percent -- effectively demonstrating that the protective benefits of late preconditioning can be induced with nitrates.

"This, therapeutically, could have significant implications because we can now use nitrates to precondition patients at risk for heart attacks or prior to cardiac surgery," Bolli says.

Going to mediation

Now that Bolli had established the role of nitric oxide in triggering preconditioning, he turned his thoughts to the mechanisms that actually protect ischemic tissue.

While nitric oxide starts the preconditioning process, actual protection doesn't begin until 12 to 24 hours later, when some kind of mediator appears at the molecular level. This mediator, which now has been identified as an enzyme, is the final product of a complex cascade of events, and it is this enzyme that actually conveys protection to the heart.

Because nitric oxide played such a pivotal role in triggering the late preconditioning process, Bolli hypothesized in 1996 that it also was somehow involved in the mediation of protection. Again, laboratory analysis proved the hypothesis correct: an inducible form of the NO-producing enzyme, called nitric oxide synthase, was directly responsible for mediating late preconditioning.

"Normally, this inducible form of nitric oxide synthase -- iNOS -- is not present in the heart, Bolli says. "But when the heart is stressed, even by rigorous exercise, it starts making iNOS to protect itself and make itself very resistant to infarction."

Bolli calls the discovery a "paradigm shift" because many researchers had regarded iNOS as "the bad guy."

"People thought that iNOS was responsible for things like inflammation, organ rejection, sepsis and so on. All of that may be true, but in those cases, iNOS is present at grotesquely elevated levels. What we found is that iNOS is upregulated very mildly after preconditioning, so at low levels it has a protective function."

The next logical step for Bolli's molecular biologists was to find a way to induce continuous iNOS production in the heart, so that cardioprotection would be non-stop. Such an approach meant gene therapy, and the researchers set about modifying a harmless virus so that it could transfer the iNOS-producing gene into mice. The genetic sleight-of-hand worked flawlessly.

"After we transferred the iNOS gene into the mice, the gene began making the iNOS enzyme, which then made nitric oxide," Bolli says. "So now, their hearts were making nitric oxide all the time. And sure enough, these hearts were very resistant to infarction. Basically, they looked like a preconditioned hearts."

Bolli hopes some day to test his gene therapy in human patients as a superior alternative to any drug treatments that might be developed to promote preconditioning.

"If we can find a gene therapy to protect the patient, it's going to be much more effective than drugs, because drugs usually work only if you give them before the heart attack," Bolli notes.

"We can't do that in patients, because you never know when the heart attack will come. It may come tonight at 9 o'clock, or it may come five years from now. So a patient could take drugs every day for 20 years, waiting for the heart attack to come. Or a patient could forget to take his drugs. That's a little cumbersome as a life-long therapy to minimize heart attacks.

"On the other hand, if we can put a gene in the heart that's there all the time, that means that heart will be protected for life.

"If it works, it would be really dramatic," adds Bolli, who notes that coronary artery disease is the leading cause of death in every Western country.

Looking forward

Bolli's research into preconditioning, which has consumed nearly his entire tenure at UofL, has been extraordinarily productive. His iNOS model is now another proven hypothesis, and Bolli's UofL team is regarded around the world as the definitive authority on myocardial ischemia. Moreover, several researchers have begun applying Bolli's iNOS discoveries to other tissues, exploring the role of iNOS in preconditioning such organs as the liver and kidney.

He's also won enough prestigious awards to last a lifetime, including the American Heart Association's 2001 Basic Research Prize. Past recipients include two Nobel Laureates and seven members of the National Academy of Sciences.

But Bolli, whom Redinger describes as "extremely hardworking," is not the kind of man to spend much time looking back.

"Bolli's past accomplishments have been groundbreaking, and he's got many fields coming to harvest," Redinger says. "I think that tells you the dimension of the man. But he always has so much more to see and do. There is no burn-out for Roberto Bolli."

Eric Olson, Ph.D., agrees.

"Dr. Bolli has chosen to extend his deep knowledge of cardiology and human disease to a molecular level, and that's a difficult thing to do," says Olson, chairman of molecular biology at the University of Texas Southwestern Medical Center and a member of the National Academy of Sciences.

"It requires a special type of person who doesn't want to sit back and rest on his accomplishments, but instead wants to push on the envelope. And that is exactly what he's doing."

First on Bolli's agenda is the development of a cross-disciplinary Institute of Molecular Cardiology, which UofL's Board of Trustees just approved.

"At the moment, the institute is made up essentially of the people in the Division of Cardiology, but I'm hoping that other departments like physiology, biochemistry and surgery can come join us."

And, of course, Bolli hopes to recruit even more scientists for his ever-expanding cardiology research program. Next up: cardiac regeneration, which holds the promise of regrowing heart tissue damaged by ischemia and infarction.

"It's very clear now that you can take adult stem cells from bone marrow and make them differentiate into other organs -- liver, brain or heart," Bolli explains.

"We've already begun working on that here, but we've not yet published any of our findings."

If past success is any indication-and with Roberto Bolli, why wouldn't it be? -- the human heart will continue telling him its secrets for many years to come.