Self Defense for the Heart
Roberto Bolli and his world-class team help the heart protect itself
Story by Dale Greer, photos by John Lair
Like all cardiologists, Dr. Roberto Bolli, chief of the Division of Cardiology in the University of Louisville School of Medicine, has made a career out of listening to the human heart.
But Bolli, who is also director of U of L's Institute of Molecular Cardiology and the Jewish Hospital Heart and Lung Institute Distinguished Chair in Cardiology, hears more than rhythmic beats and pulsing blood. He also hears answers that explain some of the heart's most fundamental mysteries.
 Dr. Roberto Bolli |
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 U of L 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," says Bolli, whose Institute of Molecular Cardiology is one of the most respected cardiology research teams in the world. "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."
Record Grant
Bolli and his fellow researchers recently were awarded as $11.7 million grant from the National Heart, Lung and Blood Institute--part of the National Institutes of Health (NIH)--to build on their research into ways the heart can protect itself from tissue damage at the molecular and cellular level.
The award is the largest nationally competitive NIH grant ever given to U of L.
"The research being done here at U of L by Dr. Bolli and the team at the Institute of Molecular Cardiology has tremendous potential to help millions of Americans at risk for heart disease," says U of L President James Ramsey. "This kind of groundbreaking research is important for the university, the community and ultimately the world."
In awarding the grant, NIH reviewers used highly unusual language, calling the proposal "a paradigm of what a program project grant should be."
The program has four major components. Bolli is leading research into ways to build on late preconditioning to provide sustained protection against tissue damage during a heart attack. Using genetic therapy, he hopes to develop ways to make the heart permanently resistant to tissue damage in patients at high risk for heart attack.
Another component of the research examines the role of three key genes in heart failure after a heart attack to determine whether they play a part in protecting against tissue damage. Led by biophysicist Dr. Sumanth Prabhu, a nationally recognized expert in heart failure, researchers are examining whether these protective genes work to sustain or improve heart function in the long-term.
Dr. Yu-Ting Zuan, a specialist in cellular biology, leads a team examining the role of a particular signaling pathway in heart attacks and the molecular and genetic factors that determine how the heart may protect itself against tissue damage in the event of a heart attack.
The final component of the research program examines the role of mitochondria in cell death and how it can be prevented. Because mitochondria act as engines that provide fuel for the cell's biological processes, dysfunctional mitochondria lead to cell death.
Building on his NIH-funded research in environmental cardiology, Aruni Bhatnagar, M.D., and his research team are examining how several biochemical mechanisms may influence mitochondrial function.
Astonishing Productivity
Recruited to U of L 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.
Less than 12 years later, more than 70 faculty researchers, research associates, fellows and support staff work in Bolli laboratories. Collectively, they have brought in more than $50 million in NIH grants to the university and currently hold 16 NIH grants, totaling approximately $10 million a year.
"Dr. Bolli's progress since coming to U of L has been quite incredible," says Dr. Richard Redinger, 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, Dr. Eduardo Marban, 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 Marban, chief of cardiology at John Hopkins University and an occasional collaborator of Bolli's. "His visionary research is truly groundbreaking."
Cardioprotection
A native of Perugia, Italy, Bolli spent his childhood engrossed in study. Even his playtime activities--chemistry sets and reagents--foreshadowed a life of science.
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.
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"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."
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, which, again, means brief loss of blood flow.
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.
The NIH research failed to find any protective benefit of the anti-inflammatory agents, but it did pique Bolli's inherent curiosity. What, he wondered, is the root cause of cell death during ischemia and how can it be prevented?
Building from Scratch
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 in animals 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."
When Bolli's fellowship ended, he carefully considered his career options and decided to stay put, beginning an 11-year tenure at Baylor. His resources were modest--he had limited physical space (500 square feet) 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 irregular 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.
 Dr. Bolli and researchers. |
"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 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 clarifying 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, but it's very rare for a hypothesis to be confirmed by several labs and finally become accepted," he notes. "Only a tiny fraction of initial hypotheses survive the test over time."
With that 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 up to four days. Bolli was especially intrigued by the discovery because it signaled the involvement of genetic reprogramming at the molecular level.
Bolli and his scientific team have begun exploring gene therapy as a way to convey around-the-clock protection for the heart.
"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, U of L 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.
On to Louisville
"We conducted a national search, and Dr. Bolli was by far the best candidate," recalls Redinger, U of L's medicine department chair. "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 a similar 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 on which to draft a 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 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 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."
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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 U of L team then extended its research to humans, giving intravenous nitroglycerin to patients who were scheduled to undergo balloon angioplasty the next day.
"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 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.
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.
Bolli's research into nitric oxide represented a "paradigm shift," he says.
"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."
This discovery was important because many researchers had regarded iNOS as "the bad guy."
"People thought that iNOS was responsible for things like inflammation, organ rejection, septic shock 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 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 p.m. 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 lifelong therapy.
"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. It would be like vaccinating the heart against heart attacks."
If it works, it would be quite dramatic, adds Bolli, noting 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 U of L, has been extraordinarily productive. His iNOS model is now another proven hypothesis, and Bolli's U of L 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 enzyme's role in preconditioning organs such as the liver and kidney.
He's also won many prestigious awards, including a $2.5 million MERIT Award from the NIH (the first in Kentucky), the 2004 Lucian Award ($40,000) from McGill University, the Research Achievement Award ($30,000) from the International Society for Heart Research, the 2004 Ken Bowman Award from the Institute of Cardiovascular Sciences at the University of Manitoba and the 2005 Howard Morgan Award for Distinguished Achievements in Cardiovascular Research from the International Academy of Cardiovascular Sciences. He was also the recipient of 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, a member of U of L and Jewish Hospital's Cardiovascular Innovation Institute, is not the kind of man to spend much time looking back.
"Dr. Bolli's past accomplishments have been groundbreaking, and he's got many fields coming to harvest," Redinger says. "That tells you the dimension of the man. He always has so much more to see and do. There is no burn-out for Roberto Bolli."
Dr. Eric Olson, chairman of molecular biology at the University of Texas Southwestern Medical Center and a member of the National Academy of Sciences, 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," Olson says. "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."
