USF Health preclinical study suggests boosting cardiac SIRT1/SIRT3 levels in older heart attack patients may help protect against ischemia-reperfusion injury

Tampa, FL (Aug. 20, 2021) — Sirtuins are a family of anti-aging proteins that help regulate cellular lifespan, metabolism, and resistance to stress. The potential protective effect of these sirtuin enzymes in age-related diseases, including cardiovascular diseases, remains an area of intense investigation.

Principal investigator Ji Li, PhD, is a professor of surgery and member of the USF Health Heart Institute at the USF Health Morsani College of Medicine. | Photo by Allison Long, USF Health Communications

Now, a new preclinical study led by University of South Florida Health (USF Health) researchers has determined that sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3) levels decline in aging hearts, disrupting the ability of cardiac muscle cells (cardiomyocytes) to contract in response to ischemia-reperfusion injury (also known as reperfusion injury). Furthermore, age-related SIRT1 and SIRT3 deficiency can impair cardiac function by altering mitochondrial dynamics, which play an important role in metabolic health and inflammatory response, the researchers report.

The findings were published online July 3 in Aging Cell.

“We discovered that age-related changes in mitochondrial dynamics are caused by SIRT1/SIRT3 deficiency, specifically in the cardiomyocytes,” said principal investigator Ji Li, PhD, professor of surgery in the USF Health Morsani College of Medicine. “You need a strong presence of SIRT1 and SIRT3 to keep mitochondrial dynamics healthy in the heart. Otherwise, the heart’s pumping function becomes weak.”

Diastolic functions assessment of a mouse heart imaged with ultrasound echocardiography | Photo by Allison Long, USF Health Communications

Mitochondria produce the energy needed to drive nearly all processes in living cells. Cardiac muscle cells contain more mitochondria than any other cells, because the heart needs large amounts of energy to constantly pump blood throughout the body. Stabile mitochondrial dynamics maintain a healthy balance between the constant division (fission) and merging (fusion) of mitochondria and help ensure the quality of these specialized structures known as the “powerhouse” of the cell.

Reperfusion, a common treatment following acute heart attack, restores blood flow (and thus oxygen) to a region of the heart damaged by a blood clot blocking the coronary artery. Paradoxically, in some patients this necessary revascularization procedure triggers further injury to heart muscle tissue surrounding the initial heart attack site. No effective therapies currently exist to prevent reperfusion injury.

Research associate Di Ren, PhD (left) works with the heart perfusing system in the Department of Surgery physiology laboratory as USF undergraduate student Julia Fedorova watches. | Photo by Allison Long, USF Health Communications

To help analyze the response of cardiac mitochondria to ischemia-reperfusion stress, the USF Health researchers deleted SIRT1 or SIRT3 in cardiac muscle cells of mouse hearts, and examined the mitochondrial response to ischemic stress by restricted blood flow. They found that the mitochondria in mouse hearts lacking cardiomyocyte SIRT3 were more vulnerable to reperfusion stress than the mouse hearts with SIRT3 intact. The cardiac mitochondrial dynamics (including shape, size, and structure of mitochondria) in these knockout mice physiologically resembled that of aged wildtype (normal) mice retaining cardiac SIRT3.

Furthermore, the young mice with SIRT1 or SIRT3 removed had measurably weaker cardiomyocyte contractions and exhibited aging-like heart dysfunction when ischemia-reperfusion stress was introduced. In essence, without SIRT1/SIRT3 the hearts of these otherwise healthy young mice looked and behaved like old hearts.

“We started this study trying to understand why older people have higher incidences of heart attacks than younger people, and why they die more often even if they receive maximum treatment. Younger people are much more likely to recover from heart attacks and less likely to suffer from ischemia-reperfusion injury,” said Dr. Li, a member of the USF Health Heart Institute. “Our research suggests that one reason could be that both SIRT1 and SIRT3 are downregulated with aging. Younger people have higher levels of these proteins needed to make mitochondrial dynamics healthier.”

Dr. Li’s research team (pictured here) focuses on understanding the molecular mechanisms of coronary artery disease, the most common cause of age-related heart disease. | Photo by Allison Long, USF Health Communications

The study also suggests that, before surgically opening blocked coronary arteries to restore blood flow in older patients, administering a treatment to “rescue” (improve) their diminished SIRT1/ SIRT3 levels may increase tolerance to cardiac muscle reperfusion stress, thereby reducing heart attack complications and deaths, Dr. Li said. Such a cardioprotective treatment might apply a genetic approach to increase SIRT1/SIRT3 production, or an agonist (drug) to activate SIRT1/ SIRT3, he added.

If their mouse model findings translate to human hearts, Dr. Li’s group wants to work with companies interested in developing and testing SIRT1/SIRT3 activators to mitigate heart attack-related reperfusion injury.

“Our ultimate goal is to identify ideal targets for the treatment of heart attack, especially in older patients,” said Dr. Li, whose research is supported by grants from the National Heart, Lung, and Blood Institute, the National Institute on Aging, and the National Institute of General Medical Sciences.

USF Health’s Hua Pan, PhD, MBA, won first place for her moderated poster presentation titled “Anti-thrombin nanoparticles limit ischemia-reperfusion injury and no-reflow in myocardial infarction” on Sept. 2 at the annual European Society of Cardiology Congress in Paris. The Congress draws some 35,000 scientists from more than 100 countries over five days.

As part of the highly competitive presentation moderated by two chairpersons, Dr. Pan, a biomedical engineer and assistant professor of cardiovascular sciences at the USF Health Morsani College of Medicine, was required to present a 5-minute talk on the research poster in front of a group of audiences.  Her prize – free registration at next year’s Congress – was awarded for the poster session covering innovations in cardiac magnetic resonance Imaging.

USF Health Heart Institute’s Hua Pan, PhD, MBA

Dr. Pan and colleagues at the USF Health Heart Institute developed antithrombosis perfluorocarbon nanoparticles, which act as “smart Band-Aids” to find and stay only at the injured area in the heart to deliver treatment. These nanoparticles were evaluated in a rat model for heart attack and their treatment effect was visualized by MRI.  The study showed that the treatment limited further vascular damage from ischemia-reperfusion injury (IRI), a common complication following acute treatment of heart attacks.

Ironically, when blood flow is restored to the region of the heart injured by a blood clot that blocks the coronary artery, this blood reflow can expand injury to tissue surrounding the initial heart attack and lead to congestive heart failure. In explaining IRI, Dr. Pan compares an obstructed blood vessel to a clogged water pipe, already weakened by prior damage, which may leak once the pipe is unclogged and water (blood) flows freely again.

“The antithrombin nanoparticles we developed acted locally to preserve the blood vessels (pipes) in the heart, so that the restored blood reached areas where the treatment was needed, without leaking into areas where it could cause more harm,” Dr. Pan said.  The precision nanoparticle treatment calmed unnecessary inflammation as well as inhibiting the thrombin from forming more blood clots leading to blood vessel obstructions in the heart, she added. It did so without causing the bleeding risk associated with existing anticoagulant drugs.

Because the antithrombin nanoparticles “do not prolong bleeding times or coagulation parameters beyond approximately 30 to 60 minutes after injection, yet maintain prolonged surveillance against activated thrombin locally in the injured area, they represent a potentially useful therapy for cardiac IRI,” Dr. Pan and her poster co-authors concluded.

USF Health researcher Mack Wu, MD, studies what happens when the microvascular endothelial barrier controlling blood-tissue exchange is compromised during ischemia-reperfusion injury, a condition that can lead to irreversible tissue damage. He also investigates the molecular control of gut permeability, also known as “leaky gut,” in tissue injuries caused by trauma and severe burns.

His group’s work has broad implications for a variety of conditions including stroke, heart attack, thrombosis, sepsis, trauma or other inflammatory diseases associated with microvascular injury.

Mack Wu, MD, is a professor of surgery and molecular medicine at USF Health Morsani College of Medicine and a research physiologist at James A. Haley Veterans’ Hospital. On the monitor next to him are images of microvessels in the small intestine injected with fluorescent dye.

The closely connected endothelial cells lining the interior of blood vessel walls play a critical role in limiting the how much fluid, proteins and small molecules cross the wall of the tiny blood vessels, or microvessels. However when this protective endothelial barrier is damaged, excessive amounts of blood fluid, proteins and molecules leak outside the microvessels into nearby body tissue – a process known as microvascular hyperpermeability. If this breech of endothelial barrier is associated with a body-wide inflammatory response, it can trigger a chain of events leading to edema (swelling), shock from severe blood and fluid loss (hypovolemic shock), and ultimately multiple organ failure.

Pinpointing potential solutions for ischemia-reperfusion injury

Previous research by Dr. Wu’s laboratory and other groups discovered that ischemia-reperfusion injury can cause endothelial barrier damage leading to vascular hyperpermeability, or abnormally leaky blood vessels.

Ischemia-reperfusion injury is typically associated with conditions like organ transplantation, stroke, heart attack, or cardiopulmonary bypass where blood supply to a vital organ is temporarily cut off (ischemia), resulting in oxygen deprivation. For instance, a period of ischemia occurs while a donor organ is transported to a recipient in the operating room, or when a clot interrupts blood circulation to the brain. When blood supply is re-established with new blood returned to the previously oxygen-deprived area (reperfusion), tissue injury can worsen because the reperfusion itself causes inflammation and oxidative damage rather than restoring normal function. It its severest form, ischemia-reperfusion injury can result in multiple organ failure, or even death.

“I believe endothelial barrier injury is one of the key elements of ischemia-reperfusion injury, so my group is trying to find out which molecule is ultimately responsible for the endothelial barrier damage,” said Dr. Wu, a professor of surgery and molecular medicine at USF Health Morsani College of Medicine and a research physiologist at James A. Haley Veterans’ Hospital.

Dr. Wu with some members of his laboratory team. From left, Rebecca Eitnier, research assistant; Shimin Zhang, Department of Molecular Medicine graduate student; Ricci Haines, research associate; and Fang Wang, research assistant.

With the support of a $1.49-million, four-year R01 grant from the National Heart, Lung and Blood Institute, Dr. Wu’s team is zeroing in on a molecule known as focal adhesion kinase, or FAK, an enzyme that may play a role in weakening the microvascular endothelial barrier during ischemia-reperfusion injury.   Using cell models and a newly developed mouse model in which the endothelial-specific gene for FAK is knocked out, the USF researchers are testing whether selectively inhibiting FAK activity can rescue the endothelial barrier from such injury.

The work is critical because no FDA-approved treatment exists to prevent tissue damage following reperfusion. Identifying a new mechanism for the injury would provide potential targets for drug development, Dr. Wu said. So for instance, he said, after an initial stroke a new intravenously administered drug selectively targeting endothelial cells in the brain’s microvessels might stop further harmful swelling of the brain caused by stroke.

Defining molecular control of “leaky gut” in severe burn trauma

A second grant from the U.S. Department of Veterans Affairs funds Dr. Wu’s studies to define the underlying molecular mechanisms of leaky guts induced by traumatic injury associated with thermal (fire, scald or chemical) burns.  Massive burn trauma is a significant cause of injury and death in American soldiers. With a $960,000 VA Merit Award, Dr. Wu focuses on how intestinal epithelial barrier damage happens during severe burns, with the aim of developing targeted therapies to prevent posttraumatic complications.  In particular, he is working to determine the pathways by which the protein palmitoylation in gut epithelial cells are stimulated by burn injury.

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Epithelial cells line the interior of the small intestines, and after severe burn injury, this protective epithelial barrier commonly breaks down, causing bacteria and toxins to flow from the intestine into the circulating blood.  The result of this abnormal epithelial permeability, or “leaky gut,” can be deadly if sepsis ensues – a bacterial infection in the bloodstream sets up a body-wide inflammatory response leading to multiple organ failure.

While the role gut barrier failure plays in posttraumatic complications is well recognized, its cellular and molecular mechanisms remain poorly understood.  Currently, pushing IV fluids to help prevent hypovolemic shock and administering antibiotics and anti-inflammatories are the only therapies, mostly supportive, Dr. Wu said.

“More effective early therapeutic interventions to prevent leaky gut and systemic inflammatory response will be key to preventing sepsis,” he added, whether in soldiers with trauma or VA patients with inflammatory bowel diseases.

From industry to academia

Dr. Wu joined USF Health and the Haley VA Hospital in 2011.  He came from Sacramento, Calif, where he was an associate professor of surgery at the University of California at Davis School of Medicine and a research physiologist at Sacramento VA Medical Center.   Previously, Dr. Wu was a faculty member in the Department of Medical Physiology at Texas A&M University Health Science Center. He screened pharmaceutical compounds as a toxicologist in a biotechnology laboratory before joining Texas A&M, moving from industry to academia in 1995.

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Dr. Wu received his MD degree from Second Military Hospital in Shanghai, China, and conducted an internship at Shanghai Second Hospital.

One of his earliest and most highly cited studies, published in the American Journal of Physiology (1996), was first to report nitric oxide’s role in contributing to cardiovascular injury. The study showed an increase in nitric oxide induces vascular endothelial growth factor (VEGF) to promote leakage in tiny coronary veins.

Another more recent study in Shock (2012) provided direct evidence that thermal burn injury causes intestinal barrier disruption and inflammation characterized by intestinal mucosal permeability (leakage) and an infiltration of immune system cells known as neutrophils.

Something you may not know about Dr. Wu:

He loves deep-sea fishing. Dr. Wu has fished for sharks off the Golf coast of Texas, rockfish off the Pacific coast of California, and grouper off the west coast of Florida.

Dr. Wu is a member of the USF Health Heart Institute. His team’s work has broad implications for a variety of conditions including stroke, heart attack, thrombosis, sepsis, trauma or other inflammatory diseases associated with microvascular injury.

Photos by Eric Younghans, USF Health Communications and Marketing