02/15 - Mighty Mitochondria - MSMR: What A Year!

Did you know that the human body includes over 600 muscles? Muscles are involved in every movement that we make, from breathing to running a marathon. In order to function properly, muscles, like all cells, need energy, which is produced by mitochondria inside the cells. As with all biological processes, though, the production of energy is quite complicated. One missing element can be disastrous.

In this month's story, the missing element is the protein, Tafazzin, or TAZ for short. TAZ is an enzyme that is required in the production of the lipid called cardiolipin, which is an essential element in energy production in mitochondria. Mutation of the gene encoding TAZ causes a disease known as Barth syndrome, a rare genetic disorder. Barth syndrome is characterized by delayed growth and muscle weakness, which results in varying degrees of physical disability. Children with Barth syndrome are often below average for height and weight compared to their peers, although their growth phase can last longer than other children so they often attain normal size. Children with Barth syndrome may tire easily when they participate in aerobic exercise. These children are not usually diagnosed, however, until they begin having problems with the most important muscle in the body—the heart.

Dr. William Pu of Harvard Medical School, a pediatric cardiologist by training, first met children with Barth syndrome while training for his specialty in cardiology nearly two decades ago. "I was deeply affected by a Barth syndrome patient, and others with poorly characterized genetic disorders," recalls Dr. Pu. "I wanted to figure out what was happening inside their bodies, to help them lead normal lives." Dr. Pu still sees patients, but he now spends most of his time in the laboratory trying to do just that.

Research into heart muscle diseases has been difficult because it is rare to obtain muscle samples from patients, and these samples only last for a few days.

Research into heart muscle diseases has been difficult because it is rare to obtain muscle samples from patients, and these samples only last for a few days. Dr. Pu's research changed dramatically when researchers led by Dr. Shinya Yamanaka in Kyoto, Japan, building on the work of Dr. John Gurdon in London, England, showed that adult cells could be re-programmed to become stem cells and then programmed again to become any other type of cell in the body. Dr. Yamanaka and Dr. Gurdon were jointly awarded the Nobel Prize in 2012 for their discoveries, which resulted in a technology known as induced pluripotent stem cells, or iPSCs . Dr. Pu wondered whether he could use iPSC technology to overcome the limited availability of heart muscle samples and create a model of Barth syndrome in a dish. "There was only way to find out," Dr. Pu commented. In his subsequent research, Dr. Pu successfully reprogrammed the cells of patients with Barth syndrome, and also made some remarkable discoveries about Barth syndrome, our muscle cells, and, by extension, our overall health.

To begin, Dr. Pu obtained skin cells from two unrelated patients with Barth syndrome. Using the techniques pioneered by Drs. Yamanaka and Gurdon, he and his team then converted these skin cells into stem cells. In the right environment, these stem cells could become any other type of cell in the body. The correct environment for the production of heart cells had already been established by previous research.

Building on this work, Dr. Pu treated the skin-turned-stem cells with three growth factors to promote their transformation into heart cells. The researchers knew they had succeeded when the skin-turned-stem cells-turned heart cells started beating on the petri dish.

Dr. Pu used genetic analysis and mass spectroscopy to confirm that the heart muscle cells generated from the patient-derived iPSCs had the TAZ mutation and the abnormal cardiolipin production associated with the disease. They also observed that the mitochondria of the re-programmed heart cells were smaller than the mitochondria of healthy cells. Mitochondria are known as the power-houses of the cell. They are the site of energy production, a process known as oxidative phosphorylation, which turns sugar into energy in the form of adenosine triphosphate, or ATP.

Mitochondria are known as the power-houses of the cell.

The observed changes in mitochondrial shape led Dr. Pu and his team to further investigate mitochondrial function in the reprogrammed heart cells. When the researchers measured the rate of oxygen consumption in Barth syndrome mitochondria, they found it was elevated. Further chemical analysis of ATP production in these cells revealed a very inefficient process. The mitochondria consumed more oxygen to generate less ATP. Some of the consumed oxygen was converted abnormally into reactive intermediates known as reactive oxygen species , or ROS. The ROS damaged other proteins necessary for muscle function, leading to the muscle problems experienced by patients with Barth syndrome.

"If you put these findings in the context of what we know right now about cell function and disease," commented Dr. Pu, "these results are quite remarkable." That's because, although researchers have identified other mitochondrial diseases, these diseases have all been thought to be the result of a lack or reduction of ATP production. This research shows that in Barth syndrome, and possibly some other mitochondrial diseases, the mitochondria are producing the same amount of ATP. But they are also producing excessive amounts of toxic ROS—extra, highly reactive, forms of oxygen — that are the cause of muscle weakness in Barth cells. ROS makes the cells unable to contract efficiently. These findings, then, open up a whole new field of research into mitochondrial diseases.

These findings, then, open up a whole new field of research into mitochondrial diseases.

There is still much work to be done. Dr. Pu's ultimate goal is not only to better understand Barth syndrome, but to be able to effectively treat it. First, he needed to confirm that his results were caused by the TAZ mutation in Barth cells and not from the process of making iPSCs. To do this, Dr. Pu and his team introduced TAZ mutations into otherwise healthy cell lines. They took iPSCs from healthy patients and introduced the genetic mutation found in Barth syndrome in the TAZ gene. The researchers found that the mutations in the TAZ gene were sufficient to cause abnormal cardiolipin and excessive ROS production characteristic of Barth heart cells.

Next, the researchers re-introduced the normal TAZ gene into the skin-turned-stem cells-turned-Barth heart cells and found that they were able to restore cardiolipin production to normal levels and to correct the excessive ROS production. These findings confirmed that the TAZ mutation causes the abnormal cellular processes associated with Barth syndrome and that these abnormalities are reversed if the mutation is corrected.

By comparing the results of these experiments in Barth heart cells with healthy heart cells, Dr. Pu and his team observed further abnormalities. In order to beat properly, the contracting units of muscle cells, sarcomeres, must line up correctly. Dr. Pu and his team observed that the sarcomeres in Barth heart cells didn't function properly. "They aligned irregularly, which made their contractions ineffective," remarked Dr. Pu.

To further investigate sarcomere function, Dr. Pu reached out to his colleague Dr. Kevin Kit Parker, also at Harvard Medical School. What A Year highlighted Dr. Parker's research in the November 2007 story " A Film Strip that Could Save Your Life. " Dr. Adam Feinberg, a researcher in Dr. Parker's laboratory, had created strips of heart muscle that could be used to test heart function under a variety of conditions. The strips have a base of very flexible plastic, and are lined with a protein called fibronectin that holds the heart cells together. If left alone, heart cells will grow sporadically in all different directions. On these plastic strips, however, the fibronectin tells the heart cells exactly how to grow. When the heart cells line up along the lines of fibronectin on the plastic strips, they create small beating chips. Researchers can measure the strength of the heart cells based on how far they bend the plastic during contraction.

In his current research, Dr. Pu collaborated with researchers in Dr. Parker's lab to create similar plastic chips that allow him to measure the strength of the sarcomeres in Barth and normal heart cells generated from iPSCs. Using these strips, known as "heart-on-chip assays," Dr. Pu found that Barth heart cells were not strong enough to contract the chip at all. Control heart cells, on the other hand, exhibited normal contraction.

When the TAZ gene was re-introduced into Barth heart cells, however, sarcomere function was restored: normal contractions were observed on the plastic chip. Next, Dr. Pu and his team used a variety of treatments in an attempt to restore normal function to Barth cells. In particular, they found that chemicals that soak up excessive ROS also restored sarcomere organization and contraction strength just as well as re-introducing the normal TAZ gene.

This finding is relevant not only to Barth syndrome but to potentially many other heart ailments as well because elevated ROS levels have been associated with heart failure and the aging process. Therapies that limit ROS production could potentially lead to treatments for Barth syndrome and all sorts of heart conditions. "Of course, this is still a long way away," cautioned Dr. Pu. This research was done on cells in a controlled environment.

In his most recent experiments, Dr. Pu is testing his hypotheses in mouse models. It could be years before clinical trials in humans become possible. But Dr. Pu is hopeful. "I think we could have something in the next decade," he commented. Dr. Pu also wants to conduct more experiments to further understand the relationship between mitochondria, muscle development, and the heart. "There is so much more to learn," concluded Dr. Pu.

Dr. William Pu is an Associate Professor at Harvard Medical School and Associate in the Department of Cardiology at Boston Children's Hospital. He is a pediatric cardiologist by training, which means that after medical school he completed a fellowship first in pediatrics followed by a second fellowship in cardiology. Dr. Pu's laboratory focuses on the regulation of gene expression in healthy and diseased hearts. Areas of emphasis include Barth syndrome and mitochondrial regulation of heart development. When not in the laboratory, Dr. Pu enjoys spending time with his family.