By David F. Salisbury
March 22. 2002

Vanderbilt scientists have created a new strain of mouse that exhibits cardiac hypertrophy — an enlargement of the heart similar to that which causes heart failure in millions of Americans each year — and may help explain why men are subject to this fatal condition while women are spared until menopause.

The new mice were created using genetic engineering techniques that allow researchers to disable specific genes in an animal's genome. In this case, the mice were created by "knocking out" the gene that expresses a protein named FKBP12.6 that binds to special receptors in heart cells that control the release of calcium ions into the cells' interior. Regular spikes in calcium concentrations within cardiac muscle cells cause the heart to beat.

What makes these mice particularly interesting, say the researchers, is that they exhibit sex differences in the development of cardiac hypertrophy similar to those in humans. The male mice develop enlarged hearts but the females do not. However, when the females are given a drug that blocks the action of the female hormone, estrogen, their hearts enlarge as well, the scientific team from Vanderbilt and Cornell universities report in the March 21 issue of the journal Nature.

Currently, there is no cure for severe heart enlargement

"Once people develop severe cardiac hypertrophy, they have about four to five years to live. It's a condition for which there is no cure," says Sidney Fleischer, professor of biological sciences and pharmacology at Vanderbilt. "We don't understand the events that take place at the molecular level that cause the heart to become enlarged. If we knew the molecular signals that cause such an enlargement then we should be able to come up with ways to prevent it and perhaps reverse it."

Fleischer led the research effort working with postdoctoral researchers Hong-Bo Xin, who helped initiate the knock-out mouse project, and later with Dong-Sheng Cheng. The Cornell group, led by Michael Kotlikoff and joined by Xin, focused its studies on the changes in the mouse heart at the level of cardiac muscle cells, or cardiomyocytes.

The new strain of mouse is a valuable new tool for studying the molecular signals that induce heart cells to enlarge, Fleischer maintains. The mice show about a 25 percent enlargement in their hearts compared to normal mice. That is enough to study the condition but it is not enough to kill the animals. That allows the researchers to use these mice to explore the molecular signals that trigger changes in heart cell size.

Added support for hypothesis that calcium ion regulation is involved in cardiac hypertrophy

The fact that knocking out a gene associated with calcium regulation produces hypertrophy adds additional support to the hypothesis that calcium may be involved in pathological heart enlargement. This was first suggested in 1998 when Eric Olson and his colleagues at the University of Texas Southwestern Medical Center in Dallas found that mice genetically engineered to over-produce a calcium-controlled protein, called calcineurin, can induce massive hypertrophy.

When Fleischer and his colleagues created the knock-out mice with the assistance of the Vanderbilt Transgenic/ES Cell Shared Resource Lab directed by Mark Magnuson, they were not thinking about hypertrophy. They were pursuing the latest lead in a 30-year study into the nature of the system that triggers skeletal muscle contraction and relaxation that allows animals to move and manipulate their environment as well as powering the heart beat.

Since 1970, biologists have known that muscle contractions are triggered by sharp increases in the concentration of calcium ions in the cell's interior. In cardiomyocytes, about 15 percent of the calcium comes from the region outside of the cell through special, regulated pores in the cell membrane. Scientists knew that most of the calcium ions are released from an internal storehouse called the sarcoplasmic reticulum, but they did not know the nature of the calcium-ion release mechanism.

Fleischer's lab discovers molecular machinery that releases calcium ions into cell interior

Then, in 1987, Fleischer's lab found a drug called ryanodine that binds to the complex protein that performs this vital function. The drug enabled them to identify and isolate the protein, called the intercellular calcium release channel (ICRC), which creates an opening in the sarcoplasmic reticulum membrane. The channel protein, also known as the ryanodine receptor, acts like a microscopic valve that either opens or closes the opening depending on a number of factors, including which proteins are bound to it. The finding stimulated the field of calcium signaling in muscle, Fleischer pointed out.

The discovery was originally made in skeletal muscle but the Fleischer lab soon found the analogous structure in cardiac cells. The ICRC's amino acid sequence was determined in Shosaku Numa's laboratory at Kyoto University and David MacLennan's lab at the University of Toronto. A colleague — Terry Wagenknecht of the Wadsworth Center of the New York Department of Health in Albany — worked out the protein's 3-D structure shortly thereafter.

In the process of matching the sequence and the structure of the ICRC, which happens to be the most complex protein yet sequenced, they used special enzymes to break the ryanodine receptor down into short amino-acid sequences, called peptides. Fleischer and one of his collaborators, Andrew Marks, now at Columbia University in New York, found a sequence that did not match that of the channel protein. Several years later another group of researchers found a protein, called FK Binding Protein 12, involved in the action of drugs that block immune rejection of transplanted organs that contained this same sequence at one end.

FK Binding Protein proves to be key to opening calcium channel

This alerted Fleischer's group that FKBP12 could be a key protein associated with the calcium channel protein. When they tested the ryanodine receptor, they confirmed that FKBP12 was tightly bound to the calcium channel protein. Moreover, they discovered that, when it is present, the ICRC remains closed and when it is removed the channel is activated. When they looked at cardiac muscle cells, however, they found that a slightly different protein — one that differed by only 18 amino acids out of a total 108 — was involved. However, when they added and removed this protein, called FKBP12.6, from the ICRC they did not see a similar effect.

So Fleischer and his colleagues decided to create a knock-out mouse to determine FKBP12.6's function.

"After we knocked out the gene and we observed the animals, they looked healthy," Fleischer says. "We thought they would fall over when we put them on a treadmill, but they ran equally as well as normal animals. So we were getting pretty depressed about finding a role for FKBP12.6 until we got the idea of looking at the dynamic action of the heart and discovered that the mice's hearts were indeed abnormal."

Knock-out mice look healthy but have abnormal hearts

Working with Tadashi Inagami and Takaaki Senbonmatsu in Vanderbilt's department of biochemistry, the researchers used a technique called echocardiography to measure the properties of the knock-out mouse's heart. This technique is used in humans to analyze heart activity and to measure cardiac hypertrophy. The researchers were surprised to find that the male mice exhibited hypertrophy, but the females did not. In the male mice, they found that the wall that divides the left and right ventricles in the heart was enlarged by about 25 percent and that the ratio of the weight of their hearts to their bodies was significantly higher than in normal mice.

While these studies were going on, Kotlikoff and his co-workers, who are experts in the study of cardiomyocytes, compared the heart cells from the normal and knockout mice. They found that the uptake and release of calcium were abnormal in the knock-out strain, and that this held true for both males and females.

The observation of the sex difference in cardiac hypertrophy led the researchers to administer a drug that blocks the action of estrogen to the female knock-out mice. When this was done, they found that the females soon developed hypertrophy comparable to that of the males.

"With the knock-out mice, we've got the calcium signaling defined in a way that causes cardiac hypertrophy," says Fleischer. "So we should be able to sort out the molecular signaling events that lead to this condition. It may also prove possible to slow down and even reverse the hypertrophy. And, chances are, it will be similar in humans."

-VU-

Nature paper [membership required]

Picture of hypertrophic heart: eAtlas of Pathology

Calcineurin and Cardiac Disease, The Scientist, Jul. 10, 2000 [membership required]

Online information and research description of Sidney Fleischer

Online biography of Michael Kotlikoff