BERKELEY, CA — The first study using robotic microarray technology to screen whole heart tissue from mice found 55 genes involved in cardiac hypertrophy; 30 genes were associated with the condition for the first time, and over half of these were completely novel. Moreover, in a discovery that would not have been made using standard biochemical methods, eight genes were found to be associated only with recovery from hypertrophy (regression).

CARL FRIDDLE (LEFT) AND JIM BRISTOW WITH THE MICROARRAY SYSTEM

"Cardiac hypertrophy happens when a heart is stressed -- as by high blood pressure, or when some heart cells die and others must overcompensate," explains Carl Friddle of the Department of Energy's Lawrence Berkeley National Laboratory, where the study was performed. "Instead of dividing, the cells grow larger. Initially this compensation is helpful, but excessive hypertrophy can kill some cells, which increases the stress, causing the surviving cells to grow larger still -- a runaway cycle that can end in heart failure."

"Hypertrophy is independent of other heart-disease risk factors such as high blood pressure," says James Bristow, a staff scientist at Berkeley Lab and an associate professor of pediatrics at the University of California at San Francisco. "You can treat high blood pressure, and when blood pressure goes down, hypertrophy may or may not regress completely. Patients might benefit if we could treat it directly."

To understand the relationship of genes involved in all phases of hypertrophy and regression, Bristow and Friddle applied the microarray techniques that Friddle had learned in Pat Brown's laboratory at Stanford University. Later, working in Edward Rubin's Genome Sciences Department in Berkeley Lab's Life Sciences Division, Friddle used the technique to monitor the expression of up to 10,000 genes at a time.

"Of an estimated 100,000 genes in the mouse genome, more than 4,000 have been characterized so far, and in the near future we expect another 8,000 to be well described," Friddle says. "Microarray technology can look at all of these known genes at once and see which are being expressed at various stages of hypertrophy. Eventually we will be able to look at the whole genome."

Cardiac hypertrophy was induced in young male mice by administering the drugs angiotensin II or isoproteronol over a period of one to two weeks. When the drugs were withdrawn, heart weights returned to normal in these groups. Mice in a matched group, serving as controls, were given only saline solution.

To test each stage of hypertrophy and recovery, RNA was extracted from heart tissues at daily intervals, including the RNA representing all the active genes in each of the groups.

PINS OF THE MICROARRAYER POSITION GENE FRAGMENTS ON GLASS SLIDES

To see which genes were active, the pins of the robotic microarrayer positioned dots of gene fragments from the Genome Sciences Department's library of cloned genes on glass microscope slides. Each gene fragment is capable of binding to the messenger RNA (mRNA) that codes for a particular gene's protein product.

Messenger RNA extracted from heart tissue was placed on each slide. The mRNA from hypertrophied hearts was labeled with red-fluorescing dye, that from normal hearts with green-fluorescing dye. The mRNA from both kinds of tissues was mixed together and allowed to react with the thousands of gene-fragment strands on the microscope slide.

A laser scanner excited the dyes. A spot that glowed more red than green marked a gene that was active at that stage of hypertrophy or recovery. A spot that glowed green marked a gene strongly expressed by the normal heart. A computer compared images of the slides to identify the various active genes: spots that didn't glow, or glowed only faintly, marked genes expressed weakly or not at all by heart cells.

"Because cardiac hypertrophy has been extensively studied, some 300 genes are already known to be associated with it," Bristow says. "Although we found just 55, more than half of these were new."

Friddle notes that "the main reason we didn't identify more, even though we were looking at only about five percent of the mouse genome, is that we wanted to make sure we didn't have any false positives. The question isn't how big an expression effect is, but whether expression can be replicated."

The researchers screened for such factors as gene expression caused by the drugs, not the hypertrophy itself. They also eliminated numerous sources of variability in expression, "including some so random you might as well blame them on the phase of the moon," Bristow says. "But by using microarray analysis in-house at Berkeley Lab, we were able to do numerous replications -- when we saw changes in the expression of a gene repeatedly, we knew it was real."

An important result of the whole-tissue approach, using repeated microarray analyses over time, was the identification of genes expressed only in the regression phase. "Hypertrophy has been studied to death," Bristow says, "yet surprisingly little attention has been paid to regression. If we know which genes are expressed during recovery from cardiac hypertrophy, we may be able to stimulate them to overexpress -- one of the first things we want to investigate is possible leads for modifying regression therapeutically."

Friddle, Bristow, and their colleagues Teiichiro Koga and Edward M. Rubin report the results of their study in an article titled "Expression profiling reveals distinct sets of genes altered during induction and regression of cardiac hypertrophy," in the Proceedings of the National Academy of Sciences, 6 June 2000, which has been published online. The research was supported by the National Institutes of Health and the Department of Energy.

The Berkeley Lab is a U.S. Department of Energy national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California.