Researchers at Cornell University, the University of Pittsburgh, and Japan’s RIKEN Brain Science Institute have created transgenic mice whose hearts produce a fluorescent protein that’s turned on by calcium ions. Calcium concentrations in heart cells skyrocket and then plummet during electrical signalling and muscle contraction in animals, including humans and mice. Using a fluorescence microscope, the scientists were able to film these waves of activity moving through the four chambers of the mice’s hearts with each beat. Cornell University’s Michael Kotlikoff, who led the research, calls the fluorescent protein a “molecular spy.”
[To see images of fluorescent hearts, click here.]
Biologists have long dreamed of being able to see the activities of cells in living, functioning animals. Now the techniques that will make this possible are coming to fruition. These new genetically engineered mice are providing a window into what happens during a heart beat and also illuminating the electrical signals that coordinate the beating. The mice have provided insights into the development of the mammalian heart, and are also being used to evaluate heart stem cell transplants. And, finally, the researchers are also breeding mice with glowing nerve cells to study signalling in the brain.
“The goal for all of us is to see the changes in ion concentration or other aspects of cellular function” in living animals, says Withrow Gil Wier, a professor of physiology at the University of Maryland School of Medicine, who in 1980 first showed the transience of calcium in heart muscles.
“We learn a lot by taking cells out, chopping them up, and looking at individual molecules. But it doesn’t tell us what we need to know about the complex interactions between cells,” says Kotlikoff, who is chair of the department of biomedical sciences at Cornell’s College of Veterinary Medicine.
RIKEN scientist Junichi Nakai created the fluorescent protein (called “GCaMP2”) by modifying an existing one that was not bright or stable enough to use for in vivo heart experiments. Cornell scientists genetically engineered mice that produce this protein day after day and at a steady concentration, in their hearts and nowhere else in the body.
Kotlikoff says the advantage of the new fluorescent protein over its predecessors is the speed at which it can turn on and off, like a light bulb, its stability at body temperature, and its brightness. The mouse heart beats up to 600 times per minute. “There’s so much background light and reflection and motion that you need something very bright to be able to detect these cellular signals,” he says.
To see the workings of the cells, Kotlikoff says, “We anesthetize the mouse, ventilate it, open up the chest, and shine light right on the heart.” A group led by Guy Salama at the University of Pittsburgh School of Medicine was responsible for producing movies of the beating hearts. Salama uses a high-speed camera to create clear, high-resolution images. He recorded a frame every millisecond, capturing several images of each heartbeat.
Because the researchers filmed the mice’s hearts at all stages of development, from day-old embryos to adulthood, the movies “show you what’s going on every time the mouse’s heart beats for its whole life,” Kotlikoff says.
In all mammals the heart is the first organ to start working, driven by the embryo’s need for oxygen. However, it has to start pumping before the structures that maintain the heartbeat’s steady pace have developed. Kotlikoff discovered a signalling pathway that helps the embryonic heart maintain its pace for only a few days, until the adult structure develops. He says he has also seen arrhythmias – disruptions in the heart’s steady electrical signals that lead to fast, slow, or erratic heartbeats, and that can cause sudden death.
Kotlikoff says he is breeding mice that make the new fluorescent protein only in specialized heart tissues and parts of the body where calcium is important, such as the brain, where calcium plays an important role in signalling. “We can put it where we want and listen in on specific signals that are passing between one cell and another cell,” he explains.
Kotlikoff is also using the glowing heart cells to study stem cell transplants. “We can differentiate these [glowing] cells and put them into hearts that have been injured. The cells signal when they’re activated, so we can tell how they behave in their new environment.”
Igor Efimov, associate professor of biomedical engineering at Washington University in St. Louis, who studies the disruptions in electrical signalling that cause arrhythmias, says this work is a major breakthrough: “I think it will open a new opportunity for imaging, so that we can finally express intrinsic sensors in different compartments of the heart or brain and study how impulses are conducted under normal conditions, which is very important,” he says.
While Wier cautions that this research is still in its early stages, he says it is “promising compared to what we had in the past. This [work] is getting us closer to being able to see physiological changes, in the least invasive way.”