Modified GFP enables in vivo imaging of cellular signaling in the heart
Gwynne D. Koch
The efficient functioning of the heart
— an organ that begins to operate before it is fully developed — depends
on the coordinated release and reuptake of calcium ions from organelles within cells.
Subtle dysfunctions of this process can result in irregular heartbeat and even death.
Fluorescence imaging using indicator molecules
that bind to calcium ions has provided some insight into the regulatory processes
underlying signaling events in single cells. However, these approaches have limitations
when applied to complex, multicellular organs such as the beating heart.
According to Michael I. Kotlikoff of
Cornell University in Ithaca, N.Y., there also is no way to introduce a dye into
a working heart in vivo. Although the heart can be removed from the body and perfused
with a dye, allowing a brief period of signal recording, the loading of the dye
is often uneven and the fluorescent signal cannot be isolated to target cells.
Kotlikoff and colleagues at several
other universities have created an improved calcium-ion sensor by modifying
GFP. The sensor molecule exhibits greater brightness and stability, enabling examination
of signaling events in heart cells in vivo. They genetically engineered mice to
express the fluorescent molecule when calcium concentrations in heart cells increase,
which occurs when the muscle contracts. The advantage of genetically encoding the
sensor is that the molecule is always present at the same concentration in cells
and is confined to the cells of interest.
GFP was modified to create a brighter and more stable calcium-ion
sensor that enables recording of cellular signaling in mouse hearts in vivo. The
sequential images show calcium-ion-dependent fluorescence of the sensor molecule
during a single cardiac cycle.
Calcium ions signal the heart to contract
and determine the force of the contraction. Recording the intensity of the sensor
molecule’s fluorescence enabled the researchers to monitor the pattern, rate
and force of the contractions. The molecule was excited at 488 nm with a mercury
lamp, and emission was collected above 500 nm using a cutoff filter on a wide-field
dissecting microscope from Leica Microsystems of Wetzlar, Germany.
Because mouse hearts beat rapidly —
about six to 10 times per second — images were captured at 66 and 128 fps
using a high-speed electron-multiplying CCD camera — cooled to —90 °C
— from Andor Technology of Belfast, UK.
Motion is a persistent difficulty when
imaging in vivo. To limit motion, the researchers packed the chest with gauze to
stabilize the heart and occasionally increased the anesthesia, which slowed the
heart to a more reasonable rate of five to six beats per second. They also imaged
at 128 or 256 Hz.
The study demonstrates that a genetically
encoded sensor molecule can be used to monitor cellular calcium-ion signaling
events in vivo, such as those that occur during development and disease
processes, in complex, multicellular organs. The team is experimenting with transplanting
embryonic cells from the mice into damaged tissues to determine the effectiveness
of cell-based therapies and to see what happens to the cells electrically after
transplantation. They also are engineering mice to express the sensor molecule in
neurons and in smooth-muscle, conducting and endothelial cells.
PNAS, March 21, 2006, pp. 4753-4758.
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