Transgenic Mice for cGMP Imaging
Rationale: Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown.
Objective: To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer (FRET)-based ratiometric cGMP sensor, cGi500, in cardiovascular tissues.
Methods and Results: Mouse lines with smooth muscle-specific or ubiquitous expression of cGi500 were generated by random transgenesis using a SM22α promoter fragment or by targeted integration of a Cre recombinase-activatable expression cassette driven by the cytomegalovirus early enhancer/chicken β-actin/β-globin (CAG) promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were <100 nM, while stimulation with cGMP-elevating agents, such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, ANP and CNP, evoked FRET changes corresponding to cGMP peak concentrations of ~3 µM. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, ANP and CNP. Robust NO-induced cGMP transients with peak concentrations of ~1 to >3 µM could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, using a dorsal skinfold chamber model and multi-photon FRET microscopy, NO-stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation.
Conclusions: These cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live cells, tissues, and mice under normal and pathological conditions, or during pharmacotherapy with cGMP-elevating drugs.
- transgenic mice
- FRET sensor
- smooth muscle
- atrial natriuretic factor
- cardiovascular imaging
- nitric oxide
- vascular smooth muscle
- Received January 27, 2013.
- Revision received June 20, 2013.
- Accepted June 25, 2013.