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-based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues.


METHODS AND RESULTS
Mouse lines with smooth muscle-specific or ubiquitous expression of cGi500 were generated by random transgenesis using an 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 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 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, atrial natriuretic peptide, and C-type natriuretic peptide evoked fluorescence resonance energy transfer changes corresponding to cGMP peak concentrations of ≈ 3 µmol/L. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, atrial natriuretic peptide, and C-type natriuretic peptide. Robust nitric oxide-induced cGMP transients with peak concentrations of ≈ 1 to > 3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide-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.
['Thunemann|Martin|M|', 'Wen|Lai|L|', 'Hillenbrand|Matthias|M|', 'Vachaviolos|Angelos|A|', 'Feil|Susanne|S|', 'Ott|Thomas|T|', 'Han|Xiaoxing|X|', 'Fukumura|Dai|D|', 'Jain|Rakesh K|RK|', 'Russwurm|Michael|M|', 'de Wit|Cor|C|', 'Feil|Robert|R|']
D000818:Animals D015374:Biosensing Techniques D002319:Cardiovascular System D006152:Cyclic GMP D031541:Fluorescence Resonance Energy Transfer D051379:Mice D008822:Mice, Transgenic D036641:Microscopy, Fluorescence, Multiphoton D023421:Models, Animal D009130:Muscle, Smooth D009131:Muscle, Smooth, Vascular D015398:Signal Transduction
cyclic GMP fluorescence resonance energy transfer vasodilation biosensing techniques microscopy, fluorescence, multiphoton muscle, smooth