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Circ Res. 2013 Aug 2;113(4):365-71. doi: 10.1161/CIRCRESAHA.113.301063. Epub 2013 Jun 25.

Transgenic mice for cGMP imaging.

Author information

1
Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen, Germany.

Abstract

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.

KEYWORDS:

biosensing techniques; cyclic GMP; fluorescence resonance energy transfer; microscopy, fluorescence, multiphoton; muscle, smooth; vasodilation

PMID:
23801067
PMCID:
PMC3896241
DOI:
10.1161/CIRCRESAHA.113.301063
[Indexed for MEDLINE]
Free PMC Article

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