Transgenic Mice for Real Time Visualization of cGMP in Intact Adult Cardiomyocytes
Rationale: cGMP is an important second messenger which regulates cardiac contractility and protects the heart from hypertrophy. However, due to the lack of real time imaging techniques, specific subcellular mechanisms and spatio-temporal dynamics of cGMP in adult cardiomyocytes are not well understood.
Objective: To generate and characterize a novel cGMP sensor model to measure cGMP with nanomolar sensitivity in adult cardiomyocytes.
Methods and Results: We generated transgenic mice with cardiomyocyte-specific expression of the highly sensitive cytosolic Förster resonance energy transfer (FRET)-based cGMP biosensor red cGES-DE5 and performed the first FRET measurements of cGMP in intact adult mouse ventricular myocytes. We found very low (~10 nM) basal cytosolic cGMP levels which can be markedly increased by natriuretic peptides (CNP>>ANP) and to much smaller extent by the direct stimulation of the soluble guanylyl cyclase. Constitutive activity of this cyclase contributes to basal cGMP production which is balanced by the activity of clinically established phosphodiesterase (PDE) families. The PDE3 inhibitor cilostamide showed especially strong cGMP responses. In a mild model of cardiac hypertrophy after transverse aortic constriction, PDE3 effects were not affected, while the contribution of PDE5 was significantly increased. In addition, after natriuretic peptide stimulation, PDE3 was also involved in cGMP/cAMP cross-talk.
Conclusions: The new sensor model allows visualization of real time cGMP dynamics and pharmacology in intact adult cardiomyocytes. FRET imaging suggests the importance of well-established and potentially novel PDE-dependent mechanisms which regulate cGMP under physiological and pathophysiological conditions.
- phosphodiesterase inhibitor
- cardiac myocyte
- fluorescent imaging
- transgenic mice
- Received August 21, 2013.
- Revision received February 10, 2014.
- Accepted March 4, 2014.