Question:

Cyclic GMP (cGMP) is an important signaling molecule responsible for the regulation of multiple physiological functions. In the heart, high intracellular cGMP levels have been shown to reduce the contractility and to protect the myocardium from pressure-overload induced hypertrophy. However, direct measurements of cGMP and guanylyl cyclase (GC) activity in intact adult cardiomyocytes have been challenging due to low sensitivity of the available methods.

Methods:

Here we used a highly sensitive (EC₅₀ ~40 nM) and specific Förster resonance energy transfer (FRET)-based cGMP biosensor called red cGES-DE5. We expressed this biosensor in heart muscle of transgenic mice and studied real-time dynamics of cGMP in freshly isolated adult mouse cardiomyocytes. To study changes in cGMP signaling in cardiac disease, we used cells isolated from mice 8 weeks after transverse aortic constriction (TAC).

Results:

Stimulation of single myocytes with saturating concentrations of GC-A and GC-B ligands atrial (ANP) and C-type natriuretic peptides (CNP) led to an increase of free cytosolic cGMP levels which was much more pronounced after CNP stimulation. In myocytes isolated from mice after TAC, ANP-induced cGMP signals were abrogated, whereas CNP still led to robust increases of intracellular cGMP.

Conclusions:

Measurements of free cytosolic cGMP levels in cardiomyocytes show that GC-B, followed by much less active GC-A, represents the major particulate GC isoform which, in contrast to GC-A, is not desensitized upon pressure overload induced hypertrophy in terms of its coupling to GMP production.