Abstract
The second messenger cyclic guanosine 3′5′-monophosphate (cGMP) plays an important role in the regulation of multiple physiologic processes in the cardiovascular system for example it is known to inhibit hypertrophy and to protect against ischemia-reperfusion injury. As a consequence of hypoxia in the heart, maladaptive signaling cascades are activated that can result in cardiac damage and finally lead to heart failure. However, cellular responses to hypoxia/reoxygenation (H/R) are still incompletely understood and almost nothing is known about cGMP dynamics in the context of H/R. The aim of this project was to investigate the effects of H/R on cGMP dynamics in mammalian cardiomyocytes. Therefore transgenic mice with cardiomyocyte-specific expression of the cytosolic Förster resonance energy transfer (FRET)-based cGMP sensor red cGES-DE5 were used to study cGMP dynamics in single adult cardiomyocytes exposed to H/R. In addition, a Langendorff system was used for FRET measurements in whole-heart. Basal cGMP levels in single adult cardiomyocytes exposed to H/R were increased. This increase was generated already during the hypoxic period and was maintained during reoxygenation. PDE3 protein expression and activity were significantly downregulated after H/R, however RNA expression level of PDE3 was not significantly changed. At the same time, protein levels of the soluble guanylyl cyclase β-subunit showed a tendency towards downregulation during H/R. In whole heart measurements, we could show that cGMP levels increase during anoxia and decrease during reoxygenation. This indicates that there is also an influence of other cell-types on cGMP dynamics or on cGMP cell-cell transfer. In conclusion, we developed FRET-based cGMP measurements in single cardiomyocytes and whole hearts in context of H/R and found an increase of intracellular cGMP in hypoxia. This offers great opportunities to dissect the molecular mechanism of cGMP signaling regulation during ischemic injury and should help to distinguish between direct protective effects on cardiomyocytes and indirect mechanisms such as cell-cell interactions in cGMP signaling during and after H/R.
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