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The importance of the oncogene Ras in cardiac hypertrophy is well appreciated. The hypertrophic effects of the constitutively active mutant Ras-Val12 are revealed by clinical syndromes due to the Ras mutations and experimental studies. We examined the possible anti-hypertrophic effect of Ras inhibition in vitro using rat neonatal cardiomyocytes (NRCM) and in vivo in the setting of pressure-overload left ventricular (LV) hypertrophy (POH) in rats. Ras functions were modulated via adenovirus directed gene transfer of active mutant Ras-Val12 or dominant negative mutant N17-DN-Ras (DN-Ras). Ras-Val12 expression in vitro activates NFAT resulting in pro-hypertrophic and cardio-toxic effects on NRCM beating and Z-line organization. In contrast, the DN-Ras was antihypertrophic on NRCM, inhibited NFAT and exerted cardio-protective effects attested by preserved NRCM beating and Z line structure. Additional experiments with silencing H-Ras gene strategy corroborated the antihypertrophic effects of siRNA-H-Ras on NRCM. In vivo, with the POH model, both Ras mutants were associated with similar hypertrophy two weeks after simultaneous induction of POH and Ras-mutant gene transfer. However, LV diameters were higher and LV fractional shortening lower in the Ras-Val12 group compared to control and DN-Ras. Moreover, DN-Ras reduced the cross-sectional area of cardiomyocytes in vivo, and decreased the expression of markers of pathologic cardiac hypertrophy. In isolated adult cardiomyocytes after 2weeks of POH and Ras-mutant gene transfer, DN-Ras improved sarcomere shortening and calcium transients compared to Ras-Val12. Overall, DN-Ras promotes a more physiological form of hypertrophy, suggesting an interesting therapeutic target for pathological cardiac hypertrophy.

Background RV and LV have different embryologic, structural, metabolic, and electrophysiologic characteristics, but whether interventricular differences exist in β‐adrenergic (β‐ AR ) responsiveness is unknown. In this study, we examine whether β‐ AR response and signaling differ in right ( RV ) versus left ( LV ) ventricles. Methods and Results Sarcomere shortening, Ca 2+ transients, I Ca,L and I Ks currents were recorded in isolated dog LV and RV midmyocytes. Intracellular [ cAMP ] and PKA activity were measured by live cell imaging using FRET‐based sensors. Isoproterenol increased sarcomere shortening ≈10‐fold and Ca 2+ ‐transient amplitude ≈2‐fold in LV midmyocytes (LVMs) versus ≈25‐fold and ≈3‐fold in RVMs. FRET imaging using targeted Epac2camps sensors revealed no change in subsarcolemmal [ cAMP ], but a 2‐fold higher β‐AR stimulation of cytoplasmic [ cAMP ] in RVMs versus LVMs. Accordingly, β‐AR regulation of I Ca,L and I Ks were similar between LVMs and RVMs, whereas cytoplasmic PKA activity was increased in RVMs. Both PDE3 and PDE4 contributed to the β‐AR regulation of cytoplasmic [ cAMP ], and the difference between LVMs and RVMs was abolished by PDE3 inhibition and attenuated by PDE4 inhibition. Finally LV and RV intracavitary pressures were recorded in anesthetized beagle dogs. A bolus injection of isoproterenol increased RV dP /dt max ≈5‐fold versus 3‐fold in LV. Conclusion Canine RV and LV differ in their β‐AR response due to intrinsic differences in myocyte β‐AR downstream signaling. Enhanced β‐AR responsiveness of the RV results from higher cAMP elevation in the cytoplasm, due to a decreased degradation by PDE3 and PDE4 in the RV compared to the LV.

International audience

International audience; Cyclic AMP regulates a multitude of cellular responses and orchestrates a network of intracellular events. In the heart, cAMP is the main second messenger of the β-adrenergic receptor (β-AR) pathway producing positive chronotropic, inotropic and lusitropic effects during sympathetic stimulation. Whereas short term stimulation of β-AR/cAMP is beneficial for the heart, chronic activation of this pathway triggers pathological cardiac remodelling which may ultimately lead to heart failure (HF). Cyclic AMP is controlled by two families of enzymes with opposite actions: adenylyl cyclases which control cAMP production and phosphodiesterases which control its degradation. The large number of families and isoforms of these enzymes, their different localization within the cell and their organization in macromolecular complexes leads to a high level of compartmentation, both in space and time, of cAMP signaling in cardiac myocytes. Here, we review the expression level, molecular characteristics, functional properties and roles of the different adenylyl cyclase and phosphodiesterase families expressed in heart muscle and the changes that occur in cardiac hypertrophy and failure.

Objectives This study investigated whether myocardial phosphodiesterase-2 (PDE2) is altered in heart failure (HF) and determined PDE2-mediated effects on beta-adrenergic receptor (β-AR) signaling in healthy and diseased cardiomyocytes. Background Diminished cyclic adenosine monophosphate (cAMP) and augmented cyclic guanosine monophosphate (cGMP) signaling is characteristic for failing hearts. Among the PDE superfamily, PDE2 has the unique property of being able to be stimulated by cGMP, thus leading to a remarkable increase in cAMP hydrolysis mediating a negative cross talk between cGMP and cAMP signaling. However, the role of PDE2 in HF is poorly understood. Methods Immunoblotting, radioenzymatic- and fluorescence resonance energy transfer–based assays, video edge detection, epifluorescence microscopy, and L-type Ca2+ current measurements were performed in myocardial tissues and/or isolated cardiomyocytes from human and/or experimental HF, respectively. Results Myocardial PDE2 expression and activity were ∼2-fold higher in advanced human HF. Chronic β-AR stimulation via catecholamine infusions in rats enhanced PDE2 expression ∼2-fold and cAMP hydrolytic activity ∼4-fold, which correlated with blunted cardiac β-AR responsiveness. In diseased cardiomyocytes, higher PDE2 activity could be further enhanced by stimulation of cGMP synthesis via nitric oxide donors, whereas specific PDE2 inhibition partially restored β-AR responsiveness. Accordingly, PDE2 overexpression in healthy cardiomyocytes reduced the rise in cAMP levels and L-type Ca2+ current amplitude, and abolished the inotropic effect following acute β-AR stimulation, without affecting basal contractility. Importantly, PDE2-overexpressing cardiomyocytes showed marked protection from norepinephrine-induced hypertrophic responses. Conclusions PDE2 is markedly up-regulated in failing hearts and desensitizes against acute β-AR stimulation. This may constitute an important defense mechanism during cardiac stress, for example, by antagonizing excessive β-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular antiadrenergic therapeutic strategy in HF.