Cyclic nucleotides mediated signaling determines the regulation of many cardiac function. It is generally admitted that the cyclic nucleotides degradation is due to phosphodiesterases. Recently, cAMP and cGMP were also shown to be extruded from the cell by an active efflux transporter, called MRP4. We investigated whether inhibition of MRP4 may independently control cyclic nucleotides levels and modify functions related to cyclic nucleotides signalling pathways in cardiac myocytes.

We used silencing RNA or adenovirus encoding MRP4 shRNA (Ad-ShMRP4) to inhibit MRP4 expression in rat cardiomyocytes in vitro. We then performed physiological evaluation of cardiac morphology and function in knock-out MRP4 mice. As a model of adrenergically induced cardiac hypertrophy, miniosmotic pumps containing isoproterenol delivering 20 ìg/g/day for 2 weeks were implanted subcutaneously into 3-months old MRP4 KO and wild type mice. As a model of physiological hypertrophy, animals were housed in a cage with free access to a running monitored wheel.

MRP4 is present in human, mouse and rat cardiomyocytes and over-expressed in case of increased cardiac intracellular cAMP. Using a FRET technique MRP4 inhibition was shown to increase intracellular cAMP level. Adult rat cardiomyocytes infected with Ad-shMRP4 demonstrated a significant increase in the calcium current density and in cell size compared to cells infected with the negative control Ad-shLuciferase, suggesting an activation of the cAMP/PKA pathway. While unchallenged young (3 months) MRP4 KO mice displayed normal cardiac parameters, MRP4 KO mice progressively developed significant cardiac hypertrophy by 9-12 months of age (HW/BW ; 5.15 vs 4.46 in WT mice). Isoproterenol-treated MRP4 KO mice displayed a significant increase in cardiac hypertrophy compared to stimulated WT mice (HW/BW: 6±0.38 vs 5.29±0.34 ; p=0.001). In contrast to the regulation of pathological cardiac hypertrophy, MRP4 inhibition did not affect the physiological cardiac growth response associated with physical training (HW/BW: 4.83±0.01, vs 4.84±0.13, p=NS), indicating the absence of a regulatory role of MRP4 in physiological cardiac hypertrophy.

These results reveal a unique and important function for MRP4 in stress-dependent cardiac growth by controlling cyclic nucleotides signalling pathways in cardiomyocytes.