Abstract 3198: MRP4 Acts in Balance With Phosphodiesterases to Regulate cAmp-dependent Signalling Pathway in Cardiac Myocytes
Background. MRP4 and MRP5, ATPases of the ABCC family, are able to extrude cyclic nucleotides from various cells. The goal of the present study was to determine the role of cAMP extrusion by MRPs in the physiology of the heart.
Results. MRP4 and MRP5 were detected in total rat hearts but only MRP4 was present at the membrane of cardiomyocytes and was up-regulated during pressure-overload induced cardiac hypertrophy. In isolated rat ventricular myocytes, MRP4 silencing by an adenovirus encoding a short hairpin RNA resulted in the phosphorylation of the protein kinase A target proteins troponin I (TNI) and ryanodine receptor (RyR2). MRP4 silencing was also associated with a marked increased in L-type Ca2+ current density with a leftward shift of its activation as observed following cAMP-dependent phosphorylation of channels. The cardiac function was then studied in homozygous MRP4 ablated mice (MRP4 KO) and compared to Wild-Type (WT) controls. Under basal conditions, 3 month-old animals displayed normal cardiac function and morphology (HW/TL: 7.75±0.15 in MRP4 KO vs 7.59±0.14 in WT, p=NS). No changes in TNI and RyR2 phosphorylation were observed. However, we observed that PDE3A and PDE4A were up-regulated in cardiac extracts from MRP4 KO mice compared to controls. Whereas cardiac rhythm was normal under basal conditions (RR (ms): 101±3 in MRP4 KO vs 108±4.9 in WT, p=NS), milrinone (1mg/kg), a PDE3 inhibitor, induced a greater decrease in RR interval in MRP4 KO than in WT mice (−51.3±12.9 vs −17.66±5.5 respectively, p<0.01). We then found that MRP4 KO mice displayed age-dependent cardiac hypertrophy (HW/TL in 9-months old mice: 9.8±0.57 MRP4 KO vs 7.82±0.13 in WT, p<0.001) with PKA-associated RyR2 hyperphosphorylation, calstabin2 dissociation from the RyR complex and increased sparks frequency. Chronic β-adrenergic stimulation using isoproterenol (20 μg/g/day for 2 weeks) infusion or by thoracic aortic constriction exacerbated hypertrophy in MRP KO mice and expression of hypertrophic molecular markers.
Conclusion: MRP4 is a new regulator of cAMP metabolism in cardiac myocytes. MRP4 operates in parallel to PDEs to control cAMP concentration and regulate cAMP-dependent signalling pathways.