Endothelin downregulates SERCA2 gene and protein expression in adult rat ventricular myocytes: regulation by pertussis toxin-sensitive G_iprotein and cAMP

Abstract

Hilal-Dandan R, He H, Martin JL, Brunton LL, Dillmann WH. Endothelin downregulates SERCA2 gene and protein expression in adult rat ventricular myocytes: regulation by pertussis toxin-sensitive G i protein and cAMP. Am J Physiol Heart Circ Physiol 296: H728 -H734, 2009. First published January 16, 2009 doi:10.1152/ajpheart.00584.2008.-Downregulation of the sarcoplasmic reticulum calcium ATPase (SERCA2) is associated with diastolic dysfunction in the failing heart. Elevated plasma endothelin-1 (ET) levels are correlated with congestive heart failure suggesting that ET may play a pathophysiological role. We have investigated the ability of ET to regulate SERCA2 gene expression in isolated adult rat ventricular myocytes. We find that ET enhances net protein synthesis by ϳ40% but significantly downregulates SERCA2 mRNA expression, time dependently, by ϳ30 -50%, and the expression of SERCA2 protein by ϳ 50%. In myoyctes, ET binds to ET A receptor that couples to G q and Gi proteins. Inhibition of Gq-PLC-induced phosphoinositide (PI) hydrolysis with U73122 (1 M) or inhibition of G i protein with pertussis toxin (PTX) abolishes the ability of ET to downregulate SERCA2 mRNA gene expression. Further investigation suggests that ET coupling to PTX-sensitive G i with consequent lowering of cAMP is required for downregulation of SERCA2 mRNA levels. Increasing intracellular cAMP quantity using cAMP-specific PDE inhibitor Ro20-1724 or cAMP analog dibutyryl-cAMP reverses ET-induced downregulation of SERCA2 mRNA levels. The data indicate that, in adult myocytes, ET downregulates SERCA2 mRNA and protein levels, and the effect requires cross-talk between G q and PTX-sensitive G i pathways.hypertrophy; sarcoplasmic reticulum calcium ATPase; Gi; Gq THE CONTRACTION AND RELAXATION CYCLE of cardiac muscle is tightly regulated by the release and re-uptake of Ca 2ϩ . During excitation-contraction coupling, extracellular Ca 2ϩ enters the myocyte through Ca 2ϩ channels and induces Ca 2ϩ release from the sarcoplasmic reticulum (SR) to enhance contraction; subsequent Ca 2ϩ uptake via the SR Ca 2ϩ -ATPase (SERCA2) reduces intracellular Ca 2ϩ levels and facilitates muscle relaxation or diastole (4, 39). Thus cardiac muscle performance depends on the adequacy of SR and SERCA2 function.Evidence in the literature suggests that alterations in SR function and expression levels of SERCA2 mRNA correlate with prolonged and incomplete diastole in the hypertrophied and failing heart muscle (1,2,5,6,30,35,36). Decreased expression of the mRNA encoding the SERCA2 gene is associated with slower removal of cytosolic Ca 2ϩ (1,2,5,6,9,30,35,36), whereas upregulation of SERCA2 mRNA expression, induced by thyroid hormone, is associated with an increase in the rate of tension development and enhancement in the rate of relaxation (9, 34). Furthermore, increasing the expression of SERCA2 gene in transgenic animals, or in myocytes through adenovirus expressing the SERCA2 gene, results in enhanced relaxation (8,13,15).Mechanical, hormonal, and neuronal factors may c...

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