Reversal of isoproterenol-induced downregulation of phospholamban and FKBP12.6 by CPU0213-mediated antagonism of endothelin receptors

Abstract

The downregulation of phospholamban (PLB) and FKBP12.6 as a result of beta- receptor activation is involved in the pathway(s) of congestive heart failure. We hypothesized that the endothelin (ET)-1 system may link to downregulated PLB and FKBP12.6. Rats were subjected to ischemia/reperfusion (I/R) to cause heart failure (HF). 1 mg/kg isoproterenol (ISO) was injected subcutaneously (sc) for 10 d to worsen HF. 30 mg/kg CPU0213 (sc), a dual ET receptor (ETAR/ETBR) antagonist was given from d 6 to d 10. On d 11, cardiac function was assessed together with the determination of mRNA levels of ryanodine receptor 2, calstabin-2 (FKBP12.6), PLB, and sarcoplasmic reticulum Ca2+-ATPase. Isolated adult rat ventricular myocytes were incubated with ISO at 1X10(-6) mol/L to set up an in vitro model of HF. Propranolol (PRO), CPU0213, and darusentan (DAR, an ETAR antagonist) were incubated with cardiomyocytes at 1X10(-5) mol/L or 1X10(-6) mol/L in the presence of ISO (1X10(-6) mol/L). Immunocytochemistry and Western blotting were applied for measuring the protein levels of PLB and FKBP12.6. The worsened hemodynamics produced by I/R were exacerbated by ISO pretreatment. The significant downregulation of the gene expression of PLB and FKBP12.6 and worsened cardiac function by ISO were reversed by CPU0213. In vitro ISO 1X10(-6) mol/L produced a sharp decline of PLB and FKBP12.6 proteins relative to the control. The downregulation of the protein expression was significantly reversed by the ET receptor antagonist CPU0213 or DAR, comparable to that achieved by PRO. This study demonstrates a role of ET in mediating the downregulation of the cardiac Ca2+-handling protein by ISO.