Messenger RNA expression and immunological quantification of phospholamban and SR-Ca(2+)-ATPase in failing and nonfailing human hearts.

Abstract

Human heart failure is associated with prolonged relaxation and prolonged Ca2+ transients which indicates an impaired function of the sarcoplasmic reticulum (SR) and may be detrimental for cardiac function. Controversy exists whether the altered SR function is accompanied by changes in the expression of phospholamban (PLB) and cardiac SR-Ca(2+)-ATPase (SERCA2) on mRNA and/or protein levels.We determined mRNA and/or protein levels for PLB and SERCA2 in the same left ventricular tissue of patients undergoing heart transplantation due to idiopathic dilated cardiomyopathy (IDC) or ischemic cardiomyopathy (ICM) in comparison to left ventricular tissue from nonfailing human hearts (NF). Total protein extracts were prepared and subjected to SDS gel electrophoresis. PLB and SERCA2 were identified with specific antibodies. Total RNA was isolated and hybridized with 32P-labeled cDNAs for human PLB and rat SERCA2.Hybridization revealed the three expected mRNAs with the PLB probe (3.3 kb, 1.9 kb and 0.6 kb) and a single band with the SERCA2 probe (4.5 kb). Determination of respective proteins by immunoblotting revealed unchanged protein levels for PLB and SERCA2, whereas the mRNA levels for PLB and SERCA2 were reduced by about 30% and 50%, respectively.These data show the level of SERCA2 and PLB protein and mRNA in the same hearts. The reduced mRNA level of SERCA2 and PLB is in accordance with previous data. However, the unchanged protein levels may indicate that the diminished RNA expression is not accompanied by a corresponding decrease for these proteins in human heart failure. These data also show that the altered SR function in human heart failure cannot be explained by altered protein levels of PLB and SERCA2. Furthermore, it is suggested that extrapolations from cardiac mRNA levels to protein expression may be misleading.