UNCHANGED PROTEIN EXPRESSION OF SARCOPLASMIC-RETICULUM CA2-ATPASE, PHOSPHOLAMBAN, AND CALSEQUESTRIN IN TERMINALLY FAILING HUMAN MYOCARDIUM()

The enhanced diastolic Ca2+ levels observed In cardiac myocytes from p atients with idiopathic dilated cardiomyopathy (DCM) may be either a c onsequence of functional impairment of sarcoplasmic reticulum calcium- ATPase (SERCA 2) and its regulator protein phospholamban or due to a r eduction in the number of SERCA 2 proteins. As different myocardial me mbrane preparations may lead to different accumulation of proteins, th e present study evaluated two different membrane preparations, in huma n failing and nonfailing myocardium for comparison of SERCA 2 activity and the protein expression of SERCA 2 and phospholamban. Crude membra nes and tissue homo-genates without any centrifugation steps were prep ared from human nonfailing hearts (donor hearts, NF, n=18) and termina lly failing hearts (heart transplant, DCM, n=18). Calsequestrin protei n expression was used as an internal control for overall protein expre ssion. In both crude membranes and homogenates maximal SERCA 2 activit y (V-max) was significantly reduced in failing heart preparations (NF crude membranes, 130+/-8;; DCM crude membranes, 102+/-5 nmol ATP/mg pr otein per minute). In contrast, the protein expression of SERCA 2 (NF crude membranes, 488+/-35; DCM crude membranes, 494+/-42; P=0.92), pho spholamban (NF crude membranes, 497+/-51; DCM crude membranes, 496+/-4 5; P=0.98) and calsequestrin (NF crude membranes, 109+/-06; DCM crude membranes, 107+/-08; P=0.84) was unchanged in NF and DCM hearts in bot h preparation methods. This was also the case when the protein express ion was normalized to calsequestrin protein levels. Preparation of sar coplasmic reticulum in crude membranes led to enhanced purification an d consequently higher SERCA 2; phospholamban, and calsequestrin protei n levels in crude membranes than in the homogenates, which was paralle led by an increase in SERCA 2 enzyme activity. In conclusion, the alte red Ca2+ handling in DCM may be a consequence of reduced SERCA 2 enzym e activity and not the result of differences in protein expression of the Ca2+ regulating proteins SERCA 2, phospholamban, and calsequestrin in human myocardium. The present study emphasizes the importance of d ifferent myocardial membrane preparations with respect to quantitative investigations of protein expression and function.