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 patients with idiopathic dilated cardiomyopathy (DCM) may be either a consequence of functional impairment of sarcoplasmic reticulum calcium-ATPase (SERCA 2) and its regulator protein phospholamban or due to a reduction in the number of SERCA 2 proteins. As different myocardial membrane preparations may lead to different accumulation of proteins, the present study evaluated two different membrane preparations, in human failing and nonfailing myocardium for comparison of SERCA 2 activity and the protein expression of SERCA 2 and phospholamban. Crude membranes and tissue homo-genates without any centrifugation steps were prepared from human nonfailing hearts (donor hearts, NF, n=18) and terminally failing hearts (heart transplant, DCM, n=18). Calsequestrin protein expression was used as an internal control for overall protein expression. In both crude membranes and homogenates maximal SERCA 2 activity (Vmax) was significantly reduced in failing heart preparations (NF crude membranes, 130+/-8; DCM crude membranes, 102+/-5 nmol ATP/mg protein per minute). In contrast, the protein expression of SERCA 2 (NF crude membranes, 488+/-35; DCM crude membranes, 494+/-42; P=0.92), phospholamban (NF crude membranes, 497+/-51; DCM crude membranes, 496+/-45; 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 both preparation methods. This was also the case when the protein expression was normalized to calsequestrin protein levels. Preparation of sarcoplasmic reticulum in crude membranes led to enhanced purification and consequently higher SERCA 2, phospholamban, and calsequestrin protein levels in crude membranes than in the homogenates, which was paralleled by an increase in SERCA 2 enzyme activity. In conclusion, the altered Ca2+ handling in DCM may be a consequence of reduced SERCA 2 enzyme 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 different myocardial membrane preparations with respect to quantitative investigations of protein expression and function.