Reduced Ca(2+)-sensitivity of SERCA 2a in failing human myocardium due to reduced serin-16 phospholamban phosphorylation.
It is still a matter of debate, whether decreased protein expression of SERCA 2a and phospholamban (PLB), or alterations in the phosphorylation state of PLB are responsible for the reduced SERCA 2a function in failing human myocardium. Thus, in membrane preparations from patients with terminal heart failure due to idiopathic dilated cardiomyopathy (NYHA IV. heart transplants) and control hearts (NF), SERCA 2a activity was measured with an NADH coupled assay with as well as without stimulation with protein kinase A (PKA). The protein expression of SERCA 2a, PLB and calsequestrin as well as the phosphorylation status of PLB (Back-phosphorylation technique: Serine-16-PLB specific antibody) were analysed using Western blotting technique and specific antibodies. In NF, the maximal activity (Vmax) and the Ca(2+)-sensitivity of SERCA 2a activity were significantly higher compared to NYHA IV. Protein expression of SERCA 2a, PLB and calsequestrin were unchanged, whereas both, the phosphorylation status of PLB as well as serine-16-PLB-phosphorylation, were significantly reduced in NYHA IV. After stimulation with PKA only the Ca(2+)-sensitivity, but not Vmax increased concentration-dependently. Therefore, in human myocardium, the Ca(2+)-sensitivity but not the Vmax of SERCA 2a is regulated by cAMP-dependent phosphorylation of phospholamban at position serine-16. Threonine-17-PLB-phosphorylation or direct phosphorylation of SERCA 2a may be candidates for regulation of maximal SERCA 2a activity in human myocardium.
Schwinger
R H
RH
Laboratory of Muscle Research and Molecular Cardiology, University of Cologne, Germany.
Münch
G
G
Bölck
B
B
Karczewski
P
P
Krause
E G
EG
Erdmann
E
E
D000328:Adult; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003720:Densitometry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006333:Heart Failure / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D009206:Myocardium / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D017868:Cyclic AMP-Dependent Protein Kinases; D010761:Phosphorus Radioisotopes; D000099211:Phospholamban