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Human heart failure: cAMP stimulation of SR Ca(2+)-ATPase activity and phosphorylation level of phospholamban
U Schmidt1, R J Hajjar, C S Kim
1Integrated Physiology Research Laboratories, Cardiovascular Division, Department of Cardiovascular Medicine, Boston University School of Medicine, Boston 02118, MA, USA.
Insights
In human heart failure, reduced sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity is linked to impaired phospholamban phosphorylation. This suggests a key mechanism contributing to SR dysfunction in end-stage heart failure.
Area of Science:
- Cardiology
- Molecular Biology
- Biochemistry
Background:
- Failing human myocardium exhibits reduced sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity.
- Controversy exists regarding whether altered regulation or expression changes of SR Ca(2+)-ATPase or phospholamban cause this reduction.
Purpose of the Study:
- To investigate if altered cAMP-dependent phosphorylation of phospholamban contributes to reduced SR Ca(2+)-ATPase activity in human heart failure.
Main Methods:
- Western blot analysis to quantify protein levels of phospholamban and SR Ca(2+)-ATPase.
- Assessing SR Ca(2+)-ATPase activity responsiveness to cAMP and protein kinase A.
- Backphosphorylation technique to measure cAMP-dependent phospholamban phosphorylation levels.
Main Results:
- Protein levels of phospholamban and SR Ca(2+)-ATPase were unchanged in failing versus nonfailing myocardium.
- Failing myocardium showed decreased responsiveness of SR Ca(2+)-ATPase activity to cAMP and protein kinase A activation.
- A significant decrease (20 +/- 2%) in cAMP-dependent phospholamban phosphorylation was observed in failing myocardium.
Conclusions:
- Impaired SR function in human end-stage heart failure may be partly attributed to reduced cAMP-dependent phosphorylation of phospholamban.
- This suggests a regulatory mechanism, rather than expression changes, is involved in SR Ca(2+)-ATPase dysfunction in heart failure.
Abstract:
Failing human myocardium has been associated with decreased sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity. There remains controversy as to whether the regulation of SR Ca(2+)-ATPase activity is altered in heart failure or whether decreased SR Ca(2+)-ATPase activity is due to changes in SR Ca(2+)-ATPase or phospholamban expression. We therefore investigated whether alterations in cAMP-dependent phosphorylation of phospholamban may be responsible for the reduced SR Ca(2+)-ATPase activity in human heart failure. Protein levels of phospholamban and SR Ca(2+)-ATPase, detected by Western blot, were unchanged in failing compared with nonfailing human myocardium. There was decreased responsiveness to the direct activation of the SR Ca(2+)-ATPase activity by either cAMP (0.01-100 micromol/l) or protein kinase A (1-30 microgram) in failing myocardium. Using the backphosphorylation technique, we observed a decrease of the cAMP-dependent phosphorylation level of phospholamban by 20 +/- 2%. It is concluded that the impaired SR function in human end-stage heart failure may be due, in part, to a reduced cAMP-dependent phosphorylation of phospholamban.