Accurate quantitation of phospholamban expression and phosphorylation in biological samples

Abstract

Phospholamban (PLB) reversibly inhibits the sarcoplasmic reticulum calcium ATP-ase (SERCA) in cardiomyocytes. When SERCA is active, it pumps calcium into the sarcoplasmic reticulum (SR) to reduce cytosolic [Ca++]. Calcium efflux from the cytosol reduces the Ca++ available to the cytosolic contractile apparatus so that the heart can relax during diastole. The extent of relaxation depends on the amount of calcium that SERCA removes from the cytosol during diastole, while the contractile force depends on the magnitude of the end-diastolic calcium transient. Thus SERCA inhibition affects both the contractile and relaxation phases of the cardiac cycle. Unphosphorylated PLB (uPLB) inhibits SERCA at low Ca++ concentration and phosphorylated PLB (pPLB) is less inhibitory, so myocardial physiology and pathology depend critically on the mole fraction of pPLB, Xp, equal to pPLB/(uPLB+pPLB), the concentrations of total PLB (tPLB) and SERCA, and uPLB/SERCA. Prior to our assay, neither Xp nor tPLB could be measured accurately. Previous measurements relied on radioactive tracers, which only measured changes in these parameters, or immunoblots, which did not provide acceptable precision or accuracy. The fundamental problems with immunoblots were due to the lack of (a) accurate standards for pPLB and uPLB, (b) antibodies completely specific for pPLB and uPLB, and (c) a mathematical relationship between the antibody selectivity, the intensities of the samples and Xp. I have solved these problems using purified uPLB and pPLB standards, produced by solid-phase peptide synthesis, by performing two parallel immunoblots with antibodies partially specific for uPLB and pPLB, and deriving accurate equations for calculating Xp and tPLB. When this method was applied to mixtures of known composition, it measured both Xp and tPLB with ≥ 96% accuracy. I used this assay on samples of pig cardiac SR and found that Xp varied widely among four animals, from 0.08 to 0.38, but there was remarkably little variation in the ratios of Xp/tPLB and uPLB/SERCA, suggesting that PLB phosphorylation is tuned to maintain homeostasis in SERCA regulation. I have extended this method to measure accurately the mole fractions of PLB phosphorylated at Ser16, Thr17 and bisphospho-Ser16-Thr17 in biological samples, and to analyze the PLB phosphorylation status of cardiac tissue samples obtained from human patients with specific cardiomyopathies. This assay can be adapted to any phospho-protein, and with any other posttranslational modification where purified standards and partially specific antibodies are available.