The use of phosphate-affinity SDS-PAGE to measure the cardiac troponin I phosphorylation site distribution in human heart muscle.
We have used phosphate affinity SDS-PAGE to separate the phosphorylated species of cardiac troponin I (cTnI). To test the method we phosphorylated pure cTnI with protein kinase A catalytic subunit and observed up to six bands corresponding to 0, 1P, 2P, 3P, 4P and 5P phospho-species. We examined the phospho-species of cTnI in human heart myofibrillar extracts by phosphate affinity SDS-PAGE and Western blotting with a non-specific troponin I (TnI) antibody. In donor heart samples the bis-phosphorylated species of cTnI predominated and no more highly phosphorylated species were not detectable (0P was 10.3±1.9%, 1P, 17.5±3.5%, 2P, 72.2±4.7%, 11 samples). Total phosphorylation was 1.62±0.06 molsPi/mol TnI. In myofibrils from end-stage failing hearts, the unphosphorylated cTnI species predominated (0P was 78.5±1.8%, 1P, 17.5±1.9%, 2P, 4.0±0.7%, total phosphorylation 0.26±0.02 molsPi/mol TnI, five samples). Muscle from patients with hypertrophic obstructive cardiomyopathy was also largely unphosphorylated (0P was 76.6±3.1%, 1P, 17.5±2.7%, 2P, 5.9±0.8%, total phosphorylation 0.29±0.04 molsPi/mol TnI, 19 samples). Using a range of phospho-specific antibodies we demonstrated that 3/4 of the bis-phosphorylated band of donor heart cTnI is phosphorylated at Ser22 and Ser23 in approximately equal amounts and that phosphorylation of Ser43 and Thr142 was not detected.
National Heart and Lung Institute, Imperial College London, London, UK. a.messer@imperial.ac.uk.
Andrew E
AE
Messer
Clare E
CE
Gallon
William J
WJ
McKenna
Cristobal G
CG
Dos Remedios
Steven B
SB
Marston