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.