Analysis of Cardiac Myofibrillar Troponin I Phosphorylation in Normal and Failing Human Hearts Using Phos-Tags

Recently, we have used phosphate-affinity SDS-PAGE gels containing Phos-tag-acrylamide (a phosphate-chelating molecule), to determine the level of cardiac troponin I (cTnI) phosphorylation in human myofibrillar extracts. The Phos-tag moiety binds to, and retards, the mobility of phosphoproteins through the gel and results in the separation of the phosphoprotein bands according to their phosphorylation level.Samples from time-courses of in-vitro PKA catalytic subunit-treated recombinant human cTnI and myofibrillar extracts from non-failing donor, hypertrophic obstructive cardiomyopathy (HOCM) and end-stage failing human heart tissue were analysed by phosphate-affinity SDS-PAGE. Separate gel bands corresponding to 1P, 2P, 3P, 4P and 5P cTnI were observed for the PKA-treated recombinant cTnI. Western blotting probed with the anti-cTnI antibody 14G5 and several different site specific phospho-cTnI antibodies demonstrated that all five of these phospho-species bound to a Ser24P-specific antibody, while a Thr144P-specific antibody only reacted with the 3P, 4P and 5P phospho-species of cTnI.We observed 3 phospho-species of cTnI in the human heart tissue extracts, which correspond to 0P, 1P and 2P cTnI. Ratios of 0P cTnI were significantly higher in failing and HOCM (both 63±4%) compared to donor (8±2%) while ratios of 2P were significantly lower (failing = 6±2%, HOCM = 8±2%, donor 73±6%). Western blots demonstrated that in human heart cTnI phosphorylation of Ser23/24 was mainly present in the 2P species (with a very small proportion in the 1P species) and that there was no phosphorylation at Thr144. Calculated levels of total cTnI phosphorylation in both HOCM (0.37±0.03, n=50, p<0.0001) and failing heart (0.38±0.03, n=24, p<0.0001) were significantly reduced from levels in donor heart (1.65±0.04, n=38) and were comparable to previously determined measurements obtained from Pro-Q Diamond phosphoprotein gel staining.