The use of phosphate‐affinity SDS‐PAGE to measure the cardiac troponin I phosphorylation site distribution in human heart muscle

Abstract

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.

Molecular Mechanism of the E99K Mutation in Cardiac Actin (ACTC Gene) That Causes Apical Hypertrophy in Man and Mouse

“…These septal samples are characterized by a low level of phosphorylation of MyBP-C and troponin I (29,30,(55)(56)(57). In contrast, the two samples examined here, from free wall and from the atrial-septal junction region, showed high levels of phosphorylation similar to those in donor hearts (Fig.…”

Impact of site-specific phosphorylation of protein kinase A sites Ser23and Ser24of cardiac troponin I in human cardiomyocytes

“…phosphorylated, cTnI-DA mimics Ser 23 phosphorylated and Ser 24 unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca 2ϩ concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes.…”

“…This positive lusitropic effect may be induced by a decrease in myofilament Ca 2ϩ sensitivity (32,35,51) and by enhanced cross-bridge cycling kinetics (11,20,33). It is well established (mainly from studies in rodents) that phosphorylation of cTnI at the PKA sites Ser 23 and Ser 24 leads to a decrease in myofilament Ca 2ϩ sensitivity, through a conformational change of the troponin complex. This structural change reduces the affinity of Ca 2ϩ binding to cTnC (16,30).…”

“…The collective effects are important for in vivo function, but to understand the effects in both healthy and diseased myocardium, information is required of each of the contributing factors. Finally, theoretically, four different PKA-phosphorylated cTnI forms could coexist: one unphosphorylated, two monophosphorylated (either on Ser 23 or Ser 24 ), and one bisphosphorylated (18,34). A previous study (52) in rodents suggested that bisphosphorylation of cTnI is required for the reduction in myofilament Ca 2ϩ sensitivity, but knowledge of the functional consequences of monophosphorylated cTnI also is important since recent stud-ies (48,50) in human postmortem control hearts and fresh donor transplant hearts with normal cardiac function revealed that ϳ40% of cTnI is monophosphorylated at Ser 23 .…”

“…These cTn complexes contained unphosphorylated PKA sites, pseudo-monophosphorylated cTnI, and pseudo-bisphosphorylated cTnI. Ser 23 and/or Ser 24 were mutated into aspartic acid and into alanine to mimic phosphorylation and dephosphorylation, respectively. This approach allowed us to study the effect of cTnI phosphorylation on active [maximal force (F max )] and passive isometric force (F pas ), the force-Ca 2ϩ relation, and the rate of force redevelopment (k tr ; a measure of cross-bridge kinetics).…”