Microanalysis and distribution of cardiac troponin I phospho species in heart areas

Sequential phosphorylation and dephosphorylation of cTnI by the cAMP dependent protein kinase and by protein phosphatase 2A, respectively, produce the non-, mono- and bisphosphorylated species (Jaquet et al., 1995, Eur. J. Biochem. 231, 486-490). The aim of this study was to determine these forms even in small tissue samples, e.g. in biopsy probes of approximately 30 mg which would allow to define the phosphorylation state of cTnI in heart areas. In order to do so a micro isolation procedure for cTnI had to be established. cTnI is extracted from small bovine, rabbit and human heart tissue samples (30-100 mg) under special conditions avoiding dephosphorylation and is isolated by affinity chromatography on cTnC Sepharose. All three species, the bis-, mono- and dephospho cTnI, are precipitated quantitatively by acetone, then they are separated by non-equilibrium isoelectric focusing and quantified by scanning densitometry. The method presented here allows to quantify the three cTnI species reproducibly. No other phosphorylated species are detected. Truncated cTnI forms of each phospho species are found in human biopsy samples due to removal of a approximately 36 amino acid peptide from the C-terminus. In bovine, human and rabbit heart the pattern of the three cTnI phospho species is characteristic for left and right atrium, left and right ventricle and septum.