Multiple Reaction Monitoring to Identify Site-Specific Troponin I Phosphorylated Residues in the Failing Human Heart

Abstract

Background Human cardiac troponin I is known to be phosphorylated at multiple amino acid residues by several kinases. Advances in mass spectrometry allow sensitive detection of known and novel phosphorylation sites and measurement of the level of phosphorylation simultaneously at each site in myocardial samples. Methods and Results On the basis of in silico prediction and liquid chromatography/mass spectrometry data, 14 phosphorylation sites on cardiac troponin I, including 6 novel residues (S4, S5, Y25, T50, T180, S198), were assessed in explanted hearts from end-stage heart failure transplantation patients with ischemic heart disease or idiopathic dilated cardiomyopathy and compared with samples obtained from nonfailing donor hearts (n=10 per group). Thirty mass spectrometry–based multiple reaction monitoring quantitative tryptic peptide assays were developed for each phosphorylatable and corresponding nonphosphorylated site. The results show that in heart failure there is a decrease in the extent of phosphorylation of the known protein kinase A sites (S22, S23) and other newly discovered phosphorylation sites located in the N-terminal extension of cardiac troponin I (S4, S5, Y25), an increase in phosphorylation of the protein kinase C sites (S41, S43, T142), and an increase in phosphorylation of the IT-arm domain residues (S76, T77) and C-terminal domain novel phosphorylation sites of cardiac troponin I (S165, T180, S198). In a canine dyssynchronous heart failure model, enhanced phosphorylation at 3 novel sites was found to decline toward control after resynchronization therapy. Conclusions Selective, functionally significant phosphorylation alterations occurred on individual residues of cardiac troponin I in heart failure, likely reflecting an imbalance in kinase/phosphatase activity. Such changes can be reversed by cardiac resynchronization.

Length-dependent activation is modulated by cardiac troponin I bisphosphorylation at Ser23 and Ser24 but not by Thr143 phosphorylation

“…Tachampa et al (33) showed that substitution of a single amino acid at position 143 of cTnI (from Thr to Pro) greatly diminished the lengthdependent increase in myofilament Ca 2ϩ sensitivity. Since Thr143 is a well-known PKC-mediated phosphorylation site that has been implicated in heart failure (25,32,43), we investigated if phosphorylation of Thr143 may modify LDA of the myofilaments in human cardiomyocytes. We show that pseudo-phosphorylation of Thr143 does not affect the lengthdependent increase in maximal force and Ca 2ϩ sensitivity irrespective of the level of cTnI phosphorylation at PKA sites.…”

“…Our data in donor cardiomyocytes confirm a previous study (16) that indirectly demonstrated that phosphorylation of cTnI-Ser23/24 enhances the increase in Ca 2ϩ sensitivity upon an increase in sarcomere length. However, while monophosphorylation of Ser23 or Ser24 has been reported in the human heart (42,43), no functional effect of monophosphorylation has been found (39,44). Therefore, we investigated if monophosphorylation of Ser23 or Ser24 would affect LDA of the myofilaments.…”

“…Interestingly, Thr143 is a well-known target of protein kinase C (PKC), and its phosphorylation has been implicated in heart failure (25,32,43) where an increase in PKC-mediated cTnI phosphorylation has been found together with a decreased phosphorylation of the protein kinase A (PKA) sites (43). PKC-mediated Thr143 phosphoryation may, either alone or in concert with downregulated PKA-mediated phosphorylation, underlie changes in LDA in the heart.…”

Cellular and Molecular Aspects of Dyssynchrony and Resynchronization

“…This is opposite that observed with synchronous heart failure, which increases calcium sensitivity in humans [69] and canines [68,70], changes thought due to a decline of PKA-phosphorylation of cardiac troponin I (TnI) at serines 22 and 23 [71]. This over-sensitization can result in diastolic dysfunction [72] and arrhythmias [73].…”

Transmural Heterogeneity of Cellular Level Power Output Is Reduced in Human Heart Failure

“…Total cTnI phosphorylation was not correlated with pCa 50 either (r=−0.011, p=0.942, data not shown). cTnI undergoes many complex posttranslational modifications -the human cardiac isoform has 14 phosphorylation sites [24]. It is therefore possible that more detailed analysis might have revealed significant relationships.…”