Cardiac Troponin I Phosphorylation at ser149 by Protein Kinase A: A Potential Modulator of Myocardial Contractility

Abstract

In the heart, stimulation of β-adrenergic pathway and subsequent activation of protein kinase A (PKA) is known to increase myocardial contractility. The increase in contractility is, in part, due to target phosphorylation of troponin I (TnI). In this study, we sought to identify novel target sites for PKA that could potentially contribute to this increase in contractility. To induce phosphorylation of TnI, cardiac and fast skeletal muscle from 3-4 month old Sprague Dawley rats was mechanically disrupted and demembranated followed by incubation with the catalytic subunit of PKA (50U PKA/ 3mg tissue, 0-30 min). To identify target specific phosphorylation on fast skeletal (fsTnI) or cardiac (cTnI) TnI, western blot analysis with phospho-specific antibodies was performed. PKA treatment increased phosphorylation of cTnI at ser22/23, as expected, but also at ser149. Similarly, PKA treatment increased phosphorylation of fsTnI at ser117, which is the equivalent to ser149 in cTnI. Accordingly, fsTnI demonstrated no observable phosphorylation at ser22/23. Adenosine-monophosphate activated kinase (AMPK) has been shown to target ser149 of cTnI. Therefore, to validate PKA-dependent phosphorylation of cTnI at ser149, hearts were excised and perfused with AICAR, a known activator of AMPK. AICAR-perfused hearts demonstrated a time-dependent increase in phosphorylation of cTnI at ser149. These results demonstrate that PKA-dependent phosphorylation can target ser149 in cTnI and, equivalently, ser117 in fsTnI. The functional consequence of this target site phosphorylation and how it impacts contractility is currently under investigation.