Abstract 1478: AMP-Activated Protein Kinase Phosphorylates Cardiac Troponin I and Alters Contractility in Murine Ventricular Myocytes
In the heart, AMP-activated protein kinase (AMPK) is critical in the regulation of energy balance and in myocardial signaling, and is activated especially during ischaemia and exercise. It is a αβγ heterotrimer, with α being the catalytic subunit. Mutations affecting the regulatory γ2 subunit have been shown to cause a cardiac phenotype of hypertrophy and conduction disease, thus suggesting a specific role for this subunit in the heart. The γ isoforms are highly conserved at their C-termini but very different at their N-termini; hence we have investigated whether the unique N-terminus of γ2 could be involved in conferring substrate specificity or in determining intracellular localization. We have undertaken a GAL4-based yeast two-hybrid assay to screen a human heart cDNA library using the N-terminal 273 residues of γ2 as bait. Five proteins were identified as true interactors in the yeast assay, one of these being cardiac troponin I (cTnI). In vitro studies showed that cTnI (isolated or reconstituted as troponin complex) is a good substrate for AMPK. Studies using site-specific cTnI mutants and mass spectrometry identified Ser-150 as the principal residue phosphorylated by AMPK. Actomyosin ATPase assays showed that phosphorylation at this residue resulted in increased Ca2+ sensitivity of contractile regulation (ΔpCa50=+0.07). Treatment of cardiomyocytes with the AMPK activator AICAR for 15 min resulted in prolonged relaxation (time to 50% relaxation, TR50) in field stimulated left ventricular myocytes of C57B/6 mice (TR50 in ms: 29.8±1.4 in control and 38.0±2.4 with AICAR, n=11, 3 Hz and 35oC). There was a tendency towards an increase in contraction with AICAR. Pretreatment of myocytes with AMPK inhibitor compound C (10 μM) abolished the effects of AICAR on myocytes (TR50 in ms: 30.9±1.7 before AICAR and 31.5±2.6 after AICAR, n=9). The effect of AICAR was mediated without altering the amplitude of the Ca2+ transient, suggesting that it may be caused by a change in myofilament Ca2+-sensitivity and consistent with AMPK phosphorylation of cTnI. We hypothesize that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.