Abstract 1462: Titin Phosphorylation by Protein Kinases A and G in Normal and Failing Human Hearts Decreases Myocardial Passive Stiffness
Background: Left-ventricular (LV) function can be altered via increased protein kinase-A (PKA) activity following beta-adrenoceptor stimulation or by promoting the cGMP-dependent signaling pathway, which accelerates relaxation and reduces diastolic tone through activation of protein kinase-G (PKG). PKA was shown to phosphorylate the giant protein titin and decrease the passive stiffness of human donor heart tissue. Whether titin phosphorylation and titin stiffness can be regulated by PKG had been unknown.
Methods: We used 2% SDS-PAGE and autoradiography to study PKA and PKG mediated titin phosphorylation in skinned LV tissue of 6 human control donor and 8 end-stage failing non-ischemic DCM hearts (NYHA class III–IV, EF<30%). We also tested if recombinantly expressed segments from the elastic titin region can be phosphorylated by the two kinases. Further, we measured the passive force-sarcomere length (SL) relationships, employing skinned LV-fiber and isolated myofibril mechanics, to determine how titin phosphorylation affects passive stiffness.
Results: Autoradiographic analysis demonstrated that PKA and PKG phosphorylate the two main cardiac titin isoforms, N2B and N2BA, as well as the recombinantly expressed N2B-unique sequence, a cardiac-specific titin region; PKG produced relatively stronger phosphorylation signals than PKA. Dephosphorylation/back-phosphorylation assays showed high inherent PKA-dependent titin phosphorylation, but low levels of inherent PKG-dependent titin phosphorylation, in both DCM and non-failing hearts. Both kinases reduced passive stiffness to a similar degree, on average by 15–20%, but showing substantial variability among hearts, in control donor and DCM myocardium.
Conclusions: The cardiac-specific N2B-region of human titin is established as a novel target for PKG-mediated phosphorylation. Similar to PKA, PKG phosphorylates titin in human donor and DCM hearts and significantly reduces titin-based stiffness. Altered inherent titin-phosphorylation levels could partly account for the alterations in passive LV wall stiffness observed in failing human hearts. The PKG-mediated reduction in titin stiffness could be exploited therapeutically to improve diastolic heart function.