Protein Kinase G Modulates Human Myocardial Passive Stiffness By Phosphorylation Of The Titin Springs

Abstract

The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin-isoform composition and protein kinase-A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone is lowered, left ventricular extensibility is increased, and relaxation is accelerated also by activating protein kinase-G (PKG). Here we studied using back-phosphorylation assays whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the two main cardiac titin isoforms, N2BA and N2B, in human donor and dog left ventricle. In human myofibers/myofibrils dephosphorylated prior to mechanical analysis, passive stiffness dropped 10-20% upon application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band-titin domains established that PKG phosphorylates titin's N2-B and N2-A domains. Using site-directed mutagenesis, a serine residue near the COOH-terminus of the cardiac N2-B-unique sequence (N2-Bus) was identified as a PKG-phosphorylation site. To address the mechanism of the PKG-effect on titin stiffness, single-molecule AFM force-extension experiments were performed on engineered N2-Bus-containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus, whereas phosphorylation of other titin sites could affect protein-protein interactions. Results suggest that reducing titin stiffness through PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Since failing human hearts revealed lower PKG-mediated basal titin phosphorylation than donor hearts, titin-phosphorylation deficits may contribute to diastolic dysfunction in heart failure.