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Protein kinase G modulates human myocardial passive stiffness by phosphorylation of the titin springs


The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin-isoform composition and protein kinase-A (PKA) dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left-ventricular extensibility are also altered by protein kinase-G (PKG) activation. We used back-phosphorylation assays to determine 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 and canine 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, serine residue S469 near the COOH-terminus of the cardiac N2-B-unique sequence (N2-Bus) was identified as a PKG and PKA 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; phosphorylation of other titin sites could affect protein-protein interactions.


The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure.

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Correspondence to Martina Krüger.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution 2.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Krüger, M., Kötter, S., Grützner, A. et al. Protein kinase G modulates human myocardial passive stiffness by phosphorylation of the titin springs. BMC Pharmacol 9 (Suppl 1), P37 (2009).

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