Published online before print November 20, 2008, doi: 10.1161/CIRCRESAHA.108.184408
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Submitted on July 29, 2008
Revised on October 21, 2008
Accepted on November 6, 2008
Protein Kinase G Modulates Human Myocardial Passive Stiffness by Phosphorylation of the Titin Springs
Martina Krüger ;
From the Physiology and Biophysics Unit (M.K., S.K., A.G., P.L., C.A., W.A.L.), University of Muenster, Germany; Mayo Foundation (M.M.R.), Rochester, Minn; Institute of Clinical Biochemistry and Pathobiochemistry (E.B.), University of Wurzburg, Germany; and Muscle Research Unit (C.G.d.R.), Bosch Institute, University of Sydney, Australia.
* To whom correspondence should be addressed. E-mail: wlinke{at}uni-muenster.de.
The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by 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 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. 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 atomic force microscopy 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.
Key words: cGMP • nitric oxide • diastolic function • connectin • passive tension
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