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

Martina Krüger, Sebastian Kötter, Anika Grützner, Patrick Lang, Christian Andresen, Margaret May Redfield, Elke Butt, Cris G. Dos Remedios, Wolfgang A. Linke

Research output: Contribution to journalArticle

216 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)87-94
Number of pages8
JournalCirculation Research
Volume104
Issue number1
DOIs
StatePublished - Jan 2 2009

Fingerprint

Connectin
Cyclic GMP-Dependent Protein Kinases
Phosphorylation
Myofibrils
Protein Isoforms
Phosphoserine
Atomic Force Microscopy
Site-Directed Mutagenesis
Cyclic AMP-Dependent Protein Kinases
Autoradiography
Serine
Heart Ventricles

Keywords

  • CGMP
  • Connectin
  • Diastolic function
  • Nitric oxide
  • Passive tension

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Protein kinase G modulates human myocardial passive stiffness by phosphorylation of the titin springs. / Krüger, Martina; Kötter, Sebastian; Grützner, Anika; Lang, Patrick; Andresen, Christian; Redfield, Margaret May; Butt, Elke; Dos Remedios, Cris G.; Linke, Wolfgang A.

In: Circulation Research, Vol. 104, No. 1, 02.01.2009, p. 87-94.

Research output: Contribution to journalArticle

Krüger, M, Kötter, S, Grützner, A, Lang, P, Andresen, C, Redfield, MM, Butt, E, Dos Remedios, CG & Linke, WA 2009, 'Protein kinase G modulates human myocardial passive stiffness by phosphorylation of the titin springs', Circulation Research, vol. 104, no. 1, pp. 87-94. https://doi.org/10.1161/CIRCRESAHA.108.184408
Krüger, Martina ; Kötter, Sebastian ; Grützner, Anika ; Lang, Patrick ; Andresen, Christian ; Redfield, Margaret May ; Butt, Elke ; Dos Remedios, Cris G. ; Linke, Wolfgang A. / Protein kinase G modulates human myocardial passive stiffness by phosphorylation of the titin springs. In: Circulation Research. 2009 ; Vol. 104, No. 1. pp. 87-94.
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