Protein kinase G modulates human myocardial passive stiffness by phosphorylation of the titin springs.
Circ Res
; 104(1): 87-94, 2009 Jan 02.
Article
em En
| MEDLINE
| ID: mdl-19023132
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Quinases
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Processamento de Proteína Pós-Traducional
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Proteínas Quinases Dependentes de GMP Cíclico
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Insuficiência Cardíaca Diastólica
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Ventrículos do Coração
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Proteínas Musculares
Tipo de estudo:
Prognostic_studies
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2009
Tipo de documento:
Article