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 (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.
Krüger
Martina
M
Physiology and Biophysics Unit, University of Muenster, Germany.
Kötter
Sebastian
S
Grützner
Anika
A
Lang
Patrick
P
Andresen
Christian
C
Redfield
Margaret M
MM
Butt
Elke
E
dos Remedios
Cris G
CG
Linke
Wolfgang A
WA
D000595:Amino Acid Sequence; D000818:Animals; D064211:Connectin; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D006152:Cyclic GMP / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology*; D004285:Dogs; D004548:Elasticity; D054144:Heart Failure, Diastolic / Q000378:metabolism*; D006352:Heart Ventricles / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D009210:Myofibrils / Q000187:drug effects / Q000648:ultrastructure; D009569:Nitric Oxide / Q000502:physiology; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012518:Sarcomeres / Q000378:metabolism / Q000648:ultrastructure; D013329:Structure-Activity Relationship; D020257:Ventricular Remodeling / Q000502:physiology
D064211:Connectin; D009124:Muscle Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C577285:TTN protein, human; D009569:Nitric Oxide; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP