Aim: 

The passive properties of the giant protein titin (>3 MDa) are defined by the expression ratio of the two cardiac isoforms N2BA and N2B in the sarcomere, and can be dynamically modulated via phosphorylation by cAMP or cGMP-dependent protein kinases (PKA, PKG). In cultured embryonic rat cardiomyocytes (ECM) titin phosphorylation is significantly increased upon insulin treatment. Since insulin is known to activate PKG in an NO-dependent manner, we tested whether PKG is responsible for the insulin-induced changes in titin phosphorylation.

Methods: 

Cultured embryonic rat cardiomyocytes (ECMs) were treated with insulin (175 nM) for 5–60 min in the absence or presence of specific inhibitors of the NO-dependent PKG-pathway. Titin phosphorylation was analyzed in 2% SDS PAGE using the phosphoprotein stain ProQ-Diamond. Activation of PKG was confirmed by detecting phosphorylation of its target protein vasodilator-stimulated phosphoprotein (VASP).

Results: 

Titin phosphorylation reached a maximum after 15 min of insulin treatment and was increased 1.9-fold for N2BA-titin and 1.6-fold for N2B-titin compared to untreated ECMs. The insulin-induced effect on titin phosphorylation was prevented by inhibition of nitric oxide synthase (L-NAME 100 mM), guanylate cyclase (ODQ 10 mM) or PKG (Rp-8-PET-Br-cGMP 100 nM).

Conclusion: 

We conclude that insulin modulates PKG-dependent titin phosphorylation by activating the nitric oxide-sensitive PKG pathway, and thereby modify titin-based passive stiffness.