Impact of cGMP-PKG Pathway Modulation on Titin Phosphorylation and Titin-Based Myocardial Passive Stiffness

Abstract

RATIONALE: The crucial contribution of the giant myofilament protein titin to diastolic stiffness and cardiomyocyte passive force (Fpassive) is dependent, in part, on titin isoform composition and phosphorylation. Phosphorylation of titin by cyclic guanosine monophosphate (cGMP)-dependent protein kinase G (PKG) lowers titin-based stiffness, thus mediating a mechanical signaling process that is disturbed in heart failure. OBJECTIVE: To elucidate which elements of the nitric oxide (NO) cGMP-PKG signaling network are critical for titin phosphorylation and stiffness in vivo. METHODS AND RESULTS: We employed genetic knockout (KO) mouse models deficient for enzymes of the cGMP-PKG pathway, including cardiomyocyte-specific deletion of the guanylyl cyclase (GC)-A receptor and cGMP-dependent PKG (cGKI), as well as global deletion of soluble GC (sGC). We assessed titin phosphorylation by immunoblotting using phosphoserine-specific titin antibodies and by mass spectrometry quantification using stable isotope labeling of amino acids in mixed cultures of heart tissue from either wild-type (WT) or KO mice. The Fpassive of single permeabilized cardiomyocytes was recorded before and after administration of PKG. In all three genetic models, all-titin phosphorylation was reduced compared to WT hearts. The important PKG-dependent phospho-S4080 site within the N2-Bus region of mouse titin was hypophosphorylated in all three KO models. Unexpectedly, mass spectrometry analysis revealed that most class 1 titin phospho-sites within the molecular spring segment, including the Ig-domain regions, were hyperphosphorylated. Only a few sites showed a phosphorylation deficit or remaining unchanged. Particularly in the cGKI model many class 1 phospho-sites were hyperphosphorylated compared to WT hearts, indicative of the presence of compensatory processes following loss of PKG; indeed, this was associated with upregulation of several kinases that phosphorylate titin, including CaMKII, ERK2 and PKCα and with a clear rise in Fpassive in KO vs. WT cardiomyocytes. We also found an upregulation of phosphodiesterase 3, but downregulation of phosphodiesterase 9a, which regulates natriuretic peptides rather than nitric oxide-stimulated cGMP in heart myocytes. While administration of PKG lowered Fpassive of WT and KO cardiomyocytes in all models, this effect was more pronounced in the cGKI KO. CONCLUSIONS: Multiple in vivo phosphorylated class I titin phospho-sites were identified within the molecular spring segment, some of which depended on the cGMP-PKG pathway. While cGMP-activated PKG remains an important titin-targeting kinase, many titin phospho-sites may be regulated through a network of protein kinases/phosphatases.