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 cGMP-PKG pathway enzymes, including cardiomyocyte-specific deletion of the guanylyl cyclase(GC)-A receptor and cGMP-dependent-PKG(cGKI), and global deletion of soluble GC(sGC). We assessed titin phosphorylation and Fpassive of single permeabilized cardiomyocytes recorded before/after PKG administration. In all three models, all-titin phosphorylation was reduced compared to WT hearts. The important PKG-dependent phospho-S4080 site within titin-N2-Bus 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 and a clear rise in Fpassive in KO vs. WT cardiomyocytes. 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.