QUESTION: Myocardial diastolic stiffness is dependent, in part, on signaling pathways and phosphorylation. Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII) δ and protein kinase G (PKG) are known to target titin, but it is unknown so far if PKG phosphorylates CaMKIIδ.

METHODS: Left ventricular (LV) endomyocardial biopsy samples were procured in patients with heart failure and preserved LV ejection fraction (HFpEF) and from control subjects. CaMKIIδ phosphorylation by PKG was assessed in recombinant proteins and HFpEF biopsies by autoradiography, immunoblotting and quantified in vivo by mass spectrometry (MS). To measure interaction of CaMKII with PKG, HEK293 cells were transfected with PKG and CaMKII. Biopsies were used for in vivo and recombinant CaMKIIδ for in vitro phosphorylation with PKG and subsequent MS. CaMKII oxidation was examined by immunoblotting. Acute stimulation of sGC (with BAY 41-8543) was studied on mRen2 rats characterized with diastolic dysfunction (HFpEF) and control rats. PKG mediated-CaMKII phosphorylation was studied on stimulated Ca2+ transients using confocal microscopy (Fluo-4).

RESULTS: HFpEF patients were characterized with high diastolic stiffness, increased interstitial fibrosis and elevated cardiomyocytes passive stiffness. HFpEF patients showed unchanged CaMKIIδ expression level and increased phosphorylation and oxidation compared to controls. The in vitro phosphorylation of CaMKIIδ by PKG and MS analysis revealed several phosphosites on CaMKIIδ. However, the most highly phosphorylated sites were located in the regulatory domain and the linker region of CaMKII. The luminescense signal was decreased in HEK293 cells after acute treatment with sGC stimulator, indicating association and dissociation of PKG from CaMKII. CNP resulted in a significant change in FCFP/FYFP of CaMKII compared to baseline. PKG inhibitor (Rp-8-Br-PET-cGMPS) inhibits completely CaMKII activity. Significant correlations between the loss of PKG of each individual (patient) and increased CaMKII activity and oxidative stress in the same individual was observed. Acute intravenous injection of PKG stimulator in anaesthetized HFpEF rats significantly improved LV diastolic dysfunction evident from improved ratio of peak velocity blood flow from gravity in early diastole to peak velocity flow in late diastole caused by atrial contraction, isovolumic relaxation time and time constant of isovolumetric pressure decline. In addition, the acute stimulation showed improved LV end-diastolic pressure and stiffness in HFpEF rats. sGC stimulation improved further cGMP-PKG pathway via increased cGMP concentration and PKG activity. Also, we found reduced CaMKIIδ auto-phosphorylation and oxidation, in addition to reduced oxidative stress and inflammation via reducing the pro-inflammatory cytokines. Cellular sGC stimulation caused a significant increase in Ca2+ transient amplitude in wild type (WT) mice but not in CaMKIIδc knock-out mice suggesting that the effects of sGC on calcium transient amplitude might be CaMKII dependent. In addition, sGC treatment in WT cardiomyocytes showed significant higher calcium transient amplitude compared to sGC treated cells from CaMKIIδc, suggesting the important role of PKG mediated-CaMKII phosphorylation in regulating calcium handling.