Background: 

Myofiber passive stiffness is lowered by phosphorylation of the giant sarcomeric protein titin, with beneficial effects on diastolic function. Titin can be phosphorylated by cGMP-activated protein kinase (PK)G, a pathway stimulated by B-type natriuretic peptide (BNP) or PDE-5A inhibitor (sildenafil). Whether titin phosphorylation and stiffness are affected by PKG activation in vivo had not been studied. Here we examined how dogs with experimental hypertension and diastolic dysfunction, induced by renal wrapping, respond to treatment with b-blockers, sildenafil, and BNP, in terms of altered titin phosphorylation and passive stiffness.

Methods and Results: 

Isolated permeabilized cardiomyocytes from left ventricular (LV) biopsies untreated (n=5), treated with b-blockers (n=7), followed by sildenafil (n=7) and BNP (n=7) were attached to a force transducer and the passive length tension relation (F_passive) was measured between 1.8 and 2.4mm sarcomere length (SL) before and after PKA and PKG administration. We also assessed phosphorylation of myofilamentary proteins including titin, cMyBP-C, desmin, cTnT, cTnI and MLC2 by gel electrophoresis using SYPRO Ruby (total-protein) and ProQ Diamond (phospho-protein) stain and expression of titin PEVK, phospho-PEVK, cTnI, phospho-cTnI (at Ser 23/24), PKCa and phospho-PKCa by Western blotting. Compared to untreated cardiomyocytes, F_passive was unaltered with b-blocker therapy, but significantly reduced with sildenafil; with BNP F_passive did not change further. Ex-vivo administration of PKG lowered F_passive of untreated and b-blocker cardiomyocytes to the levels measured in sildenafil and BNP samples, and additional administration of PKA did not further change F_passive. Total titin phosphorylation was low in untreated samples and with b-blockade, but significantly increased with sildenafil and BNP. Total cTnI phosphorylation and phospho-cTnI at Ser 23/24 were low in untreated compared to treated samples but were similar between b-blocker, sildenafil and BNP groups. Phospho-PEVK and phospho-PKCa were high in untreated samples and with b-blockade, but reduced with sildenafil and BNP.

Conclusions: 

Acute cGMP enhancing therapy with sildenafil and BNP improves LV diastolic function through correction of a titin phosphorylation deficit, thereby reducing cardiomyocyte passive stiffness.