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 elderly dogs with experimental hypertension and diastolic dysfunction induced by renal wrapping respond to treatment with beta-blockers, sildenafil, and BNP, in terms of altered titin phosphorylation and passive stiffness.

Methods: 

Phosphorylation of titin isoforms was studied by gel electrophoresis using SYPRO-Ruby (total-protein) and Pro-Q-Diamond (phospho-protein) stain and was reported as ratio of phosphorylated titin isoforms (P-N2BA/P-N2B) and as total titin phosphorylation. Isolated permeabilized myocytes from left ventricular (LV) tissue samples were attached to a force transducer and resting tension (F_passive) was measured over a physiological sarcomere length (SL) range. Measurements were done on LV samples of healthy control (n=8) and diastolic heart failure (DHF) dogs (n=8), as well as LV biopsies of DHF dogs treated with beta-blocker (n=4) followed by sildenafil (n=4) and BNP (n=4).

Results: 

The P-N2BA/P-N2B ratio was low in controls (1.4±0.2), increased in DHF (3.7±0.8), remained high after beta-blockade (3.1±0.3), but significantly decreased in sildenafil (1.8±0.1) and BNP-treated (1.9±0.1) dogs. Total-titin phosphorylation was low in DHF (0.40±0.01a.u.) and beta-blocker-treated (0.30±0.08a.u.) hearts, but significantly increased in sildenafil-administered dogs (0.80±0.08a.u.) and remained high with BNP (0.80±0.06a.u.). Baseline F_passive at 2.2mm SL was low in control myocytes (3.7±0.4kN/m²), elevated in DHF (7.3±1.0kN/m²) and after beta-blockade (7.8±1.0kN/m²), but returned to control levels with sildenafil (3.8±0.3kN/m²), where it was maintained with BNP (4.0±0.3kN/m²).

Conclusion: 

Acute cGMP-enhancing therapy with sildenafil and BNP improves LV diastolic function through correction of a titin-phosphorylation deficit, particularly of the stiff N2B-titin isoform, thereby reducing myocyte passive stiffness.