Abstract 14966: Cyclic GMP Enhancing Therapy Promotes Titin Phosphorylation and Corrects High Cardiomyocyte Passive Stiffness in Diastolic Heart Failure
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 PDE5A 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 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 ProQ 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 passive tension (Fpassive) was measured between 1.8 and 2.4μm sarcomere length (SL). Cells were obtained from 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, propranolol (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-blockers (3.1±0.3), but significantly decreased with sildenafil (1.8±0.1) and BNP (1.9±0.1). Total titin phosphorylation was low in DHF (0.40±0.01a.u.) and beta-blocker-treated (0.30±0.08a.u.) hearts, significantly increased in sildenafil-treated dogs (0.80±0.08a.u.), and remained high with BNP (0.80±0.06a.u.). Fpassive at 2.2μm SL was low in control myocytes (3.7±0.4kN/m2), elevated in DHF (7.3±1.0kN/m2) and after beta-blockers (7.8±1.0kN/m2), but returned to control levels with sildenafil (3.8±0.3kN/m2) and remained low with BNP (4.0±0.3kN/m2).
Conclusions: 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.
- © 2010 by American Heart Association, Inc.