Hamdani, Nazha

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 β-blockers, sildenafil, and BNP, in terms of altered titin phosphorylation and passive stiffness. Isolated permeabilized cardiomyocytes from left ventricular (LV) biopsies untreated (n=5), treated with β-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 pas ) was measured between 1.8 and 2.4µm sarcomere length (SL) before and after PKA and PKG administration. We also assessed phosphorylation of myofilament proteins including titin, cMyBPC, desmin, cTnT, cTnI and MLC2 by gel electrophoresis (SYPRO Ruby and ProQ Diamond stain) and expression of titin PEVK, phospho-PEVK, cTnI, phospho-cTnI (at Ser 23/24), PKCα and phospho-PKCα by Western blotting. Compared to untreated cardiomyocytes, F pas was unaltered with β-blocker therapy, but significantly reduced with sildenafil; with BNP F pas did not change further. Ex-vivo administration of PKG lowered F pas of untreated and β-blocker cardiomyocytes to the levels measured in sildenafil and BNP samples, and additional administration of PKA did not further change F pas . Total titin phosphorylation was low in untreated samples and with β-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 β-blocker, sildenafil and BNP groups. Phospho-PEVK and phospho-PKCα were high in untreated samples and with β-blockade, but reduced with sildenafil and BNP. Acute cGMP enhancing therapy with sildenafil and BNP improves LV diastolic function through correction of a titin phosphorylation deficit, thereby reducing cardiomyocyte passive stiffness.

Background— In vitro studies suggest that phosphorylation of titin reduces myocyte/myofiber stiffness. Titin can be phosphorylated by cGMP-activated protein kinase. Intracellular cGMP production is stimulated by B-type natriuretic peptide (BNP) and degraded by phosphodiesterases, including phosphodiesterase-5A. We hypothesized that a phosphodiesterase-5A inhibitor (sildenafil) alone or in combination with BNP would increase left ventricular diastolic distensibility by phosphorylating titin. Methods and Results— Eight elderly dogs with experimental hypertension and 4 young normal dogs underwent measurement of the end-diastolic pressure-volume relationship during caval occlusion at baseline, after sildenafil, and BNP infusion. To assess diastolic distensibility independently of load/extrinsic forces, the end-diastolic volume at a common end-diastolic pressure on the sequential end-diastolic pressure-volume relationships was measured (left ventricular capacitance). In a separate group of dogs (n=7 old hypertensive and 7 young normal), serial full-thickness left ventricular biopsies were harvested from the beating heart during identical infusions to measure myofilament protein phosphorylation. Plasma cGMP increased with sildenafil and further with BNP (7.31±2.37 to 26.9±10.3 to 70.3±8.1 pmol/mL; P <0.001). Left ventricular diastolic capacitance increased with sildenafil and further with BNP (51.4±16.9 to 53.7±16.8 to 60.0±19.4 mL; P <0.001). Changes were similar in old hypertensive and young normal dogs. There were no effects on phosphorylation of troponin I, troponin T, phospholamban, or myosin light chain-1 or -2. Titin phosphorylation increased with sildenafil and BNP, whereas titin-based cardiomyocyte stiffness decreased. Conclusion— Short-term cGMP-enhancing treatment with sildenafil and BNP improves left ventricular diastolic distensibility in vivo, in part by phosphorylating titin.