Sildenafil and B-type natriuretic peptide acutely phosphorylate titin and improve diastolic distensibility in vivo

Abstract

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.

Figure 1. End-Diastolic and End-Systolic Pressure Volume Relationships

Top panels show representative EDPVRs from an OH and a YN dog, defined during acute caval occlusion at baseline and after sequential treatment with sildenafil and BNP. The dotted line shows point of intersection of each curve with mid-LVEDP of PVR at baseline. The lower panels show ESPVRs from an OH and a YN dog, at baseline and after sequential treatment with sildenafil and BNP. The dotted line shows point of intersection of each curve with mid-LVESP of PVR at baseline. There is a rightward shift in the PVRs with serial therapy.

Figure 2. Diastolic and Systolic Capacitance

Left panel shows group data for diastolic capacitance during sequential therapy indexed to baseline for 8 OH and 4 YN dogs. Right panel shows group data for systolic capacitance during sequential therapy indexed to baseline. ANOVA P<0.05 for effect of serial therapy on diastolic and systolic capacitance. † P <0.05 vs. baseline. ‡ P<0.05 vs. sildenafil.

Figure 3. Passive Force Measurements in Isolated Skinned Cardiomyocytes from OH and YN dogs

Fpassive was higher in cardiomyocytes from OH dogs but in both YN and OH dogs, Fpassive was lower in cardiomyocytes obtained after sildenafil and BNP treatment compared to cardiomyocytes obtained from LV tissue at baseline. † P<0.05 vs. baseline.

Figure 4. Phosphorylation of Troponin I by Western blotting and 2D SDS-PAGE

A. Multiplex Western blots using a serine 23/24 phosphospecific antibody and a total TnI antibody in OH and YN dogs. A common standard (STD) was loaded alongside the baseline (BL), sildenafil (SIL) and BNP biopsy samples to allow for comparisons across gels. B. Group data for phosphorylation ratio of serine 23/24 TnI to total TnI indexed to baseline. P-value is non-significant. C. Total protein stain of a sample homogenate resolved by 2D SDS-PAGE. Two phosphorylated isoelectric variants, 1-P and 2-P, of TnI are observed. D. Proportion of the 1-P and 2-P isoelectric variants of TnI with serial therapy. P-value is non-significant.

Figure 5. Phosphorylation of Troponin T, MLC-1, and MLC-2

A. Representative total protein stain of a LV homogenate resolved by 2D SDS-PAGE. Three prominent TnT isoforms are identified. Isoform 1 demonstrated 3 isoelectric variants indicating three (bi-, mono- and non-) phosphorylation states, whereas the less abundant isoforms 2 and 3 were each present in two (non- and mono-) phosphorylated forms. MLC-1 is present as two (non- and mono-) phosphorylated forms whereas MLC-2 is present as three (non-, mono- and a rarely detected bi-) phosphorylated form. B. The distribution of isoelectric variants of the abundant TnT isoform (isoform 1) is plotted at baseline and in response to sildenafil and BNP treatment. There was no change in the less abundant isoforms of TnT (isoforms 2 and 3, not shown). C. The percentage of non- and mono-phosphorylated MLC-1 and MLC-2 at baseline and after sequential treatment with sildenafil and BNP are shown. P-values were non-significant.

Figure 6. Phosphorylation of Phospholamban

Representative Western blot for serine 16 phosphorylated (A) and total (B) phospholamban pentamer and monomer. Phospholamban was dephosphorylated following baseline autonomic blockade (BL) and did not change with sequential sildenafil or BNP therapy. Phosphorylation of phospholamban in the standard (STD) is evident.

Figure 7. Phosphorylation of Titin

A. Representative phosphoprotein stain (ProQ diamond) and total protein stain (Sypro Ruby) of a titin gel at baseline and after sequential treatment with sildenafil and BNP. B. Group data (n=7 per group) for the ratio of phosphorylated titin (ProQ diamond) to total titin (Spyro Ruby) indexed to baseline. C. Group data (n=7 per group) for the ratio of phosphorylated N2B isoform to N2BA isoform (ProQ diamond). D. Back phosphorylation assay by autoradiography to assess titin phosphorylation: Skinned fibers were either phosphorylated by adding PKG or first dephosphorylated with a phosphatase and then treated with PKG (PP+PKG) to assess inherent phosphorylation (difference in the phosphorylation signal between PKG and PP+PKG). Control sample was not treated with PKG. Assessed for N2B isoform and T2 degradation product. Signal from N2BA band on the autoradiograms was too faint to allow for a meaningful analysis. E. Group data (n=5) for inherent phosphorylation of N2B isoform. F. Group data (n=5) for inherent phosphorylation of T2 degradation product. † P-value<0.05 vs. baseline