Sildenafil prevents and reverses transverse-tubule remodeling and Ca(2+) handling dysfunction in right ventricle failure induced by pulmonary artery hypertension

Abstract

Right ventricular (RV) failure (RVF) is the main cause of death in patients with pulmonary artery hypertension (PAH). Sildenafil, a phosphodiesterase type 5 inhibitor, was approved recently for treatment of PAH patients. However, the mechanisms underlying RV contractile malfunction and the benefits of sildenafil on RV function are not well understood. We aimed to investigate the following: (1) the ultrastructural and excitation-contraction coupling alterations underlying PAH-induced RVF; (2) whether the ultrastructural changes are reversible; and (3) the mechanisms underlying the therapeutic benefits of sildenafil in PAH-RVF. We used a single injection of monocrotaline in Wistar rats to induce pulmonary vascular proliferation, which led to PAH and RVF. RV myocytes displayed severe transverse (T)-tubule loss and disorganization, as well as blunted and dys-synchronous sarcoplasmic reticulum Ca(2+) release. Sildenafil prevented and reversed the monocrotaline-induced PAH and LV filling impairment. Early intervention with sildenafil prevented RV hypertrophy and the development of RVF, T-tubule remodeling, and Ca(2+) handling dysfunction. Although late treatment with sildenafil did not reverse RV hypertrophy in animals with established RVF, RV systolic function was improved. Furthermore, late intervention partially reversed both the impairment of myocyte T-tubule integrity and Ca(2+) handling protein and sarcoplasmic reticulum Ca(2+) release function in monocrotaline-treated rats. In conclusion, PAH-induced increase in RV afterload causes severe T-tubule remodeling and Ca(2+) handling dysfunction in RV myocytes, leading to RV contractile failure. Sildenafil prevents and partially reverses ultrastructural, molecular, and functional remodeling of failing RV myocytes. Reversal of pathological T-tubule remodeling, although incomplete, is achievable without the regression of RV hypertrophy.

Figure 1. Effects of MCT and sildenafil on vascular wall thickness in lung pulmonary arteries

A–D, Representative images of pulmonary vessels in the lungs of control (A), MCT (B), MCT rats treated with sildenafil beginning at day 1 (C) and MCT rats treated with sildenafil beginning at day 23 for two weeks (D). E, Vascular wall thickness in pulmonary vessels under 500 μm in length. **, p < 0.01; ***, p<0.001 vs. saline control; ††, p < 0.01 vs. MCT group. N=3, 3, 4, 4 lungs for each group, respectively.

Figure 2. Effects of MCT and sildenafil treatments on RV morphometric and RV systolic function

A–B, RV weight was measured after 24 hours of drying at 37°C (A) and normalized to LV+septum weight (B). N=5, 6, 7, 5 hearts per group, respectively. C–D, RV dimensions during systole (C) and diastole (D), and RV wall thickness during diastole (E). F, RV fractional shortening (FS, %), calculated as (RV dimension during diastole – RV dimension during systole) divided by RV dimension during diastole. N=11, 10, 9, 8 per group, respectively. MCT treated rats developed RVF with significant reduction in RV FS. Sildenafil early treatment prevented MCT induced RVF with maintained FS. Sildenafil late treatment improved RV systolic function, comparing to those of MCT alone. **, p < 0.001 vs. saline control; †, p<0.05; ††, p < 0.001 vs. MCT group; ‡, p<0.05 vs. Sil_MCT/D1 group.

Figure 3. MCT-induced PAH impairs LV filling

A, End diastolic volume; B, LV stroke volume; C, cardiac output. MCT induced PAH - RV afterload increase caused impairment of LV filling. Both early and late treatments with sildenafil normalized LV filling and output. D, LV ejection fraction (EF) was normal among all groups. ***, p<0.001 vs. saline control; †, p < 0.05; ††, p < 0.01 vs. MCT group. N=11, 10, 9, 8 per group, respectively.

Figure 4. Confocal imaging of Ca²⁺ transients in single isolated RV myocytes

A–D, representative Ca²⁺ transients from control, MCT, Sil_MCT/D1, and Sil_MCT/D23 groups, respectively. Shown underneath each panel of Ca²⁺ image is the spatially averaged Ca²⁺ profile. RV myocyte from MCT-treated rat (B) exhibited defects in the activation of Ca²⁺ transients (red arrows), prolonged plateau (green doublehead arrows) and slower decay phase (pink arrows). E–G, Summary data on parameters of Ca²⁺ transients, including amplitude (F/F₀), time to peak (T_peak) and decay rate (time to 50% decay, T₅₀). *, p<0.05 vs control; **, p<0.01 vs control; †, p<0.05 vs MCT; ††, p<0.01 vs MCT. n=30, 54, 61, 52 cells (from 3–6 hearts), respectively.

Figure 5. In situ confocal imaging of RV myocyte T-tubule system from Langendorff perfused intact hearts

A–D, representative T-tubule images from control, MCT, Sil_MCT/D1, and Sil_MCT/D23 group, respectively. B, MCT-treated rats exhibited severe T-tubule loss and disorganization in RV myocytes. C, Early treatment of sildenafil (Sil_MCT/D1) prevented MCT-induced T-tubule remodeling. D, Late treatment of sildenafil (Sil_MCT/D23) partially reversed MCT induced T-tubule remodeling. E. Average data of TT_power (T-tubule power, an index of the strength of T-tubule regularity, for analysis, See reference Wei S, et al, 2010). TT_power of Sil_MCT/D1 group is not different from control. TT_power of Sil_MCT/D23 group is significantly higher than that of MCT rats, but lower than those of control and Sil_MCT/D1 groups. **, p<0.01 vs control; ††, p<0.01 vs MCT; ‡, p<0.05 vs. Sil_MCT/D1 group. N=7, 9, 7, 5 hearts per group, respectively.

Figure 6. Alterations in expression of Ca²⁺ handling proteins in RV myocytes in response to sildenafil treatment

RV tissue lysates were extracted for Western blotting assay. Representative blots and quantitation of JP-2 (A), Cav1.2 (B), NCX1 (C), RyR2 (D), PLN (E) and SERCA (F) levels. *, p<0.05 vs control; **, p<0.01 vs control; †, p<0.05 vs MCT; ††, p<0.01 vs MCT; ‡, p<0.05 vs. Sil_MCT/D1 group. N=4–5 hearts for each group.