Simvastatin causes pulmonary artery relaxation by blocking smooth muscle ROCK and calcium channels: Evidence for an endothelium-independent mechanism

Figures

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  Fig 1. Confirmation of PAH by echocardiography. Echocardiographic images collected from rats immediately before injection (day 0), at 21 days after injection and immediately prior to termination. A) Parasternal short-axis views with right ventricle outlined by dotted lines. B) Pulsed-wave Doppler recordings of pulmonary outflow. Arrowheads indicate notching in the images from treated rats. C) M-mode imaging of the lateral tricuspid annulus. TAPSE was measured as the total displacement of the annulus from end-diastole (marked by lower line) to end-systole (upper line).

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  Fig 2. Simvastatin inhibits pulmonary artery reactivity. A, B) Original records of tension developed by pulmonary arteries in response to 30 nM U46619, followed by increasing concentrations of ACh (10 nM– 10 μM). Records from paired vessels treated with vehicle (left) or 5 μM simvastatin (right) from control (A) and MCT-treated (B) rats. C,D) Concentration response curves for U46619 and phenylephrine acting on control vessels, under control conditions and after incubation for 1 hour with 5 μM simvastatin. Points and bars represent mean ± s.e.m of vessels from 4 animals.

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  Fig 3. Effects of simvastatin, ROCK and calcium-channel block on basal tone of diseased arteries. Trace is original record of baseline tension before and during the application of 5 μM simvastatin. Histogram shows the mean change in tension following application of simvastatin (0.1–5 μM) or vehicle (n = 4), 10 μM Y27632 (n = 10) or 1 μM nifedipine (n = 5) to arteries from MCT rats at resting tension. *differs significantly from time-matched control (2-way repeated measures (RM) ANOVA and Sidak multiple comparisons test of each simvastatin concentration versus the vehicle control); #differs significantly from 5 μM simvastatin (one-way ANOVA with Tukey multiple comparisons test, each group compared with every other group).

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  Fig 4. ROCK inhibition partly mimics simvastatin. A) Contraction of healthy and MCT arteries to 30 nM U46619 (n = 6–7), 5 μM phenylephrine (PE, n = 6) and 50 mM KCl (n = 8) in control conditions, after 10 min exposure to 10 μM Y27632 and after 30 min of washing. * differs significantly from control by 2-way RM ANOVA with Tukey’s multiple comparisons test. B) Comparison of healthy vessel contraction to KCl, phenylephrine and U46619 in the presence of 10 μM Y27632, before and after exposure to simvastatin. * differs significantly from KCl in Y27632 by paired t-test. C) ROCK activity in arteries from 9 rats in the absence (con) or presence of 30 nM U46619 (U46), with or without the addition of 5 μM simvastatin (simv). *differs significantly from control and U46619 alone by one-way ANOVA with Tukey’s multiple comparisons test.

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  Fig 5. Simvastatin and Ca2+ influx. A) Contraction of healthy and MCT arteries to KCl (healthy n = 7, MCT n = 3), phenylephrine (n = 6–7) and U46619 (n = 5) before (control) and after 10 min exposure to 1 μM nifedipine. *p<0.05 compared with matched control (2-way RM ANOVA with Sidak post-test). B) Inset: fluorescence (380 nm) recordings from fura-2 loaded pulmonary artery smooth muscle cells in control conditions or the presence of simvastatin, before and after applying 1 mM MnCl2. Plot shows the increase in fluorescence decay upon exposure to MnCl2 in control conditions (n = 28 cells from 5 artery preparations) and after incubation with 5 μM simvastatin (n = 27 cells from 5 arteries). MnCl2 was applied at 1 mM (closed symbols) except for 7 simvastatin-treated cells exposed to 10 mM MnCl2 (open symbols). Wilcoxon matched pairs signed rank test indicates a significant difference between basal and MnCl2 in each condition* and between MnCl2 in the absence or presence of simvastatin#.

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  Fig 6. Simvastatin does not alter contraction evoked by SR Ca2+. Tension responses to 3 μM thapsigargin (A) or CPA (B), followed by 20 μM 2-APB. C, Early and sustained contraction of healthy arteries to thapsigargin or CPA (mean ± s.e.m., n = 7–9 rats), followed by tension remaining after application of 2-APB with and without exposure to 5 μM simvastatin. *differs significantly from peak and sustained responses (2-way RM ANOVA with Tukey’s multiple comparisons).

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  Fig 7. Simvastatin improves endothelial function in PAH. Concentration-response relationships for PA relaxation induced by ACh (A) and SNP (B) in arteries from control and MCT-treated rats, with and without simvastatin (5 μM) pre-treatment. Arteries were constricted with 30 nM U46629 until a stable plateau was reached then ACh (10 nM—10 μM) or SNP (1 nM—1 μM) was applied cumulatively. Relaxation was measured as percent of U46619-induced tone. Data represent mean ± s.e.m. of 6–9 animals. * differs significantly from the control response of MCT vessels at the same dilator concentration (unpaired t-test).