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Sildenafil treatment significantly inhibited both these signals in the RV and the LV (Fig 4).
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These results suggest that the RV undergoes early hypertrophy molecular changes similar to the LV in the absence of RV afterload increase at the very early stage of LV pressure-overload, and that sildenafil treatment inhibits such molecular remodeling process in both ventricles.
| Parameter | Description | Typical Value |
|---|---|---|
| Bioavailability | Percent absorbed into bloodstream | ~40% |
| Tmax (time to peak) | Time to reach maximum plasma concentration | 30-120 minutes |
| Half-life | Duration of drug activity | 4-5 hours |
| Metabolism | Main route | Liver (CYP3A4 enzyme) |
| Excretion | How the drug leaves the body | Mainly feces, some urine |
Quantification results of phosphor/total ratio (p/t ratio) normalized to sham controls are shown in the bar graphs on the right.
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TAC induced robust phosphorylation of ERK1/2 in RV as well as LV myocardium, and sildenafil prevented this activation in the RV.
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Two-day TAC induced only mild increase in the total heart weight (~10%), and this was inhibited by sildenafil treatment (Fig 1A). At this early stage, RV free wall weight and lung weight was neither impacted by TAC nor by sildenafil treatment (Fig 1A). M-mode transthoracic sildenafil softgel capsule echocardiography showed minimal change in LV chamber size (LVEDD: LV end-diastolic dimension; LVESD: LV end-systolic dimension) and function (LVFS: LV fractional shortening) (Fig 1B). These results indicate that RV was apparently unaffected at this early stage when LV developed hypertrophy in response to the loading stress. (A) Postmortal assessment of heart, ventricles, and lung.
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Heart weight (HW), RV weight (RVW), and lung weight normalized to tibial length (TL) is shown. Transverse aortic constriction (TAC) for two days increased HW, and sildenafil prevented this increase. Neither RVW nor lung weight was affected by two-day TAC with or without sildenafil treatment. LV end-diastolic dimension (LVEDD), LV end-systolic dimension (LVESD), and LV fractional shortening (LVFS) are shown. Two-day TAC altered neither LV dimensions nor function.
Two-day LV pressure overload (TAC) did not affect RV pressure or function, but prolonged LV relaxation that was ameliorated by sildenafil
Results are expressed as mean ± s.e.m. TAC 2d Veh, TAC for 2 days with vehicle treatment; TAC 2d Sil, TAC for 2days with sildenafil treatment. n.s., not significant by one-way or two-way analysis of variance; *, p<0.05 versus sham group; †, p<0.05 versus TAC 2d Veh group. We also performed hemodynamic studies using a micro-catheter placed in both ventricles for detailed hemodynamic assessment. While TAC induced significant increase in LV peak systolic pressure (LVP sys, 139mmHg), compared to sham controls (89 mmHg), RV mean pressure (RVP mean) was not yet affected (Fig 2A). RCAN1 mRNA expression was increased by TAC in both ventricles, and suppressed by sildenafil.
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As inflammation marker genes were up-regulated in both ventricles at this early stage, and were prevented by sildenafil, we further performed an immunohistochemical study and assessed macrophage infiltration in the RV and the LV.
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These results suggest the anti-inflammatory and anti-oxidative impacts of sildenafil. (A) Expression of fetal genes as markers of cardiac hypertrophy, encoding for BNP and B-MHC, normalized to GAPDH. Transverse aortic constriction (TAC) for two days induced marked increase in BNP mRNA levels in the RV myocardium as well as in the LV myocardium, which was prevented by sildenafil. Expression of B-MHC was increased by two-day TAC in LV, but was not affected by sildenafil. (B) Expression of inflammatory cytokines, IL1b and IL6, normalized to GAPDH.
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IL1b and IL6 was up-regulated by TAC in both ventricles. Sildenafil inhibited both in LV, and also attenuated both in RV. (C) Expression of genes for enzymes inducing oxidative stress, NOX2 and NOX4, normalized to GAPDH. Expression of NOX2 was increased by TAC in both ventricles, and suppressed by sildenafil in LV. TAC and sildenafil had little influence on NOX4 expression.
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n.s., not significant by one-way analysis of variance; *, p<0.05 versus sham group; ✝, p<0.05 versus the TAC 2d Veh group. We also examined RV hypertrophic signaling pathways at this early stage of LV disease, including ERK and calcineurin; the former known to contribute to concentric hypertrophy and the latter to pathologic remodeling [16–18]. For calcineurin activity, we assessed RCAN1 (regulator of calcineurin 1) gene expression levels. RCAN1 is extremely responsive to changes in calcineurin activity in vivo [19] and thus its expression levels have been used as reflecting calcineurin activity [20,21]. ERK was significantly phosphorylated in the RV as well as in the LV (Fig 4A and 4B), and calcineurin activity was also increased in the RV similarly to the LV (Fig 4C). We found that F4/80 positive cells were significantly increased in both ventricles of 2day-TAC hearts and that sildenafil significantly inhibited the increase in both ventricles (Fig 5). (A-C) Myocardium stained for F4/80+ cells in the RV and the LV of the Sham mouse (A), the TAC-2d-Veh mouse (B), and the TAC-2d-Sil mouse (C).
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Total heart tissues were fixed in Tissue-Tek UFIX (Sakura Finetek Japan, Tokyo, Japan), embedded in paraffin, and cut cross-sectionally into 4–6 μm slices. Tissue sections were deparaffinized with a series of xylene washes, and rehydrated in ethanol solutions with decreasing concentrations from 100% to 90%, 80%, and 70%. Antigen retrieval was carried out by incubating the sections with 0.1% trypsin solution (Sigma-Aldrich) for 40 minutes at 37°C. Endogenous peroxidases were quenched in 3% of hydrogen peroxide in methanol for 10 minutes at room temperature. Subsequently, slides were blocked for 1 hour at room temperature with PBS containing 1.5% of normal rabbit serum (S-5000, Vector Laboratories, Burlingame, CA), and incubated overnight at 4°C with 1:400 of rat anti-mouse F4/80 antibody (MCAP 497; Bio-Rad, formerly AbD Serotec).
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The following day, rabbit anti-rat biotinylated secondary antibody (BA-4000, Vector Laboratories) was diluted 1.5:100 in 0.5% normal rabbit serum, and the sections were incubated for 1 hour at room temperature. Then slides were treated with avidin-peroxidase conjugate (VECTASTAIN Elite ABC Kit; PK-6104; Vector Laboratories) for 30 minutes at room temperature, and the signal was developed using the 3,3’-diaminibenzidine (DAB) substrate (SK-4100; Vector Laboratories). After counterstaining with hematoxylin, slides were dehydrated through graded ethanol (95% and 100%), cleared by washing in xylene, and mounted with Mount Quick (Daido Sangyo, Tokyo, Japan). Slides were examined by light microscopy (BX51; Olympus, Tokyo, Japan) and the number of F4/80 positive cells was counted in 10 randomly selected high-power field (400x magnification) with the observer (TK) blinded to sample identity. All the images were obtained at 200x magnification using an Olympus DP70 camera.
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All values are expressed as mean ± standard error of the mean (s.e.m). Differences between multiple groups were compared by one-way or two-way analysis of variance (ANOVA) and Tukey’s post-hoc multiple-comparisons test, using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria, version 3.3.1) [14]. The other analyses were performed with Excel 2013 (Microsoft, Redmond, WA). P values less than 0.05 were considered significant. To examine early molecular alterations in the RV following LV disease, we used a mouse model of LV pressure-overload (TAC), in which LV was exposed to moderate pressure-overload created by surgical constriction of transverse aorta and examined the effect of concomitant sildenafil treatment. (D) The number of F4/80+ cells per high-power field.
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Transverse aortic constriction for two days induced F4/80+ macrophage infiltration into myocardium not only in the LV but also in the RV, which was suppressed by sildenafil.
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These results confirm the absence of RV hemodynamic load at this early stage of the LV disease caused by moderate LV pressure-overload. Concomitant sildenafil treatment did not lower systolic LV peak pressure as reported previously [15], or affect RV mean pressure (Fig 2A). Neither dP/dtmax (peak rate of ventricular pressure rise) nor dP/dtmin (peak rate of ventricular pressure decline) was altered by the moderate TAC in either ventricles at this early stage (Fig 2B). Importantly, however, relaxation time constant (tau) was prolonged in the LV, again consistent with the general notion that the impairment of LV relaxation occurs early before systolic function starts to deteriorate in heart diseases. Sildenafil treatment normalized LV tau, while the other parameters examined were not altered (Fig 2B).
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(A) Peak systolic left ventricular pressure (LVP sys) and mean right ventricular pressure (RVP mean). Transverse aortic constriction (TAC) for two days increased systolic LV pressure, but had no effects on RV pressure. (B) Peak rate of ventricular pressure rise (dP/dtmax), peak rate of ventricular decline (dP/dtmin), and relaxation time constant (tau). LV tau was prolonged by two-day TAC, which was normalized by sildenafil. n.s., not significant by one-way analysis of variance; *, p < 0.05 versus sham group; §, p = 0.05 versus sham group; ‡, p = 0.07 versus TAC 2d Veh group.
RV remained apparently normal by two-day LV pressure overload (TAC), with mild LV hypertrophy that was inhibited by sildenafil
To elucidate molecular changes in both ventricles, we first determined messenger RNA (mRNA) expression levels for brain natriuretic peptide (BNP) and beta myosin heavy chain (B-MHC), both markers for fetal gene recapitulation. Interestingly, BNP mRNA levels were markedly increased in the RV free wall myocardium as well as in the LV myocardium in two-day TAC hearts (Fig 3A). Sildenafil treatment potently inhibited BNP expression levels in both ventricles, whereas it had no impact on the latter. Interestingly, pro-inflammatory marker genes, including interleukin-1 beta (IL1b) and interleukin-6 (IL6), were also up-regulated in the RV free wall as well as in the LV myocardium (Fig 3B), associated with increased nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) but not NOX4 mRNA levels (Fig 3C), suggesting the involvement of inflammation as lovegra sildenafil well as oxidative stress in the RV and LV pathophysiology at this early stage of the LV disease. Sildenafil treatment significantly inhibited the mRNA increase in IL1b, IL6 and NOX2 in LV, and also attenuated the increase in these genes in RV (Fig 3B and 3C).
