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For more comprehensive analysis, in vivo LV and RV function were assessed by pressure catheter. Mice were anesthetized with 0.5% inhaled isoflurane, 1000 mg/kg intraperitoneal urethane, and 10 mg/kg intraperitoneal etomidate, were subjected to tracheostomy, and were ventilated with 6–7 μl/g tidal volume and 120 breaths/min. Volume loading (10% bovine albumin, 100–150 μl over 3 minutes) was provided via a 30-gauge cannula in the left external jugular vein. The LV apex was exposed by incising diaphragm and left costal arch. A 1.4-Fr pressure catheter (SPR-839; Millar Instruments, Houston, TX) was inserted through the LV apex which was in advance pricked with a 26-gauge needle, and was positioned along the longitudinal axis. And a 1-Fr pressure catheter (PVR-1035; Millar Instruments) was placed into the RV which was in advance pricked with a 27-gauge needle. All data were collected, saved to disk, and analyzed using MPVS Ultra Foundation System (ADInstruments, New South Wales, Australia) with LabChart 7 software (ADInstruments). All values were averaged over nine consecutive cardiac cycles while blood pressure was stable.

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After physiological studies, mice were euthanized by cervical dislocation, and the heart was resected and washed in phosphate buffered saline (PBS; 137.0mM NaCl, 2.68mM KCl, 8.1mM Na2HPO4, and 1.47mM KH2PO4, pH 7.4). Total heart tissues for immunohistochemistry were proceeded to fixation steps, and those for gene expression analysis and western blotting were dissected as below. After total heart weight was measured, great vessels and atriums were removed, the RV free wall was separated from the LV wall and the intraventricular septum (IVS), and RV free wall was weighed. LV apex and IVS were removed from the LV. Remained LV free wall and the RV free wall were trimmed 0.5 mm from edges. Each part of a heart was rapidly frozen in liquid nitrogen, then stored at -80°C for subsequent procedures. The lung weight and tibial length was measured. Total ribonucleic acid (RNA) was extracted from frozen heart tissue using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. The yield of RNA was estimated spectrophotometrically using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA). The same amount of RNA was reverse transcribed into complementary deoxyribonucleic acid (cDNA) using High Capacity RNA-to-cDNA kit (Thermo Fisher Scientific). Quantitative real-time PCR was carried out using SYBR Green assay or Taqman probe assay.

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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).

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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). SYBR Green assay was conducted using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) according sildenafil 100mg lowest price to the manufacturer's instructions. PCR conditions were 2 minutes at 50°C, 2 minutes at 95°C, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Taqman assay was performed using THUNDERBIRD Probe qPCR Mix (TOYOBO) with following TaqMan primers (Thermo Fisher Scientific): mouse Il1b (Assay ID: Mm00434228_m1), mouse Il6 (Assay ID: Mm00446190_m1), mouse Nox2 (Assay ID: Mm01287743_m1), mouse Nox4 (Assay ID: Mm00479246_m1), mouse Rcan1 (Assay ID: Mm01213407_m1), and mouse Gapdh (Assay ID: Mm99999915_g1).

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Thermal cycling was 2 minutes at 50°C, 10 minutes at 95°C, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. PCR reactions were performed, recorded, and analyzed by using LightCycler 480 (Roche Applied Science, Mannheim, Germany) or QuantStudio 5 (Thermo Fisher Scientific). PCR was performed in duplicate and gene expression level was normalized by Gapdh. Briefly, frozen heart tissues were homogenized in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA) with 1 mM phenylmethylsulfonyl fluoride, proteinase inhibitors cocktail (cOmplete Mini EDTA-free; Roche Applied Science), and phosphatase inhibitors cocktail (PhosSTOP; Roche Applied Science). The homogenate was centrifuged at 17,800 g for 10 minutes at 4°C, and the resulting supernatant was designated proteins fraction.

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For more comprehensive analysis, in vivo LV and RV function were assessed by pressure catheter. Mice were anesthetized with 0.5% inhaled isoflurane, 1000 mg/kg intraperitoneal urethane, and 10 mg/kg intraperitoneal etomidate, were subjected to tracheostomy, and were ventilated with 6–7 μl/g tidal volume and 120 breaths/min. Volume loading (10% bovine albumin, 100–150 μl over 3 minutes) was provided via a 30-gauge cannula in the left external jugular vein. The LV apex was exposed by incising diaphragm and left costal arch. A 1.4-Fr pressure catheter (SPR-839; Millar Instruments, Houston, TX) was inserted through the LV apex which was in advance pricked with a 26-gauge needle, and was positioned along the longitudinal axis.

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And a 1-Fr pressure catheter (PVR-1035; Millar Instruments) was placed into the RV which was in advance pricked with a 27-gauge needle. All data were collected, saved to disk, and analyzed using MPVS Ultra Foundation System (ADInstruments, New South Wales, Australia) with LabChart 7 software (ADInstruments). All values were averaged over nine consecutive cardiac cycles while blood pressure was stable. After physiological studies, mice were euthanized by cervical dislocation, and the heart was resected and washed in phosphate buffered saline (PBS; 137.0mM NaCl, 2.68mM KCl, 8.1mM Na2HPO4, and 1.47mM KH2PO4, pH 7.4). Total heart tissues for immunohistochemistry were proceeded to fixation steps, and those for gene expression analysis and western blotting were dissected as below. Protein concentration of each fraction was measured using BCA Protein Assay Reagent (Thermo Fisher Scientific), and samples were diluted to the same concentration. Each protein solution was added with NuPage LDS Sample Buffer (Thermo Fisher Scientific) and 0.1M dithiothreitol, denatured at 95°C for 10 minutes, then sample solutions were obtained.

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Sample solutions were separated on 12.5% polyacrylamide gels (Wako Pure Chemicals Industries), and transferred onto the polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA).

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Blotted membranes were blocked for 1 hour at room temperature with 5% nonfat dried milk in Tris-buffered saline solution (TBS; 20mM Tris, 500mM NaCl, pH 7.4) containing 0.1% Tween-20 (Sigma-Aldrich, St. Louis, MO). Blocked membranes were incubated overnight at 4°C with following primary antibodies: 1:1,000 of anti-Erk1/2 (#9102; Cell Signaling Technology), and 1:1,000 of anti-phospho-Erk1/2 (#9101; Cell Signaling Technology). Next day, the sildenafil oral strips membranes were incubated with horseradish peroxidase conjugated secondary antibody (1:5,000) (sc-2357; Santa Cruz Biotechnology, Dallas, TX) for 1 hour at room temperature. Immunoblot bands was quantified using ImageJ software (NIH Image, Bethesda, MD).

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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). Blotted membranes were blocked for 1 hour at room temperature with 5% nonfat dried milk in Tris-buffered saline solution (TBS; 20mM Tris, 500mM NaCl, pH 7.4) containing 0.1% Tween-20 (Sigma-Aldrich, St. Louis, MO).

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After total heart weight was measured, great vessels and atriums were removed, the RV free wall was separated from the LV wall and the intraventricular septum (IVS), and RV free wall was weighed. LV apex and IVS were removed from the LV. Remained LV free wall and the RV free wall were trimmed 0.5 mm from edges. Each part of a heart was rapidly frozen in liquid nitrogen, then stored at -80°C for subsequent procedures. The lung weight and tibial length was measured.

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Total ribonucleic acid (RNA) was extracted from frozen heart tissue using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. The yield of RNA was estimated spectrophotometrically using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA). The same amount of RNA was reverse transcribed into complementary deoxyribonucleic acid (cDNA) using High Capacity RNA-to-cDNA kit (Thermo Fisher Scientific). Quantitative real-time PCR was carried out using SYBR Green assay or Taqman probe assay. SYBR Green assay was conducted using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) according sildenafil 100mg lowest price to the manufacturer's instructions. Blocked membranes were incubated overnight at 4°C with following primary antibodies: 1:1,000 of anti-Erk1/2 (#9102; Cell Signaling Technology), and 1:1,000 of anti-phospho-Erk1/2 (#9101; Cell Signaling Technology). Next day, the sildenafil oral strips membranes were incubated with horseradish peroxidase conjugated secondary antibody (1:5,000) (sc-2357; Santa Cruz Biotechnology, Dallas, TX) for 1 hour at room temperature.

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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. Immunoblot bands was quantified using ImageJ software (NIH Image, Bethesda, MD). 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). 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. 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. 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 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. Heart weight (HW), RV weight (RVW), and lung weight normalized to tibial length sildenafil oral solution (TL) is shown.

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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. Results are expressed as mean ± s.e.m.

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TAC 2d Veh, TAC for 2 days with vehicle treatment; TAC 2d Sil, TAC for 2days with sildenafil treatment.

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PCR conditions were 2 minutes at 50°C, 2 minutes at 95°C, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Taqman assay was performed using THUNDERBIRD Probe qPCR Mix (TOYOBO) with following TaqMan primers (Thermo Fisher Scientific): mouse Il1b (Assay ID: Mm00434228_m1), mouse Il6 (Assay ID: Mm00446190_m1), mouse Nox2 (Assay ID: Mm01287743_m1), mouse Nox4 (Assay ID: Mm00479246_m1), mouse Rcan1 (Assay ID: Mm01213407_m1), and mouse Gapdh (Assay ID: Mm99999915_g1). Thermal cycling was 2 minutes at 50°C, 10 minutes at 95°C, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. PCR reactions were performed, recorded, and analyzed by using LightCycler 480 (Roche Applied Science, Mannheim, Germany) or QuantStudio 5 (Thermo Fisher Scientific). PCR was performed in duplicate and gene expression level was normalized by Gapdh.

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Briefly, frozen heart tissues were homogenized in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA) with 1 mM phenylmethylsulfonyl fluoride, proteinase inhibitors cocktail (cOmplete Mini EDTA-free; Roche Applied Science), and phosphatase inhibitors cocktail (PhosSTOP; Roche Applied Science). The homogenate was centrifuged at 17,800 g for 10 minutes at 4°C, and the resulting supernatant was designated proteins fraction. Protein concentration of each fraction was measured using BCA Protein Assay Reagent (Thermo Fisher Scientific), and samples were diluted to the same concentration. Each protein solution was added with NuPage LDS Sample Buffer (Thermo Fisher Scientific) and 0.1M dithiothreitol, denatured at 95°C for 10 minutes, then sample solutions were obtained. Sample solutions were separated on 12.5% polyacrylamide gels (Wako Pure Chemicals Industries), and transferred onto the polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). 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). These results confirm the absence of RV hemodynamic load at this early stage of the LV disease caused by moderate LV pressure-overload.

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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 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 sildenafil oral solution (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. 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).