Pluripotent stem-cell-based screening uncovers sildenafil as a mitochondrial disease therapy

cGMP (cyclic guanosine monophosphate)-phosphodiesterase 5 (PDE5) inhibitors, widely used to treat PH through their pulmonary vasorelaxation properties, have shown direct cardiac benefits, but their impacts on the RV in LV diseases are not fully determined. Here we show that RV molecular alterations occur early in the absence of RV hemodynamic changes during LV pressure-overload and are ameliorated by PDE5 inhibition. Two-day moderate LV pressure-overload (transverse aortic constriction) neither altered RV pressure/ function nor RV weight in mice, while it induced only mild LV hypertrophy. Importantly, pathological molecular features were already induced in the RV free wall myocardium, including up-regulation of gene markers for hypertrophy and inflammation, and activation of extracellular signal-regulated kinase (ERK) and calcineurin. Importantly, dexamethasone also prevented these RV molecular changes, similarly to sildenafil treatment. These results suggest the contributory role of inflammation to the early pathological interventricular interaction between RV and LV. The current study provides the first evidence for the novel early molecular cross-talk between RV and LV, preceding RV hemodynamic changes in LV disease, and supports the therapeutic strategy of enhancing cGMP signaling pathway to treat heart diseases. Citation: Imai Y, Kariya T, Iwakiri M, Yamada Y, Takimoto E (2018) Sildenafil ameliorates right ventricular early molecular derangement during left ventricular pressure overload. Editor: Vincenzo Lionetti, Scuola Superiore Sant'Anna, ITALY Received: January 16, 2018; Accepted: March 23, 2018; Published: April 5, 2018 This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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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. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by National Institute of Health Grants R01 HL-093432 (ET). Competing interests: ET has received research support from Actelion Pharmaceuticals Japan Ltd., and YI, TK, MI, and YY have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The right ventricle (RV) is a chamber of the heart that pumps out blood into pulmonary circulation under low pressure.

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While cardiac output can be maintained even in the absence of functional RV under physiological conditions, growing evidence has indicated that RV dysfunction has deleterious impacts on prognosis as well as functional capacity in heart failure [1,2]. In patients with systolic heart failure, RV ejection fraction (RVEF) <20% is an independent predictor of mortality and heart failure hospitalization [3].

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RV dysfunction is the strong predictor of death also in heart failure patients with preserved ejection fraction (HFpEF), associated with severe symptoms [4,5]. Although RV dysfunction following LV disease is thought initiated due to RV pressure-overload, which occurs sildenafil 50 mg india following elevated LV end-diastolic pressure, with the mechanisms shared by pulmonary arterial hypertension or pulmonary stenosis, studies that directly assess molecular mechanisms remain scant. Cyclic guanosine monophosphate (cGMP) is an intracellular second messenger downstream of nitric oxide and natriuretic peptides, and has been gaining attention as a key to heart failure treatment. Phosphodiesterase 5 (PDE5) inhibitors block degradation of cGMP and thus activate cGMP signaling pathways. While PDE5 inhibitors are in wide clinical use for the treatment of pulmonary hypertension, benign prostate hyperplasia and erectile dysfunction [6] through their vasorelaxation action, growing evidence has suggested that PDE5 inhibition also provide beneficial cardiac effects.

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We prepared four arms: (1) sham surgery (Sham), (2) TAC with normal soft chow (TAC 2d Veh), (3) TAC with sildenafil chow (TAC 2d Sil), and (4) TAC with dexamethasone treatment (TAC 2d DXM). Animals were anesthetized with 1% inhaled isoflurane and 10 mg/kg intraperitoneal etomidate, then intubated, and mechanically ventilated. The mediastinum was opened through dislocation of 2nd and 3rd left sternocostal joints, then transverse aorta was exposed at the back of thymus. Between the brachiocephalic trunk and the left common carotid artery a 27-gauge needle was placed alongside transverse aorta, and the aorta and the needle was tied around using 7–0 prolene suture. After the needle was withdrawn, the aorta was constricted to a diameter of 0.4 mm.

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Sham-operated animals were subjected to the same surgical procedures without aortic constriction. After the chest closure with 6–0 prolene, they were allowed to recover from anesthesia, and placed on a heating plate until full recovery of consciousness. For two days after surgery TAC-2d-Sil mice were treated with sildenafil citrate (Wako Pure Chemical Industries, Osaka, Japan; 200mg/kg/day) mixed in soft chow (Transgenic Dough Diet; Bio-Serv, Flemington, NJ; 100g/kg/day). Free sildenafil 100 mg cost plasma concentration of sildenafil with the dose in mice are comparable to those in humans using standard clinical dosing[7], because mice metabolize sildenafil at approximately 100 times higher rate than humans[13]. Sham, TAC-2d-Veh and TAC-2d-DXM animals received the soft diet without sildenafil.

Animal models

TAC-2d-DXM mice were injected intraperitoneally with dexamethasone sodium phosphate (Aspen Japan, Tokyo, Japan; 20mg/kg/day) on the day of the surgery and the following day. Total number of mice used in this study was 38. In each group (Sham, TAC 2d Veh, TAC 2d Sil), eleven mice were allocated. Out of 11, three mice for in vivo hemodynamics study, five for gene expression study, and three for western blot analysis. Five mice were allocated to TAC-2d-DXM group for gene expression study. PDE5 inhibition with sildenafil or tadalafil ameliorated experimental models of heart diseases in rodents [7,8]. Chronic sildenafil treatment improved cardiac function and clinical status in patients with systolic heart failure and diabetic cardiomyopathy [9,10]. Prior studies revealed that multiple mechanisms might contribute to such cardiac benefits, including Gq signal deactivation [8], improvement of mitochondrial energy metabolism [11] and modulation of inflammation [12]; however, the molecular impact of PDE5 inhibition on RV has not been fully determined. In the current study, employing a mouse model of LV pressure-overload, we investigated early molecular changes in the RV myocardium, and tested the impacts of concomitant sildenafil treatment. We found that pathologic molecular derangement in the RV myocardium occurred at very early stages before RV hemodynamic overload became evident, and that sildenafil ameliorated such molecular abnormalities though mechanisms involving its anti-inflammatory effects. All animal protocols were approved by the animal care and use committee of the University of Tokyo (approval number: H15-099). All experiments were performed on C57BL/6J male mice (7–10 weeks old; CLEA Japan, Tokyo, Japan). They were housed in controlled environment with a 12h light/ 12h dark cycle at a maintained temperature, and kept with free access to food and water throughout the whole experiment period.

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Pressure overload was posed by transverse aortic constriction (TAC) [7].

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• Avoid alcohol consumption when using sildenafil, especially at high doses.

We prepared four arms: (1) sham surgery (Sham), (2) TAC with normal soft chow (TAC 2d Veh), (3) TAC with sildenafil chow (TAC 2d Sil), and (4) TAC with dexamethasone treatment (TAC 2d DXM). Animals were anesthetized with 1% inhaled isoflurane and 10 mg/kg intraperitoneal etomidate, then intubated, and mechanically ventilated. The mediastinum was opened through dislocation of 2nd and 3rd left sternocostal joints, then transverse aorta was exposed at the back of thymus. Between the brachiocephalic trunk and the left common carotid artery a 27-gauge needle was placed alongside transverse aorta, and the aorta and the needle was tied around using 7–0 prolene suture. After the needle was withdrawn, the aorta was constricted to a diameter of 0.4 mm. Sham-operated animals were subjected to the same surgical procedures without aortic constriction.

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cGMP (cyclic guanosine monophosphate)-phosphodiesterase 5 (PDE5) inhibitors, widely used to treat PH through their pulmonary vasorelaxation properties, have shown direct cardiac benefits, but their impacts on the RV in LV diseases are not fully determined. Here we show that RV molecular alterations occur early in the absence of RV hemodynamic changes during LV pressure-overload and are ameliorated by PDE5 inhibition. Two-day moderate LV pressure-overload (transverse aortic constriction) neither altered RV pressure/ function nor RV weight in mice, while it induced only mild LV hypertrophy. Importantly, pathological molecular features were already induced in the RV free wall myocardium, including up-regulation of gene markers for hypertrophy and inflammation, and activation of extracellular signal-regulated kinase (ERK) and calcineurin. Importantly, dexamethasone also prevented these RV molecular changes, similarly to sildenafil treatment.

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These results suggest the contributory role of inflammation to the early pathological interventricular interaction between RV and LV. The current study provides the first evidence for the novel early molecular cross-talk between RV and LV, preceding RV hemodynamic changes in LV disease, and supports the therapeutic strategy of enhancing cGMP signaling pathway to treat heart diseases. Citation: Imai Y, Kariya T, Iwakiri M, Yamada Y, Takimoto E (2018) Sildenafil ameliorates right ventricular early molecular derangement during left ventricular pressure overload. Editor: Vincenzo Lionetti, Scuola Superiore Sant'Anna, ITALY Received: January 16, 2018; Accepted: March 23, 2018; Published: April 5, 2018 This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files.

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Funding: This work was supported by National Institute of Health Grants R01 HL-093432 (ET). Competing interests: ET has received research support from Actelion Pharmaceuticals Japan Ltd., and YI, TK, MI, and YY have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The right ventricle (RV) is a chamber of the heart that pumps out blood into pulmonary circulation under low pressure. While cardiac output can be maintained even in the absence of functional RV under physiological conditions, growing evidence has indicated that RV dysfunction has deleterious impacts on prognosis as well as functional capacity in heart failure [1,2]. After the chest closure with 6–0 prolene, they were allowed to recover from anesthesia, and placed on a heating plate until full recovery of consciousness. For two days after surgery TAC-2d-Sil mice were treated with sildenafil citrate (Wako Pure Chemical Industries, Osaka, Japan; 200mg/kg/day) mixed in soft chow (Transgenic Dough Diet; Bio-Serv, Flemington, NJ; 100g/kg/day). Free sildenafil 100 mg cost plasma concentration of sildenafil with the dose in mice are comparable to those in humans using standard clinical dosing[7], because mice metabolize sildenafil at approximately 100 times higher rate than humans[13].

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In patients with systolic heart failure, RV ejection fraction (RVEF) <20% is an independent predictor of mortality and heart failure hospitalization [3]. RV dysfunction is the strong predictor of death also in heart failure patients with preserved ejection fraction (HFpEF), associated with severe symptoms [4,5]. Although RV dysfunction following LV disease is thought initiated due to RV pressure-overload, which occurs sildenafil 50 mg india following elevated LV end-diastolic pressure, with the mechanisms shared by pulmonary arterial hypertension or pulmonary stenosis, studies that directly assess molecular mechanisms remain scant. Cyclic guanosine monophosphate (cGMP) is an intracellular second messenger downstream of nitric oxide and natriuretic peptides, and has been gaining attention as a key to heart failure treatment. Phosphodiesterase 5 (PDE5) inhibitors block degradation of cGMP and thus activate cGMP signaling pathways.

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While PDE5 inhibitors are in wide clinical use for the treatment of pulmonary hypertension, benign prostate hyperplasia and erectile dysfunction [6] through their vasorelaxation action, growing evidence has suggested that PDE5 inhibition also provide beneficial cardiac effects. PDE5 inhibition with sildenafil or tadalafil ameliorated experimental models of heart diseases in rodents [7,8]. Chronic sildenafil treatment improved cardiac function and clinical status in patients with systolic heart failure and diabetic cardiomyopathy [9,10]. Prior studies revealed that multiple mechanisms might contribute to such cardiac benefits, including Gq signal deactivation [8], improvement of mitochondrial energy metabolism [11] and modulation of inflammation [12]; however, the molecular impact of PDE5 inhibition on RV has not been fully determined. In the current study, employing a mouse model of LV pressure-overload, we investigated early molecular changes in the RV myocardium, and tested the impacts of concomitant sildenafil treatment.

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We found that pathologic molecular derangement in the RV myocardium occurred at very early stages before RV hemodynamic overload became evident, and that sildenafil ameliorated such molecular abnormalities though mechanisms involving its anti-inflammatory effects. All animal protocols were approved by the animal care and use committee of the University of Tokyo (approval number: H15-099). All experiments were performed on C57BL/6J male mice (7–10 weeks old; CLEA Japan, Tokyo, Japan). They were housed in controlled environment with a 12h light/ 12h dark cycle at a maintained temperature, and kept with free access to food and water throughout the whole experiment period. Pressure overload was posed by transverse aortic constriction (TAC) [7]. Sham, TAC-2d-Veh and TAC-2d-DXM animals received the soft diet without sildenafil. TAC-2d-DXM mice were injected intraperitoneally with dexamethasone sodium phosphate (Aspen Japan, Tokyo, Japan; 20mg/kg/day) on the day of the surgery and the following day. Total number of mice used in this study was 38. In each group (Sham, TAC 2d Veh, TAC 2d Sil), eleven mice were allocated. Out of 11, three mice for in vivo hemodynamics study, five for gene expression study, and three for western blot analysis.

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Five mice were allocated to TAC-2d-DXM group for gene expression study.

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Before and two days after surgery cardiac function was assessed by transthoracic, two-dimensional guided M-mode echocardiography in conscious mice using Vevo2100 (FUJIFILM VisualSonics, Toronto, Ontario, Canada) with 30 MHz linear-array transducer. M-mode LV end-systolic diameter (LVESD) and LV end-diastolic diameter (LVEDD) were measured in the short-axis view. LV fractional shortening (LVFS) was calculated as follows: LVFS = (LVEDD-LVESD)/LVEDD. Studies and analysis were performed by the same investigator (TK). For more comprehensive analysis, in vivo LV and RV function were assessed by pressure catheter.

S1 Fig. Western blot of p-ERK1/2 in the RV myocardium.

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.

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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. 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. Before and two days after surgery cardiac function was assessed by transthoracic, two-dimensional guided M-mode echocardiography in conscious mice using Vevo2100 (FUJIFILM VisualSonics, Toronto, Ontario, Canada) with 30 MHz linear-array transducer.

Medication Class	Interaction Effect	Notes
Nitrates	Severe hypotension risk	Do not combine
Alpha-blockers	Potentiates blood pressure lowering	Use with caution
CYP3A4 inhibitors	Increases sildenafil levels	Adjust dosage accordingly
Other PDE5 inhibitors	Increased risk of side effects	Usually not combined

M-mode LV end-systolic diameter (LVESD) and LV end-diastolic diameter (LVEDD) were measured in the short-axis view. LV fractional shortening (LVFS) was calculated as follows: LVFS = (LVEDD-LVESD)/LVEDD. Studies and analysis were performed by the same investigator (TK).

• High-dose sildenafil should only be used in rare cases and under strict medical supervision.
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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.

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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. 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.

• Storage of sildenafil should be in a cool, dry place away from direct sunlight.
• Common side effects include dizziness, upset stomach, or vision changes.
• The dosage of sildenafil should be tailored to individual health needs.
• Sildenafil can interact with other medications, affecting its safety and efficacy.
• Always seek medical guidance before using sildenafil, especially at high doses.

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.

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