How Sildenafil Works in the Body

For MT-ATP6 immunoblotting, pooled NPCs (at least 1.5 x 106 NPCs) were homogenized in RIPA buffer and sonicated in an ultrasonic bath (Branson 1800-E) for 1 min, incubated in ice for 30 min and then centrifuged at 10,000 x g for 15 min at 4 °C. Protein concentration was determined with the Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific). 30 μg of proteins were run through a 4 % separating / 10 % resolving polyacrylamide gel with stain-free technology (Bio-Rad laboratories), to allow detection and quantification.

• Sildenafil 100g is rarely prescribed and considered an experimental or off-label dose.
• Like all medications, it should only be used as directed by a healthcare professional.
• Overdose signs include severe hypotension, vision loss, or chest pain.
• Use with caution in patients with liver or kidney impairment.
• Avoid alcohol consumption when using sildenafil, especially at high doses.

Proteins were then electroblotted onto a PVDF membrane. After 1 h of blocking with 3 % BSA in TBS-Tween at RT, the membranes were incubated with the primary antibodies overnight at 4 °C and subsequently probed with HRP-conjugated secondary antibodies for 2 h at RT. Chemiluminescence-based immunostaining (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific) was performed. Images were acquired with the ChemiDoc MP Imaging system. Data analysis and quantification was performed using Image Lab software, 6.1 (Bio-Rad Laboratories).

• Sildenafil was initially developed to treat hypertension and angina.
• The typical prescribed dose is up to 100mg, but only under medical supervision.
• Recreational misuse of high doses can result in serious health issues.
• Educate yourself about the potential risks and proper use of sildenafil.
• The drug works best when taken about 30-60 minutes before activity.

The following primary antibody was used ATP6 (rabbit, 1:500, Immunological Science), with the appropriate secondary antibodies: anti-rabbit, HRP Conjugate (1:5000, Promega) For preparation of mitochondrial-enriched fractions for Blue Native PAGE, approximately 2 x 107 cells were resuspended in 400 μl of ice-cold Medium A (0.32 M sucrose, 10 mM Tris-HCl pH 7.4, 1 mM EDTA), homogenized in a glass/glass dounce homogenizer with a tight pestle by 10 strokes and centrifuged at 800 x g for 5 min at 4 °C to remove cell debris. Supernatants were collected and centrifuged at 9,000 x g for 10 min at 4 °C to obtain the enriched mitochondrial fractions and resuspended in 50 μl of cold Medium A to quantify the protein yield by Bicinchoninic Acid (BCA) protein assay Kit (Thermo Fisher Scientific). Supernatants were collected, supplemented with 5 μl of Sample Buffer (750 mM aminocaproic acid, 50 mM Bis-Tris/HCl pH 7, 0.5 mM EDTA, and 5 % Serva Blue G-250) and stored at -80 °C until use.

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For Blue Native Gel Electrophoresis (1D-BNGE) and 2D SDS-PAGE, about 40 μg of solubilized mitochondria were separated by pre-cast NativePAGE 3-12 % BisTris gels (Invitrogen) and run in a Mini Gel Tank (Thermo Fisher Scientific) at 4 °C. The upper buffer chamber was filled with Dark Blue Cathode buffer (50 mM Tricine, 15 mM Bis-Tris pH 7, 0.02 % Serva Blue G-250), and the lower buffer chamber with Anode buffer (50 mM Bis-Tris pH 7). Samples were run at 100 V for 30 min, then at 200 V. Dark Blue Cathode buffer was replaced with Light Blue Cathode Buffer (50 mM Tricine, 15 mM Bis-Tris pH 7, 0.002 % Serva Blue G-250) at half run. Samples were then electroblotted onto a PVDF membrane and blocked for 1 h (3 % BSA in TBS-Tween) at RT before proceeding with the immunodetection. In 1D-BNGE, the antibody against ATP5B was used to visualize complex V holoenzyme and intermediate subassemblies; complex III (detected with antibody against UQCRFS1) was shown as loading control. Denaturing 2D-BNGE was used to confirm the presence of different subassemblies of complex V. For complex V and complex III visualization, we used antibodies against ATP5B (1:1,000, Sigma-Aldrich) and UQCRSF1 (1:1,000, Abcam). For 2D-PAGE, the gel strips of the 1D-BNGE were excised and covered with a denaturing solution (1 % SDS, 1 % lovegra sildenafil β- Mercaptoethanol) for 1 h at RT. Each gel strip was run through a 4 % stacking / 10 % resolving polyacrylamide gel electrophoresis (Bio-Rad), electroblotted onto a PVDF membrane, and blocked for 1 h at RT with 3 % BSA in TBS-Tween. The following antibodies were used: ATP5B (1:1,000, Sigma-Aldrich), NDUFA9 (1:1,000, Invitrogen), and SDH-B (1:800, Abcam). Isolated total RNA was transcribed into complementary DNA (cDNA). In the Mastercycler X50s (Eppendorf), the mixture was heated to 65 °C for 5 min and then quickly chilled on ice.

1 μl (200 units) of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) was added to the reaction tube and mixed. The reaction mixture was incubated for 50 min at 37 °C, and inactivated by heating at 70 °C for 15 min. For the qPCR reaction, the synthesized cDNA was mixed with 10 μM primer mix (primers designed with Integrated DNA technologies, Table S5) and SYBR™ Green PCR Master Mix (Applied Biosystems) diluted in water in a 96-well plate. The plate was transferred to CFX96™ Real-Time System qPCR machine (Bio-Rad) and the following program was started using CFX96 software: 2 min at 50 °C, 10 min at 95 °C, 39 x (15 s at 95 °C, 30 s at 62 °C, 30 s at 72 °C), 15 min at 95 °C, 5 s at 62 °C, and 50 s at 95 °C. To check the quality of the qPCR products, melting curves were assessed first.

For this, the CT values of the genes of interest were subtracted from the CT values of the housekeeping genes Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and/or ornithine decarboxylase antizyme 1 (OAZ1). To investigate the differentiation ratio of brain organoids, we used SOX2/CTIP2 ratio. For this, the qPCR method was applied to RNA samples from each five pooled day 70 cortical brain organoids (CTRL_1, CTRL_2, ATP6_4, ATP6_7) with or without 10 μM sildenafil treatment for 24 hours. Control iPSCs (CTRL_8) and LS iPSCs (ATP6_2) were differentiated into brain capillary endothelial cells (BCECs) following previously described protocols.149,150 Single cells were isolated using Accutase (Thermo Fisher Scientific) and seeded onto Matrigel-coated 6-well plates (Nunc™, Thermo Fisher Scientific) in 2 ml/well mTeSR™ Plus supplemented with 10 μM Y-27632 (STEMCELL Technologies). The starting cell number was optimized for both cell lines respectively. After 3 days, medium was changed to 2 ml/well unconditioned medium (UM) when the optimal cell density of 2 - 4 x 105 cells/cm2 was reached (referred to sildenafil citrate drug as day 0). UM was composed of 78.5 % DMEM/F12 (Thermo Fisher Scientific), 20 % KnockOut serum replacement (Thermo Fisher Scientific), 1% MEM NEAA (Thermo Fisher Scientific), 0.5 % L-glutamine (Capricorn), and 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific). UM was changed daily for the following five days to initiate co-differentiation of BCECs and neuronal cells. On day 6, medium was changed to 4 ml/well endothelial cell (EC) medium, composed of Human Endothelial-SFM (Thermo Fisher Scientific) and 0.5 % B27 Supplement (Thermo Fisher Scientific), supplemented with 20 ng/ml hFGF and 10 μM retinoic acid (RA) for BCEC expansion.

BCECs were adapted to EC medium without hFGF and RA at day 9 for 24 h.

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To investigate the differentiation ratio of brain organoids, we used SOX2/CTIP2 ratio. For this, the qPCR method was applied to RNA samples from each five pooled day 70 cortical brain organoids (CTRL_1, CTRL_2, ATP6_4, ATP6_7) with or without 10 μM sildenafil treatment for 24 hours. Control iPSCs (CTRL_8) and LS iPSCs (ATP6_2) were differentiated into brain capillary endothelial cells (BCECs) following previously described protocols.149,150 Single cells were isolated using Accutase (Thermo Fisher Scientific) and seeded onto Matrigel-coated 6-well plates (Nunc™, Thermo Fisher Scientific) in 2 ml/well mTeSR™ Plus supplemented with 10 μM Y-27632 (STEMCELL Technologies). The starting cell number was optimized for both cell lines respectively. After 3 days, medium was changed to 2 ml/well unconditioned medium (UM) when the optimal cell density of 2 - 4 x 105 cells/cm2 was reached (referred to sildenafil citrate drug as day 0).

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UM was composed of 78.5 % DMEM/F12 (Thermo Fisher Scientific), 20 % KnockOut serum replacement (Thermo Fisher Scientific), 1% MEM NEAA (Thermo Fisher Scientific), 0.5 % L-glutamine (Capricorn), and 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific). UM was changed daily for the following five days to initiate co-differentiation of BCECs and neuronal cells. On day 6, medium was changed to 4 ml/well endothelial cell (EC) medium, composed of Human Endothelial-SFM (Thermo Fisher Scientific) and 0.5 % B27 Supplement (Thermo Fisher Scientific), supplemented with 20 ng/ml hFGF and 10 μM retinoic acid (RA) for BCEC expansion. BCECs were adapted to EC medium without hFGF and RA at day 9 for 24 h. We measured transendothelial electrical resistance (TEER) values using an electrode to evaluate the integrity of the in vitro BBB.

S2 Fig. Western blot of t-ERK1/2 in the RV myocardium.

Only BBBs with TEER values ≥ 1000 Ω∗cm2 at day 10 were included. To monitor monolayer integrity for both iPSC-derived BBB models, 10 μM sodium fluorescein (Sigma-Aldrich) was added to the apical compartment of two reference inserts for liquid sildenafil citrate each BCEC differentiation. The intensity of the fluorescent tracer molecule was measured in pooled samples of both compartments respectively using a fluorescent plate reader (Infinite M1000 Pro, TECAN, excitation: 490 nm, emission: 525 nm). We applied 10 μM sildenafil, sildenafil citrate, or reference compounds in EC medium to the apical compartment of two inserts each and incubated the cells for 1 h at 37 °C, 5 % CO2 on an orbital shaker (100 rpm). Diazepam and atenolol were used as internal controls given their known high and low BBB permeability, as previously demonstrated.151,152 Media from both the apical (A) and basolateral (B) compartments were collected, pooled and stored at -80 °C until quantification. We measured transendothelial electrical resistance (TEER) values using an electrode to evaluate the integrity of the in vitro BBB. Only BBBs with TEER values ≥ 1000 Ω∗cm2 at day 10 were included. To monitor monolayer integrity for both iPSC-derived BBB models, 10 μM sodium fluorescein (Sigma-Aldrich) was added to the apical compartment of two reference inserts for liquid sildenafil citrate each BCEC differentiation. The intensity of the fluorescent tracer molecule was measured in pooled samples of both compartments respectively using a fluorescent plate reader (Infinite M1000 Pro, TECAN, excitation: 490 nm, emission: 525 nm). We applied 10 μM sildenafil, sildenafil citrate, or reference compounds in EC medium to the apical compartment of two inserts each and incubated the cells for 1 h at 37 °C, 5 % CO2 on an orbital shaker (100 rpm). Diazepam and atenolol were used as internal controls given their known high and low BBB permeability, as previously demonstrated.151,152 Media from both the apical (A) and basolateral (B) compartments were collected, pooled and stored at -80 °C until quantification.

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For MT-ATP6 immunoblotting, pooled NPCs (at least 1.5 x 106 NPCs) were homogenized in RIPA buffer and sonicated in an ultrasonic bath (Branson 1800-E) for 1 min, incubated in ice for 30 min and then centrifuged at 10,000 x g for 15 min at 4 °C. Protein concentration was determined with the Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific). 30 μg of proteins were run through a 4 % separating / 10 % resolving polyacrylamide gel with stain-free technology (Bio-Rad laboratories), to allow detection and quantification. Proteins were then electroblotted onto a PVDF membrane. After 1 h of blocking with 3 % BSA in TBS-Tween at RT, the membranes were incubated with the primary antibodies overnight at 4 °C and subsequently probed with HRP-conjugated secondary antibodies for 2 h at RT.

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Chemiluminescence-based immunostaining (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific) was performed. Images were acquired with the ChemiDoc MP Imaging system. Data analysis and quantification was performed using Image Lab software, 6.1 (Bio-Rad Laboratories). The following primary antibody was used ATP6 (rabbit, 1:500, Immunological Science), with the appropriate secondary antibodies: anti-rabbit, HRP Conjugate (1:5000, Promega) For preparation of mitochondrial-enriched fractions for Blue Native PAGE, approximately 2 x 107 cells were resuspended in 400 μl of ice-cold Medium A (0.32 M sucrose, 10 mM Tris-HCl pH 7.4, 1 mM EDTA), homogenized in a glass/glass dounce homogenizer with a tight pestle by 10 strokes and centrifuged at 800 x g for 5 min at 4 °C to remove cell debris. Supernatants were collected and centrifuged at 9,000 x g for 10 min at 4 °C to obtain the enriched mitochondrial fractions and resuspended in 50 μl of cold Medium A to quantify the protein yield by Bicinchoninic Acid (BCA) protein assay Kit (Thermo Fisher Scientific).

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Supernatants were collected, supplemented with 5 μl of Sample Buffer (750 mM aminocaproic acid, 50 mM Bis-Tris/HCl pH 7, 0.5 mM EDTA, and 5 % Serva Blue G-250) and stored at -80 °C until use. For Blue Native Gel Electrophoresis (1D-BNGE) and 2D SDS-PAGE, about 40 μg of solubilized mitochondria were separated by pre-cast NativePAGE 3-12 % BisTris gels (Invitrogen) and run in a Mini Gel Tank (Thermo Fisher Scientific) at 4 °C. The upper buffer chamber was filled with Dark Blue Cathode buffer (50 mM Tricine, 15 mM Bis-Tris pH 7, 0.02 % Serva Blue G-250), and the lower buffer chamber with Anode buffer (50 mM Bis-Tris pH 7). Samples were run at 100 V for 30 min, then at 200 V. Dark Blue Cathode buffer was replaced with Light Blue Cathode Buffer (50 mM Tricine, 15 mM Bis-Tris pH 7, 0.002 % Serva Blue G-250) at half run. Peak areas were used to calculate the apparent permeability (Papp) of each substance using equation: Quantification of sildenafil, diazepam and atenolol in apical and basolateral media samples was performed using LC-MS/MS. The settings were maintained except for minor adjustments.

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Propidium iodide (PI) (Invitrogen) was diluted in RPMI to 1.5 mM and administered at a final concentration of 500 nM. Imaging and analysis were performed using the Incucyte live-cell analysis system. For Glycostress test, LS iPSC-CMs (ATP6_6 and ATP6_7) were seeded in 24-well Seahorse assay plates at a density of 100,000 cells/well ten days prior the experiment. Chromatographic separation of atenolol (tR = 3.5 min), sildenafil (tR = 4.3 min) and diazepam (tR = 5.5 min) was achieved with gradient elution within 10 min of total run-time.

• Sildenafil operates by increasing blood flow to the penis during arousal.
• It is a phosphodiesterase type 5 (PDE5) inhibitor used in sexual medicine.
• The standard dose ranges from 25mg to 100mg, but 100g refers to a very high, rare dose.
• Users should always adhere to prescribed doses to avoid health risks.
• Never attempt to self-medicate with high doses like 100g without professional advice.

After positive electrospray ionization, the MS/MS detector recorded three mass transitions per drug compound (fragmentor voltage [FV] and collision energies [CE] in parentheses): Sildenafil: m/z 475.2 → 58.0 (FV: 220 V, CE: 68 eV, quantifier), m/z 475.2 → 100.0 (FV: 220 V, CE: 28 eV), m/z 475.2 → 283.1 (FV: 220 V, CE: 44 eV); Diazepam: m/z 285.1 → 154.1 (FV: 144 V, CE: 28 eV), m/z 285.1 → 193.1 (FV: 144 V, CE: 36 eV, quantifier), m/z 285.1 → 222.1 (FV: 144 V, CE: 28 eV) and Atenolol: m/z 267.2 → 56.0 (FV: 128 V, CE: 32 eV), m/z 267.2 → 74.1 (FV: 128 V, CE: 24 eV), m/z 267.2 → 144.9 (FV: 128 V, CE: 28 eV, quantifier). Peak areas were determined with MassHunter Quantitative Analysis software (version 10.1, Agilent Technologies) and the three drugs were quantified using matrix-matched external calibration in the concentration range of 0.001 to 10 μM.

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Samples were then electroblotted onto a PVDF membrane and blocked for 1 h (3 % BSA in TBS-Tween) at RT before proceeding with the immunodetection. In 1D-BNGE, the antibody against ATP5B was used to visualize complex V holoenzyme and intermediate subassemblies; complex III (detected with antibody against UQCRFS1) was shown as loading control. Denaturing 2D-BNGE was used to confirm the presence of different subassemblies of complex V. For complex V and complex III visualization, we used antibodies against ATP5B (1:1,000, Sigma-Aldrich) and UQCRSF1 (1:1,000, Abcam). For 2D-PAGE, the gel strips of the 1D-BNGE were excised and covered with a denaturing solution (1 % SDS, 1 % lovegra sildenafil β- Mercaptoethanol) for 1 h at RT.

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Each gel strip was run through a 4 % stacking / 10 % resolving polyacrylamide gel electrophoresis (Bio-Rad), electroblotted onto a PVDF membrane, and blocked for 1 h at RT with 3 % BSA in TBS-Tween. The following antibodies were used: ATP5B (1:1,000, Sigma-Aldrich), NDUFA9 (1:1,000, Invitrogen), and SDH-B (1:800, Abcam). Isolated total RNA was transcribed into complementary DNA (cDNA). In the Mastercycler X50s (Eppendorf), the mixture was heated to 65 °C for 5 min and then quickly chilled on ice. 1 μl (200 units) of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) was added to the reaction tube and mixed.

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The reaction mixture was incubated for 50 min at 37 °C, and inactivated by heating at 70 °C for 15 min. For the qPCR reaction, the synthesized cDNA was mixed with 10 μM primer mix (primers designed with Integrated DNA technologies, Table S5) and SYBR™ Green PCR Master Mix (Applied Biosystems) diluted in water in a 96-well plate. The plate was transferred to CFX96™ Real-Time System qPCR machine (Bio-Rad) and the following program was started using CFX96 software: 2 min at 50 °C, 10 min at 95 °C, 39 x (15 s at 95 °C, 30 s at 62 °C, 30 s at 72 °C), 15 min at 95 °C, 5 s at 62 °C, and 50 s at 95 °C. To check the quality of the qPCR products, melting curves were assessed first. For this, the CT values of the genes of interest were subtracted from the CT values of the housekeeping genes Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and/or ornithine decarboxylase antizyme 1 (OAZ1). iPSC-CMs lines were obtained through WNT signaling modulation followed by metabolic selection, as previously described.153,154 The resulting iPSC-CMs were maintained under feeder-free, serum-free culture conditions until day 60 post-differentiation. Confirmation of iPSC-CM identity was performed by immunostaining against alpha-actinin (ACTN2) (Sigma-Aldrich) and mitochondria were visualized with MitoSpy (BioLegend). To assess cell viability, 100,000 LS iPSC-CMs (ATP6_7) per well were seeded in 24-well plates. For imaging acquisition, the Calcein AM Viability Dye (Invitrogen) was dissolved in DMSO at 1 mg/ml, and applied at a final concentration of 1 μM in RPMI for 30 min at 37 °C.

• Sildenafil's patent expired in many countries, leading to generic versions.
• The cost of sildenafil can vary depending on the dose and brand.
• Use of sildenafil in women is not approved, and its safety in females is unestablished.
• Consultation with a healthcare provider is crucial before starting high doses.
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After incubation, cells were washed twice with 1 x DPBS. Propidium iodide (PI) (Invitrogen) was diluted in RPMI to 1.5 mM and administered at a final concentration of 500 nM.

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Peak areas were used to calculate the apparent permeability (Papp) of each substance using equation: Quantification of sildenafil, diazepam and atenolol in apical and basolateral media samples was performed using LC-MS/MS. The settings were maintained except for minor adjustments. Chromatographic separation of atenolol (tR = 3.5 min), sildenafil (tR = 4.3 min) and diazepam (tR = 5.5 min) was achieved with gradient elution within 10 min of total run-time. After positive electrospray ionization, the MS/MS detector recorded three mass transitions per drug compound (fragmentor voltage [FV] and collision energies [CE] in parentheses): Sildenafil: m/z 475.2 → 58.0 (FV: 220 V, CE: 68 eV, quantifier), m/z 475.2 → 100.0 (FV: 220 V, CE: 28 eV), m/z 475.2 → 283.1 (FV: 220 V, CE: 44 eV); Diazepam: m/z 285.1 → 154.1 (FV: 144 V, CE: 28 eV), m/z 285.1 → 193.1 (FV: 144 V, CE: 36 eV, quantifier), m/z 285.1 → 222.1 (FV: 144 V, CE: 28 eV) and Atenolol: m/z 267.2 → 56.0 (FV: 128 V, CE: 32 eV), m/z 267.2 → 74.1 (FV: 128 V, CE: 24 eV), m/z 267.2 → 144.9 (FV: 128 V, CE: 28 eV, quantifier). Peak areas were determined with MassHunter Quantitative Analysis software (version 10.1, Agilent Technologies) and the three drugs were quantified using matrix-matched external calibration in the concentration range of 0.001 to 10 μM.

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iPSC-CMs lines were obtained through WNT signaling modulation followed by metabolic selection, as previously described.153,154 The resulting iPSC-CMs were maintained under feeder-free, serum-free culture conditions until day 60 post-differentiation. Confirmation of iPSC-CM identity was performed by immunostaining against alpha-actinin (ACTN2) (Sigma-Aldrich) and mitochondria were visualized with MitoSpy (BioLegend). To assess cell viability, 100,000 LS iPSC-CMs (ATP6_7) per well were seeded in 24-well plates. For imaging acquisition, the Calcein AM Viability Dye (Invitrogen) was dissolved in DMSO at 1 mg/ml, and applied at a final concentration of 1 μM in RPMI for 30 min at 37 °C. After incubation, cells were washed twice with 1 x DPBS. Imaging and analysis were performed using the Incucyte live-cell analysis system. For Glycostress test, LS iPSC-CMs (ATP6_6 and ATP6_7) were seeded in 24-well Seahorse assay plates at a density of 100,000 cells/well ten days prior the experiment.