How Sildenafil Works in the Body

All cytosolic ROIs were of identical size and remained inside the cytosol during the full duration of the recording. Cytosolic fura-2 signals were first individually background-corrected for each excitation wavelength, after which the ratio value (340/380 nm) was calculated. 10 μM ROCK inhibitor was added to the final media after splitting to promote neuron survival. Experiments and quantification were performed as previously described.81 Neurons generated for neurite outgrowth quantification were treated with 10 μM sildenafil in 0.1 % DMSO or 0.1 % DMSO alone following media changes on days 8, 10, and 12. On day 14, the neurons were split and seeded onto black-wall, flat-clear-bottom 96-well plates (Greiner) at a density of 5,000 cells/well, and treated once more with Sildenafil or DMSO. Neurons were then cultured until day 16, when they were fixed with PFA, blocked, and stained for TUJ1 and Hoechst.

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Water (eluent A) and acetonitrile (eluent B), both acidified with 0.1 % formic acid, were pumped with 0.4 ml/min. Elution of sildenafil and its internal standard was achieved with a 10-min linear gradient from 5 % to 90 % eluent B. Total run-time was 17 min including re-equilibration of the LC system. MS/MS analyses were carried out using an Ultivo triple-quadrupole mass spectrometer (Agilent Technologies) operating in the positive electrospray ionization mode (ESI+). The following ion source parameters were set: sheath gas temperature, 400 °C; sheath gas flow, 12 l/min of nitrogen; nebulizer pressure, 20 psi; drying gas temperature, 100 °C; drying gas flow, 7 l/min of nitrogen; capillary voltage, 3.0 kV; nozzle voltage, 0 kV. Neurite images were then acquired using the Operetta CLS high content imaging system (Revvity) the TUJ1 and Hoechst signals at 20 x. Following acquisition, the neurite images were analyzed using CellProfiler (version 4.2.5). Briefly, the Hoechst signal was used to identify the nucleus as a primary object and the TUJ1 signal was used to identify the neurites as secondary objects. The nuclear objects were then expanded to serve as a proxy for the cell body and subtracted from the TUJ1 signal to leaving only the individual neurites. The results were exported as CSV for data analysis. Mean neurite length was calculated as the mean over the median per experiment. On day 12, neurons were transfected with 10 nM of PRKG1 siRNA (Ambion) or scrambled siRNA (Ambion) by adding the transfection mix directly to the well. On day 14, after re-seeding the neurons onto the 96-well plates, neurons were transfected again with 10 nM of PRKG1 siRNA or scramble siRNA. On day 16, PRKG1 knock-down efficiency was evaluated by qPCR and neurite length was quantified with our HCA protocol.81 Sildenafil citrate (Sigma-Aldrich) was dissolved in DMSO, then diluted with tap water to obtain the final concentration of 400 mg/l drinking water and given ad libitum. Untreated mice (10 females, 8 males) were compared to sildenafil-treated Ndufs4 KO mice (6 females, 6 males). Electrocardiography (ECG) recordings were recorded from conscious mice, using the INDUS Rodent Surgical Monitoring system without anesthesia. Transthoracic echocardiograms to measure ventricular size, wall thickness, and ejection fraction were performed on mice using the Vevo 2,700 VisualSonics System.

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Intraperitoneal injection with 0.1 mg midazolam was applied for anxiolytic muscle relaxant effects, without any anesthetic agent that may cause cardiac suppression. Standard 2D, M-mode, Doppler, Tissue Doppler images were obtained using a 30-MHz linear array transducer, including the parasternal short and long axis. All image analysis was performed by Vevo 2,100 analysis software (v.1.5) after rapid image collection. The diastolic function was measured using tissue Doppler imaging of the mitral annulus (E`) and conventional mitral inflow (E wave). Metabolic chamber by Promethion (Sable Systems International) was applied to characterize the metabolism of whole-animal energy expenditure.145 The 16-cage metabolic chamber system directly measures various parameters over a 72-hour period, such as heat production, oxygen consumption (VO2), CO2 production, and XY activity movement. The respiratory exchange ratio (RER) is calculated as the ratio of carbon dioxide production (VCO2) to oxygen consumption. This parameter is useful to estimate the metabolic status. The xy axis detection of animal motion measure activity during day-time and night-time. The formula applied was Heat = (1.232∗VCO2)+(3.815∗VO2). For generation of SURF1 KO pigs, we followed a protocol for somatic cell nuclear transfer (SCNT) based on our previous method11 with minor modifications.

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All cytosolic ROIs were of identical size and remained inside the cytosol during the full duration of the recording. Cytosolic fura-2 signals were first individually background-corrected for each excitation wavelength, after which the ratio value (340/380 nm) was calculated. 10 μM ROCK inhibitor was added to the final media after splitting to promote neuron survival. Experiments and quantification were performed as previously described.81 Neurons generated for neurite outgrowth quantification were treated with 10 μM sildenafil in 0.1 % DMSO or 0.1 % DMSO alone following media changes on days 8, 10, and 12. On day 14, the neurons were split and seeded onto black-wall, flat-clear-bottom 96-well plates (Greiner) at a density of 5,000 cells/well, and treated once more with Sildenafil or DMSO.

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Neurons were then cultured until day 16, when they were fixed with PFA, blocked, and stained for TUJ1 and Hoechst. Neurite images were then acquired using the Operetta CLS high content imaging system (Revvity) the TUJ1 and Hoechst signals at 20 x. Following acquisition, the neurite images were analyzed using CellProfiler (version 4.2.5). Briefly, the Hoechst signal was used to identify the nucleus as a primary object and the TUJ1 signal was used to identify the neurites as secondary objects. The nuclear objects were then expanded to serve as a proxy for the cell body and subtracted from the TUJ1 signal to leaving only the individual neurites.

Quantitative real-time polymerase chain reaction (PCR)

The results were exported as CSV for data analysis. Mean neurite length was calculated as the mean over the median per experiment. On day 12, neurons were transfected with 10 nM of PRKG1 siRNA (Ambion) or scrambled siRNA (Ambion) by adding the transfection mix directly to the well. On day 14, after re-seeding the neurons onto the 96-well plates, neurons were transfected again with 10 nM of PRKG1 siRNA or scramble siRNA. On day 16, PRKG1 knock-down efficiency was evaluated by qPCR and neurite length was quantified with our HCA protocol.81 Sildenafil citrate (Sigma-Aldrich) was dissolved in DMSO, then diluted with tap water to obtain the final concentration of 400 mg/l drinking water and given ad libitum. Briefly, ovaries from pubertal gilts of domestic pig, large white background were collected at a local abattoir sildenafil citrate tablet and transported to the lab at 31-33 °C. Follicles larger than 3 mm were aspirated, and cumulus-oocyte complexes (COCs) were selected and matured in vitro at 38.5 °C under 5 % CO2 for 42 h. One day before SCNT, donor cells were induced into quiescence via serum starvation (0.5 % FCS). On the day of SCNT, cells were trypsinized, washed, and resuspended in SOF-Hepes medium. Matured oocytes (with extruded polar body) were selected and zona pellucida was removed using 0.5 % pronase in DPBS. All manipulations were performed in SOF-Hepes with 10 % FCS.

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Sildenafil was always administered at the same time and dissolved in milk. The veterinarian ensured that the entire dose was consumed. For biochemical and immunoblot experiments, we isolated the basal ganglia of newborn male pigs: 3 SURF1 KO, 3 SURF1 KO treated with sildenafil (2.1 mg/kg/day), and 3 sex and age-matched WT. Spectrophotometric analyses of complex IV (CIV) and sildenafil pink pill citrate synthase (CS) activities were carried out as previously described.135 CIV activity was normalized to CS activity, an index of mitochondrial mass. For histological analysis, tissues were removed immediately after death, post-fixed in 4 % PFA at 4 °C for 48 h, cryoprotected in DPBS containing 30 % sucrose for 24 h at 4 °C, embedded in optimal cutting temperature (OCT) compound, and frozen on dry ice.

Western blot analysis

Cryostat sections (10 μm thick) were then mounted on gelatin-coated glass slides. The sections were stained with hematoxylin and eosin (H&E). Images were acquired using a Nikon Eclipse E400 light microscope at 20 x or 40 x magnification. The sildenafil plasma concentration was monitored in three treated LS patients. EDTA blood was drawn in the morning before the first dose (trough level) and one hour after oral sildenafil application (peak level).

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Depicted time points in Figure 6D were separated by more than 3 months for each patient. The quantification was performed by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). The EDTA-blood sample was centrifuged at 3,000 x g for 10 min at 4 °C and the plasma was aliquoted and frozen at -80 °C until measurement. Proteins were precipitated by vortexing for 1 min and subsequent storage for 60 min at -20 °C. Supernatants were obtained by centrifugation at 16,000 x g at 4 °C for 10 min and subjected to LC-MS/MS sildenafil quantification applying the multiple reaction monitoring (MRM) approach. Enucleated cytoplasts were exposed to phytohemagglutinin and fused with donor cells via double DC-pulse (1.2 kV/cm), then returned to maturation medium.

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Untreated mice (10 females, 8 males) were compared to sildenafil-treated Ndufs4 KO mice (6 females, 6 males). Electrocardiography (ECG) recordings were recorded from conscious mice, using the INDUS Rodent Surgical Monitoring system without anesthesia. Transthoracic echocardiograms to measure ventricular size, wall thickness, and ejection fraction were performed on mice using the Vevo 2,700 VisualSonics System. Intraperitoneal injection with 0.1 mg midazolam was applied for anxiolytic muscle relaxant effects, without any anesthetic agent that may cause cardiac suppression. Standard 2D, M-mode, Doppler, Tissue Doppler images were obtained using a 30-MHz linear array transducer, including the parasternal short and long axis.

ERK and calcineurin were activated in the RV as well as in the LV, and were inhibited by sildenafil treatment

All image analysis was performed by Vevo 2,100 analysis software (v.1.5) after rapid image collection. The diastolic function was measured using tissue Doppler imaging of the mitral annulus (E`) and conventional mitral inflow (E wave). Metabolic chamber by Promethion (Sable Systems International) was applied to characterize the metabolism of whole-animal energy expenditure.145 The 16-cage metabolic chamber system directly measures various parameters over a 72-hour period, such as heat production, oxygen consumption (VO2), CO2 production, and XY activity movement. The respiratory exchange ratio (RER) is calculated as the ratio of carbon dioxide production (VCO2) to oxygen consumption. This parameter is useful to estimate the metabolic status.

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The xy axis detection of animal motion measure activity during day-time and night-time. The formula applied was Heat = (1.232∗VCO2)+(3.815∗VO2). For generation of SURF1 KO pigs, we followed a protocol for somatic cell nuclear transfer (SCNT) based on our previous method11 with minor modifications. Briefly, ovaries from pubertal gilts of domestic pig, large white background were collected at a local abattoir sildenafil citrate tablet and transported to the lab at 31-33 °C. Follicles larger than 3 mm were aspirated, and cumulus-oocyte complexes (COCs) were selected and matured in vitro at 38.5 °C under 5 % CO2 for 42 h. As nuclear donor, we used a cell clone obtained by homologous direct recombination (HDR)11 that harbors a 106 bp deletion on the left homology arm of exon 3 as well as a puromycin-resistance cassette in both alleles of the SURF1 gene. Embryos were cultured in modified mSOF medium with amino acids and 4 mg/ml BSA, using a modified Well-of-the-Well system. Sows were synchronized with 12 mg/day of altrenogest for 15 days. On day 13, they received 0.15 mg PgF2α, followed by 1000 IU hCG 96 h after the last altrenogest dose.

• 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.

On day 6 of SCNT embryo development (compacted morula to early blastocyst stage), embryos were surgically transferred via mid-ventral laparotomy, five days after standing estrus. Pregnancy was assessed via ultrasound on day 30 and confirmed on day 60 post-transfer. Farrowing was induced with PgF2α on days 114-115 of gestation. For body temperature detection we used a Thermal Imaging Camera (Dongguan Xintai Instrument Co., Ltd, China). SURF1 KO male piglets were treated immediately after birth with sildenafil citrate (n=3 animals with 2.1 mg/kg/day, n=4 animals with 0.5 mg/kg/day).

• 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.
• Always verify your medication's source to ensure authenticity and safety.

At birth, the animals were separated from the sow and transferred to an incubator kept at 35 °C. Sildenafil was always administered at the same time and dissolved in milk. The veterinarian ensured that the entire dose was consumed. For biochemical and immunoblot experiments, we isolated the basal ganglia of newborn male pigs: 3 SURF1 KO, 3 SURF1 KO treated with sildenafil (2.1 mg/kg/day), and 3 sex and age-matched WT.

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

Spectrophotometric analyses of complex IV (CIV) and sildenafil pink pill citrate synthase (CS) activities were carried out as previously described.135 CIV activity was normalized to CS activity, an index of mitochondrial mass. For histological analysis, tissues were removed immediately after death, post-fixed in 4 % PFA at 4 °C for 48 h, cryoprotected in DPBS containing 30 % sucrose for 24 h at 4 °C, embedded in optimal cutting temperature (OCT) compound, and frozen on dry ice. Cryostat sections (10 μm thick) were then mounted on gelatin-coated glass slides.

• 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 sections were stained with hematoxylin and eosin (H&E).

Side Effects

One day before SCNT, donor cells were induced into quiescence via serum starvation (0.5 % FCS). On the day of SCNT, cells were trypsinized, washed, and resuspended in SOF-Hepes medium. Matured oocytes (with extruded polar body) were selected and zona pellucida was removed using 0.5 % pronase in DPBS. All manipulations were performed in SOF-Hepes with 10 % FCS. Enucleated cytoplasts were exposed to phytohemagglutinin and fused with donor cells via double DC-pulse (1.2 kV/cm), then returned to maturation medium.

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As nuclear donor, we used a cell clone obtained by homologous direct recombination (HDR)11 that harbors a 106 bp deletion on the left homology arm of exon 3 as well as a puromycin-resistance cassette in both alleles of the SURF1 gene. Embryos were cultured in modified mSOF medium with amino acids and 4 mg/ml BSA, using a modified Well-of-the-Well system. Sows were synchronized with 12 mg/day of altrenogest for 15 days. On day 13, they received 0.15 mg PgF2α, followed by 1000 IU hCG 96 h after the last altrenogest dose. On day 6 of SCNT embryo development (compacted morula to early blastocyst stage), embryos were surgically transferred via mid-ventral laparotomy, five days after standing estrus.

How does sildenafil work (mechanism of action)?

Pregnancy was assessed via ultrasound on day 30 and confirmed on day 60 post-transfer. Farrowing was induced with PgF2α on days 114-115 of gestation. For body temperature detection we used a Thermal Imaging Camera (Dongguan Xintai Instrument Co., Ltd, China). SURF1 KO male piglets were treated immediately after birth with sildenafil citrate (n=3 animals with 2.1 mg/kg/day, n=4 animals with 0.5 mg/kg/day). At birth, the animals were separated from the sow and transferred to an incubator kept at 35 °C. Images were acquired using a Nikon Eclipse E400 light microscope at 20 x or 40 x magnification. The sildenafil plasma concentration was monitored in three treated LS patients. EDTA blood was drawn in the morning before the first dose (trough level) and one hour after oral sildenafil application (peak level). Depicted time points in Figure 6D were separated by more than 3 months for each patient. The quantification was performed by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). The EDTA-blood sample was centrifuged at 3,000 x g for 10 min at 4 °C and the plasma was aliquoted and frozen at -80 °C until measurement. Proteins were precipitated by vortexing for 1 min and subsequent storage for 60 min at -20 °C. Supernatants were obtained by centrifugation at 16,000 x g at 4 °C for 10 min and subjected to LC-MS/MS sildenafil quantification applying the multiple reaction monitoring (MRM) approach. Water (eluent A) and acetonitrile (eluent B), both acidified with 0.1 % formic acid, were pumped with 0.4 ml/min. Elution of sildenafil and its internal standard was achieved with a 10-min linear gradient from 5 % to 90 % eluent B. Total run-time was 17 min including re-equilibration of the LC system. MS/MS analyses were carried out using an Ultivo triple-quadrupole mass spectrometer (Agilent Technologies) operating in the positive electrospray ionization mode (ESI+).

Side Effect	Severity	Notes
Headache	Common	Usually mild
Flushing	Common	Transient
Dizziness	Common	Particularly when standing up quickly
Vision disturbances	Less common	Temporary, includes blue tint or blurred vision
Priapism	Rare	Needs urgent medical attention

The following ion source parameters were set: sheath gas temperature, 400 °C; sheath gas flow, 12 l/min of nitrogen; nebulizer pressure, 20 psi; drying gas temperature, 100 °C; drying gas flow, 7 l/min of nitrogen; capillary voltage, 3.0 kV; nozzle voltage, 0 kV. The following mass transitions were recorded (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); d3-sildenafil: m/z 478.2 → 60.9 (FV: 220 V, CE: 68 eV, quantifier), m/z 478.2 → 103.0 (FV: 220 V, CE: 28 eV), m/z 478.2 → 283.1 (FV: 220 V, CE: 44 eV). Peak areas were determined with MassHunter Software (Agilent Technologies) and sildenafil was directly quantified via its internal standard d3-sildenafil that was concentrated to 8 nM in the samples. Heteroplasmy levels and mtDNA haplotypes in the whole exome sequencing (WES) datasets were determined using the software MToolBox v.1.146,147 Results were visualized in the IGV viewer v2.16.148 Heteroplasmy levels of the MT-ATP6 mutations in fibroblasts, iPSCs, and NPCs were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, as described before.27 Genomic DNA was isolated using the Nucleo-Spin Tissue kit (Macherey-Nagel).

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The following mass transitions were recorded (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); d3-sildenafil: m/z 478.2 → 60.9 (FV: 220 V, CE: 68 eV, quantifier), m/z 478.2 → 103.0 (FV: 220 V, CE: 28 eV), m/z 478.2 → 283.1 (FV: 220 V, CE: 44 eV). Peak areas were determined with MassHunter Software (Agilent Technologies) and sildenafil was directly quantified via its internal standard d3-sildenafil that was concentrated to 8 nM in the samples. Heteroplasmy levels and mtDNA haplotypes in the whole exome sequencing (WES) datasets were determined using the software MToolBox v.1.146,147 Results were visualized in the IGV viewer v2.16.148 Heteroplasmy levels of the MT-ATP6 mutations in fibroblasts, iPSCs, and NPCs were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, as described before.27 Genomic DNA was isolated using the Nucleo-Spin Tissue kit (Macherey-Nagel). We used restriction enzyme StuI (NEB, R0187, 10,000 units/ml) for the m.9185T>C mutation (wild-type: 24+90 pb, mutation: 114 pb), HpaII (NEB, R0171, 10,000 units/ml) for m.8993T>C and m.8993T>G (wild type: 25+155 bp; mutant: 180 bp), XbaI (NEB, R0145, 10,000 units/ml) for m.9176 T>G (wild type: 24+155 bp, mutant: 179 bp). The percentage of cleaved versus uncleaved fragments was determined by capillary electrophoresis and laser detection of the FAM-labelled RFLP-fragments using the 3,500 Series Genetic Analyzer (Applied Biosystems, RRID:SCR_021901) and normalized to a standard curve of known degrees of heteroplasmy. We used restriction enzyme StuI (NEB, R0187, 10,000 units/ml) for the m.9185T>C mutation (wild-type: 24+90 pb, mutation: 114 pb), HpaII (NEB, R0171, 10,000 units/ml) for m.8993T>C and m.8993T>G (wild type: 25+155 bp; mutant: 180 bp), XbaI (NEB, R0145, 10,000 units/ml) for m.9176 T>G (wild type: 24+155 bp, mutant: 179 bp). The percentage of cleaved versus uncleaved fragments was determined by capillary electrophoresis and laser detection of the FAM-labelled RFLP-fragments using the 3,500 Series Genetic Analyzer (Applied Biosystems, RRID:SCR_021901) and normalized to a standard curve of known degrees of heteroplasmy.