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AggreWell plates were centrifuged at 100 x g for 3 min and left at 37 °C and sildenafil from canada 5 % CO2 for 24 h.

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On day 1, spheroids were transferred to a petri dish using a cut P1000 tip and placed on a shaker.

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Until day 6, ES medium was changed daily.

Condition	Description	Recommended Dose
Erectile Dysfunction	Difficulty achieving/maintaining erection	50-100 mg before activity
Pulmonary Arterial Hypertension	Improves blood flow in lungs	20 mg three times daily
Off-label Uses	Other possible benefits	Under medical supervision

Neurospheres were fed with Neural Differentiation Medium (NDM) consisting of Neurobasal A medium (Gibco), GlutaMAX, penicillin-streptomycin, MycoZAP and 2 % B27 without vitamin A (Gibco).

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From day 6-15, medium was changed daily and supplemented with 20 ng/ml EGF (R&D Systems) and 20 ng/ml FGF2 (R&D Systems).

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From day 70 on, organoids were kept in cortical differentiation medium IV (CDMIV) that additionally was supplemented with 1 % B27 with vitamin A. The second protocol was based on a different publication.63 For this, AggreWell plates (STEMCELL Technologies) were prepared by incubation with Anti-Adherence Rinsing Solution (STEMCELL Technologies) for 15 min. iPSCs were detached from the 6-well plates using Accutase as described above. The cell suspension was pelleted at 200 x g for 4 min and resuspended in iPS Brew (Miltenyi Biotec) with 10 μM ROCK inhibitor. Cells were counted with CytoSmart Cell Counter (Greiner Bio-One) and 2.75 million cells were seeded in 1.5 ml per AggreWell. For long-term maturation after day 46, NDM medium was prepared with 2 % B27 with vitamin A, and organoids were fed according to their needs.

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Images of brain organoids were acquired using the Eclipse Ts2 light microscope (Nikon), and at later growth stages, when the organoids were larger, using a DMS1000 microscope (Leica). The size of the organoids was determined by area calculation using ImageJ. For MMP quantification, we applied a high-content analysis (HCA)-based live-cell detection assay that we previously established.31 Briefly, we seeded NPCs onto black-wall, clear-bottom 96-well plates (SCREENSTAR, Greiner) pre-coated with Geltrex (Thermo Fisher Scientific) at a density of 1.5 x 105 cells/cm2, and incubated them in NPC medium overnight at 37 °C, 5 % CO2.

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

The next day, NPCs were treated with either dimethylsulfoxide (DMSO) or sildenafil dilution series in 100 μl and incubated for 16 h at 37°C, 5% CO2.

Side Effects

We obtained written informed consent to use patient material and health data from the patients and their guardians according to the Declaration of Helsinki. On day 6, we transferred the suspended cells onto Matrigel-coated well plates using: the same media without SB-431542 and dorsomorphin, but with the addition of 150 μM ascorbic acid (Sigma-Aldrich). We maintained NPCs on this media and used them for experiments between passage 7 and passage 30. Differentiation into neuronal cultures containing dopaminergic neurons was performed as previously described.25 Briefly, starting from NPCs at low confluence (10-30 %), we switched to media containing Neurobasal:DMEM/F12 (1:1), 0.5 x N2, 0.5 x B27 with vitamin A, 1 x MycoZap Plus-CL, with the addition of 200 mM vitamin C, 100 ng/ml FGF8 (R&D Systems) and 1 mM PMA. After 8 days, we used the media Neurobasal:DMEM/F12 (1:1), 0.5 x N2, 0.5 x B27 with vitamin A, 1 x MycoZap Plus-CL, supplemented with 200 mM vitamin C, 0.5 mM PMA, 500 mM cAMP (STEMCELL Technologies), 10 ng/mL BDNF (MACS Miltenyi), 10 ng/ml GDNF (MACS Miltenyi) and 1ng/mL TGF-beta 3 (MACS Miltenyi).

Clinical monitoring

After ten days, the medium was changed to Neurobasal:DMEM/F12 (1:1), 0.5 x N2, 0.5 x B27 with vitamin A, 1 x MycoZap Plus-CL, supplemented with 200 mM vitamin C, 500 mM cAMP, 10 ng/ml BDNF, 10 ng/ml GDNF, and 1 ng/ml TGF-beta 3. Cortical brain organoids were generated following two protocols (Figure S7A). The first protocol was based on our modifications62 of a previous publication.134 Briefly, on day 0, iPSCs at 80 % confluence were seeded into low-attachment U-bottom 96-well plates (Corning) to induce neurosphere formation. iPSCs were washed with DPBS (Gibco) and detached with Accutase (Gibco) for 5 min at 37 °C. Cell pellets were resuspended in cortical differentiation medium I (CDMI) consisting of Glasgow-MEM, 20 % Knockout Serum Replacement, MEM-NEAA, sodium pyruvate, 2-mercaptoethanol and penicillin-streptomycin (all from Gibco). After 30 min incubation, cells were washed with 100 μl NPC medium without phenol red.

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At day 30 after birth, pigs were transferred to the breeding cages and gradually switched from reconstituted milk to pelleted feed. Experiments in pigs were carried out according to the ARRIVE guidelines and approved by the Local Ethics Committee of Avantea, in accordance with the Italian Law (D.Lgs 26/2014) and EU directive 2010/63/EU regulating animal experimentation and approved by relevant authorities (Ministry of Health project # 367/2022-PR). Big-conductance calcium-activated potassium (BKCa) KO human U87 glioblastoma lines with stably deletion of the KCNMA1 gene, encoding for BKCa,80 were kindly provided by Prof. Adam Szewczyk (Nencki Institute, Warsaw). WT and BKCa KO glioblastoma (U-87 MG) cells were grown in DMEM medium (Thermo Fisher Scientific) with 10 % fetal bovine serum (FBS) (Gibco) 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco) in a humidified atmosphere of 5 % CO2 at 37 °C.80 The off-label individual basis treatment with sildenafil citrate (Revatio®) in LS patients carrying MT-ATP6 variants was registered on ClinicalTrials.gov (ID: NCT06967831).

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The treatment was performed as “individual basis treatment", also known as “named-patient basis (NPB) therapy”, which refers to pre-authorization access to unauthorized medicines requested by doctors for specific patients with serious unmet needs. In this treatment, the physicians carry an individual responsibility and there is no requirement of formal approval by EMA or a local IRB. This process is governed by national legislation within each EU Member State, under Article 5(1) of Directive 2001/83/EC. Patients were of Caucasian ethnicity and included both male and female individuals (according to sildenafil gel sex assigned at birth). Details of patient features and clinical outcomes are reported in Tables 1, S3, and S4. NPCs were kept in 100 μl medium (w/o phenol red) for the duration of the assay.

Storage Condition	Details	Temperature Range
Cool, Dry Place	Keep away from light and moisture	15-30°C
Keep Out of Reach of Children	To prevent accidental ingestion	-
Packaging	Original tightly sealed containers	-

For confirming MMP depolarizing effect, we used the mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) in combination with the mitochondrial complex III inhibitor antimycin A (AA).31 Imaging was performed with an Operetta CLS high content imaging system (Revvity) with 20 x water objective in confocal mode.

• 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.

Imaging time did not exceed 30 min.

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Petra Böhme); 21 microsatellite loci were amplified using PCR and tagged products were analyzed using GeneMapper ID v.3.2.1 (Applied Biosystems). The use of iPSCs in pseudoanonymized manner was approved by the local ethic committees of Charité-Universitätsmedizin Berlin (EA2/131/13 and EA2/107/14) and Heinrich Heine University Düsseldorf (Study number 2020-967_5). Germline Ndufs4 KO mice were obtained from the University of Washington.132 All mice were on the C57/BL6/J background and were fed the regular diet from Harlan Teklad.132 Sildenafil citrate treatment was initiated soon after weaning and genotyping, between day 25-30 of age. Treatment studies were carried out in 16 female mice and 14 male mice. Mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Johns Hopkins University, USA (M023M236).

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Domestic pigs of large white background were used to study SURF1 KO. Male KO animals were compared to sex and age-matched WT animals. 7 SURF1 KO male animals were treated with sildenafil citrate starting immediately after birth. On the first day after birth, pigs were fed reconstituted milk (NeoPig Rescue, Provimi, 10-12 ml every hour) via gastric tube. By the second day, WT piglets and almost all sildenafil-treated SURF1 KO piglets began drinking milk independently from an automatic feeder connected to the incubator. TMRM and Hoechst 33342 intensity within the cells was calculated using Columbus software (Revvity, version 2.9.0).

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The supplemented seeding suspension was distributed to low attachment U-bottom 96-well plates by adding 100 μl per well and incubated at 37 °C and 5 % CO2. On day 18, the developing organoids were transferred to 100 mm petri dishes filled with cortical differentiation medium II (CDMII) consisting of DMEM/ F12, 1 % GlutaMAX, 1 % N2 supplement, 1 % chemically defined lipid concentrate and penicillin-streptomycin (Gibco). The petri dishes were placed on an orbital shaker at 70 rpm at 37 °C and 5 % CO2. On day 35, CDMII was replaced with cortical differentiation medium III (CDMIII) that in addition contains 10 % FBS (Gibco) and heparin (Merck). CDMIII was changed every two to four days, depending on the colour of the medium. To assess the effect of KT5823 on MMP in presence and absence of sildenafil, NPCs were seeded, incubated as described above, and pre-treated for 1 h with either DMSO alone or 1 μM KT5823 dissolved in DMSO. After the incubation time, 10 μM sildenafil dissolved in DMSO was added on top and incubated for 16 h at 37°C, 5% CO2.

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We cultured all iPSC lines on Matrigel (Corning)-coated plates using StemMACS iPS-Brew XF medium, supplemented with MycoZap (Lonza). CTRL_8 and ATP6_2 iPSCs were cultured in mTeSR Plus Medium (STEMCELL Technologies) prior to BCEC differentiation. Information on sex and age of the individuals from which the iPSCs were derived is provided in Figure S10H. We cultivated all iPSCs in a humidified atmosphere of 5 % CO2 at 37 °C and 5 % oxygen. iPSCs were split at 70-80 % confluence with 0.5 μM EDTA (Invitrogen) in 1 x PBA (Gibco).

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We added 10 μM ROCK inhibitor (Enzo Biochem Inc) after splitting to promote survival. Cultures were routinely monitored for mycoplasma contamination by PCR using nine primers to amplify the six most common mycoplasma strains (Table S5). The positive control and internal contro were kindly provided by Dr. Cord Uphoff (DZMS, Germany). We monitored the identity of iPSCs using STR analysis, which was performed by the Forensic Department of the University Hospital Düsseldorf (Dr.