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Property	Description	Value
Molecular Formula	-	C22H30N6O4S
Molecular Weight	-	474.58 g/mol
Melting Point	-	187°C
Solubility	In water	Very low
pKa	-	6.8

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 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. 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 each BCEC differentiation.

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However, using this later approach does not change the conclusions from the model. The additional Ca2+ flux (Jx) was added to capture the many experimental results, and particularly the observations that mitochondrial Ca2+ does not drop to zero in MCU knocked-down cells.70,164 The effects of pyruvate dehydrogenase (PDH)-catalyzed reaction, glycolytic pathway (vGLY), and the TCA cycle (reduction of [NAD+]m into NADH) is formulated as: The aspartate-glutamate carrier (AGC) is part of MAS NADH shuttle system defined as: The rate at which NADH is oxidized in the ETC and the rate at which protons are extruded from the mitochondria are combined into one equation as: The activity of adenine nucleotide translocator (ANT) in electrogenic exchange of ATP or ADP across the inner mitochondrial membrane is given by:where and denote the fact that only a fraction of nucleotides has access to the transporter. The dependence of the membrane potential is due to the negatively charged ADP and ATP. The rate of ATP synthesis by F1F0 ATPase is modeled as: JHYD represents the rate of ATP consumption (ATP hydrolysis) in the cytosol. The first term encodes the link between Ca2+ activity and ATP consumption in the cytosol whereas the second term captures ATP-consuming processes in the cytosol.

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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).

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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. 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.

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

Chromatographic sildenafil pills 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).

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After three baseline acidification rate recordings, 10 mM of glucose was injected, followed by a sequential injection of 1 μM ATP synthase inhibitor oligomycin A, and 100 mM 2-deoxy-D-glucose. Measurements of extracellular acidification were obtained via a Seahorse XFe24 Analyzer. Total protein content served as normalization for oxygen consumption rate (OCR) in each well. To determine the effect of impaired calcium signaling on mitochondrial function in LS, we applied a mathematical model that couples cytosolic calcium dynamics with mitochondrial function.70 The cytosolic calcium equations were modified to incorporate calcium exchange between the cytoplasm and extracellular space through plasma membrane calcium ATPase (PMCA) and store-operated calcium channels (SOCC). In this model, both PMCA and sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) fluxes depend on the ATP levels to capture the potential rise in cytosolic calcium due to reduced ATP levels in cells with MT-ATP6 variants.

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Parameters used in the model are based on previous works70,156,157,158,159,160,161,162 and listed in Table S2. We converted the calcium fluorescence traces from cBOS (Figure 4B) into corresponding cytosolic calcium concentration () using the formula developed before.163 That is,Where is the change in , = 0.1 μM is the baseline cytosolic Ca2+ concentration, = 0.2 mM in the dissociation constant for OBG-1 (used to label cytoplasmic calcium in cBOS), and . Fmax and Fmin are the maximum and minimum fluorescent values in a trace, respectively. The instantaneous change in fluorescence is , where = 0.01 is the baseline fluorescence. Finally, with Fmax as lovegra sildenafil the maximum fluorescence in the trace.

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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).

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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, sildenafil gel 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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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.

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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 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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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 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). Imaging and analysis were performed using the Incucyte live-cell analysis system.

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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. A Glycostress test was used to evaluate glycolysis activity, following a previously established protocol.155 One hour before assay initiation, the cell culture medium was replaced with glucose-free Agilent XF assay medium and iPSC-CMs were incubated for 20 min at 37 °C in the absence of CO2. After three baseline acidification rate recordings, 10 mM of glucose was injected, followed by a sequential injection of 1 μM ATP synthase inhibitor oligomycin A, and 100 mM 2-deoxy-D-glucose. Measurements of extracellular acidification were obtained via a Seahorse XFe24 Analyzer.

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Total protein content served as normalization for oxygen consumption rate (OCR) in each well.

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To determine the effect of impaired calcium signaling on mitochondrial function in LS, we applied a mathematical model that couples cytosolic calcium dynamics with mitochondrial function.70 The cytosolic calcium equations were modified to incorporate calcium exchange between the cytoplasm and extracellular space through plasma membrane calcium ATPase (PMCA) and store-operated calcium channels (SOCC). In this model, both PMCA and sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) fluxes depend on the ATP levels to capture the potential rise in cytosolic calcium due to reduced ATP levels in cells with MT-ATP6 variants. Parameters used in the model are based on previous works70,156,157,158,159,160,161,162 and listed in Table S2. We converted the calcium fluorescence traces from cBOS (Figure 4B) into corresponding cytosolic calcium concentration () using the formula developed before.163 That is,Where is the change in , = 0.1 μM is the baseline cytosolic Ca2+ concentration, = 0.2 mM in the dissociation constant for OBG-1 (used to label cytoplasmic calcium in cBOS), and .

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Fmax and Fmin are the maximum and minimum fluorescent values in a trace, respectively.

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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. 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 sildenafil pills 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).

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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. 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.

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After incubation, sildenafil gel 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. 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. A Glycostress test was used to evaluate glycolysis activity, following a previously established protocol.155 One hour before assay initiation, the cell culture medium was replaced with glucose-free Agilent XF assay medium and iPSC-CMs were incubated for 20 min at 37 °C in the absence of CO2. The instantaneous change in fluorescence is , where = 0.01 is the baseline fluorescence. Finally, with Fmax as lovegra sildenafil the maximum fluorescence in the trace.

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To match the smaller timestep used in simulating mitochondrial bioenergetics, the time trace was interpolated. The resulting traces were used for fitting the model to the observations in cells from MT-ATP6 and MT-ATP6 + Sil experiments. These equations were solved in MATLAB 2023b using ode15s method, and are given as:Where Ca2+ fluxes from the endoplasmic reticulum (ER) to the cytoplasm through IP3 receptors (JIP3), from the cytoplasm to the ER through SERCA (JSERCA), from the cytoplasm to the extracellular space (ECS) through PMCA (JPMCA), from the ECS to the cytoplasm through SOCC (JSOCC), from the cytoplasm to mitochondria through mitochondrial calcium uniporter (MCU) (JMCU), and from mitochondria to the cytoplasm through Na+/Ca2+ exchangers (NCX) (JNCX) are given as: We modeled metabolic stress by a rise in IP3 concentration through the phospholipase C (PLC) pathway due to the rise in glutamate during metabolic stress, which is formulated by equation (11). Metabolic stress also impairs the fluxes through PMCA and SERCA through a decrease in [ATP]c, which is already incorporated in their respective equations. One can also model metabolic stress by decreasing the rate at which NADH is oxidized in the electron transport chain (ETC) (and/or decreasing the rate of glycolytic pathway and other complexes in ETC) by itself or in combination with a rise in IP3 concentration. However, using this later approach does not change the conclusions from the model. The additional Ca2+ flux (Jx) was added to capture the many experimental results, and particularly the observations that mitochondrial Ca2+ does not drop to zero in MCU knocked-down cells.70,164 The effects of pyruvate dehydrogenase (PDH)-catalyzed reaction, glycolytic pathway (vGLY), and the TCA cycle (reduction of [NAD+]m into NADH) is formulated as: The aspartate-glutamate carrier (AGC) is part of MAS NADH shuttle system defined as: The rate at which NADH is oxidized in the ETC and the rate at which protons are extruded from the mitochondria are combined into one equation as: The activity of adenine nucleotide translocator (ANT) in electrogenic exchange of ATP or ADP across the inner mitochondrial membrane is given by:where and denote the fact that only a fraction of nucleotides has access to the transporter. The dependence of the membrane potential is due to the negatively charged ADP and ATP. The rate of ATP synthesis by F1F0 ATPase is modeled as: JHYD represents the rate of ATP consumption (ATP hydrolysis) in the cytosol. The first term encodes the link between Ca2+ activity and ATP consumption in the cytosol whereas the second term captures ATP-consuming processes in the cytosol.