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Another key responder was PRKG1, a master regulator and downstream target of PDE5,56 which was highlighted by the multi-omics map of the sildenafil rescue signature (Figure 2D). To monitor the expression of putative sildenafil-responsive genes, we treated LS NPCs grown in physiologically low glucose with 1 or 10 μM sildenafil for 6 and 24 h (Figures S6E and S6F). The treatments modulated the expression of sildenafil targets over time, including proliferation-related genes BRD4, STAT3, and NOTCH157,58; synapsis-associated NRG159; neuroinflammation-associated P2RX460; and glucose metabolism-related SLC37A4.61 We used cortical brain organoids to explore the impact of sildenafil on human neurodevelopment.

In agreement with previous studies in LS brain organoids,25,29,30 MT-ATP6 variants impaired neurogenic zone formation (Figure 3A) and altered the ratio of early neurons to neural progenitors (Figure 3B). Two different protocols for generating cortical brain organoids showed defective growth rates (Figure S7A).

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We applied chemical manipulation of the PDE5 pathway to prove its involvement in the MMP rescue of LS NPCs (Figure 4I). The cGMP analog 8-Br-cGMP recapitulated the MMP amelioration seen with sildenafil (Figure 4J). Inhibition of the downstream target PRKG1 with KT5823 blunted the effect of sildenafil on MMP normalization (Figure 4K). We quantified the effect of sildenafil on neuronal outgrowth81 of dopaminergic neurons (Figure S10C). In agreement with previous findings,25,30 MT-ATP6 mutant neurons and SURF1 mutant neurons exhibited reduced neurite length (Figures 4N, 4O, and S10D).

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Sildenafil promoted neurite outgrowth in LS neurons (Figures 4N, 4O, and S10D). This improvement was specific to LS neurons (carrying MT-ATP6, SURF1, or NDUFS4 variants) (Figure 4P) and was not observed in control neurons (Figure S10E). PRKG1 signal was reduced in LS neurons (Figure S10F), and PRKG1 knockdown in control neurons recapitulated the neurite growth defects seen in LS neurons (Figure 4Q). Because PRKG1 is involved in both calcium homeostasis and neurite outgrowth,82,83,84,85,86 its dysregulation in LS might underscore its role as a therapeutic target that could contribute to the sildenafil response (Figure S10G). To address the therapeutic potential of sildenafil in vivo, we employed the germline Ndufs4 KO mouse.14,15 Sildenafil was added to the drinking water of the animals starting on day 25. One protocol62 showed size defects in LS organoids after 50 days in culture (Figure S7B); another protocol,63 allowing initial homogeneous organoid shape, resulted in earlier growth defects in LS organoids that became less pronounced over time (Figure S7B). The results suggest that MT-ATP6 variants might affect neural progenitor development.

We treated LS brain organoids with sildenafil for either 24 h (acute paradigm) or 45 days (chronic paradigm) (Figure 3C). MT-ATP6 variants disrupted BPs related to synapses and neuronal projections (Figure S7E) and CCs related to neuronal cell bodies and synapses (Figure S7F). The acute sildenafil signature modulated pathways related to embryonic development and Wingless-related integration site (WNT) signaling (Figure 3F) and corrected gene defects (Figures S7C and S7D), including WDR45B, which was downregulated in LS brain organoids (Table S1) and upregulated by sildenafil (Figure S7D), and whose variants are associated with neurodevelopmental disorders.64 Sildenafil also rescued the ratio of early neurons to neural progenitors (Figure 3D) and upregulated DBX1 (Figure 3E). To dissect the cell populations affected by MT-ATP6 variants and chronic sildenafil, we performed single-nucleus RNA sequencing (snRNA-seq) (Figure 3C). Unsupervised clustering highlighted 9 clusters (Figure S8A): clusters 0 and 3 for radial glia, clusters 4 and 8 for progenitors, cluster 6 for proliferating progenitors, cluster 5 for immature neurons, cluster 2 for FOXG1-positive neurons, cluster 1 for FOXG1-negative neurons, and cluster 7 for other cell types (Figure S8B). This annotation revealed that MT-ATP6 variants impaired neuronal commitment, with alterations in radial glia, progenitors, and FOXG1-positive neurons (Figures 3G and 3H).

The disease signature involved the downregulation in the progenitor population of PRKG1 and NLGN1, a regulator for synapse development65 (Figure 3I), and the downregulation in the neuronal population of genes impacting neurite outgrowth, such as STMN266 (Figure S8D; Table S1).

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The sildenafil signature upregulated neuronal outgrowth-associated 150mg sildenafil for sale genes, such as RGS667 (Figures S8C and S8D; Table S1).

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The effect of sildenafil was mostly evident in radial glia and progenitors (Figure S8C; Table S1). Whereas STMN2 was mainly present in FOXG1-positive neurons and immature neurons, RGS6 was found mainly in radial glia, and PRKG1 in radial glia and progenitors (Figures 3J and S8D).

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To dissect the cell populations affected by MT-ATP6 variants and chronic sildenafil, we performed single-nucleus RNA sequencing (snRNA-seq) (Figure 3C). Unsupervised clustering highlighted 9 clusters (Figure S8A): clusters 0 and 3 for radial glia, clusters 4 and 8 for progenitors, cluster 6 for proliferating progenitors, cluster 5 for immature neurons, cluster 2 for FOXG1-positive neurons, cluster 1 for FOXG1-negative neurons, and cluster 7 for other cell types (Figure S8B). This annotation revealed that MT-ATP6 variants impaired neuronal commitment, with alterations in radial glia, progenitors, and FOXG1-positive neurons (Figures 3G and 3H). The disease signature involved the downregulation in the progenitor population of PRKG1 and NLGN1, a regulator for synapse development65 (Figure 3I), and the downregulation in the neuronal population of genes impacting neurite outgrowth, such as STMN266 (Figure S8D; Table S1). The sildenafil signature upregulated neuronal outgrowth-associated 150mg sildenafil for sale genes, such as RGS667 (Figures S8C and S8D; Table S1).

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The effect of sildenafil was mostly evident in radial glia and progenitors (Figure S8C; Table S1). Whereas STMN2 was mainly present in FOXG1-positive neurons and immature neurons, RGS6 was found mainly in radial glia, and PRKG1 in radial glia and progenitors (Figures 3J and S8D). The glycolytic signature, suggested to be an indicator of brain organoid stress,68 was not altered, indicating that neither MT-ATP6 variants nor sildenafil posed additional stress (Figure S8E). Altogether, LS disrupted brain organoid development by impairing early neuronal organization of radial glia and progenitor populations, and sildenafil specifically affected those populations. We examined the functional consequences of sildenafil in LS neural cells. The glycolytic signature, suggested to be an indicator of brain organoid stress,68 was not altered, indicating that neither MT-ATP6 variants nor sildenafil posed additional stress (Figure S8E). Altogether, LS disrupted brain organoid development by impairing early neuronal organization of radial glia and progenitor populations, and sildenafil specifically affected those populations. We examined the functional consequences of sildenafil in LS neural cells. Given the reported calcium dysregulation26,27 and the known deterioration of LS patients upon metabolic decompensation,3 we induced acute metabolic stress in LS brain organoids to monitor their intracellular calcium response. We dissected brain organoids on days 70–74 to prepare cortical brain organoid slices (cBOSs),69 which we grew until day 129 and then treated with sildenafil for 24 h before applying acute metabolic stress (2 min of glucose deprivation and inhibition of glycolysis [GLY] and OXPHOS) (Figure 4A). The calcium response to metabolic stress was more pronounced and premature in LS cBOS compared with control cBOS, suggesting increased susceptibility to metabolic imbalance (Figures 4B and 4C). Pre-treatment with sildenafil in LS cBOS reduced their calcium load after metabolic stress and also their peak calcium amplitude (Figures 4B and 4C). Hence, sildenafil might prevent excessive decompensation in LS neuronal cells under acute metabolic stress.

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The modeling thus suggested that sildenafil’s action may be mimicked by increasing CV activity together with sildenafil tablet manforce 50 mg decreasing calcium release from the endoplasmic reticulum (ER), in turn improving cellular bioenergetics. In the absence of extracellular calcium, pre-treatment with sildenafil in LS NPCs increased the thapsigargin-induced cytoplasmic calcium signal (Figures 4D and 4E), possibly indicating higher ER calcium storage or modulation of mitochondrial calcium homeostasis.74,75 A faster decline of calcium in sildenafil-treated cells (Figure 4D) could imply reduced calcium release from the ER through IP3R, as suggested by the model, or enhanced cytosolic calcium efflux to the extracellular space through the sodium-calcium exchanger (NCX) or the PMCA because of increased cytosolic ATP levels by sildenafil or augmented uptake into mitochondria. To indirectly address mitochondrial calcium, we investigated big-conductance calcium-activated potassium (BKCa) channels, which are localized in the plasma and mitochondrial membrane76 and have been implicated in sildenafil’s action.77,78 As previously shown,79 human BKCa KO cells80 displayed increased MMP compared with wild type (WT) (Figures 4F and 4G). Sildenafil normalized MMP when BKCa KO cells were incubated in DMSO (Figure 4F) but not when they were acutely exposed to the CV inhibitor oligomycin (Figure 4G). The results suggest that BKCa channels may contribute to the action of sildenafil on MMP when cells lack a functional CV and that residual CV activity is necessary for the restorative effect of sildenafil, as indicated by the model.

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

These findings raised the possibility that MMP normalization by sildenafil may not be due to direct mitochondrial uncoupling. To test this, we employed NPCs carrying a variant in the nuclear gene SURF125 (Figure S10H). The MMP of SURF1 mutant NPCs was depolarized compared with that of isogenic control NPCs (Figure 4H). Nonetheless, sildenafil still restored MMP (Figure 4H). Hence, sildenafil might also be beneficial in other forms of LS where MMP is depolarized. To investigate the interplay between calcium and bioenergetics in treated LS neural cells, we applied a mathematical model70 that coupled cytosolic calcium dynamics with mitochondrial function, incorporating F1F0 ATPase activity (F1F0), GLY, the adenine nucleotide translocator (ANT), NADH production via the aspartate-glutamate carrier (AGC), and NADH oxidation in ETC (o). Based on our previous calcium modeling,71,72,73 we integrated additional fluxes through the plasma membrane calcium ATPase (PMCA), the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and IP3 receptors (IP3Rs) (Table S2). Following global sensitivity analysis70 (Figure S9A), the model recapitulated the pattern of calcium traces measured in LS cBOS (Figure S9B). Combining a high rate of F1F0 with a low rate of IP3R (Figure S9C) reproduced the reduction of calcium peak in sildenafil-treated LS cBOS under stress (Figures 4B and 4C) and key features observed in sildenafil-treated LS NPCs, including MMP reduction (Figures 1D and 1E), slight NADH reduction (Figure S1D), and ATP increase (Figure 1G). Other combinations failed to recapitulate sildenafil effects (Figures S9D and S9E). The modeling thus suggested that sildenafil’s action may be mimicked by increasing CV activity together with sildenafil tablet manforce 50 mg decreasing calcium release from the endoplasmic reticulum (ER), in turn improving cellular bioenergetics. In the absence of extracellular calcium, pre-treatment with sildenafil in LS NPCs increased the thapsigargin-induced cytoplasmic calcium signal (Figures 4D and 4E), possibly indicating higher ER calcium storage or modulation of mitochondrial calcium homeostasis.74,75 A faster decline of calcium in sildenafil-treated cells (Figure 4D) could imply reduced calcium release from the ER through IP3R, as suggested by the model, or enhanced cytosolic calcium efflux to the extracellular space through the sodium-calcium exchanger (NCX) or the PMCA because of increased cytosolic ATP levels by sildenafil or augmented uptake into mitochondria. To indirectly address mitochondrial calcium, we investigated big-conductance calcium-activated potassium (BKCa) channels, which are localized in the plasma and mitochondrial membrane76 and have been implicated in sildenafil’s action.77,78 As previously shown,79 human BKCa KO cells80 displayed increased MMP compared with wild type (WT) (Figures 4F and 4G). Sildenafil normalized MMP when BKCa KO cells were incubated in DMSO (Figure 4F) but not when they were acutely exposed to the CV inhibitor oligomycin (Figure 4G).

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Another key responder was PRKG1, a master regulator and downstream target of PDE5,56 which was highlighted by the multi-omics map of the sildenafil rescue signature (Figure 2D). To monitor the expression of putative sildenafil-responsive genes, we treated LS NPCs grown in physiologically low glucose with 1 or 10 μM sildenafil for 6 and 24 h (Figures S6E and S6F). The treatments modulated the expression of sildenafil targets over time, including proliferation-related genes BRD4, STAT3, and NOTCH157,58; synapsis-associated NRG159; neuroinflammation-associated P2RX460; and glucose metabolism-related SLC37A4.61 We used cortical brain organoids to explore the impact of sildenafil on human neurodevelopment. In agreement with previous studies in LS brain organoids,25,29,30 MT-ATP6 variants impaired neurogenic zone formation (Figure 3A) and altered the ratio of early neurons to neural progenitors (Figure 3B). Two different protocols for generating cortical brain organoids showed defective growth rates (Figure S7A).

RV remained apparently normal by two-day LV pressure overload (TAC), with mild LV hypertrophy that was inhibited by sildenafil

One protocol62 showed size defects in LS organoids after 50 days in culture (Figure S7B); another protocol,63 allowing initial homogeneous organoid shape, resulted in earlier growth defects in LS organoids that became less pronounced over time (Figure S7B). The results suggest that MT-ATP6 variants might affect neural progenitor development. We treated LS brain organoids with sildenafil for either 24 h (acute paradigm) or 45 days (chronic paradigm) (Figure 3C). MT-ATP6 variants disrupted BPs related to synapses and neuronal projections (Figure S7E) and CCs related to neuronal cell bodies and synapses (Figure S7F). The acute sildenafil signature modulated pathways related to embryonic development and Wingless-related integration site (WNT) signaling (Figure 3F) and corrected gene defects (Figures S7C and S7D), including WDR45B, which was downregulated in LS brain organoids (Table S1) and upregulated by sildenafil (Figure S7D), and whose variants are associated with neurodevelopmental disorders.64 Sildenafil also rescued the ratio of early neurons to neural progenitors (Figure 3D) and upregulated DBX1 (Figure 3E). The results suggest that BKCa channels may contribute to the action of sildenafil on MMP when cells lack a functional CV and that residual CV activity is necessary for the restorative effect of sildenafil, as indicated by the model. These findings raised the possibility that MMP normalization by sildenafil may not be due to direct mitochondrial uncoupling.

• Storage of sildenafil should be in a cool, dry place away from direct sunlight.
• Common side effects include dizziness, upset stomach, or vision changes.
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To test this, we employed NPCs carrying a variant in the nuclear gene SURF125 (Figure S10H). The MMP of SURF1 mutant NPCs was depolarized compared with that of isogenic control NPCs (Figure 4H). Nonetheless, sildenafil still restored MMP (Figure 4H). Hence, sildenafil might also be beneficial in other forms of LS where MMP is depolarized. We applied chemical manipulation of the PDE5 pathway to prove its involvement in the MMP rescue of LS NPCs (Figure 4I). The cGMP analog 8-Br-cGMP recapitulated the MMP amelioration seen with sildenafil (Figure 4J). Inhibition of the downstream target PRKG1 with KT5823 blunted the effect of sildenafil on MMP normalization (Figure 4K). We quantified the effect of sildenafil on neuronal outgrowth81 of dopaminergic neurons (Figure S10C).

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Given the reported calcium dysregulation26,27 and the known deterioration of LS patients upon metabolic decompensation,3 we induced acute metabolic stress in LS brain organoids to monitor their intracellular calcium response. We dissected brain organoids on days 70–74 to prepare cortical brain organoid slices (cBOSs),69 which we grew until day 129 and then treated with sildenafil for 24 h before applying acute metabolic stress (2 min of glucose deprivation and inhibition of glycolysis [GLY] and OXPHOS) (Figure 4A). The calcium response to metabolic stress was more pronounced and premature in LS cBOS compared with control cBOS, suggesting increased susceptibility to metabolic imbalance (Figures 4B and 4C). Pre-treatment with sildenafil in LS cBOS reduced their calcium load after metabolic stress and also their peak calcium amplitude (Figures 4B and 4C). Hence, sildenafil might prevent excessive decompensation in LS neuronal cells under acute metabolic stress.

S4 Fig. Western blot of t-ERK1/2 in the LV myocardium.

To investigate the interplay between calcium and bioenergetics in treated LS neural cells, we applied a mathematical model70 that coupled cytosolic calcium dynamics with mitochondrial function, incorporating F1F0 ATPase activity (F1F0), GLY, the adenine nucleotide translocator (ANT), NADH production via the aspartate-glutamate carrier (AGC), and NADH oxidation in ETC (o). Based on our previous calcium modeling,71,72,73 we integrated additional fluxes through the plasma membrane calcium ATPase (PMCA), the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and IP3 receptors (IP3Rs) (Table S2). Following global sensitivity analysis70 (Figure S9A), the model recapitulated the pattern of calcium traces measured in LS cBOS (Figure S9B). Combining a high rate of F1F0 with a low rate of IP3R (Figure S9C) reproduced the reduction of calcium peak in sildenafil-treated LS cBOS under stress (Figures 4B and 4C) and key features observed in sildenafil-treated LS NPCs, including MMP reduction (Figures 1D and 1E), slight NADH reduction (Figure S1D), and ATP increase (Figure 1G). Other combinations failed to recapitulate sildenafil effects (Figures S9D and S9E). In agreement with previous findings,25,30 MT-ATP6 mutant neurons and SURF1 mutant neurons exhibited reduced neurite length (Figures 4N, 4O, and S10D). Sildenafil promoted neurite outgrowth in LS neurons (Figures 4N, 4O, and S10D).

This improvement was specific to LS neurons (carrying MT-ATP6, SURF1, or NDUFS4 variants) (Figure 4P) and was not observed in control neurons (Figure S10E). PRKG1 signal was reduced in LS neurons (Figure S10F), and PRKG1 knockdown in control neurons recapitulated the neurite growth defects seen in LS neurons (Figure 4Q).

• 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.

Because PRKG1 is involved in both calcium homeostasis and neurite outgrowth,82,83,84,85,86 its dysregulation in LS might underscore its role as a therapeutic target that could contribute to the sildenafil response (Figure S10G). To address the therapeutic potential of sildenafil in vivo, we employed the germline Ndufs4 KO mouse.14,15 Sildenafil was added to the drinking water of the animals starting on day 25. The treatment extended the lifespan of Ndufs4 KO mice (Figure 5A) and alleviated muscle weakness and ataxia by partially correcting defective energy expenditure (Figure 5B). Upon placing LS mice in metabolic chambers (Figure S11A), we observed improved oxygen consumption and carbon dioxide production with sildenafil (Figure 5C), possibly indicating enhanced metabolic fitness.

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The treatment extended the lifespan of Ndufs4 KO mice (Figure 5A) and alleviated muscle weakness and ataxia by partially correcting defective energy expenditure (Figure 5B). Upon placing LS mice in metabolic chambers (Figure S11A), we observed improved oxygen consumption and carbon dioxide production with sildenafil (Figure 5C), possibly indicating enhanced metabolic fitness. Cardiac bradyarrhythmia and dysfunction were also ameliorated (Figure S11B).

S3 Fig. Western blot of p-ERK1/2 in the LV myocardium.

Cardiac bradyarrhythmia and dysfunction were also ameliorated (Figure S11B).