Sodium disrupts mitochondrial energy metabolism to execute NECSO

Domain: biology
Published: 2025-12-13
Editorial outcome: major_revision

Abstract
Na+ influx is a critical pathological event in various conditions such as ischemia, hyperosmotic stress, and organ failure. Although persistent activation of the transient receptor potential cation channel subfamily M member 4 (TRPM4) by chemical agonist Necrocide 1 (NC1) triggers necrosis by sodium overload (NECSO), the underlying mechanism remains to be elucidated. Here, we demonstrate that Na+ influx promotes necrosis by suppressing mitochondrial energy production. TRPM4-mediated Na⁺ entry elevates mitochondrial Na⁺ and reduces mitochondrial Ca²⁺ via NCLX, inhibiting oxidative phosphorylation and the Trichloroacetic acid (TCA) cycle, leading to severe energy depletion. This results in Na/K-ATPase inactivation, loss of ion gradients, cellular swelling and lysis. Our study reveals how sodium overload in NECSO disrupts mitochondrial metabolism to cause energy failure, potentially underlying diseases with elevated Na⁺.

Introduction
Na+ is the most abundant cation in extracellular fluid. Normal concentrations of Na+ in extracellular fluid (ECF) range from 135 to 145 mmol/L, while the concentrations of Na+ inside cells (intracellular fluid) are ~10–12 mmol/L1. Maintaining this transmembrane concentration gradient is crucial for establishing the resting membrane potential and facilitating nutrient (such as glucose, amino acids, etc.) transport across cell membranes2. The Na+ gradient is also pivotal in osmoregulation and water balance because Na+, accounting for 90% of ECF osmolality in combination with associated anions in mammalian cells3, drives water via osmosis to the direction of elevated Na+ concentration. Therefore, the maintenance of the Na+ gradient is vital for cellular homeostasis and the preservation of cell volume. In order to stabilize Na+ gradients, organisms have evolved complex mechanisms, including ion transport proteins (e.g., Na+/Ca2+ exchanger)4, ion channels (e.g., TRP channels)5, and ion pumps like Na/K-ATPase6. Central to this regulation, Na/K-ATPase is pivotal in maintaining the transmembrane gradients of Na+ and K+. By harnessing the energy from hydrolyzing one ATP molecule, the pump expels three Na+ from the cell and imports two K+ into the cell6. A significant portion of the cellular energy expenditure is devoted to sustaining the normal operation of the Na/K-ATPase, particularly in neurons7, underscoring the importance of Na+ homeostasis in cellular function.

The collapse of the Na+ gradient and subsequent swelling of the cells seem to be a common feature in various types of necrosis. In the process of necroptosis, MLKL polymers form pores in the cellular membrane8,9. During pyroptosis, the GSDM-NT fragment binds to and induces pore formation in the cellular membrane10,11. In the case of ferroptosis, lipid peroxidation increases membrane tension, leading to the activation of Piezo112. These distinct programmed cell death pathways ultimately result in a similar consequence: a substantial influx of Na+ and water into the cell. The prevailing understanding from current research suggests that Na+ influx occurs predominantly at the late stage of cell death, subsequent to plasma membrane damage, and its role in executing cell death is largely attributed to the disruption of osmotic balance. However, a key question that requires further elucidation centers on the potential of Na+ influx to operate not just as a consequence, but as an initiating regulator in the cell death cascade.

The consequence of Na+ influx in the cytoplasm remains elusive. It has been shown that hypoxia can induce Na+ entry into mitochondria via the mitochondrial Na+/Ca2+ exchanger (NCLX). Once inside the mitochondria, Na+ interacts with phospholipids, thereby reducing the fluidity of the inner mitochondrial membrane and impeding the migration of free ubiquinone between Complex II and Complex III. This process inhibits oxidative phosphorylation (OXPHOS) function and fosters the production of reactive oxygen species (ROS)13. This research implies the potential of Na+ to influence mitochondrial energetics directly. However, the exact role of mitochondrial Na+ in promoting cell death requires further investigation. Under normal conditions, the concentration of Na+ inside mitochondria is lower than that in the cytosol. In respiring mitochondria, the concentration of Na+ in the mitochondrial matrix can be as much as 8 times lower than that in the cytosol14,15. NCLX, located on the inner mitochondrial membrane16, serves as a pivotal pathway for Na+ influx into the mitochondria13. In studies using isolated mitochondria, it has been demonstrated that an elevation in extra-mitochondrial Na+ concentration can activate the transport activity mediated by NCLX17. Nevertheless, the role of NCLX in mediating the effects of increased cytosolic Na+ on mitochondrial function and cell death is yet to be fully understood.

In our previous study, we identified a form of necrosis driven by Na+ overload, termed necrosis induced by sodium overload (NECSO)18,19. Unlike conventional forms of necrosis, NECSO is triggered by the activation of the monovalent cation channel Transient Receptor Potential Melastatin 4 (TRPM4), leading to substantial Na+ influx19. What distinguishes NECSO is its independence from well-characterized cell death pathways, such as apoptosis, necroptosis, ferroptosis, and pyroptosis, with Na+ acting as the key effector of cell death. Notably, classic features of apoptosis, such as chromatin fragmentation and caspase activation, are not observed in NECSO19. Moreover, inhibitors of apoptosis, necroptosis, and ferroptosis cannot block NECSO. Similarly, genetic depletion of key mediators like BAX, BAK, MLKL, ACSL4, and GSDMD does not prevent NECSO either18. However, TRPM4 knockout completely abolished Na+ influx and the subsequent necrotic process19.

In this study, we aim to uncover how Na+ influx, specifically through TRPM4 activation, influences mitochondrial functionality and contributes to necrosis. Utilizing the chemical agonist Necrocide 1 (NC1) to selectively activate TRPM4 provides a potent tool to further investigate the mechanisms underlying Na+ influx-induced necrosis. We propose that, by disrupting mitochondrial energy homeostasis and ionic balance, Na+ functions as an active initiator of necrosis, rather than merely a late-stage player in osmotic disruption. This study not only expands our understanding of necrosis but also potentially reveals the regulatory role of Na+ in pathological injuries characterized by elevated sodium levels.

Results
Sodium overload triggers energy depletion and mitochondrial ionic imbalance prior to cell rupture in NECSO
We initially utilized inductively coupled plasma-mass spectrometry (ICP-MS) to measure the intracellular concentration of Na+ in MCF7 cells that were treated with 100 nM NC1 at different time intervals, enabling us to investigate the dynamic changes in Na+ levels attributed to the NC1 treatment. We observed that a 30-min treatment with NC1 is sufficient to elevate intracellular sodium levels, which escalated over time with extended NC1 exposure (Fig. 1a). This indicates that sodium influx is initiated early upon NC1 treatment and persists over time.

To further confirm that sodium influx is an early event in NECSO, we subsequently monitored the time course of cell death in NC1-treated MCF7 cells. Concurrently, we assessed cellular ATP levels by CellTiter Glo and membrane integrity through propidium iodide (PI) staining positivity and lactate dehydrogenase (LDH) release. Interestingly, we found a marked decline in intracellular ATP levels prior to an increase in membrane permeability, implying a potential causal relationship (Fig. 1b). Specifically, following NC1 treatment for 2 h, a decline of ~10% in ATP levels was observed. After 4 h of NC1 treatment, ATP levels dropped to 60%, while <10% of the cells were PI positive. After 18 h, the percentage of cells staining positive for PI approached 100%. LDH release lagged behind PI positivity (Fig. 1b). The delayed release of LDH, given its large molecular size (135–145 kDa), may be attributed to the requirement for more extensive membrane damage to form larger pores than those permeable to PI. Similarly, in MB468 cells, our findings showed that the decline in intracellular ATP levels, post NC1 treatment, initiated earlier and proceeded at a faster rate compared to the increase in membrane permeability (Supplementary Fig. 1a). As a control, when MB468 cells were treated with 1 μM RSL3 to induce ferroptosis, we observed no discernible difference in the timing or rate of decline in intracellular ATP levels compared to the increase of membrane permeability (Supplementary Fig. 1b). Furthermore, a distinguishing feature was noted: in NECSO, LDH release was delayed compared to PI positivity, while in ferroptosis, LDH release closely followed PI positivity (Supplementary Fig. 1a, b), suggesting that the membrane damage in NECSO was more gentle and gradual compared with what observed in ferroptosis. In summary, our findings establish that sodium influx occurs during the earliest period post-NC1 treatment and suggest that energy depletion may contribute to subsequent membrane rupture. We further investigated the role of compounds that promote ATP generation, such as creatine and lipoic acid, in NECSO. Creatine is phosphorylated by creatine kinase to produce phosphocreatine (PCr), a high-energy compound. Upon degradation, PCr releases energy that is used to regenerate ATP from ADP and inorganic phosphate (Pi), thereby rapidly replenishing cellular ATP reserves and maintaining energy homeostasis20. α-Lipoic acid, an essential cofactor for mitochondrial metabolism, enhances the activity of mitochondrial enzymes involved in energy metabolism and ATP production21. Notably, both creatine and α-lipoic acid were found to inhibit LDH release in NECSO (Fig. 1c).

Next, it is imperative to ascertain whether the depletion of cellular energy is a direct consequence of sodium influx. To block sodium influx, we used a Na⁺-free isotonic extracellular buffer solution (EBS) without serum or amino acids, in which Na⁺ was replaced by equimolar non-permeable NMDG⁺ to preserve iso-osmolarity. A standard Na⁺-containing EBS served as control. The treatment was limited to 8 h to elicit NC1’s effect while minimizing starvation-induced cell death. The ICP-MS analysis confirmed that sodium influx was completely abolished in NC1-treated MCF7 cells cultured in Na+-free medium (Supplementary Fig. 1c). Treatment with 100 nM NC1 in MCF7 cells cultured in Na+-free medium resulted in constant intracellular ATP levels, implying the significance of sodium influx in mediating ATP fluctuations. Accordingly, there was no detection of PI staining or LDH efflux, suggesting that the cellular membrane remained intact. In contrast, MCF7 cells treated with NC1 in Na⁺-containing EBS showed a decrease in ATP levels at an early stage, followed by positive PI staining and LDH release (Supplementary Fig. 1e, f). Similarly, NC1 treatment of MB468 cells also induced early sodium influx, followed by a sodium-dependent decline in ATP levels and subsequent membrane rupture (Supplementary Fig. 1g–i).

Further, we used Seahorse XF technology to quantify cellular ATP production rate in MCF7 cells treated with NC1. The findings revealed that in the control group, a majority of the ATP in MCF7 cells is derived from mitochondrial oxidative phosphorylation (mitoATP). Notably, following treatment with NC1, the mitoATP production rate decreased while the ATP production rate by glycolysis (glycoATP) increased compensatorily. However, the total ATP production rate declined (Fig. 1d). The data confirm that NC1 induces energy depletion, pointing to mitochondria as the main reason for energy production shortfall. To further investigate whether sodium influx contributes to the decrease in ATP production rate, we measured the ATP synthesis rate following NC1 treatment in both Na⁺-containing and Na⁺-free EBS. In Na⁺-containing EBS, NC1 treatment led to a significant reduction in the overall ATP production rate, primarily due to decreased mitoATP generation, accompanied by a compensatory increase in glycoATP production (Supplementary Fig. 2a). In contrast, NC1 treatment in Na⁺-free EBS had no notable effect on ATP synthesis rate (Supplementary Fig. 2b).

Thus, we further investigated whether NC1 could cause damage to the mitochondria. We employed transmission electron microscopy (TEM) to examine the ultrastructural alterations of mitochondria in MCF7 cells following treatment with NC1. We observed that, as early as 30 min post-NC1 treatment, mitochondrial swelling commenced. After one hour of NC1 exposure, mitochondrial swelling became more pronounced. At three hours, severe cristae damage occurred, with inner membrane blebs and lowered matrix density. By five hours, cristae were largely disassembled, and the intermembrane space was notably expanded. These findings demonstrate that mitochondrial swelling progresses progressively with extended exposure to NC1 (Supplementary Fig. 2c). However, when Na+ was equimolarly substituted with NMDG+, NC1 treatment did not induce mitochondrial swelling; rather, shrinkage of the mitochondria was observed following 3 and 5 h of treatment with NC1 (Supplementary Fig. 2c). This may attribute to the displacement of Na+ by NMDG+, which blocks NC1-induced Na+ influx (Supplementary Fig. 1c) but does not block NC1-induced K+ efflux (Supplementary Fig. 1d) as potassium homeostasis is the main regulator of the mitochondrial matrix volume22. Mitochondria possess various K+ channels that permit K+ to move along their electrochemical gradient23, and the efflux of K+ from mitochondria leads to shrinkage of the matrix. We also employed Mitotracker Red to label mitochondria, finding that under conditions with Na+ present, NC1 treatment led to a weakening in Mitotracker Red signal, indicative of mitochondrial damage. In contrast, in the absence of extracellular Na+, Mitotracker Red signal was maintained following NC1 treatment (Fig. 1e, f).

Given the importance of mitochondrial ionic imbalance to mitochondrial swelling, we studied whether NC1 disturbs mitochondrial ionic balance. Exposure of MCF7 cells to NC1 in Na⁺-containing EBS resulted in elevated [Na⁺]mito (Fig. 1g) and reduced [Ca²⁺]mito, as measured using both the genetically encoded sensor mito-RGECO1 (Fig. 1H, I) and the Rhod-2 AM probe (Supplementary Fig. 2d–f). However, the changes of [Na+]mito and [Ca2+]mito were prevented by Na+ depletion (Fig. 1g–i, Supplementary Fig. 2d–f). Collectively, these data demonstrate that NC1 induces energy depletion and mitochondrial ionic imbalance prior to cell rupture, which depends on sodium influx.

Mitochondrial respiration is inhibited in NECSO
To further assess mitochondrial function, we evaluated mitochondrial respiratory capacity by measuring the oxygen consumption rate (OCR). Seahorse profile revealed that maximal respiration of MCF7 cells significantly reduced after NC1 exposure. As the duration of NC1 treatment on cells increases, the extent of suppression of maximal respiration gradually intensifies. After NC1 treatment of MCF7 cells for 5 hours, the basal respiration was also significantly inhibited (Fig. 2a–c). Consistently, in MB468 cells, OCR was also observed to decrease upon processing of NC1 (Supplementary Fig. 3a–c). We next used tetramethylrhodamine ethyl ester (TMRE) to test whether NC1 led to changes in mitochondrial membrane potential (Ψm). The live cell images showed that NC1 led to a progressive collapse of Ψm (Fig. 2d). The decrease of Ψm was also confirmed by FACS (Fig. 2e).

Recent studies have suggested that Na+ can inhibit the activity of mitochondrial Complex II+III13,24,25. Since NC1 triggers sodium influx, we explored whether the Na⁺ accumulation in the cytoplasm, induced by NC1, could attenuate the function of the mitochondrial Electron Transport Chain (ETC). We initially assessed the activity of Complex II in MCF7 cells following treatment with NC1. The results showed that the activity of Complex II decreased after treatment with NC1 (Fig. 2f). To further substantiate the direct impact of elevated cytoplasmic Na+ on ETC and rule out potential confounding effects of intracellular compensatory mechanisms, we assessed the activity of isolated mitochondrial Complex II+III under escalating concentrations of NaCl within a cell-free system. The result showed that high salt inhibited Complex II+III activity in intact mitochondria in a dose-dependent manner (Fig. 2g).

Mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis in the cytosol are both major pathways for cellular energy production. To assess the contribution of mitochondrial metabolism as an energy source in NECSO, we substituted galactose for glucose in the culture medium. Galactose, which is processed more slowly via the Leloir pathway than glucose in glycolysis26 and yields no net ATP in glycolysis27, effectively dampens glycolysis and shifts metabolism towards greater mitochondrial reliance28. The results indicated that cells with a greater dependence on mitochondrial oxidative phosphorylation are more sensitive to NC1 than cells utilizing glycolysis (Fig. 2h), implying that the suppression of mitochondrial energy generation by NC1 promotes NECSO.

TCA cycle is inhibited in NECSO
Mitochondria, in addition to playing a crucial role in energy production, have significant functions in cellular metabolism. To uncover metabolic features of NC1-treated cells, we employed liquid chromatography–mass spectrometry (LC–MS)-based targeted metabolomics to investigate intracellular metabolite alterations in MCF7 cells after NC1 treatment. Notably, pathway analysis revealed that the TCA cycle was significantly altered by NC1 exposure (Fig. 3a and Supplementary Data 1).

Further, we specifically analyzed changes in TCA cycle-related metabolites at various time points post NC1 treatment. Metabolite profiling revealed that prolonged NC1 exposure led to a time-dependent accumulation of key TCA cycle intermediates, including isocitrate, α-ketoglutarate, succinate, fumarate, and malate (Fig. 3b and Supplementary Data 1). Such a concurrent increase signifies a substantial obstruction in the TCA cycle’s flow. Since mitochondrial Ca2+ is crucial for activating mitochondrial metabolism and three key TCA cycle enzymes—pyruvate dehydrogenase (PDH)29, isocitrate dehydrogenase (IDH)30, and α-ketoglutarate dehydrogenase (AKGDH)31, the suppression of the TCA cycle observed in NC1-treated cells is likely due to a decline in [Ca2+]mito (Fig. 1h, Supplementary Fig. 2d). Supportively, inhibition of MCU, the main channel for mitochondrial calcium uptake, by either siRNAs or inhibitors (DS16570511, RuRed) further exacerbated NECSO (Supplementary Fig. 4a, b). Pyruvate is converted to acetyl-CoA in the mitochondrial matrix by the pyruvate dehydrogenase complex (PDHc), fueling the TCA cycle (Fig. 3c). The PDHc comprises three domains: pyruvate dehydrogenase (PDH, E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). Thiamine diphosphate (TPP) is an essential cofactor for pyruvate dehydrogenase32. The accumulation of pyruvate and TPP indicates a disruption in pyruvate metabolism. Moreover, phosphorylation of the E1 alpha 1 subunit (PDHA1) at Ser293, which inactivates PDHc33, increased progressively with NC1 treatment duration (Supplementary Fig. 4c). This provides further evidence that PDHc activity is attenuated during NECSO.

In Fig. 3b, succinate exhibited the most pronounced accumulation, with a noticeable increase in its levels even at an early stage (30 min) after NC1 treatment. Considering that the accumulation of succinate might be due to suppression of the conversion of succinate to fumarate catalyzed by succinate dehydrogenase (SDH, also known as Mitochondrial Complex II)34, we next evaluated the succinate/fumarate ratio. SDH serves as the hub connecting the TCA cycle and oxidative phosphorylation, which facilitates the transfer of electrons from succinate to ubiquinone while reducing succinate to fumarate (Fig. 3c). Results shown in Fig. 3d indicate that the succinate/fumarate ratio progressively increases with the extension of NC1 treatment time. This finding aligns with and supports the observation of reduced Complex II activity presented in Fig. 2f. Conclusively, the metabolic profiling provides compelling evidence for mitochondrial dysfunction in NC1-treated cells.

Sodium overload-induced mitochondrial dysfunction depends on mitochondrial NCLX
NCLX has been identified as a pivotal pathway mediating Na+ influx into mitochondria by exchanging mitochondrial Ca2+ to cytoplasm16. To ascertain whether NCLX mediates NC1-induced mitochondrial damage, we employed a specific inhibitor of NCLX (CGP37157, CGP)35 in subsequent experiments. Notably, the NCLX inhibition significantly mitigated the mitochondrial swelling induced by NC1, as shown by electron microscopy analysis (Fig. 4a). CGP markedly reversed not only the NC1-induced rise of [Na+]mito (Fig. 4b), but also the reduction of [Ca2+]mito (Fig. 4c), suggesting that NCLX plays a pivotal role in mediating the NC1-induced disturbance of mitochondrial ionic balance. Furthermore, CGP alleviated the decline of Ψm induced by NC1 (Fig. 4d). Seahorse profile also revealed that the inhibition of NCLX significantly rescued both maximal respiration and basal respiration reduction in cells treated with NC1 (Fig. 4e–g). In addition, CGP ameliorated the decrease in Complex II+III activity caused by high salt (Fig. 4h). Consistently, CGP mitigated the accumulation of TCA cycle metabolites (Supplementary Fig. 5a and Supplementary Data 2) and reduced the elevated succinate/fumarate ratio (Supplementary Fig. 5b) induced by NC1. In line with these data, CGP augmented both the mitoATP synthesis rate and the overall ATP production rate in cells that were subjected to NC1 treatment (Fig. 4i). Obviously, CGP inhibited NC1-induced cell death (Fig. 4j). In summary, these results indicate that NC1-induced sodium influx exacerbates mitochondrial damage and facilitates cell death through NCLX.

Cellular energy depletion exacerbates monovalent cation gradients collapse by inhibiting the Na/K-ATPase in NECSO
One of the primary usages of cellular energy is to sustain the normal function of the Na/K-ATPase. The Na/K-ATPase maintains cellular osmotic pressure and homeostasis by pumping 3 Na+ out of the cells in exchange for 2 K+, utilizing the energy derived from the hydrolysis of a single ATP. Insufficient cellular energy supply can impair the activity of the Na/K-ATPase36. We therefore investigated whether Na/K-ATPase activity was inhibited in NECSO. We quantified the portion of ATPase activity that is sensitive to ouabain (a well-known Na/K-ATPase inhibitor)37 in a membrane-rich sample of cells, by employing malachite green to detect the phosphate liberated from ATP. As illustrated in Fig. 5a, the activity of Na/K-ATPase in MCF7 cells was inhibited after 1 hour of NC1 treatment, and further suppressed as NC1 exposure extended. Ouabain amplifies the NC1-induced ionic imbalance, characterized by an accumulation of Na+ (Fig. 5b) and a reduction in K+ (Fig. 5c). Furthermore, even a very low concentration of ouabain (10 nM) can exacerbate NC1-induced cell death (Fig. 5d, e). These results suggest that Na/K-ATPase inhibition plays a crucial role in the collapse of ion gradients and cell death induced by NC1.

To determine whether the suppression of Na/K-ATPase activity is caused by NC1-induced sodium influx, we assessed Na/K-ATPase activity in cells treated with NC1 under Na⁺-free conditions, with Na⁺-containing EBS serving as the control. Our findings revealed that in the absence of extracellular sodium, NC1 did not lead to inhibition of Na/K-ATPase activity (Fig. 5f), thereby implicating sodium influx as a prerequisite for this effect. We further confirmed in MB468 cells that NC1 inhibited Na/K-ATPase activity in a sodium influx-dependent manner (Fig. S6a, b), while ouabain promoted NECSO (Supplementary Fig. 6c, d).

TRPM4 is required for mitochondrial dysfunction and energy depletion in NECSO
Consistent with our previous work19, we have demonstrated that TRPM4 mediates the sodium influx (Fig. 6a) and necrosis induced by NC1 (Fig. 6b and c). In order to further confirm that NC1-induced mitochondrial damage is a consequence of elevated cytosolic sodium levels rather than direct NC1 action on mitochondria, we investigated whether NC1 retains its capacity to induce mitochondrial injury in TRPM4-knockout cells. Transmission electron microscopy observations of NC1-treated TRPM4-knockout cells revealed no significant differences in mitochondrial morphology compared to control cells (Fig. 6d). Furthermore, no discernible changes were observed in [Na+]mito and [Ca2+]mito following the administration of NC1 to TRPM4-knockout cells (Fig. 6e, f). Additionally, TRPM4-knockout completely abolished NC1-induced decrease of Ψm (Fig. 6g), oxygen consumption rate (Fig. 6h, i) and Complex II activity (Fig. 6j). Importantly, the administration of NC1 to TRPM4-knockout cells had no impact on either the ATP production rate (Fig. 6k) or the activity of the Na/K-ATPase (Fig. 6l). Consequently, the detailed mechanism illuminated by our investigation suggests that NC1-induced sodium influx, mediated through TRPM4, initiates a series of events wherein NCLX facilitates the influx of Na+ into mitochondria while promoting the efflux of Ca2+ from the matrix. This imbalance in mitochondrial ionic homeostasis dampens mitochondrial energy production, which directly contributes to the inhibition of Na/K-ATPase activity.

Osmoprotectants and AQP4 inhibition suppress NECSO
Next, we want to clarify how, following the collapse of the ion gradients due to energy deficiency, NECSO is ultimately executed. The collapse of the ion gradients elevates osmotic pressure, resulting in water influx, cell swelling, and the development of nanometer-scale membrane defects. Osmoprotectants with diameters larger than the membrane defects remain in the extracellular medium, where they counterbalance the increased intracellular osmolarity, thereby preventing water influx, swelling, and lysis (Fig. 7a). In contrast, osmoprotectants with smaller diameters do not exhibit this protective effect. Thus, we employed a series of osmoprotectants with different hydrated diameters, including sucrose (~0.9 nm), PEG 2K (~2.6 nm), PEG 4K (~3.2 nm), and PEG 8K (~4 nm)38,39. The inhibition of LDH release by osmoprotectants was size-dependent, with PEG8K almost completely inhibiting NC1-induced LDH release (Fig. 7b). Moreover, PEG8K almost entirely inhibits NC1-induced cell swelling and PI uptake (Fig. 7c). This suggests that in NECSO, osmotic cell swelling leads to the formation of size-varying membrane defects, ultimately resulting in cell lysis. Intriguingly, aquaporins (AQPs) mediate water flow, among which AQP4 has been reported to form heterodimers with TRPM440. We therefore tested the effect of the AQP4 inhibitor AER-271 and AQP4 siRNAs in NECSO. The results showed that both pharmacological and genetic inhibition of AQP4 significantly reduced NC1-induced LDH release (Fig. 7d and e), suggesting that AQP4 plays a role in mediating water influx during NECSO.

In summary, our study reveals that NC1 activates TRPM4, increasing sodium influx and intracellular sodium levels. This stimulates NCLX-mediated Na+ import and Ca2+ export from mitochondria, disrupting mitochondrial ionic balance and impairing energy generation via the TCA cycle and oxidative phosphorylation. Reduced ATP synthesis impairs Na/K-ATPase function, collapsing ion gradients. Ultimately, osmotic imbalance leads to cell swelling and membrane rupture, and AQP4 mediates the influx of water (Fig. 7f).

Methods
Cell culture
MCF7 and MB468 cells were cultured in Sigma Aldrich’s DMEM, supplemented with 4.5 g/L glucose, L-glutamine, 10% Gibco FBS, and 1% penicillin–streptomycin. The cells were incubated at 37 °C in a 5% CO2 humidified environment. Routine mycoplasma testing ensured the cell line was uncontaminated.

The Na+-containing medium formulation is as follows: 150 mM NaCl, 5 mM KCl, 10 mM Glucose, 2 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES. In Na+-free medium, NaCl is replaced with an equimolar concentration of NMDG-Cl, while all other components remain unchanged.

Gene editing
The knockout of TRPM4 was achieved using CRISPR gene editing technology. CRISPR sgRNAs targeting TRPM4 were designed with the online tool from the Zhang lab (http://crispr.mit.edu/). The oligo sequence is 5‘-CCTAGGGCTCGTTCCCTGCG-3’.

Seahorse XF assays
Real-time monitoring of the oxygen consumption rate (OCR) was executed with the employment of an XF-96 Extracellular Flux Analyzer (102416-100, Agilent). MCF7 cells were seeded into XF-96 cell culture microplates (Agilent) at a density of 8000 cells per well one day in advance to allow for overnight adhesion and attachment. After the treatment with NC1, cells were rinsed with Seahorse XF DMEM (Agilent), which had been added with 10 mM glucose (Agilent), 1 mM sodium pyruvate, and 2 mM L-glutamine (Agilent). Then, the cells were placed in an incubator at 37 °C without CO2 for 30–60 min. A mitochondrial stress test, involving 4 cycles of 3-min mixing and subsequent 3-min OCR measurements per phase, was employed. This protocol quantified basal OCR and the cellular response to 1.5 μM oligomycin (Agilent), 2 μM FCCP (Agilent), and 0.5 μM Rotenone/Antimycin A (Agilent).

For quantifying cellular ATP production rates, we introduced a real-time ATP rate assay. This involved concurrent measurements of extracellular acidification rate (ECAR) and OCR under baseline conditions and post-exposure to 1.5 μM oligomycin and 0.5 μM Rotenone/Antimycin A (all from Agilent). The measurements obtained were converted into ATP production rates.

After both assays, nuclear staining with Hoechst (B2261, Sigma) was required. The cell number was determined by microscopic observation to normalize the OCR readings within each well.

Determination of intracellular ATP
Intracellular ATP levels were determined using the CTG Luminanfescent Assay Kit (G7570, Promega). Following the addition of 10 μl of the reagent to each well of the 96-well plate, plates were gently shaken for 10 min to facilitate cell lysis. Subsequently, luminescence signals, indicative of ATP levels, were read using the ATPlite Luminescence Assay System (PerkinElmer).

Cell death assays
In the LDH release assay, the culture supernatant was mixed with the LDH assay solution (CK12, Dojindo) and incubated at room temperature for 30 min. Afterward, a stop solution was added, and the absorbance was read at 490 nm on a microplate reader. A standard curve, created from a series of dilutions of the LDH control samples, was used to determine the percentage of LDH released from the treated cells. In PI staining, cells were incubated with 2 μg/ml PI for 15 min, then observed under a microscope. The percentage of PI-positive cells was quantified using ImageJ software.

Mitochondrial membrane potential measurements
Live cell imaging: Cells cultured in four-well Lab-Tek II Chambered Cover glass imaging chambers (Thermo Fisher Scientific) were pre-loaded with 300 nM TMRE (T669, Thermo Fisher Scientific) for 30 min and subsequently rinsed with PBS twice. Then, the cells were positioned in FluoroBrite™ DMEM medium (A1896701, Thermo Fisher Scientific) and observed under the microscope. After identifying the desired field of view, NC1 was added, and imaging commenced. Fluorescence and differential interference contrast (DIC) images were captured every 5 min using a Multipoint Confocal Live Cell Imaging System (Nikon). The acquired images were analyzed using NIS Elements (Nikon) and ImageJ (NIH) software.

Flow cytometry: Cells cultured in 12-well plates, stained with 500 nM TMRE and treated with NC1, were detached with Accutase, collected in 4% FBS-PBS. TMRE fluorescence (Ex/Em=549/575 nm) was quantified by flow cytometry.

Mitotracker analysis
MCF7 cells were seeded overnight in four-well Lab-Tek II Chambered Cover glass imaging chambers (Thermo Fisher Scientific). For mitochondrial labeling, cells were incubated with MitoTracker Red (M7512, Invitrogen) and nuclear dye Hoechst 33342 for 30 min at 37 °C. Then the cells were washed and incubated in either Na⁺-containing or Na⁺-free EBS. Thereafter, 100 nM NC1 was added for the indicated time points. Fluorescence signals at each time point were captured using a confocal microscope (Olympus).

Mitochondrial calcium imaging
Live cell imaging: MCF7 cells cultured in four-well Lab-Tek II Chambered Cover glass imaging chambers (Thermo Fisher Scientific) were pre-transfected with CMV-mito-R-GECO1 (Addgene plasmid #46021) for 24 h. Following transfection, cells were washed and then incubated in either Na⁺-containing or Na⁺-free EBS. Subsequently, cells were treated with 100 nM NC1 in the respective EBS conditions. Fluorescence and differential interference contrast (DIC) images were captured every 2 min by a Multipoint Confocal Live Cell Imaging System (Nikon).

Confocal imaging: MCF7 cells were cultured in 4-well Lab-Tek II Chambered Cover glass imaging chambers (Thermo Fisher Scientific). Prior to imaging, cells were preloaded with 2 μM Rhod-2 AM (R1244, Thermo Fisher Scientific), 200 nM MitoTracker Green (M7514, Thermo Fisher Scientific), and the nuclear dye Hoechst 33342 for 30 min at 37 °C under 5% CO₂ in the dark. Following incubation, the cells were washed with either Na⁺-containing or Na⁺-free EBS and then maintained in the respective buffer conditions. NC1 was subsequently added, and fluorescence signals were captured at defined time points using a confocal microscope (Olympus).

Flow cytometry: Cells cultured in 12-well plates were stained with 2 μM Rhod-2, AM, and then treated with NC1. The cells were detached using Accutase (A1110501, Thermo Fisher Scientific) and collected in PBS containing 4% FBS. Rhod-2 fluorescence (Ex/Em=549/575 nm) was quantified by flow cytometry. In certain experiments, cells were pretreated with 20 mM CGP-37157 (T14941, Targetmol).

Cytosolic sodium imaging
Cells grown on a glass-bottom dish were stained with 5 μM CoroNa Green, AM (c36676, Thermo Fisher Scientific) for 30 min and then treated with NC1 as indicated. CoroNa Green fluorescence (Ex/Em=492/516 nm) was observed under a confocal microscope (Olympus). The images were analyzed using ImageJ.

Electron transport chain complex assays
To assess the activity of Complex II, we utilized the Complex II Enzyme Activity Microplate Assay Kit (ab109908, Abcam). After the treatment of NC1, protein extraction of MCF7 cells was performed with a detergent solution, adjusted to the optimal concentration for assay loading. Samples were then loaded into a 96-well plate pre-coated with anti-Complex II monoclonal antibody, followed by a 2-h incubation at room temperature. After the washing step, an activity solution control buffer was added to the appropriate wells of the plate. Optical density readings at 600 nm were taken over 60 min in kinetic mode, with 30-s interval measurements, using a Microplate Reader (PerkinElmer).

The MitoTox Complex II+III OXPHOS Activity Assay Kit (ab109905, Abcam) was used to evaluate Complex II/III activity. Activity solution was combined with a range of NaCl concentrations (from +64 mM down to +0.0625 mM via serial dilutions). Following the addition of bovine heart mitochondria, kinetic absorbance readings at 550 nm were taken using a Microplate Reader (PerkinElmer). Complex II/III activity levels were normalized against a solvent-only control. In certain experiments, 20 mM CGP-37157 was included.

Metabolite extraction and quantification
After NC1 treatment, two million MCF7 cells were washed twice with ice-cold saline, lysed with 1 ml ice-cold 80% methanol in water, and quickly scraped into Eppendorf tubes and centrifuged at 15,000 × g for 15 min at 4 °C. The supernatant containing the soluble metabolic compounds was dried, reconstituted in 50 μl LC–MS grade water with 0.03% formic acid, vortexed, and centrifuged again. Clear supernatants were transferred to HPLC vials for subsequent LC–MS/MS analysis.

Samples were randomized and blinded before being analyzed by LC–MS/MS. Chromatographic separation was achieved on an ACQUITY UPLC HSS T3 column (2.1 × 150 mm, 1.8 µm) using an ACQUITY UPLC I-Class system (Waters Corporation, Milford, MA, USA). The mobile phases employed were 0.03% formic acid in water (A) and 0.03% formic acid in acetonitrile (B) and the gradient program was as follows: 0–3 min, 1% B; 3–15 min, 1%–99% B; 15–17 min, 99% B; 17–17.1 min, 99%–1% B; 17.1–20 min, 1% B. The flow rate was 0.25 ml min−1, the column was at 35 °C, and the samples in the autosampler were at 4 °C. Mass spectrometry was performed using an AB QTRAP 6500+ triple quadrupole mass spectrometer (Applied Biosystems SCIEX) in multiple reaction monitoring (MRM) mode. Chromatogram review and peak area integration were performed using MultiQuant 3.0.2 (Applied Biosystems SCIEX), and the peak area of each detected metabolite was normalized to the sum of peak areas of all detected metabolites in that sample to correct for any variations introduced by sample handling through instrument analysis. The normalized data were used as variables for the multivariate and univariate statistical analysis. The analysis of enriched pathways associated with the differential metabolites was performed using Metaboanalyst v. 6.0 (https://www.metaboanalyst.ca/). Univariate statistical differences of the metabolites between the two groups were analyzed using a two-tailed Student’s t-test.

Mitochondrial Na+ detection
Mitochondria were isolated according to the mitochondrial isolation kit’s (89874, Thermo Fisher Scientific) instructions and suspended in 100 μl HBSS. Samples were split even; one portion was incubated with 7.5 μM SBFI (MX4509, MKbio) for 45 min, while the other was used for protein quantification via BCA. SBFI fluorescence (Excitation: 340/380 nm; Emission: 520 nm) was measured on a Microplate Reader (PerkinElmer), calculating the F340/F380 ratio as a proxy for [Na+], corrected by protein concentration.

Transmission electron microscopy
Transmission electron microscopy (TEM) analysis followed standard protocols: MCF7 cells treated with 100 nM NC1 at various time points were fixed in 2% paraformaldehyde, 3% glutaraldehyde, and 0.1 M cacodylate buffer (pH 7.3), washed, and treated with 0.1% tannic acid. Post-fixed with 1% osmium tetroxide, cells were stained with 1% uranyl acetate, dehydrated with graded Epon, and embedded. Ultrathin sections, cut on a Leica EM UC7, were mounted on formvar-coated grids, stained with 3% uranyl acetate and lead citrate, and examined under an 80 kV TEM (H-7650, HITACHI).

Inductively coupled plasma-mass spectrometry (ICP-MS)
Samples were weighed into 50 ml metal-free tubes, 2 ml of 69% nitric acid was added, and tubes were placed on a digestion block (DigiPREP Jr., SCP-Science). Heated to 120 °C for 1 h with gentle swirling, then cooled before adding 0.5 ml H2O2. After reheating at 120 °C for 30 min, tubes were cooled, volumes adjusted to 20 ml with ultrapure water. Calibration standards were made by diluting 1000 mg/L ICP-MS standard solutions (Spex CertiPrep) with 10% nitric acid. Elements detected by ICP-MS (NexION 2000G, PerkinElmer) using external calibration and 103Rh as an internal standard. Quality control used a certified liver reference (T07243QC, FAPAS).

In vitro Na/K-ATPase activity assay
MCF7 cells were plated at a density of 4 × 106 cells per 10 cm2 dish and allowed to adhere overnight. Following adhesion, cells were treated with 50 nM NC1 at the indicated time points. Then, cells were harvested using ice-cold homogenizing buffer consisting of 50 mM HEPES (pH 7.4), 250 mM sucrose, 1 mM EGTA, and a protease inhibitor cocktail. Protein extraction was achieved via Dounce homogenization with 35 strokes, followed by a low-speed centrifugation (3000 × g for 10 min at 4 °C). The supernatant was collected and subjected to ultracentrifugation (100,000 × g for 30 min at 4 °C). The resulting pellet was resuspended in 50 μl of homogenizing buffer and assayed for ATPase activity in a reaction mixture containing 50 mM HEPES (pH 7.4), 50 mM NaCl, 25 mM KCl, 5 mM MgCl2, and 5 mM ATP, including or excluding 2 mM ouabain at 37 °C for 30 min while shaking. The enzymatic reaction was terminated by the addition of 10% trichloroacetic acid (T0699, Sigma), and samples were centrifuged to eliminate any precipitated debris (15,000 × g, 5 min). Enzyme activity was quantified by measuring the inorganic phosphate ([Pi]) released, using an Amalachite assay kit (MAK307, Sigma), while the protein concentration was measured using a BCA assay kit (23225, Thermo Fisher Scientific). To determine Na/K-ATPase (ouabain-sensitive) activity, the equation ([Pi] without Ouabain − [Pi] with ouabain)/quantity of protein was utilized. Experimental data are presented as relative enzyme activity (mean ± SD from three independent experiments).

Western blotting
Protein extracts from cells were prepared in RIPA buffer with a protease and phosphatase inhibitor cocktail (K1007, Apexbio), quantified using a BCA assay. Proteins (20 μg) were separated by 10% SDS–PAGE, blotted onto PVDF membranes, and blocked with 5% milk. Membranes were incubated overnight with primary antibodies, then incubated with HRP-conjugated secondary antibodies. Chemiluminescence was used for detection. The following primary antibodies were used: anti-α-tubulin (66031-1-1g, Proteintech, diluted 1:10,000), anti-TRPM4 (A10146, Abclonal, diluted 1:1000), anti-AQP4 (A2887, Abclonal, diluted 1:1000), anti-MCU (A22525, Abclonal, diluted 1:500), and anti-Phospho-PDHA1-S293 (AP1022, Abclonal, diluted 1:1000).

siRNA transfection
The following siRNA sequences were used to knockdown MCU: 1#: 5’-GGGAAUUGACAGAGUUGCUTT-3’, 2#: 5’-CGACCUAGAGAAAUACAAUTT-3’.

The siRNA sequences used to knockdown AQP4 were as follows: 1#: 5’-GAGCAGGAAUCCUCUAUCUTT-3’, 2#: 5’-CACUGGCUCAAUAGCUUUATT-3’.

All siRNAs were synthesized by Shanghai GenePharma Co. Ltd. MCF7 cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) following the manufacturer’s protocol, and knockdown efficiency was verified by western blot.

Flow cytometry
MCF7 cells grown in 12-well plates were stained with fluorescent probes and subsequently treated with NC1. The Cells were detached using Accutase and collected in 4% FBS-PBS. The fluorescence of stained cells was analyzed by a CytoFlex S flow cytometer (Beckman Coulter), with data processed using FlowJo.

Statistics and reproducibility
All experiments were independently replicated more than two times with consistent results. All microscopic, biochemical, and biological assays were independently repeated at least three times. GraphPad Prism 9.0 was used for statistics. Unpaired or paired two-tailed Student’s t-test was used when comparing data from two groups. Statistical analysis for multiple group comparisons was conducted using one-way ANOVA or two-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± s.d. and p < 0.05 was considered statistically significant. Adobe Illustrator was used for Illustrations.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this MS the author\ninterrogate a process that they claim is a new of cell death triggered by a novel cell death Na+ dependent Pathway\n(NESCO) they use an agent termed NC1 that trigger this process and suggest that it lead in the MCF7 cell line energy\ndepletion followed by death a and that this process can be reversed in a Na+ free buffer. They further suggest that this\nprocess is mediated by NCLX activity which leads to inhibition of oxidative phosphorylation . They also suggest that NC1\neffect is linked to inhibition of the Na/K ATPase, leads cell membrane pore formation and is triggering activation of TRPM4.\nWhile of interest this study it has several fundamental flaws some of them as described below:\n1) The model that they are using is comparison of cell fate in the presence of Na+ Vs Na+ free medium for many hours . Na+\nis an essential cation and provides the driving force for ion and metabolites transport and is also controlling membrane\npotential of cell membrane having also an effect on intracellular membrane properties . Notably for example cytosolic Ca2+\nwill be strongly affected by Na + and the activity of the Na+/K+ ATPase as well . Yet the author conduct only a single\nmeasurements of total cellular Na+ content instead of time dependent dynamic analysis of cytosolic and mitochondrial Na+\nK+ and Ca2+ all cations that are expected to change by multiple pathways by the lack of Na+ . Indeed the authors cite\nmitochondrial swelling triggered by NC1 a hallmark of mitochondrial Ca2+ overload but don’t monitor this effect .\n2) The author focuses mostly on a single cell line MCF7. If NC1 has such a profound effect on TRPM4 it should strongly\naffect for example neurons where TRPM 4 is abundant . Moreover if NC1 has an antitumorigenic effect the authors should\nhave used in vivo models where the administration of NC1 should have a strong effect on primary tumor growth survival etc..\n3) The authors propose that NC1 interacts with several often contradictory targets of the Na+K+ ATPase that it also\ngenerates nano holes in the membrane and is activating TMRM4 . They do not provide any model functional structural or\nmolecular dynamics to explain the puzzling and pleiotropic effect of this compound. As such it raises the concern that NC1\nsimply has a non selective disruptive effect on cell membrane organization .", "reviewer_2": "Summary:\nThe manuscript “Sodium disrupts mitochondrial energy metabolism to execute NECSO” by Qiao et al explores the\nmechanism by which cytosolic sodium overload elicits necrotic cell death, a phenomenon that this group first described in\nanother manuscript currently in revision. Here, the authors use in vitro cell culture models to demonstrate that activation of\nsodium influx into the cell via pharmacologic activation of the TRPM4 channel impairs mitochondrial oxidative\nphosphorylation and ATP production and leads to necrotic cell death. They use pharmacologic inhibition to implicate the\nmitochondrial sodium/calcium exchanger, NCLX, in this process, and propose that mitochondrial calcium depletion in the\ncontext of cytosolic sodium overload limits mitochondrial metabolism. Finally, the authors use an enzymatic assay to\ndemonstrate that the activity of the plasma membrane sodium/potassium ATPase is diminished in this setting of energy\ndepletion and suggest that the resulting perturbation of cytosolic ionic homeostasis causes osmotic imbalance, plasma\nmembrane rupture, and cell death.\nMajor concerns:\n1) A central claim of the study is that cellular ATP depletion mechanistically links Na+ overload and altered mitochondrial\nfunction to impaired Na+/K+-ATPase function that collapses plasma membrane ion gradients leading to necrotic cell death.\nIf so, can the cell death observed with cytosolic Na+ overload be rescued by boosting glycolytic ATP production, or even by\nproviding exogenous ATP (ex, via patch clamp) to the cells?\n2) Fig. 1D: Can the effects of NC1 on ATP production rates be blocked in experiments using Na+-free media?\n3) Along these lines the Na+ free control experiment only reports data out to 8h, a time right at which there is an increase in\nPI staining and drop in ATP. Please show the Na+ free condition out to at least 16hr. i.e., is the cell death just delayed and, if\nso, for how long?\n4) The mitotracker staining in Fig 1E, and the Rhod-2 staining in Fig. 1G look non-mitochondrial. I’ve never seen a mito\nnetwork as shown in 1F and Rhod2 looks to be cytosolic and is also staining the nucleolus.\n5) After redoing the experiments in Fig. 1E and Fig. S2E: Provide quantification and statistical comparison of the drop in\nmitotracker signal with NC1 treatment. Is this effect truly attenuated when the experiment is conducted in Na+-free media,\nlike the drop in Rhod-2 fluorescence shown in Fig. 1G?\n6) I have some concerns about the use of Rhod-2 fluorescence to measure mitochondrial Ca2+ in Fig. 1G. First, this dye\ntypically labels the mitochondria in a manner that is dependent on mitochondrial membrane potential. Therefore, it is difficult\nto distinguish whether the drop in Rhod-2 fluorescence seen with Na+ overload directly represents changes in mitochondrial\nCa2+ levels, or instead reflects diminished mitochondrial staining due to a reduction in mitochondrial membrane potential.\nUse of an alternative approach to assess mitochondrial calcium content, such as a genetically-encoded mito-calcium\nreporter, would help resolve this question. Second, quantification and statistical analysis of mitochondrial calcium levels\nshould be provided for this experiment.\n7) Fig. 1E: A relevant mechanistic question is whether the Na+-dependent mitochondrial swelling observed in Fig. 1E might\nreflect classical mitochondrial permeability transition, which could trigger necrotic cell death. Can this mito swelling effect of\nNa+-overload, or any of the other consequences (energy depletion, plasma membrane rupture, etc.) be alleviated by\npharmacologic inhibition of permeability transition with cyclosporin A or with deletion/loss of Ppif/CypD?\n8) A major outstanding question is whether the apparently NCLX-dependent effects of cytosolic Na+ overload (leading to\nmito Na+ loading and mito Ca2+ depletion) are direct effects of increased mitochondrial Na+, or rather direct effects of\ndiminished free mitochondrial Ca2+. How do the authors distinguish these two possibilities?\n9) Fig. 2F, Fig. 6J: Measurements of Complex II activity were performed using isolated protein immunoadsorbed onto assay\nplates for 2hrs prior to the start of the assay. This will necessarily remove CII from its native environment, as well as the local\nmitochondrial Na+ and Ca2+ concentrations affected by treatment with NC1. How, then, do the authors propose that NC1\nand the resulting cellular/mitochondrial sodium overload and/or mitochondrial Ca2+ depletion exerts this apparently “long-\nlasting” effect on CII activity?\n10) Fig. 3A, B, D: Are any of the changes in mitochondrial metabolism shown with NC1 treatment in Fig. 3 attenuated with\nNCLX inhibition or knockdown?\n11) Fig. 3B-C: the authors attempt to link mitochondrial calcium depletion with NC1 treatment to impaired pyruvate\ndehydrogenase activity, based on the accumulation of thiamine diphosphate. As PDH phosphatase is sensitive to changes\nin mitochondrial Ca2+, is the phosphorylation status of PDH (Ser293) altered under these conditions?\n12) Figure 4 is missing CGP controls throughout all experiments.\n13) NCLX loss or inhibition results in severe mitochondrial calcium overload, mitochondrial swelling, and necrotic cell death\nthat can be rescued by CypD inhibition. How is 5h of NCLX inhibition protective in the context of this cell system? What\nhappens if the authors target MCU?\nMinor concerns:\n1) Fig. 1D and Fig. 4H: Statistical comparisons should be provided to support claims of the effects of the various treatments\non mitochondrial, glycolytic, and total ATP production rates.\n2) Fig. S2C-D: provide statistical comparisons between experimental groups for ATP content and LDH release.\n3) Fig. 3B: citrate is noted in the text as accumulating following NC1 treatment, but it is not included in the heatmap of\nmetabolites in Fig. 3B. Please add it.\n4) Fig. 4C-D and Fig. 6 F-G: Y-axes of these graphs should be labelled for each treatment condition, and statistical\ncomparisons between groups should be provided to support the authors’ claims that NCLX inhibition or TRPM4 KO reverses\nNC1’s effects on mitochondrial Ca2+ and membrane potential.\n5) Fig. 5 D-E: The dose-response curves in these graphs should be compared statistically to support the claim that inhibition\nof the Na+/K+-ATPase with ouabain sensitizes cells to the effects of sodium overload.\n6) Fig. 7 C and E: add statistical comparisons for the dose-response curves.", "reviewer_3": "Na+ influx is a common event in necrotic cell death and if it was regulated during a given necrosis process, it must be\nessential for the control of the necrosis. Qiao et al used Necrocide 1, an agonist of monovalent cation non-selective channel\nTRPM4 to induce cell necrosis, and analyzed intracellular changes after Necrocide 1 treatment. Instead of the common\nthought that the collapse of Na+ gradient would directly lead to swelling of the cell and subsequent plasma membrane\ndisruption, the authors stated that sodium influx is via inhibiting mitochondrial energy production/energy depletion to\ninactivate Na/K-ATPase, resulting in ion gradient collapse, pore formation, water influx, and eventual necrotic cell death.\nThe conclusion is astonishing, but whether energy depletion is a trigger or simply a concomitant phenomenon of Necrocide\n1-caused cell necrosis has not been demonstrated unambiguously. The authors need to provide evidence that the cell\nnecrosis is not directly caused by Na+ influx. This reviewer also has a few specific comments.\n1. The requirement of AQP4 in Necrocide 1-induced necrosis needs to be demonstrated by genetic deletion of AQP4.\n2. Since AQP4 interacts with TRPM4, it could function together with TRPM4 in Necrocide 1 induced Na+ influx rather than in\na later stage of cell necrosis. This can be easily evaluated.\n3. This study has no linkage with ischemia or organ failure."}, "author_response": "Point-by-point response to reviewers’ comments\nReviewer #1 (Remarks to the Author):\nIn this MS the authors interrogate a process that they claim is a new form of cell death triggered by\na novel cell death Na+ dependent Pathway (NESCO) they use an agent termed NC1 that trigger this\nprocess and suggest that it lead in the MCF7 cell line energy depletion followed by death a and that\nthis process can be reversed in a Na+ free buffer. They further suggest that this process is mediated\nby NCLX activity which leads to inhibition of oxidative phosphorylation. They also suggest that\nNC1 effect is linked to inhibition of the Na/K ATPase, leads cell membrane pore formation and is\ntriggering activation of TRPM4.\nResponse: We appreciate the reviewer's detailed summary and constructive feedback, and we\napologize for potential confusion and misunderstandings. We would like to clarify that our\nhypothesis does not involve TRPM4 activation via Na/K-ATPase inhibition. Instead, we have\ndemonstrated that NC1 directly activates TRPM4 in our previous work (Fu, W. et al. Nature\nChemical Biology, 2025)1. Specifically, we performed a cellular thermal shift assay (CETSA) to\nassess the interaction between NC1 and TRPM4 in HEK293T cells stably expressing TRPM4. NC1\nstabilized TRPM4 at elevated temperatures, while DMSO or its inactive stereoisomer (NC1i) did\nnot (Fig.1a in this letter, Fig. 1e in the reference1). Surface plasmon resonance (SPR) confirmed\ndirect binding between NC1 and TRPM4, with a binding affinity of 0.88 μM (Fig.1b in this letter,\nFig. 1f1). Whole-cell patch-clamp recordings showed that NC1 induced robust currents in wild-type\n(WT) MCF7 cells, which were abolished in TRPM4 knockout (KO) cells (Fig.1c in this letter,\nFig.1g1). Molecular docking using the hTRPM4 structure (PDB: 6BQV) revealed that NC1 binds\nwithin a cavity in the transmembrane domain (Fig.1d in this letter, Fig. 4a1). This model predicted\ninteractions between NC1 and key residues in this cavity near the Ca²⁺-binding site, including F936,\nW864, and V904, L907, and H908 (Fig.1e, f in this letter, Fig. 4b, c1). Mutation of W864, H908 or\nF936 to alanine disrupted NC1 binding (Fig.1g in this letter, Fig. 4d1), TRPM4 channel activation\n(Fig.1h, i in this letter, Fig. 4e, f1) and NECSO sensitivity (Fig.1j in this letter, Fig. 4g1).\nand reduces mitochondrial Ca2+ via NCLX, thereby impairing mitochondrial energy production.\nThis energy deficit subsequently decreases Na/K-ATPase activity, leading to secondary ionic\ndisturbances, altered osmotic pressure, and AQP4-dependent water influx, resulting in cell swelling.\nWith regard to \"pore formation\", our current evidence is limited. Therefore, we have toned down the\nrelated claims in the revised manuscript (Manuscript lines 363-383, 389-391, 467-482). Instead, we\nnow suggest that NC1 disrupts ion homeostasis, increasing intracellular osmotic pressure, leading\nto cell swelling and subsequent membrane injury-effects that are attenuated by osmoprotectants.\nCollectively, our findings demonstrate that Na⁺ actively initiates necrosis by impairing\nmitochondrial energy homeostasis, thereby elucidating the underlying mechanism of NECSO.\nFigure 1. NC1 directly targets TRPM4.\n2\n(a) CETSA of NC1 or NC1i in HEK293T cells stably expressing TRPM4-EGFP. The graph represents\nthe relative TRPM4 band intensities of the indicated samples as the mean ± s.d. from three independent\nexperiments.\n(b) Purification with size exclusion chromatography of hTRPM4 (inset). Purified protein was loaded in\ndose-dependent concentrations (Conc.) and stained with Coomassie brilliant blue (CBB). NC1 binds to\nthe hTRPM4 protein as shown by SPR measurements.\n(c) The whole-cell current density-voltage relation of MCF7 WT and TRPM4-KO cells activated by NC1.\n(d) Surface representation showing the overall structure of the molecular modeling TRPM4-NC1\ncomplex. MW, molecular weight. Inset: a close-up view of the NC1-binding site. Individual TRPM4\nsubunits are colored cyan or gray, whereas NC1 is highlighted with yellow. Lower right: chemical\nstructure of NC1.\n(e) Specific recognition of NC1-bound hTRPM4. A 3D diagram showing that NC1 binds a deep cavity\nof TRPM4 between S3, S4, S5′ and S6′ helix; Ca2+ is colored purple\n(f) A 2D diagram showing that NC1 interacts with the key residues of TRPM4 through a mixture of\nhydrogen bonds, hydrophobic interactions and π–π stacking.\n(g) CETSA of NC1 and TRPM4 mutants of key residues in b, c. ΔTm =Tm (NC1) -Tm (DMSO). Melting\ntemperatures (Tm) were calculated by TRPM4 band intensities of the indicated samples in western blot\nwith GraphPad. The graph represents mean ± s.d. from three independent experiments.\n(h) The current density–voltage relationship of hTRPM4 mutants involving key residues for NC1 binding\nwas examined in HeLa cells.\n(i) Summary plot showing the maximum amplitude of current densities activated by NC1 in the TRPM4\nWT channel compared to the aforementioned mutants.\n(j) Dose-dependent cytotoxicity assay of NC1 in TRPM4-KO MCF7 cells with re-expression of WT\nTRPM4 or TRPM4 mutants (W864A, H908A, F936A).\nWhile of interest this study it has several fundamental flaws some of them as described below:\n1. The model that they are using is comparison of cell fate in the presence of Na+ Vs Na+ free\nmedium for many hours. Na+ is an essential cation and provides the driving force for ion and\nmetabolites transport and is also controlling membrane potential of cell membrane having also an\neffect on intracellular membrane properties. Notably for example cytosolic Ca2+ will be strongly\n3\naffected by Na + and the activity of the Na+/K+ ATPase as well. Yet the authors conduct only a\nsingle measurements of total cellular Na+ content instead of time dependent dynamic analysis of\ncytosolic and mitochondrial Na+ K+ and Ca2+ all cations that are expected to change by multiple\npathways by the lack of Na+.\nResponse: Thank you for this insightful point. We proposed that TRPM4 mediated sodium influx\ntriggered NECSO. To block sodium influx, we used a Na⁺-free isotonic extracellular buffer solution\n(EBS) without serum or amino acids, in which Na⁺ was replaced by equimolar non-permeable\nNMDG⁺ to preserve iso-osmolarity. A standard Na⁺-containing EBS served as control. The treatment\nwas limited to 8 hours to elicit NC1’s effect while minimizing starvation-induced cell death.\nWe have dynamically monitored cytosolic Na+, K+, Ca2+ changes following NC1 treatment in cells\nexposed to either Na+-containing or Na+-free EBS. We used the Inductively Coupled Plasma Mass\nSpectrometry (ICP-MS) to measure cytosolic Na+ and K+ levels, and employed the genetically\nencoded calcium indicator R-GECO1 to monitor cytosolic Ca2+ concentrations. Specifically, in Na⁺-\ncontaining EBS, NC1 treatment led to a significant increase in cytosolic Na⁺ (this letter Fig. 2a,\nManuscript Fig. S1C) and Ca²⁺ levels (Fig. 2c), accompanied by a decrease in cytosolic K⁺ (this\nletter Fig. 2b, Manuscript Fig. S1D). In contrast, in Na⁺-free EBS, the NC1-induced elevations in\ncytosolic Na⁺ (this letter Fig. 2a, Manuscript Fig. S1C) and Ca²⁺ (Fig. 2c) were blocked, whereas the\ndecline in cytosolic K⁺ remained unchanged (this letter Fig. 2b, Manuscript Fig. S1D). To clarify\nwhether the elevation of intracellular sodium, the decrease in potassium, or the increase in\ncytoplasmic calcium is the primary driver of NECSO, we have conducted a series of experiments in\nour previous work (Fu, W. et al. Nature Chemical Biology, 2025)1. In the EBS-based system, we\nselectively manipulated ion fluxes as follows: Na⁺ influx was blocked by replacing NaCl with\nNMDG, K⁺ efflux was inhibited by increasing extracellular K⁺ from 10 mM to 25 mM, and Ca²⁺\ninflux was prevented by using Ca²⁺-free EBS. Our results demonstrated that only the blockade of\nNa⁺ influx significantly suppressed NECSO. Moreover, the incidence of NECSO gradually\ndecreased as the extracellular Na⁺ concentration in EBS was reduced (this letter Fig. 2e, Fig. 2g in\nthe reference1), further supporting the critical dependence of NECSO on intracellular Na⁺\naccumulation. In contrast, neither inhibition of K⁺ efflux nor suppression of Ca²⁺ influx had a notable\n4\neffect on NECSO occurrence (this letter Fig. 2f, Fig. 2e in the reference1). Collectively, these\nfindings indicate that the rise in intracellular Na⁺ concentration is the key initiating factor in NECSO.\nThe observed decreases in K⁺ and increases in Ca²⁺ appear to be secondary, accompanying events\nrather than primary causative factors.\nFurthermore, we dynamically monitored mitochondrial Na⁺ and Ca²⁺ changes in NC1-treated cells\nunder both Na⁺-containing and Na⁺-free EBS conditions. Mitochondrial Na⁺ levels were assessed\nusing the fluorescent indicator SBFI after mitochondrial isolation, while mitochondrial Ca²⁺\ndynamics were tracked in real time with the genetically encoded sensor mito-R-GECO1. In Na⁺-\ncontaining EBS, NC1 induced mitochondrial Na⁺ accumulation (this letter Fig. 2g, Manuscript Fig.\n1G) and a corresponding decrease in mitochondrial Ca2+ (this letter Fig. 2h-1i, Manuscript Figures\n1H-1I). However, in Na+-free EBS, NC1 had no significant effect on either mitochondrial Na⁺ (this\nletter Fig. 2g, Manuscript Fig. 1G) or mitochondrial Ca²⁺ levels (this letter Fig. 2h-i, Manuscript Fig.\n1H-I). Due to our current technical limitations, we lack data on mitochondrial potassium.\nImportantly, the changes in mitochondrial Na⁺ and Ca²⁺ levels during NECSO are secondary to the\nelevation of cytoplasmic Na⁺ concentration. As our study demonstrates, the accumulation of\nmitochondrial Na⁺ and the concomitant decrease in mitochondrial Ca²⁺ promote NECSO by\nimpairing mitochondrial energy production. These findings partially explain how the influx of Na⁺\ninto the cytoplasm leads to the occurrence of NECSO.\nIn summary, using the EBS system, we demonstrate that the elevation of intracellular Na⁺\nconcentration is a key driver of NECSO, whereas changes in cytoplasmic Ca²⁺ and K⁺ levels are\nsecondary, accompanying events. Furthermore, we showed that the rise in cytoplasmic Na⁺ promotes\nNECSO by inducing mitochondrial Na⁺ accumulation and a corresponding decrease in\nmitochondrial Ca²⁺.\nfollowing NC1 treatment.\n(a) [Na+] and (b) [K+] determined by ICP-MS for MCF7 cells treated with 100 nM NC1 over time in\nNa+-containing EBS and Na+-free EBS.\n(c) MCF7 cells expressing R-GECO1 were treated with 100 nM NC1 in Na+-containing or Na+-free EBS.\nRepresentative still images from time-lapse cytosolic calcium imaging are presented.\n(d) Detection of mitochondrial Na+ content of MCF7 cells exposed to Na+-containing EBS or Na+-free\nEBS with 100 nM NC1 for 0.5,1 or 3 hours. The treatment groups in different media were compared to\ntheir respective untreated controls (0 h).\n(e) Cell viability of MCF7 cells treated with NC1 for 6 h in EBSs containing varying concentrations of\nNa+ (osmotic pressure was maintained by an equal concentration of NMDG+).\n6\n(f) Cell viability of MCF7 cells treated with NC1 for 6 h in different EBSs: normal, increased\nconcentration of KCl to 25 mM, Ca2+-free and Na+-free (osmotic pressure was maintained by an equal\nconcentration of NMDG-Cl).\n(g) Detection of mitochondrial Na+ content of MCF7 cells exposed to Na+-containing EBS or Na+-free\nEBS with 100 nM NC1 for 0.5,1 or 3 hours. The treatment groups in different media were compared to\ntheir respective untreated controls (0 h).\n(h) MCF7 cells were transfected with mito-R-GECO1 24 hours prior to treatment and then exposed to\nNa⁺-containing or Na⁺-free medium containing 100 nM NC1. Representative still images from time-lapse\nmitochondrial calcium imaging are presented.\n(i) Statistical analysis of mito-R-GECO1 fluorescence signals in (h). The treatment groups in different\nmedia were compared to their respective untreated controls (0 h).\nStatistical analysis in (d), (g) and (i) was conducted using a one-way ANOVA. Data are shown as mean\n± SD and n = 3 independent experiments.\nIndeed the authors cite mitochondrial swelling triggered by NC1 a hallmark of mitochondrial Ca2+\noverload but don’t monitor this effect.\nResponse: We apologize for the confusion. Regarding mitochondrial swelling, our results indicate\na reduction, rather than an overload, in mitochondrial calcium levels following NC1 treatment, as\nmeasured by both the genetically encoded sensor mito-R-GECO1 and the Rhod-2 AM probe.\nMito-R-GECO1 is a genetically encoded calcium sensor targeted to mitochondria via an N-terminal\nmitochondrial targeting sequence (MTS). Binding of Ca²⁺ induces interaction between calmodulin\n(CaM) and the M13 peptide, triggering a conformational change in the fused red fluorescent protein\nthat enhances chromophore fluorescence, enabling real-time monitoring of mitochondrial Ca²⁺\ndynamics2, 3. In mito-R-GECO1-transfected MCF7 cells, NC1 treatment under normal Na⁺\nconditions resulted in a time-dependent decrease in red fluorescence, indicating reduced\nmitochondrial Ca²⁺, while this effect was absent under Na⁺-free conditions (This Letter Fig. 3a-b;\nManuscript Fig. 1H-I).\ncharged form, and accumulates in mitochondria in a ΔΨm-dependent manner. Upon Ca²⁺ binding,\nintramolecular quenching is relieved, resulting in enhanced red fluorescence4. Co-staining with\nMitoTracker Green confirmed mitochondrial localization. In MCF7 cells, NC1 treatment reduced\nRhod-2 fluorescence in Na⁺-containing EBS but not in Na⁺-free EBS (This Letter Fig. 3c-3e;\nManuscript Fig. S2D-S2F), further supporting the notion that NC1 decreases mitochondrial Ca²⁺ in\nan extracellular Na⁺-dependent manner.\nFigure 3. NC1 triggered a decrease in mitochondrial Ca²⁺.\n8\n(a) MCF7 cells were transfected with mito-R-GECO1 24 hours prior to treatment and then exposed to\nNa⁺-containing or Na⁺-free medium containing 100 nM NC1. Representative still images from time-lapse\nmitochondrial calcium imaging are presented.\n(b) Statistical analysis of mito-R-GECO1 fluorescence signals in (a). The treatment groups in different\nmedia were compared to their respective untreated controls (0 h). Statistical analysis was conducted using\na one-way ANOVA. Data are shown as mean ± SD and n = 3 independent experiments.\n(c) MCF7 cells were preloaded with Rhod-2 AM and Mitotracker Green, then exposed to Na⁺-containing\nor Na⁺-free EBS with 100 nM NC1 for the indicated time points. Representative confocal microscopy\nimages are shown.\n(d) One-way ANOVA was used to analyze Rhod-2 fluorescence signals in NC1-treated MCF7 cells under\nNa⁺-containing EBS condition in (c).\n(e) One-way ANOVA was used to analyze Rhod-2 fluorescence signals in NC1-treated MCF7 cells under\nNa⁺-free EBS condition in (c).\n2) The author focuses mostly on a single cell line MCF7. If NC1 has such a profound effect on\nTRPM4 it should strongly affect for example neurons where TRPM4 is abundant.\nResponse: We appreciate the reviewer for the constructive feedback. In our previous work (Fu, W.\net al. Nature Chemical Biology, 2025)1, we have demonstrated a positive correlation between\nTRPM4 expression levels and NC1 sensitivity across multiple cell lines (this letter Fig. 4a, Fig. 3a\nin the reference1). We selected a TRPM4-high-expressing cell line (MB468) for further validation.\nNC1 induced early sodium influx in MB468 cells (this letter Fig. 4b, Manuscript Fig. S1G), resulting\nin sodium-dependent ATP depletion, plasma membrane rupture, as evidenced by PI positivity and\nLDH release (this letter Fig. 4c-4d, Manuscript Fig. S1H, S1I). Seahorse XF analysis further\nrevealed significant suppression of mitochondrial respiration by NC1 in MB468 cells (this letter Fig.\n4e-4g, Manuscript Fig. S3A-S3C), indicating impaired mitochondrial function.\nIn addition to tumor cell lines, we also conducted experiments in the human cardiac cell line AC16\n(cardiomyocyte) and iPS cell-derived cardiomyocytes (iPSC-CMs). The results showed that NC1\ncan induce a TRPM4-dependent decrease in cell viability in AC16 cells (Fig. 4h, 4i). Furthermore,\n9\niPS cell-derived cardiomyocytes (iPSC-CMs), with comparable TRPM4 expression levels with\nAC16 (Fig. 4j), were also sensitive to NC1(Fig. 4k). These results further strengthened our proposed\nconnection between NECSO and TRPM4 expression. However, due to the current difficulty in\nobtaining neuronal cells, we are unable to provide relevant data. We are working to address this issue\nand will continue to study the role of NECSO in the nervous system in the future.\nFigure 4. NC1 induces NECSO in MB468 cells and AC16 cells.\n(a) TRPM4 protein expression profiles for several cell lines and corresponding heat map for sensitivity\nto NC1-induced cell death.\n(b) MB468 cells were preloaded with CoroNa Green AM probe and then treated with 200 nM NC1 at\ndifferent time points as indicated. Cells are collected for FACS analysis to detect cytosolic sodium.\n(c) Time-course analysis of ATP levels, PI uptake, and LDH release in MB468 cells treated with 200 nM\nNC1 in Na⁺-containing EBS.\n(d) Time-course analysis of ATP levels, PI uptake, and LDH release in MB468 cells treated with 200 nM\nNC1 in Na⁺-free EBS.\n10\n(e) OCR of MB468 cells treated with 100 nM NC1 for 2 or 4 hours.\n(f) Statistical analysis of basal OCR in (e) (n=12 from 3 independent experiments).\n(g) Statistical analysis of Maximal OCR in (e) (n=12 from 3 independent experiments). All values were\ncompared to the 0 h group using one-way ANOVA in (f) and (g).\n(h) TRPM4 expression levels in AC16 and AC16-TRPM4 KO cells.\n(i) Dose-dependent cytotoxicity of NC1 in AC16-WT or TRPM4-KO cells was assessed 24 h after\ntreatment using CTG.\n(j) Immunoblot analysis of TRPM4 expression levels in AC16 and iPSC-CM cells.\n(k) Dose-dependent cytotoxicity of NC1 in iPSC-CM cells was assessed 24 h after treatment using CTG.\nMoreover if NC1 has an antitumorigenic effect the authors should have used in vivo models where\nthe administration of NC1 should have a strong effect on primary tumor growth survival etc.\nResponse: Based on TRPM4 expression levels shown in Figure 4a, we selected two TRPM4-high-\nexpressing cell lines (MCF7 and MB468) and two TRPM4-low-expressing cell lines (A549 and\nSW620) to establish xenograft tumor models in nude mice. Treatment with NC1 exhibited\nsignificant antitumor activity in tumors derived from MCF7 and MB468 cells (Fig. 5a and 5b),\nwhereas no notable therapeutic effect was observed in tumors derived from A549 (Fig. 5c) or SW620\n(Fig. 5d).\nCollectively, these results suggest that NC1 may exert potent antitumor effects in tumor tissues with high\nTRPM4 expression.\n(a) Statistical plots and images compare tumor growth, weights, and appearances between NC1-treated\n(100 mg/kg) and control groups in the MCF7 xenograft model in nude mice.\n(b) Statistical plots and images compare tumor growth, weights, and appearances between NC1-treated\n(100 mg/kg) and control groups in the MB468 xenograft model in nude mice.\n(c) Statistical plots and images compare tumor growth, weights, and appearances between NC1-treated\n(100 mg/kg) and control groups in the A549 xenograft model in nude mice.\n(d) Statistical plots and images compare tumor growth, weights, and appearances between NC1-treated\n(100 mg/kg) and control groups in the SW620 xenograft model in nude mice.\n3) The authors propose that NC1 interacts with several often contradictory targets of the Na+K+\nATPase that it also generates nano holes in the membrane and is activating TMRM4. They do not\nprovide any model functional structural or molecular dynamics to explain the puzzling and\n12\npleiotropic effect of this compound. As such it raises the concern that NC1 simply has a non-selective\ndisruptive effect on cell membrane organization.\nResponse: We sincerely regret any confusion that may have resulted from the fact that our related\nwork (Fu, W. et al. Nature Chemical Biology, 2025) had not yet been published at the time of\nsubmission. It is now available in Nature Chemical Biology1. That work shows that NC1 directly\ninteracts with and activates TRPM4. Specifically, we performed a cellular thermal shift assay\n(CETSA) to assess the interaction between NC1 and TRPM4 in HEK293T cells stably expressing\nTRPM4. NC1 stabilized TRPM4 at elevated temperatures, while DMSO or its inactive stereoisomer\n(NC1i) did not (Fig.1a in this letter, Fig. 1e in the reference1). Surface plasmon resonance (SPR)\nconfirmed direct binding between NC1 and TRPM4, with a binding affinity of 0.88 μM (Fig.1b in\nthis letter, Fig. 1f1). Whole-cell patch-clamp recordings showed that NC1 induced robust currents in\nwild-type (WT) MCF7 cells, which were abolished in TRPM4 knockout (KO) cells (Fig.1c in this\nletter, Fig.1g1). Molecular docking using the hTRPM4 structure (PDB: 6BQV) revealed that NC1\nbinds within a cavity in the transmembrane domain (Fig.1d in this letter, Fig. 4a1). The model\npredicted interactions between NC1 and key residues in this cavity near the Ca²⁺-binding site,\nincluding F936, W864, and V904, L907, and H908 (Fig.1e, f in this letter, Fig. 4b, c1). Mutation of\nW864, H908 or F936 to alanine disrupted NC1 binding (Fig.1g in this letter, Fig. 4d1), TRPM4\nchannel activation (Fig.1h, i in this letter, Fig. 4e, f1) and NECSO sensitivity (Fig.1j in this letter,\nFig. 4g1).\nIn this study, we propose that activation of TRPM4 drives Na⁺ influx, which increases cytosolic Na⁺\nlevels and activates NCLX, leading to mitochondrial sodium overload and a reduction in\nmitochondrial calcium. This ultimately impairs mitochondrial energy production. The energy deficit\nreduces Na/K-ATPase activity, amplifying ionic imbalance. All of the secondary effects (including\nNCLX activation and Na/K-ATPase inhibition) induced by NC1 are due to its targeting of TRPM4,\nas established through biochemical assays, electrophysiological measurements, and structural\nmodeling in our prior work (Fu, W. et al. Nature Chemical Biology, 2025)1. No other target of NC1\nhas been found in our analysis. Furthermore, we still don’t know the nature of nano holes at this\nmoment, therefore we tone down this statement in the revised manuscript (Manuscript lines 363-\n13\n383, 389-391, 467-482). In the revised manuscript, we propose that NC1-induced disruption of ion\nhomeostasis leads to elevated intracellular osmotic pressure, thereby causing cell swelling and\nsubsequent membrane injury - effects that can be attenuated by osmoprotectants.\nFigure 1. NC1 directly targets TRPM4.\n(a) CETSA of NC1 or NC1i in HEK293T cells stably expressing TRPM4–EGFP. The graph represents\nthe relative TRPM4 band intensities of the indicated samples as the mean ± s.d. from three independent\nexperiments.\n(b) Purification with size exclusion chromatography of hTRPM4 (inset). Purified protein was loaded in\ndose-dependent concentrations (Conc.) and stained with Coomassie brilliant blue (CBB). NC1 binds to\nthe hTRPM4 protein as shown by SPR measurements.\n14\n(c) The whole-cell current density–voltage relation of MCF7 WT TRPM4-KO cells activated by NC1.\n(d) Surface representation showing the overall structure of the molecular modeling TRPM4-NC1\ncomplex. MW, molecular weight. Inset: a close-up view of the NC1-binding site. Individual TRPM4\nsubunits are colored cyan or gray, whereas NC1 is highlighted with yellow. Lower right: chemical\nstructure of NC1.\n(e) Specific recognition of NC1-bound hTRPM4. A 3D diagram showing that NC1 binds a deep cavity\nof TRPM4 between S3, S4, S5′ and S6′ helix; Ca2+ is colored purple\n(f) A 2D diagram showing that NC1 interacts with the key residues of TRPM4 through a mixture of\nhydrogen bonds, hydrophobic interactions and π–π stacking.\n(g) CETSA of NC1 and TRPM4 mutants of key residues in e,f ΔTm =Tm (NC1) −Tm (DMSO). Melting\ntemperatures (Tm) were calculated by TRPM4 band intensities of the indicated samples in western blot\nwith GraphPad. The graph represents mean ± s.d. from three independent experiments.\n(h) The current density–voltage relationship of hTRPM4 mutants involving key residues for NC1 binding\nwas examined in HeLa cells.\n(i) Summary plot showing the maximum amplitude of current densities activated by NC1 in the TRPM4\nWT channel compared to the aforementioned mutants.\n(j) Dose-dependent cytotoxicity assay of NC1 in TRPM4-KO MCF7 cells with re-expression of WT\nTRPM4 or TRPM4 mutants (W864A, H908A, F936A).\nReviewer #2 (Remarks to the Author):\nSummary:\nThe manuscript “Sodium disrupts mitochondrial energy metabolism to execute NECSO” by Qiao\net al explores the mechanism by which cytosolic sodium overload elicits necrotic cell death, a\nphenomenon that this group first described in another manuscript currently in revision. Here, the\nauthors use in vitro cell culture models to demonstrate that activation of sodium influx into the cell\nvia pharmacologic activation of the TRPM4 channel impairs mitochondrial oxidative\nphosphorylation and ATP production and leads to necrotic cell death. They use pharmacologic\ninhibition to implicate the mitochondrial sodium/calcium exchanger, NCLX, in this process, and\npropose that mitochondrial calcium depletion in the context of cytosolic sodium overload limits\n15\nmitochondrial metabolism. Finally, the authors use an enzymatic assay to demonstrate that the\nactivity of the plasma membrane sodium/potassium ATPase is diminished in this setting of energy\ndepletion and suggest that the resulting perturbation of cytosolic ionic homeostasis causes osmotic\nimbalance, plasma membrane rupture, and cell death.\nResponse: We sincerely appreciate the reviewer’s thorough and thoughtful review of our manuscript,\nas well as the valuable and constructive feedback you have provided. The reviewer’s insightful\ncomments have significantly contributed to the refinement and strengthening of our work. In\nresponse, we have carefully addressed each point in our detailed rebuttal letter and have incorporated\nthe appropriate revisions into the revised manuscript. We deeply appreciate the reviewer’s expertise,\ntime, and valuable input, which have significantly enhanced the quality and impact of this study.\nMajor concerns:\n1) A central claim of the study is that cellular ATP depletion mechanistically links Na+ overload and\naltered mitochondrial function to impaired Na+/K+-ATPase function that collapses plasma\nmembrane ion gradients leading to necrotic cell death. If so, can the cell death observed with\ncytosolic Na+ overload be rescued by boosting glycolytic ATP production, or even by providing\nexogenous ATP (ex, via patch clamp) to the cells?\nResponse: Thanks for the suggestions. In the revision, we have tested compounds such as creatine\nand lipoic acid, which enhance energy production, and found that they significantly increased\nmitoATP and total ATP synthesis rates (Fig. 6a) while suppressing NC1-induced LDH release (this\nletter Fig. 6b, Manuscript Fig. 1C). In contrast, glycolysis-promoting compounds, including DASA-\n58 and ML-265 (activators of PKM2), showed limited efficacy-only partially enhancing glycoATP\nwithout improving mitoATP, total ATP production rates (Fig. 6c) or inhibiting LDH release (Fig. 6d).\nThis aligns with NC1’s primary effect on mitochondrial energy production and suggests that\nincreased total ATP production inhibits NECSO. In addition, given that TRPM4 activity is known\nto be inhibited by ATP (Extended Data Fig. 7c in the reference5, this letter Fig. 6e) and that NECSO\nis TRPM4-dependent (Fig. 6f in this letter, Fig. 6C in Manuscript), therefore, our results strongly\nindicate that exogenous ATP supplementation can effectively suppress TRPM4 activity and prevent\nFigure 6. Effects of energy agonists on NECSO.\n(a) MCF7 cells were pretreated with 2 mM α-lipoic acid (ALA) or 0.2 mM creatine, followed by\ntreatment with 50 nM NC1 for 3 h. ATP production rates were then measured. P values marked with #\ncompare the blank control with the groups treated with ALA or creatine alone; those marked with *\ncompare the NC1 group with the NC1 + ALA or NC1 + creatine groups. Color coding: red (mitoATP),\nblue (glycoATP), black (total ATP).\n(b) MCF7 cells were pretreated with 2 mM α-lipoic acid or 0.2 mM creatine, followed by NC1 treatment\nfor 24 hours. The effect of these compounds on NC1-induced LDH release was assessed. Statistical\nanalysis was conducted using a two-way ANOVA. Data are shown as mean ± SD of triplicate samples.\n(c) MCF7 cells were pretreated with 50 μM DASA58 or ML-265, followed by treatment with 50 nM\nNC1 for 3 h. ATP production rates were then measured. P values marked with # compare the blank control\nwith the groups treated with DASA58 or ML-265 alone; those marked with * compare the NC1 group\nwith the NC1 + DASA58 or NC1 + ML-265 groups. Color coding: red (mitoATP), blue (glycoATP),\nblack (total ATP).\n(d) MCF7 cells were pretreated with 50 μM DASA58 acid or ML-265, followed by NC1 treatment for\n24 hours. The effect of these compounds on NC1-induced LDH release was assessed.\n(e) Sample I-V curves of TRPM4 at various concentrations of cytosolic free ATP. Channels were\n17\nactivated by 300 μM Ca2+ and 10 μM PtdIns (4,5) P2 in the bath solution. Normalized currents (I/Imax)\nat 100 mV were used to generate the inhibition curves. Imax is the current at 100 mV without ATP.\n(f) LDH release of MCF7 WT cells and TKO cells treated with NC1 for 24 hours (n = 3 wells from 1\nrepresentative of 2 independent experiments). Statistical analysis was performed using a two-way\nANOVA. The above figure shows RPM4 protein levels in MCF7 WT and TKO cells.\n2) Fig. 1D: Can the effects of NC1 on ATP production rates be blocked in experiments using Na+-\nfree media?\nResponse: Yes. To further investigate whether sodium influx contributes to the decrease in ATP\nproduction rate, we measured the ATP synthesis rate following NC1 treatment in both Na⁺-\ncontaining and Na⁺-free EBS. In Na⁺-containing EBS, NC1 treatment led to a significant reduction\nin the overall ATP production rate, primarily due to decreased mitoATP generation, accompanied by\na compensatory increase in glycoATP production (this letter Fig. 7a, Manuscript Fig. S2A). In\ncontrast, NC1 treatment in Na⁺-free EBS had no notable effect on mitoATP or totalATP production\nrate (this letter Fig. 7b, Manuscript Fig. S2B).\nFigure 7. The effect of NC1 on ATP production rates is dependent on sodium influx\n(a) MCF7 cells were treated with 100 nM NC1 for 1 or 3 hours in Na⁺-containing EBS. MitoATP (red),\nglycoATP (blue), and total ATP (black) production rate at different NC1 treatment time points were each\ncompared to the control group using one-way ANOVA.\n(b) MCF7 cells were treated with 100 nM NC1 for 1 or 3 hours in Na⁺-free EBS. MitoATP (red),\nglycoATP (blue), and total ATP (black) production rate at different NC1 treatment time points were each\ncompared to the control group using one-way ANOVA.\n18\n3) Along these lines the Na+ free control experiment only reports data out to 8h, a time right at which\nthere is an increase in PI staining and drop in ATP. Please show the Na+ free condition out to at least\n16hr. i.e., is the cell death just delayed and, if so, for how long?\nResponse: We sincerely appreciate the reviewer’s constructive suggestions, which have greatly\nimproved the clarity and quality of our manuscript. To specifically examine the role of sodium influx\nin NC1-induced cell death, we employed an extracellular buffer solution (EBS) containing only ions\nand glucose (free of amino acids and serum). In the Na⁺-containing condition, extracellular Na⁺ was\nmaintained at 150 mM; in the Na⁺-free condition, Na⁺ was replaced by equimolar non-permeable\nNMDG⁺ to preserve iso-osmolarity.\nAs suggested by the reviewer, we treated cells with NC1 for 16 hours in both conditions and\ndynamically monitored the progression of NECSO. In the Na⁺-containing EBS, cells treated with\nNC1 for 8 hours showed a reduction in ATP levels to approximately 20% of baseline, with around\n60% of cells exhibiting positive PI staining, and LDH release reaching about 40% of the maximum\nlevel. These effects became more pronounced with prolonged NC1 treatment (Fig. 8a). In contrast,\nunder Na⁺-free condition, no significant changes in ATP levels, PI staining, or LDH release were\nobserved within 8 hours. However, by 12 hours, signs of ATP depletion and membrane rupture (PI\nstaining and LDH release) began to emerge. This delayed onset of cell death markers may be\nattributed to prolonged nutrient starvation.\nTherefore, in the revised manuscript, we have focused on the time window up to 8 hours to better\nreflect the specific contribution of sodium influx to NECSO (this letter Fig. 8c-8d, Manuscript Fig.\nS1E-S1F).\n(a) Time-course analysis of ATP levels, PI uptake, and LDH release in MCF7 cells treated with 100 nM\nNC1 in Na⁺-containing EBS (0-16 h).\n(b) Time-course analysis of ATP levels, PI uptake, and LDH release in MCF7 cells treated with 100 nM\nNC1 in Na⁺-free EBS (0-16 h).\n(c) Time-course analysis of ATP levels, PI uptake, and LDH release in MCF7 cells treated with 100 nM\nNC1 in Na⁺-containing EBS (0-8 h).\n(d) Time-course analysis of ATP levels, PI uptake, and LDH release in MCF7 cells treated with 100 nM\nNC1 in Na⁺-free EBS (0-8 h).\n4) The mitotracker staining in Fig 1E, and the Rhod-2 staining in Fig. 1G look non-mitochondrial.\nI’ve never seen a mito network as shown in 1F and Rhod2 looks to be cytosolic and is also staining\nthe nucleolus.\nResponse: We sincerely appreciate your thorough evaluation of our figures, which has remarkably\ncontributed to enhancing the clarity and quality of our work. We acknowledge that in our previous\nMitotracker Red staining, cell fixation after dye labeling resulted in somewhat diffuse signals. To\naddress this limitation, we have now performed Mitotracker Red staining in live cells, and the\nimproved results are shown in Fig. 1E-F of the revised manuscript (this letter Fig. 9a-b). In addition,\nwe have repeated the Rhod-2 staining experiments and co-labeled the cells with Mitotracker Green\nto confirm the mitochondrial localization of Rhod-2. We observed a strong co-localization between\nHowever, we also noticed some nonspecific nuclear staining with Rhod-2 (Manuscript Fig. S2D,\nS2F, and this letter Fig. 8c-d). We have carefully evaluated this phenomenon and confirmed that it\ndoes not compromise the validity or interpretation of our main findings.\nFigure 9. MitoTracker and Rhod-2 staining in NC1-treated MCF7 cells\n(a) Representative Mitotracker Red microscopy confocal images of MCF7 cells exposed to Na+- EBS or\nNa+-free EBS with 100 nM NC1 for indicated time points.\n(b) Statistical analysis of Mitotracker Red fluorescence signals in (a). The treatment groups in different\nEBSs were compared to their respective untreated controls (0 h). Statistical analysis was conducted using\na one-way ANOVA, n=6.\n(c) MCF7 cells were preloaded with Rhod-2 AM and Mitotracker Green, then exposed to Na⁺-containing\nor Na⁺-free EBS with 100 nM NC1 for the indicated time points. Representative confocal microscopy\nimages are shown.\n(d) One-way ANOVA was used to analyze Rhod-2 fluorescence signals in NC1-treated MCF7 cells under\nNa⁺-containing EBS condition in (c).\n(e) One-way ANOVA was used to analyze Rhod-2 fluorescence signals in NC1-treated MCF7 cells under\nNa⁺-free EBS condition in (c).\n5) After redoing the experiments in Fig. 1E and Fig. S2E: Provide quantification and statistical\ncomparison of the drop in mitotracker signal with NC1 treatment. Is this effect truly attenuated when\nthe experiment is conducted in Na+-free media, like the drop in Rhod-2 fluorescence shown in Fig.\n1G?\nResponse: We appreciated the reviewer’s insightful suggestion. We have repeated the experiment\nand observed a marked reduction in Mitotracker Red staining following NC1 treatment in Na⁺-\ncontaining EBS (Manuscript Fig. 1E, this letter Fig. 10a), which is statistically significant\n(Manuscript Fig. 1F, this letter Fig.10b). Since MitoTracker Red accumulates in mitochondria in a\nmembrane potential-dependent manner6, the diminished signal provides additional evidence that\nNC1 disrupts mitochondrial membrane potential.\nFigure 10. MitoTracker Red staining in NC1-treated MCF7 cells\n(a) Representative Mitotracker Red microscopy confocal images of MCF7 cells exposed to Na+- EBS or\nNa+-free EBS with 100 nM NC1 for indicated time points.\n(b) Statistical analysis of Mitotracker Red fluorescence signals in (a). The treatment groups in different\nEBSs were compared to their respective untreated controls (0 h). Statistical analysis was conducted using\n22\na one-way ANOVA, n=6.\n6) I have some concerns about the use of Rhod-2 fluorescence to measure mitochondrial Ca2+ in\nFig. 1G. First, this dye typically labels the mitochondria in a manner that is dependent on\nmitochondrial membrane potential. Therefore, it is difficult to distinguish whether the drop in Rhod-\n2 fluorescence seen with Na+ overload directly represents changes in mitochondrial Ca2+ levels, or\ninstead reflects diminished mitochondrial staining due to a reduction in mitochondrial membrane\npotential. Use of an alternative approach to assess mitochondrial calcium content, such as a\ngenetically-encoded mito-calcium reporter, would help resolve this question. Second, quantification\nand statistical analysis of mitochondrial calcium levels should be provided for this experiment.\nResponse: Thank you for your constructive suggestion. As suggested, we employed the genetically\nencoded mito-calcium sensor mito-R-GECO1 and transfected it into MCF7 cells to monitor\nmitochondrial calcium dynamics following NC1 treatment using live-cell imaging. The results\nshowed that in Na⁺-containing EBS, NC1 induced a significant decrease in mitochondrial calcium\nlevels, whereas this effect was not observed in Na⁺-free EBS (this letter Fig. 3a, Manuscript Fig.\n1H). Statistical analysis confirmed the significance of these findings (this letter Fig. 3b, Manuscript\nFig. 1I).\nIn addition, we performed experiments using the Rhod-2 probe, co-stained with MitoTracker Green\nto confirm its mitochondrial localization. Consistently, NC1 treatment led to a reduction in Rhod-2\nsignal under Na⁺-containing condition, while the effect was diminished in the absence of\nextracellular Na⁺ (this letter Fig. 3c, Manuscript Fig. S2D). The results were statistically significant\n(this letter Fig. 3d-e, Manuscript Fig. S2E-F).\nIn summary, our results demonstrate that NC1 treatment leads to a decrease in mitochondrial calcium\nlevels, which is dependent on extracellular sodium, consistent with our hypothesis.\n(a) MCF7 cells were transfected with mito-R-GECO1 24 hours prior to treatment and then exposed to\nNa⁺-containing or Na⁺-free medium containing 100 nM NC1. Representative still images from time-lapse\nmitochondrial calcium imaging are presented.\n(b) Statistical analysis of mito-R-GECO1 fluorescence signals in (a). The treatment groups in different\nmedia were compared to their respective untreated controls (0 h). Statistical analysis was conducted using\na one-way ANOVA. Data are shown as mean ± SD and n = 3 independent experiments.\n24\n(c) MCF7 cells were preloaded with Rhod-2 AM and Mitotracker Green, then exposed to Na⁺-containing\nor Na⁺-free EBS with 100 nM NC1 for the indicated time points. Representative confocal microscopy\nimages are shown.\n(d) (e) One-way ANOVA was used to analyze Rhod-2 fluorescence signals in NC1-treated MCF7 cells\nunder Na⁺-containing EBS (d) or Na⁺-free EBS (e) condition in (c).\n7) Fig. 1E: A relevant mechanistic question is whether the Na+-dependent mitochondrial swelling\nobserved in Fig. 1E might reflect classical mitochondrial permeability transition, which could trigger\nnecrotic cell death. Can this mito swelling effect of Na+-overload, or any of the other consequences\n(energy depletion, plasma membrane rupture, etc.) be alleviated by pharmacologic inhibition of\npermeability transition with cyclosporin A or with deletion/loss of Ppif/CypD?\nResponse: Thank you for the insightful suggestion. To evaluate the role of cyclophilin D (CypD) in\nNECSO, we employed both cyclosporin A (CsA), a specific CypD inhibitor, and siRNA-mediated\nCypD knockdown. Both pharmacological and genetic inhibition of CypD reduced NC1-induced\nLDH release (Fig. 11b). Moreover, CsA attenuated NC1-induced mitochondrial swelling, as shown\nby electron microscopy (Fig. 11c). Consistently, our previous work (Zhang, J. et al. Cell Death Dis.\n2023)7 also showed that CypD knockdown alleviates the decline in intracellular ATP levels\nfollowing NC1 treatment (Fig. 11a in this letter; Fig. 5E in reference7). These findings collectively\nsuggest that CypD contributes, at least partially, to NECSO.\nFigure 11. Inhibition of CypD partially suppresses NECSO\n25\n(a) Inhibition of NC1-induced killing by knockdown of cyclophilin D (CypD). Cells were subjected to\nthe knockdown of CypD using suitable siRNAs. Forty-eight hours post-transfection the cells were treated\nwith NC1 (50 nM, left) to measure cellular ATP levels, and the efficacy of the knockdown was determined\nby immunoblot (right).\n(b) In MCF7 cells, pretreatment was performed with either cyclosporin A (CsA, 10 μM) for 1 hour\n(DMSO served as the control), or with CypD siRNAs for 24 hours (siNC used as the control). Following\npretreatment, all cells were treated with 50 nM NC1 for 20 hours, and LDH release was measured. The\nright panel shows western blot validation of CypD siRNA knockdown efficiency.\n(c) Representative transmission electron microscopy images of MCF7 cells treated with 100 nM NC1 for\n5 hours with or without 10 μM CsA pretreatment.\n8) A major outstanding question is whether the apparently NCLX-dependent effects of cytosolic\nNa+ overload (leading to mito Na+ loading and mito Ca2+ depletion) are direct effects of increased\nmitochondrial Na+, or rather direct effects of diminished free mitochondrial Ca2+. How do the\nauthors distinguish these two possibilities?\nResponse: It remains unclear whether the mitochondrial dysfunction mediated by NCLX following\nTRPM4-mediated sodium overload is primarily driven by increased mitochondrial sodium levels or\nby reduced mitochondrial calcium levels.\nSodium accumulation in mitochondria impairs respiratory function by inhibiting the activity of\nmitochondrial complexes II and III, leading to a reduction in oxidative phosphorylation capacity\n(OXPHOS) and ATP production. Mitochondrial Ca²⁺ plays a critical role in energy metabolism by\nactivating three key dehydrogenases in the TCA cycle8-10, accelerating NADH production11. This,\nin turn, promotes electron transport chain (ETC) activity, increases the proton motive force, and boosts\nATP synthesis12, 13. Consequently, a decrease in mitochondrial Ca²⁺ compromises energy generation\ncapacity. Therefore, it is reasonable to conclude that both elevated mitochondrial sodium and\nreduced mitochondrial calcium contribute to mitochondrial injury. Importantly, both of these events\nare ultimately triggered by TRPM4-mediated sodium influx.\n26\n9) Fig. 2F, Fig. 6J: Measurements of Complex II activity were performed using isolated protein\nimmunoadsorbed onto assay plates for 2hrs prior to the start of the assay. This will necessarily\nremove CII from its native environment, as well as the local mitochondrial Na+ and Ca2+\nconcentrations affected by treatment with NC1. How, then, do the authors propose that NC1 and the\nresulting cellular/mitochondrial sodium overload and/or mitochondrial Ca2+ depletion exerts this\napparently “long-lasting” effect on CII activity?\nResponse: Na+ is known to interact with phospholipids in the mitochondrial inner membrane (IMM),\nsuch as phosphatidylcholine (PC) at the level of its carbonyl group, at a ratio of 3:1 (PC: Na+)14-16.\nThis interaction has been shown to reduce membrane fluidity, thereby impairing the diffusion of\ncoenzyme Q (CoQ) through the inner mitochondrial membrane (IMM). As a consequence, electron\ntransfer from complex II (CII) to complex III (CIII) is hindered, ultimately leading to a reduction in\nthe activity of the CII+CIII (this letter Fig. 12a, Fig.3e in the reference14). It has also been reported\nthat hypoxia-induced mitochondrial calcium overload can activate NCLX, leading to elevated\nintramitochondrial sodium levels. Consistently, inhibition of complexes II+III activity has been\nobserved in mitochondria isolated from hypoxic MEF cells, and this effect is attenuated by NCLX\nknock out (this letter Fig. 12b, Fig.3f in the reference14). In our case, NC1-induced cytosolic sodium\noverload activates NCLX, thereby facilitating the influx of sodium into mitochondria, where it\naccumulates and suppresses complex II activity through a mechanism likely analogous to that\ndescribed above.\nTherefore, the \"long-lasting\" inhibition of complex II activity after NC1 treatment, or of complexes\nII+III activity under hypoxic condition (this letter Fig. 12b, Fig.3f in the reference14), may result\nfrom the incomplete dissociation of Na+ from phospholipids during the pre-assay incubation period.\n(a) Succinate-cytochrome c (CytC) activity in mitochondrial membranes from BAECs with or without\nNaCl (n = 4).\n(b) Succinate-cytochrome c (CytC) activity in mitochondrial membranes from hypoxic or normoxic WT\nor NCLX KO MEFs (1% O ; n = 3).\n2\n10) Fig. 3A, B, D: Are any of the changes in mitochondrial metabolism shown with NC1 treatment\nin Fig. 3 attenuated with NCLX inhibition or knockdown?\nResponse: Yes, we pretreated MCF7 cells with the NCLX inhibitor CGP37157 (CGP) before NC1\ntreatment. The results showed that CGP attenuated the accumulation of TCA cycle metabolites,\nincluding pyruvate, isocitrate, α-ketoglutarate, succinate, fumarate and malate (see this letter Fig.\n13a; Manuscript Fig. S5A), and reduced the elevated succinate/fumarate ratio induced by NC1 (this\nletter Fig. 13b; Manuscript Fig. S5B).\nFigure 13. Inhibition of NCLX attenuates the accumulation of TCA cycle metabolites in NECSO.\n(a) MCF7 cells were pretreated with 20 μM CGP37157 (CGP) followed by treatment with 100 nM NC1\nfor 3 hours. A heatmap shows differentially regulated TCA cycle metabolites, normalized to 0 h control\nvalues (n = 4 per group).\n28\n(b) An unpaired two-tailed t-test was used to analyze the succinate-to-fumarate ratio from (A). Data are\npresented as mean ± SD (n = 4).\n11) Fig. 3B-C: the authors attempt to link mitochondrial calcium depletion with NC1 treatment to\nimpaired pyruvate dehydrogenase activity, based on the accumulation of thiamine diphosphate. As\nPDH phosphatase is sensitive to changes in mitochondrial Ca2+, is the phosphorylation status of\nPDH (Ser293) altered under these conditions?\nResponse: Thanks for the constructive suggestion. We assessed PDHα1 (PDHA1) phosphorylation\nat serine 293 in MCF7 cells following NC1 treatment. Our data demonstrate that NC1 induces a\ntime-dependent increase in PDHA1 ser293 phosphorylation (Manuscript Fig. S4C, this letter Fig.\n14). Since this modification is known to inhibit PDH complex activity17, our observation further\ncorroborates the notion that NC1 suppresses TCA cycle metabolism.\nFigure 14. Phosphorylation of PDHA1 at serine 293 following NC1 treatment\nMCF7 cells were treated with 50 nM NC1 for the indicated time points, and PDHA1 Ser 293\nphosphorylation was assessed.\n12) Figure 4 is missing CGP controls throughout all experiments.\nResponse: Thank you for your careful and thoughtful review. We have added the control groups to\nFig. 4 in the manuscript (this letter Fig. 15)\n(A) Representative transmission electron microscopy images of MCF7 cells treated with 100 nM NC1\nfor 5 hours with or without 20 μM CGP37157 pretreatment.\n(B) Detection of mitochondrial Na+ content of MCF7 cells treated with 50 nM NC1 for 0.5 hour with or\nwithout 20 μM CGP37157 pretreatment (n=3).\n(C) MCF7 cells were preloaded with Rhod-2 AM probe and then treated with 50 nM NC1 for 3 hours\nwith or without 20 μM CGP37157 pretreatment. Cells are collected for FACS analysis to detect\nmitochondrial calcium.\n(D) MCF7 cells were preloaded with TMRE dye and then treated with 50 nM NC1 for 3 hours with or\nwithout 20 μM CGP37157 pretreatment. Cells are collected for FACS analysis to detect mitochondrial\nmembrane potential.\n(E) OCR of MCF7 cells treated with 50 nM NC1 for 3 hours with or without 20 μM CGP37157\n30\npretreatment.\n(F) Statistical analysis of basal OCR in (E) (n=15 from 3 independent experiments).\n(G) Statistical analysis of maximal OCR in (E) (n=15 from 3 independent experiments).\n(H) Complex II+III activity of bovine heart mitochondria under condition with additional 40 mM NaCl\nin the presence or absence of 20 μM CGP37157 (n=3).\n(I) ATP production rate of MCF7 cells treated with 50 nM NC1 for 3 hours with or without 20 μM\nCGP37157 pretreatment (n=4). The P values indicated by # reflect comparisons of mitoATP, glycoATP,\nand total ATP between the CGP (-)/NC1 and CGP (-)/Ctrl groups; & indicates comparisons between the\nCGP (+)/Ctrl and CGP (+)/NC1 groups; and * signifies comparisons between the CGP (-)/NC1 and CGP\n(+)/NC1 groups. Color coding: red (mitoATP), blue (glycoATP), black (total ATP).\n(J) LDH release of NC1-treated MCF7 cells with or without 20 μM CGP37157 pretreatment. Cells were\ntreated with NC1 for 18 hours. A two-way ANOVA was performed.\nStatistical analysis in (B) (F) (G) (H) was conducted using an unpaired, two-tailed test. Data are shown\nas mean ± SD.\n13) NCLX loss or inhibition results in severe mitochondrial calcium overload, mitochondrial\nswelling, and necrotic cell death that can be rescued by CypD inhibition. How is 5h of NCLX\ninhibition protective in the context of this cell system? What happens if the authors target MCU?\nResponse: We appreciate your insightful comment. NCLX is a major mitochondrial calcium efflux\npathway. Previous study has shown that complete genetic deletion of NCLX leads to mitochondrial\ncalcium overload, which can cause cardiac injury and sudden death in mice18. Moreover, MCU is a\ncritical channel responsible for calcium entry into mitochondria.\nIn NECSO, mitochondrial calcium level is reduced. When we inhibited MCU function using either\npharmacological inhibitors (DS16570511, RuRed) or siRNAs, thereby blocking mitochondrial\ncalcium uptake, NECSO is exacerbated (this letter Fig. 16a-b, Manuscript Fig. S4A-B). Furthermore,\nknockdown of NCLX using siRNA suppresses the LDH release in NECSO (Fig. 16c). Collectively,\nthese findings support our conclusion that NC1 induces a decrease in mitochondrial calcium level,\nleading cells into a low-energy metabolic state (The relevant discussion has been added in lines 428\n31\n- 437of the Manuscript.).\nFigure 16. The role of MCU and NCLX in NECSO.\n(a) The effect of DS16570511 (25 μM) and RuRed (25 μM) on LDH release in MCF7 cells treated with\nNC1 for 18 hours was evaluated. A two-way ANOVA was used to analyze differences compared to the\ncontrol group (NC1).\n(b) The effect of MCU siRNAs on LDH release in MCF7 cells treated with NC1 for 24 hours was\nevaluated. A two-way ANOVA was used to analyze differences compared to the control group (siNC).\nThe right figure shows the expression of MCU protein level.\n(c) The effect of NCLX siRNAs on LDH release in MCF7 cells treated with 20 nM NC1 for 24 hours\nwas evaluated. The right figure shows the expression of NCLX protein level.\nMinor concerns:\n1) Fig. 1D and Fig. 4H: Statistical comparisons should be provided to support claims of the effects\nof the various treatments on mitochondrial, glycolytic, and total ATP production rates.\nResponse: We sincerely appreciate the reviewer's thorough evaluation. As suggested, we have added\nstatistical comparisons in Fig. 1D and Fig. 4H of the revised manuscript (corresponding to Figures\n17a and 17b in this response letter).\n(a) ATP generation rates from glycolysis (glycoATP) and mitochondria (mitoATP) in MCF7 cells treated\nwith 50 nM NC1 for 0, 2, 4, or 6 hours were evaluated using the Seahorse XF technology. MitoATP (red),\nglycoATP (blue), and total ATP (black) at different NC1 treatment time points were respectively\ncompared to the control group using one-way ANOVA.\n(b) ATP production rate of MCF7 cells treated with 50 nM NC1 for 3 hours with or without 20 μM\nCGP37157 pretreatment (n=4). P values marked with # compare mitoATP, glycoATP, and total ATP\nbetween NC1 and Ctrl groups; those with * compare NC1 and NC1+CGP groups. Color coding: red\n(mitoATP), blue (glycoATP), black (total ATP).\n2) Fig. S2C-D: provide statistical comparisons between experimental groups for ATP content and\nLDH release.\nResponse: Thanks for the reviewer's thorough evaluation. In the original manuscript Fig. S2C–D,\nwe aimed to demonstrate that treatment with varying concentrations of NC1 for 6 hours in Na+-\ncontaining EBS leads to a reduction in ATP levels and LDH release, whereas the same treatment in\nNa+-free EBS does not elicit these effects. In the revised version, we have adopted a new presentation\napproach (Fig. S1E–F in the revised manuscript, Fig. 17a-b in this letter), in which ATP levels, PI\nstaining, and LDH release were dynamically monitored at multiple time points following NC1\ntreatment, both in Na+-containing and Na+-free EBS conditions. The results show that in Na+-\ncontaining EBS, NC1 treatment causes an early decrease in ATP content, followed by increased PI\n33\nstaining and LDH release. In contrast, these effects are abolished in Na+-free EBS. We believe this\nupdated presentation more clearly illustrates the role of sodium influx in NECSO.\nFigure 18. Sodium-dependent cell death after NC1 Treatment.\n(a) (b) The cellular ATP levels, PI intake, and LDH release of MCF7 cells treated with 100 nM NC1 in\nNa+-containing EBS (a) or Na+-free EBS (b) were dynamically monitored.\n3) Fig. 3B: citrate is noted in the text as accumulating following NC1 treatment, but it is not included\nin the heatmap of metabolites in Fig. 3B. Please add it.\nResponse: We sincerely apologize for this typographical error. Citrate is not among the metabolites\nthat increased after NC1 treatment, and we have made the correction in the manuscript (lines 259-\n261).\n4) Fig. 4C-D and Fig. 6 F-G: Y-axes of these graphs should be labelled for each treatment condition,\nand statistical comparisons between groups should be provided to support the authors’ claims that\nNCLX inhibition or TRPM4 KO reverses NC1’s effects on mitochondrial Ca2+ and membrane\npotential.\nResponse: We have labelled the treatment condition and added statistical comparisons in Fig.4C-D\nand Fig.6F-G of the revised manuscript (corresponding to Fig. 19a-d in this response letter).\n(a) MCF7 cells were loaded with Rhod-2 AM and treated with 50 nM NC1 for 3 h, with or without 20\nμM CGP37157 pretreatment. Mitochondrial calcium levels were analyzed by FACS.\n(b) MCF7 cells were stained with TMRE and treated as in (a). Mitochondrial membrane potential was\nassessed by FACS.\n(c) MCF7 TKO cells were loaded with Rhod-2 AM and treated with 50 nM NC1 for the indicated time\nperiods. Mitochondrial calcium was measured by FACS.\n(d) MCF7 TKO cells were stained with TMRE and treated as in (c). Mitochondrial membrane potential\nwas evaluated by FACS.\n5) Fig. 5 D-E: The dose-response curves in these graphs should be compared statistically to support\nthe claim that inhibition of the Na+/K+-ATPase with ouabain sensitizes cells to the effects of sodium\noverload.\nResponse: Thanks for the thorough evaluation. We have added statistical comparisons for the dose-\nresponse curves in Fig. 5D-E of the revised manuscript (this letter Fig. 20a-b).\nFigure 20. The effect of ouabain on NECSO.\n(a) Intracellular ATP levels in NC1-treated MCF7 cells under normal DMEM conditions, with or without\n10 nM ouabain pretreatment (NC1 20 h, n = 3 wells; representative of 4 independent experiments).\n35\n(b) LDH release from NC1-treated MCF7 cells under the same conditions as in (a).\nStatistical analysis was performed by two-way ANOVA. Data are presented as mean ± SD.\n6) Fig. 7 C and E: add statistical comparisons for the dose-response curves.\nResponse: Thanks for the reviewer’s thorough evaluation. We have added statistical comparisons\nfor the dose-response curves in Fig.7C and Fig.7E of the revised manuscript (this letter Fig. 21a-b).\nFigure 21. The effects of osmoprotectants and AQP4 on NECSO\n(a) Size dependent inhibition of LDH release from NC1-treated MCF7 cells by osmoprotectants.\nOsmoprotectants were added to DMEM with a final concentration of 20 mM. NC1 treated MCF7 cells\nfor 20 hours (n = 3 wells from 1 representative of 4 independent experiments). Statistical analysis was\nperformed by two-way ANOVA to analyze the effects of different osmoprotectants compared to the NC1\ngroup.\n(b) LDH release of MCF7 cells treated with NC1 for 24 hours with or without 200 μM AER-271\npretreatment. Data were analyzed using a two-way ANOVA.\nReviewer #3 (Remarks to the Author):\nNa+ influx is a common event in necrotic cell death and if it was regulated during a given necrosis\nprocess, it must be essential for the control of the necrosis. Qiao et al used Necrocide 1, an agonist\nof monovalent cation non-selective channel TRPM4 to induce cell necrosis, and analyzed\nintracellular changes after Necrocide 1 treatment. Instead of the common thought that the collapse\nof Na+ gradient would directly lead to swelling of the cell and subsequent plasma membrane\ndisruption, the authors stated that sodium influx is via inhibiting mitochondrial energy\nproduction/energy depletion to inactivate Na/K-ATPase, resulting in ion gradient collapse, pore\nformation, water influx, and eventual necrotic cell death. The conclusion is astonishing, but whether\nenergy depletion is a trigger or simply a concomitant phenomenon of Necrocide 1-caused cell\nnecrosis has not been demonstrated unambiguously. The authors need to provide evidence that the\ncell necrosis is not directly caused by Na+ influx.\nResponse: Thank you for this thoughtful feedback. To clarify the causal relationship between energy\ndepletion and NC1-induced necrosis, we tested the effect of compounds that promote ATP\ngeneration such as alpha-lipoic acid (ALA) and creatine on NECSO (Fig. 22a). These results showed\nthat both ALA and creatine inhibit NECSO (Manuscript Fig. 1C, this letter Fig. 22b), suggesting\nthat energy depletion acts as a trigger rather than a concomitant phenomenon.\nOur findings suggest that TRPM4-mediated sodium influx initiates cell necrosis through regulatory\nsignaling pathways, rather than being a direct result of the sodium influx itself caused by TRPM4\nchannel activation. A key pathway involves the activation of NCLX following TRPM4-mediated\nsodium influx, which compromises mitochondrial ion homeostasis and impairs mitochondrial\nenergy production. This energy depletion subsequently inhibits Na/K-ATPase function, leading to\nsecondary sodium influx. The resulting increase in intracellular osmotic pressure promotes cell\nswelling and membrane damage, with AQP4 facilitating the associated water influx.\nFigure 22. Enhancing energy production suppresses NECSO.\n(a) MCF7 cells were pretreated with 2 mM α-lipoic acid (ALA) or 0.2 mM creatine, followed by\ntreatment with 50 nM NC1 for 3 h. ATP production rates were then measured. P values marked with #\ncompare the blank control with the groups treated with ALA or creatine alone; those marked with *\ncompare the NC1 group with the NC1 + ALA or NC1 + creatine groups. Color coding: red (mitoATP),\n37\nblue (glycoATP), black (total ATP).\n(b) MCF7 cells were pretreated with 2 mM α-lipoic acid or 0.2 mM creatine, followed by NC1 treatment\nfor 24 hours. The effect of these compounds on NC1-induced LDH release was assessed. Statistical\nanalysis was conducted using a two-way ANOVA. Data are shown as mean ± SD of triplicate samples.\nThis reviewer also has a few specific comments:\n1. The requirement of AQP4 in Necrocide 1-induced necrosis needs to be demonstrated by genetic\ndeletion of AQP4.\nResponse: We appreciate this constructive suggestion and as suggested by the reviewer, we used\nsiRNAs targeting AQP4, and the results showed that AQP4 knockdown inhibits NECSO (Fig. 23).\nFigure 23. AQP4 knockdown suppresses NECSO.\nThe effect of AQP4 siRNAs on LDH release in MCF7 cells treated with NC1 for 24 hours was evaluated.\nData were analyzed using a two-way ANOVA. The right figure shows the Western blot validation of\nAQP4 knockdown efficiency.\n2. Since AQP4 interacts with TRPM4, it could function together with TRPM4 in Necrocide 1\ninduced Na+ influx rather than in a later stage of cell necrosis. This can be easily evaluated.\nResponse: We thank the reviewer for this valuable suggestion. To examine whether AQP4 inhibition\naffects NC1-induced sodium influx, we employed both the AQP4 inhibitor (AER-271) and AQP4-\ntargeted siRNAs. Intracellular sodium levels were measured using the CoroNa Green AM probe.\nThe results showed that NC1 treatment in MCF7 cells leads to a significant increase in CoroNa\nGreen fluorescence, indicating sodium influx. However, neither the AQP4 inhibitor (Fig. 24a) nor\nAQP4 may not regulate TRPM4 activity.\nFigure 24. The effect of AQP4 on NC1-induced sodium influx\n(a) MCF7 cells were treated with 50 nM NC1 for 0 h, 1 h and 2 h with or without 200 μM AER-271\npretreatment, intracellular sodium was detected using CoroNa Green AM and flow cytometry.\n(b) MCF7 cells were treated with NC1 (50 nM, 3 h) after AQP4 siRNA transfection 48 hours prior.\nIntracellular sodium was detected using CoroNa Green AM and flow cytometry. The right figure shows\nthe Western blot validation of AQP4 knockdown efficiency.\n3. This study has no linkage with ischemia or organ failure.\nResponse: While our study focuses on NC1-induced necrosis in vitro, there is substantial evidence\nsuggesting that TRPM4 is involved in ischemic injuries and organ failure. Research indicates that\nTRPM4 contributes to ischemic brain injury 19, 20 and that the TRPM4 inhibitor 9-phenanthrol (9-ph)\ncan alleviate cardiac ischemia-reperfusion injury in rats21. Furthermore, an upregulation of TRPM4\nexpression has been observed in human failing heart tissue22, 23. Given that sodium overload and\nenergy depletion are hallmark features of ischemic injury24, 25 and organ failure26, 27, our findings\nsuggest the TRPM4-NCLX axis as a potential regulatory target in these conditions. We will further\nexplore the therapeutic relevance of targeting TRPM4-NCLX axis in ischemia-related conditions\nand organ failure in future studies. Notably, our group has employed the NC1-induced cell death\nsystem as a platform for screening NECSO inhibitors. We plan to further investigate the therapeutic\npotential of the identified compounds in models of ischemia and organ dysfunction.\n1. Fu, W. et al. Persistent activation of TRPM4 triggers necrotic cell death characterized by sodium\noverload. Nat Chem Biol (2025).\n2. Pérez Koldenkova, V. & Nagai, T. Genetically encoded Ca(2+) indicators: properties and evaluation.\nBiochim Biophys Acta 1833, 1787-1797 (2013).\n3. Wu, J. et al. Improved orange and red Ca²± indicators and photophysical considerations for\noptogenetic applications. ACS Chem Neurosci 4, 963-972 (2013).\n4. Fonteriz, R.I. et al. Monitoring mitochondrial [Ca(2+)] dynamics with rhod-2, ratiometric pericam\nand aequorin. Cell Calcium 48, 61-69 (2010).\n5. Guo, J. et al. Structures of the calcium-activated, non-selective cation channel TRPM4. Nature 552,\n205-209 (2017).\n6. Neikirk, K. et al. MitoTracker: A useful tool in need of better alternatives. Eur J Cell Biol 102, 151371\n(2023).\n7. Zhang, J. et al. Necrocide 1 mediates necrotic cell death and immunogenic response in human cancer\ncells. Cell Death Dis 14, 238 (2023).\n8. Denton, R.M., Randle, P.J. & Martin, B.R. Stimulation by calcium ions of pyruvate dehydrogenase\nphosphate phosphatase. Biochem J 128, 161-163 (1972).\n9. Denton, R.M., Richards, D.A. & Chin, J.G. Calcium ions and the regulation of NAD+-linked\nisocitrate dehydrogenase from the mitochondria of rat heart and other tissues. Biochem J 176, 899-\n906 (1978).\n10. McCormack, J.G. & Denton, R.M. The effects of calcium ions and adenine nucleotides on the activity\nof pig heart 2-oxoglutarate dehydrogenase complex. Biochem J 180, 533-544 (1979).\n11. Xie, N. et al. NAD(+) metabolism: pathophysiologic mechanisms and therapeutic potential. Signal\nTransduct Target Ther 5, 227 (2020).\n12. Wescott, A.P., Kao, J.P.Y., Lederer, W.J. & Boyman, L. Voltage-energized Calcium-sensitive ATP\nProduction by Mitochondria. Nat Metab 1, 975-984 (2019).\n13. Jouaville, L.S., Pinton, P., Bastianutto, C., Rutter, G.A. & Rizzuto, R. Regulation of mitochondrial\nATP synthesis by calcium: evidence for a long-term metabolic priming. Proc Natl Acad Sci U S A 96,\n13807-13812 (1999).\n14. Hernansanz-Agustín, P. et al. Na+ controls hypoxic signalling by the mitochondrial respiratory chain.\nNature 586, 287-291 (2020).\n15. Böckmann, R.A., Hac, A., Heimburg, T. & Grubmüller, H. Effect of sodium chloride on a lipid bilayer.\nBiophys J 85, 1647-1655 (2003).\n16. Cordomí, A., Edholm, O. & Perez, J.J. Effect of ions on a dipalmitoyl phosphatidylcholine bilayer. a\nmolecular dynamics simulation study. J Phys Chem B 112, 1397-1408 (2008).\n17. Fan, J. et al. Tyr-301 phosphorylation inhibits pyruvate dehydrogenase by blocking substrate binding\nand promotes the Warburg effect. J Biol Chem 289, 26533-26541 (2014).\n18. Luongo, T.S. et al. The mitochondrial Na(+)/Ca(2+) exchanger is essential for Ca(2+) homeostasis\nand viability. Nature 545, 93-97 (2017).\n19. Chen, B. et al. TRPM4-specific blocking antibody attenuates reperfusion injury in a rat model of\nstroke. Pflugers Arch 471, 1455-1466 (2019).\n20. Yan, J., Bengtson, C.P., Buchthal, B., Hagenston, A.M. & Bading, H. Coupling of NMDA receptors\nand TRPM4 guides discovery of unconventional neuroprotectants. Science 370 (2020).\n21. Wang, J., Takahashi, K., Piao, H., Qu, P. & Naruse, K. 9-Phenanthrol, a TRPM4 inhibitor, protects\nisolated rat hearts from ischemia-reperfusion injury. PLoS One 8, e70587 (2013).\n22. Ramirez Flores, R.O. et al. Consensus Transcriptional Landscape of Human End-Stage Heart Failure.\nJ Am Heart Assoc 10, e019667 (2021).\n23. Dragún, M., Gažová, A., Kyselovič, J., Hulman, M. & Máťuš, M. TRP Channels Expression Profile\nin Human End-Stage Heart Failure. Medicina (Kaunas) 55 (2019).\n24. Murphy, E., Cross, H. & Steenbergen, C. Sodium regulation during ischemia versus reperfusion and\nits role in injury. Circ Res 84, 1469-1470 (1999).\n25. Yang, J.L., Mukda, S. & Chen, S.D. Diverse roles of mitochondria in ischemic stroke. Redox Biol 16,\n263-275 (2018).\n26. Kohlhaas, M. et al. Elevated cytosolic Na+ increases mitochondrial formation of reactive oxygen\nspecies in failing cardiac myocytes. Circulation 121, 1606-1613 (2010).\n27. Lopaschuk, G.D., Karwi, Q.G., Tian, R., Wende, A.R. & Abel, E.D. Cardiac Energy Metabolism in\nHeart Failure. Circ Res 128, 1487-1513 (2021).\n41"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed all the concerns and issues that I have raised", "reviewer_2": "The authors addressed nearly all of my concerns and are congratulated on a nice study.", "reviewer_3": "The authors addressed my questions. I have no further comment."}, "author_response": ""}]

Decision letter