Improving Older Age Survival and Movement by Lowering Mitochondrial Calcium to Boost Cell Stress Response

To evaluate the broader effects of mitochondrial Ca²⁺ modulation, RNA interference (RNAi)‐mediated knockdown of mcu‐1 was initiated in N2 nematodes on day 4 of adulthood. While mcu‐1 RNAi reduced survival in early adulthood, the remaining worms experienced a significant extension in lifespan compared to controls (Figure 1a; for statistical details, see Table 1). Next, we assessed motility, measured as body bends per minute, as an indicator of health. While motility declined significantly with age, N2 nematodes treated with mcu‐1 RNAi exhibited a marked improvement in late adulthood (day 21) (Figure 1b). To pinpoint when mitochondrial Ca²⁺ modulation is most beneficial, mcu‐1 knockdown was induced during specific life stages. RNAi treatment initiated before day 14 significantly extended lifespan (Figure 1c, Table 1), while lifespan extension was not observed when mcu‐1 RNAi was applied after day 14 (Figure 1d, Table 1). These results suggest that early adulthood is a critical period for gaining the beneficial effects of reduced mitochondrial Ca²⁺ uptake. To verify reduced mitochondrial Ca²⁺ levels upon mcu‐1 knockdown, we performed live‐cell imaging in the nematode strain AQ3055 treated with control RNAi or RNAi against mcu‐1 on days 7, 14, and 21. This strain expresses the mitochondrial‐targeted Ca²⁺ biosensor YC3.60—a genetically encoded Förster resonance energy transfer (FRET)‐based sensor composed of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)—in muscle tissues of the pharynx (Figure 1e, Figure S1). We determined basal mitochondrial Ca²⁺ levels and mitochondrial Ca²⁺ uptake after stimulation with caffeine [15 mM] in worms treated with control or mcu‐1 RNAi (Figure 1f). Live‐cell imaging revealed a statistical increase in basal mitochondrial Ca²⁺ levels from day 7 to days 14 and 21. In addition, there was a significant reduction in basal mitochondrial Ca²⁺ levels at days 7, 14, and 21 through mcu‐1 knockdown (Figure 1g). Additionally, caffeine‐induced mitochondrial Ca²⁺ uptake tended to be decreased in nematodes treated with mcu‐1 RNAi (Figure S1). Quantitative real‐time PCR (qRT‐PCR) confirmed that RNAi‐induced mcu‐1 knockdown resulted in a significant reduction of mRNA expression levels at days 7, 14, and 21 (Figure 1h).

In summary, these findings suggest that reducing mitochondrial Ca²⁺ levels through RNAi‐mediated mcu‐1 knockdown extends lifespan and enhances motility in old nematodes, though it may impair survival during early adulthood.

Since mitochondrial Ca²⁺ is crucial for the activity of TCA cycle dehydrogenases (Denton and McCormack 1986) and, thus, the activity of the ETC (Mitchell 1961), we assessed the activities of the pyruvate dehydrogenase (PDH) and ETC complexes. In response to mcu‐1 RNAi treatment, we found a significant reduction in PDH activity (Figure 2a). Sequential substrate and inhibitor titrations further revealed markedly decreased activities of complexes I, II, and III, along with reduced maximal respiration (Figure 2b). Such impairments in ETC function can promote electron leakage and elevated ROS production (Zorov et al. 2014). Therefore, we performed an Amplex Red assay. H₂O₂ levels increased progressively from day 7 to day 21 in N2 nematodes. Notably, mcu‐1 RNAi treatment led to a significant rise in H₂O₂ at day 14, followed by reduced levels in late life (day 21) compared to controls (Figure 2c). To further investigate alterations in cytosolic H₂O₂ levels, live‐cell imaging was performed in the JV1 strain treated with control RNAi or RNAi against mcu‐1 on day 14. This strain expresses the H₂O₂ sensor HyPer under a ribosomal promoter and was excited at both 455 nm and 505 nm, while excitation was acquired between 500 and 521 nm (Figure 2d). The respective strain was used to assess basal cytosolic H₂O₂ levels and the response after H₂O₂ application in living nematodes at day 14 under control conditions or with mcu‐1 knockdown (Figure 2e). This approach confirmed the transient increase in cytosolic H₂O₂ levels at day 14 (Figure 2f). Consequently, we further investigated whether alterations in H₂O₂ levels are associated with lifespan extension induced by mcu‐1 RNAi. Treatment with 1 mM of the ROS scavenger N‐acetyl cysteine (NAC) (Schulz et al. 2007) abolished the lifespan‐extending effects of mcu‐1 RNAi in N2 nematodes (Figure 2g and Table 1), as did the mitochondrial‐specific antioxidant MitoTEMPO [100 nM] (Ewald et al. 2017) (Figure 2h; Table 1). These results suggest that a transient increase in ROS levels may trigger a signaling cascade that promotes longevity and improves fitness in older worms.

To investigate whether lifespan extension caused by mcu‐1 knockdown is dependent on ROS‐sensitive signaling pathways, we tested worms deficient in pmk‐1, daf‐16, and skn‐1, the respective nematode orthologs of human p38 mitogen‐activated protein kinases (MAPK) (Inoue et al. 2005), forkhead box transcription factor class O (FOXO), and nuclear factor erythroid 2‐related factor 2 (NRF2) (Tian et al. 2021). In pmk‐1‐deficient worms, mcu‐1 RNAi failed to extend lifespan (Figure 3a; Table 1), as it also did in worms deficient in daf‐16 (Figure 3b; Table 1) and skn‐1 (Figure 3c; Table 1). Notably, deficiency in AMP‐activated protein kinase (AMPK) (aak‐2) or sirtuins (sir‐2.1) could not prevent lifespan extension induced by mcu‐1 RNAi (Figure S2), suggesting that the effect is mediated by a signaling cascade triggered by transient ROS fluctuations rather than by nutrient‐sensing pathways. Since DAF‐16 and SKN‐1 promote antioxidant defense mechanisms, we also tested worms deficient in superoxide dismutase (sod‐2). Results from respective experiments demonstrate that both the lifespan‐extending (Figure 3d; Table 1) and fitness‐enhancing (Figure 3e) effects of mcu‐1 RNAi were abolished in sod‐2 deficient worms, which did not exhibit the characteristic H₂O₂ rise as seen in N2 nematodes during aging or in response to mcu‐1 RNAi at day 14 (Figure 3f). Next, the JV2 strain (Back et al. 2012) was used to assess the basal ratio of oxidized to reduced glutathione (GSSG/GSH) levels, before and after the addition of H₂O₂ in nematodes with and without knockdown of mcu‐1 (Figure 3g). During aging, the GSSG/GSH ratio increased in JV2 nematodes. Notably, GSSG levels increased during middle age in nematodes treated with mcu‐1 RNAi, while a reduced GSSG/GSH ratio was observed in old age in treated worms (Figure 3h). In parallel, we examined the expression of two established DNA damage markers, the programmed cell death activator egl‐1 and cell death protein 3 subunit p17 (ced‐3) (Greiss et al. 2008), which showed a tendency to be upregulated with age but were significantly downregulated at day 21 in worms treated with mcu‐1 RNAi, supporting the notion that ROS‐induced signaling pathways involving FOXO and NRF2 counteract age‐associated genotoxic stress (Figure S2).

In summary, these results indicate a shift in ROS homeostasis through mitochondrial Ca²⁺ modulation, involving pmk‐1, daf‐16, and skn‐1, which lead to enhanced antioxidant defense mechanisms and lifespan extension following mcu‐1 RNAi treatment.

Next, we utilized the W118 strain to test whether muscle‐specific knockdown of mcu‐1 is sufficient to extend lifespan. Indeed, overall lifespan was found to be significantly increased by mcu‐1 RNAi in this strain (Figure 4a, Table 1), while no lifespan extension was found in the VP303 strain with specific mcu‐1 knockdown in the intestine (Figure 4b, Table 1). Since muscle function is strongly relying on mitochondrial function (Gan et al. 2018; Wu et al. 2023) and NRF2 activation has been linked to increased mitochondrial biogenesis (Kraft et al. 2006), we also investigated mitochondrial structure by confocal microscopy throughout aging and in response to mcu‐1 RNAi in the newly developed worm strain MIR151 expressing mCherry under the sur‐5 promotor in the mitochondrial matrix (Figure 4c). Mitochondrial volume (Figure 4d) and surface area (Figure S3) declined, while volume‐weighted compactness (Figure 4e) and volume‐weighted sphericity (Figure S3) increased during aging, indicating a shift toward smaller, more spherical mitochondria consistent with progressive mitochondrial fragmentation during aging. Knockdown of mcu‐1 mitigated these age‐related alterations, restoring normal mitochondrial morphology by day 21 (Figures 4d,e and S3). Notably, treatment with mcu‐1 RNAi mitigated these age‐associated structural changes, maintaining larger and more interconnected mitochondria at both day 14 and day 21 (Figures 4c and S3). Functionally, mcu‐1 RNAi‐treated N2 worms showed an improved NAD⁺/NADH ratio at day 21 (Figure 4f). Moreover, mcu‐1 RNAi caused an increase in basal oxygen consumption rate (OCR) in N2 nematodes at day 21, counteracting the age‐related decrease in OCR (Figure 4g).

These data suggest that reduced mitochondrial Ca²⁺ uptake not only preserves mitochondrial structure but also enhances mitochondrial function in late adulthood.

Given the FDA's approval for the use of the MCU inhibitor mitoxantrone in humans (Fox 2004), we used this compound to test whether a low dosage of mitoxantrone could replicate the effects of mcu‐1 RNAi. Treatment with 10 nM mitoxantrone reduced lifespan during middle age significantly (days 12–14) but extended the lifespan of surviving N2 worms (Figure 5a, Table 1) and improved motility in aged nematodes at day 21 (Figure 5b, Table 1), similar to the effects observed with mcu‐1 RNAi. Like mcu‐1 RNAi, mitoxantrone significantly extended the N2 nematodes' lifespan when administered until day 14 (Figure 5c, Table 1) but was ineffective when applied after day 14 (Figure 5d, Table 1). As for mcu‐1 knockdown worms, confocal microscopy of the MIR151 strain treated with dimethyl sulfoxide (DMSO) or mitoxantrone revealed that mitoxantrone increased mitochondrial volume (Figure 5f) and surface area (Figure S4), while decreasing volume‐weighted compactness (Figure 5g) and volume‐weighted sphericity (Figure S4) on day 21, indicating a shift toward more elongated and less spherical mitochondrial morphology.

These findings indicate that the FDA‐approved drug mitoxantrone, at low doses, may mimic the effects of mcu‐1 RNAi.

To assess the translatability of our findings from nematodes to mammalian cells, we measured organellar Ca²⁺ and H₂O₂ levels in HFF‐1 cells following short‐term (3 h) treatment with a low dose [100 nM] of mitoxantrone. Live‐cell imaging of HFF‐1 cells expressing the mitochondrial Ca²⁺ sensor mtD1GO (Figure 6a) demonstrated significantly reduced basal mitochondrial Ca²⁺ levels (Figure 6b) and decreased mitochondrial Ca²⁺ uptake in response to 100 μM histamine (Figure 6c) after 3 h of mitoxantrone treatment. As in nematodes, reduced mitochondrial Ca²⁺ uptake in HFF‐1 cells was accompanied by a significant reduction in the activity of complex I, II, and III, as well as of complex IV (Figures 6d and S5). Live‐cell imaging utilizing the mitochondrial Hyper7 sensor also revealed increased mitochondrial H₂O₂ levels in HFF‐1 cells after 3 h of mitoxantrone [100 nM] treatment (Figure 6e). Similar to findings in nematodes, mitoxantrone treatment induced a transient decline in cell survival, with significantly reduced viability observed 24 h after the 3 h treatment with 100 nM mitoxantrone (Figure 6f). Proliferation analyses revealed that although many cells underwent sustained growth arrest due to mitoxantrone treatment, a subset continued to divide (Figures 6g and S5). This heterogeneous response may reflect a limited adaptive capacity that enables survival and partial regrowth within the more resistant cell fraction. As a mitohormetic response is typically characterized by enhanced antioxidant defenses following transient stress, we next assessed the expression and activity of key antioxidant enzymes after exposure to mitoxantrone. Notably, mRNA levels of SOD2 (Figure 6h), SOD1 (Figure 6i), and CAT (Figure 6j) were significantly elevated at 24 h and increased further at 48 h posttreatment. Consistently, the enzymatic activity of SODs was significantly elevated only at 48 h (Figure 6k), whereas CAT activity increased as early as 24 h and remained elevated at 48 h (Figure 6l).

These findings indicate that impaired mitochondrial Ca²⁺ uptake triggers a transient rise in ROS levels, followed by an adaptive response characterized by upregulation of the cellular antioxidant defense system. Consequently, targeting mitochondrial Ca²⁺ uptake may represent a promising therapeutic strategy to enhance health and lifespan.

C. elegans strains were obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota, US). The strains used in the present study involved N2 wild type (Bristol), KU25 (pmk‐1/km25), GA184 (sod‐2/gk257), CF1038 (daf‐16/mu86), EU31 (skn‐1/zu135), Mir248 (aak‐2/ok524), Mir14 (sir‐2.1/ok434), JV1 (jrIs1 [rpl‐17p::HyPer + unc‐119(+)] stable transgene expressing YFP‐based hydrogen peroxide sensor HyPer under ribosomal promotor) (Back et al. 2012), VP303 (kbIs7 [nhx‐2p::rde‐1 + rol‐6(su1006)]. Rollers. RNAi effective only in intestine), WM118 (neIs9 [myo‐3::HA::rde‐1 + rol‐6(su1006)] X. Transgene rescues muscle RNAi defect. Rollers.), and JV2 (jrIs2 [rpl‐17p::Grx1‐roGFP2 + unc‐119(+)] stable transgene ubiquitously expressing the roGFP2 sensor under a ribosomal promoter for in vivo estimation of GSSG/GSH ratios) (Back et al. 2012).

MIR151 (risIs10[sur‐5p::tomm20(AA1‐55)::mCherry + unc119(+)]) expressing mitochondrial mCherry sensor was generated by microparticle bombardment of strain HT1593 (unc‐119(ed3) III) with a suitable expression vector (Grigolon et al. 2022). Briefly, the expression vector was generated by Gateway cloning using pENTRY‐sur‐5p, pENTRY‐tomm20(AA1‐55)::mCherry, and pDEST‐ZK001 (modified pDEST‐MB14, lacking GFP).

AQ3055 expressing mitochondrial‐targeted YC3.60 sensor as an extrachromosomal array on the pharynx under the myo‐2 promoter (pmyo‐2::2mt8::YC3.60) was a kind gift from Javier Alvarez (Alvarez‐Illera et al. 2017).

Nematodes were grown at 20°C on agar plates with nematode growth media (NGM) with high gel strength agar (SERVA, Germany), which was spotted with Escherichia coli (E. coli), using the OP50 strain to maintain the strains as described previously (Brenner 1974; Schulz et al. 2007).

Nematodes were treated from day 4 on, after development. RNAi‐induced knockdown was performed as described (Oh et al. 2006). The HT115(DE3) E. coli strain transformed with the L4440 vector (empty vector) was used as a control, and the K02B2.3 vector with RNAi targeting mcu‐1 was used for RNAi treatment (Kamath and Ahringer 2003). Plates were supplemented with 1 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) (Roth, Austria) and ampicillin (100 mg/mL) (Roth, Austria) unless stated otherwise. The RNAi feeding protocol began on day 4 to avoid disrupting developmental processes (De‐Souza et al. 2019).

Compound treatment of nematodes was carried out on NGM agar plates containing mitoxantrone (10 nM) and control plates with the corresponding solvent, 0.001% DMSO (Roth, Austria) (Schmeisser et al. 2011). The antioxidants mitoTEMPO and NAC (Merck KGaA, Germany) were applied at a final concentrations of 100 nM and 1 mM, respectively (Zarse et al. 2012).

To obtain a synchronized nematode population, worms were allowed to lay eggs for 5 days at 20 C on agar plates and then removed with S‐buffer. Eggs were subsequently transferred to fresh NGM plates. After 4 days, 50 L4‐stage worms were manually placed on fresh NGM plates (50 worms/plate, three replicates per condition). Worms were transferred daily to new plates, and survival was monitored by counting live and dead animals. Assays were conducted at 20°C. When testing different strains, N2 worms served as controls. Statistical significance was assessed using the log‐rank test (Schulz et al. 2007).

The motility of the worms was measured on days 7, 14, and 21 using a wMicrotracker device (NemaMetrix, US) (Statzer et al. 2022). For each condition, at least three wells of a 96‐well plate were prepared, each containing 15 worms suspended in 90 μL of S‐buffer. Motility was recorded for 2 h at 20°C using the wMicrotracker software. The results were expressed as the percentage of body bends per minute over a 1 h measurement, normalized to control worms on day 7.

Total RNA from C. elegans was extracted via S‐buffer washing, 3–4 freeze–thaw cycles (liquid nitrogen/37°C), and TRIzol (1 mL; Thermo Fisher) treatment. After adding 200 μL chloroform, samples were incubated (3 min) and centrifuged (120,000 rpm); the aqueous phase was collected, RNA precipitated with 500 μL isopropanol, centrifuged (10 min, 4°C), and the pellet resuspended in 25 μL RNase‐free water (Rio et al. 2010). RNA concentration was determined with a UviLine 9400 spectrophotometer (Schott). For cDNA synthesis, 1 μg RNA was reverse‐transcribed using the Applied Biosystems kit (Thermo Fisher) per the manufacturer's protocol. mRNA levels were measured via qRT‐PCR (QuantiFast SYBR Green; Qiagen). Act‐1 and HPRT1 (Qiagen, Cat No. QT0059066) were used as reference genes for nematode and human samples, respectively. Primers (see Table 2) were obtained from Invitrogen (Austria). qRT‐PCR was performed on a LightCycler 480 (Roche), and gene expression normalized to housekeeping genes and shown as relative expression.

H₂O₂ levels were determined by the Amplex Red assay kit (Thermo Fisher Scientific, Austria), as described (Thomas et al. 2023). Synchronized and appropriately treated nematodes were incubated with 100 μM Amplex Red (Invitrogen, Carlsbad, USA) and 0.2 U/mL horseradish peroxidase in a sodium‐phosphate buffer for 3 h. Fluorescence was then measured using a CLARIOstar microplate reader (BMG Labtech, Germany). The results were normalized to protein content using the Pierce BCA protein assay (Thermo Fisher Scientific, Austria).

Synchronized nematodes were diluted in M9 buffer, anesthetized with 25 μM tetramisole hydrochloride, and plated in Seahorse XF24 culture plates with approximately 40 worms per well before measuring basal OCR as described (Thomas et al. 2023).

Nematodes were anesthetized with 5 mM levamisole in S‐buffer on empty NGM plates, then immobilized on 0.05 μm Polybead‐coated slides under a 5% agar pad. Imaging was performed on a Nikon Eclipse Ti2 microscope with a 100×/1.45 NA oil objective, standard filters, and dual Kinetix CMOS cameras (PhotoMetrics, US). Mitochondrial imaging used the MIR151 strain (mitochondrial mCherry; HT1593 background). Excitation was at 435 nm (Celesta Light Engine; Lumencor, US); emission was collected at 501–521 nm. Images (2 × 2 binning, 500 ms exposure, 0.2 μm Z‐steps) were acquired at days 7, 14, and 21 of adulthood (10 worms/condition, 5 replicates). Images were blind deconvoluted, and mitochondrial morphology was analyzed with a custom ImageJ macro. Background subtraction (rolling ball) and global/local Otsu thresholding enabled segmentation. Subsequently, the 3D ImageJ Suite plugins (Ollion et al. 2013) were used to assess mitochondrial volume, surface area, compactness, and sphericity (Gottschalk et al. 2019).

To determine GSSG/GSH ratios, the JV2 nematode strain (jrIs2[Prpl‐17p::Grx1‐roGFP2+ unc‐119(+)]) expressing the Grx1‐roGFP2 biosensor under a ribosomal promoter was used. Measurements were performed using the Nikon Eclipse Ti2 microscope with the same optical setup. Excitation was provided by 400 nm and 475 nm laser light (Celesta, Light Engine; Lumencor, US), and emission was collected at 501–521 nm. Fluorescence signals were background‐subtracted using a defined region of interest (ROI), corrected for photobleaching via exponential decay fitting, and presented as F₄₇₅/F₄₀₀ (Tawfik et al. 2022).

To image the mitochondrial Ca²⁺ sensor YC3.60, the AQ3055 strain was analyzed using an inverted Olympus IX73 microscope (Olympus, Austria) equipped with a UPlanSApo 40×/1.25 silicon objective (Olympus, Austria), a CCD QImaging Retiga R1 camera (PhotoMetrics, US), and a LedHUB illumination system (Omicron‐Laserage Laserprodukte, Rodgau‐Dudenhof, Germany) with a 455/505 nm LED and a CFP/YFP filter set (Olympus, Austria). For FRET measurements, emission was split into two channels using a dual‐channel beam splitter DV2 (PhotoMetrics, US) and a CFP/YFP beamsplitter (505DCXR, Chroma Technologies, Bellows Falls, VT, USA). Basal mitochondrial Ca²⁺ levels were measured in S‐buffer for 2 min, followed by mitochondrial Ca²⁺ uptake assessment upon caffeine [15 mM] application. Data were background‐subtracted using a defined region of interest, corrected for photobleaching using an exponential decay fit, and presented as F₅₃₅/_F480.

Cytosolic H₂O₂ levels were measured in the JV1 strain (jrIs1 [rpl‐17p::HyPer + unc‐119(+)]) on day 14. Imaging was performed using the same Olympus IX73 microscope setup with a UPlanSApo 40×/1.25 silicon objective, CCD QImaging Retiga R1 camera, and a LedHUB illumination system equipped with a 455/505 nm LED and a CFP/YFP filter set. Excitation was achieved with either 455 nm or 505 nm, and emission was collected at 501–521 nm. Background subtraction was applied using a defined ROI, and bleaching correction was performed using an exponential decay fit. H₂O₂ measurement results are expressed as F₅₀₅/F₄₅₅ (Brugger et al. 2023).

Human diploid fibroblasts (HFF‐1; ATCC SCRC‐1041) were cultured in DMEM (D5546; Sigma, Germany) with 10% heat‐inactivated FBS, 4 mM L‐glutamine, and 1% penicillin–streptomycin (Thermo Fisher, Austria) at 37°C, 5% CO₂. Cells (passages 2–15) were passaged using 0.05% trypsin–EDTA, centrifuged (1500 rpm), counted in a Neubauer chamber, and seeded at the desired density (Cavinato et al. 2021). Mitoxantrone dihydrochloride (Merck, Germany) was prepared as a 10 mM DMSO stock. Cells were treated with 100 nM mitoxantrone [0.001% DMSO] or DMSO control for 3 h before measurements.

Cell viability was measured with the fluorometric CellTiter‐Blue Cell Viability Assay (Promega, Austria), which is based on the resazurin reduction method (Chen et al. 2018). HFF‐1 cells were seeded at a density of 3200 cells per well in flat‐bottom 96‐well plates. The cells were treated for 3 h with 100 nM mitoxantrone or DMSO as a control. Afterwards, the cells were washed two times with phosphate‐buffered saline (PBS), and 100 μL fresh media was added to each well. For the assay, 20 μL of CellTiter‐Blue reagent was added to four technical repeats of four different passages of either condition, 24 h or 48 h postmitoxantrone incubation. Following a 30‐min incubation at 37°C, fluorescence (excitation at 560 nm/emission at 590 nm) was measured, and cell viability was normalized to the respective control.

Cell proliferation of fibroblasts was monitored using the CellCyte X—Live Cell Imager and Analyzer (Cytena GmbH, Germany) (Kalinova et al. 2025). HFF‐1 were seeded at a density of 40,000 cells per well in 12‐well plates. The cells were treated for 3 h with 100 nM mitoxantrone or DMSO as a control. Afterwards, the cells were washed twice with PBS, and 2 mL of fresh media was added to each well. Subsequently, the plates were placed into the CellCyte X—Live Cell Imager and Analyzer within an incubator (37°C, 5% CO₂). Enhanced contour pictures of the fibroblasts were taken every 6 h, and the confluency was analyzed with the CellCyte Studio Software (Cytena GmbH, Germany).

For live‐cell imaging, cells were seeded on 30 mm glass coverslips in 6‐well plates. Transfection with 0.5 μg mitoHyPer7 plasmid (Pak et al. 2020) and 3 μL Polyjet (SignaGen, US) in 1 mL serum−/antibiotic‐free medium was performed 2 days before imaging. To normalize maximal mitochondrial H₂O₂ levels, 150 μM H₂O₂ was added at the end of each measurement. Imaging was performed on a Nikon Eclipse Ti2 microscope (Nikon, Austria) with a 100×/1.45 NA oil objective, standard filters, and two Kinetix CMOS cameras (PhotoMetrics, US). Excitation was at 400 nm or 475 nm (Celesta Light Engine; Lumencor, US), and emission was collected at 501–521 nm. Data were background‐subtracted using a ROI and bleaching‐corrected via exponential decay fitting. H₂O₂ levels are shown as F₄₇₅/F₄₀₀ (Tawfik et al. 2022).

To assess mitochondrial Ca²⁺ levels, HFF‐1 were transfected via viral transduction with the FRET‐based mitochondrial Ca²⁺ sensor mtD1GO‐Cam (Waldeck‐Weiermair et al. 2012). After 2 days, the basal Ca²⁺ level and the response upon histamine stimulation were assessed on an inverted wide‐field microscope (Observer.A1; Carl Zeiss GmbH, Austria) as described previously (Waldeck‐Weiermair et al. 2012). Emission was collected with a 505dcxr beamsplitter on two sides of the camera (CCD camera, Coolsnap Dyno; PhotoMetrics, US). The biosensor mtD1Go‐Cam was excited with a wavelength of 477 nm (440AF21; Omega Optical, US), and emission was captured at 560 and 510 nm (480AF30 and 535AF26; Omega Optical, US). Results are shown as F₅₆₀/_F510.

HFF‐1 cells (100,000/well) were seeded in 6‐well plates and treated for 3 h with 100 nM mitoxantrone or DMSO (control). After two PBS washes, 2 mL fresh medium was added. At 24 h and 48 h posttreatment, cells were washed with ice‐cold PBS, scraped, and centrifuged (4 C, 2000 rpm, 8 min). Pellets were resuspended in 150 μL cold lysis buffer (50 mM potassium phosphate, 0.1 mM EDTA, 0.5% Triton X‐100), subjected to two freeze–thaw cycles, and centrifuged (12,000 rcf, 5 min, 4°C). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher). For SOD (MAK528) and catalase (219265) activity assays (Sigma‐Aldrich, Austria), 6 μg protein was used per reaction. Activities were measured according to the manufacturer's instructions, calculated via standard curves, and normalized to controls.

Nematodes were harvested from S‐buffer liquid culture on day 7, following treatment from day 4 with 25 μM 5‐fluorodeoxyuridine, 1 mM IPTG, and 100 μg/mL ampicillin (Roth, Austria), and the respective bacteria.

Worm pellets were resuspended in 5 mL IMBc buffer (200 mM sucrose, 10 mM Tris/MOPS, 10 mM EGTA/Tris, pH 7.4) and homogenized by passing them six times through an Isobiotec cell homogenizer (12 μm clearance; Isobiotec, Germany) using a 5 mL syringe. After centrifugation at 600 × g (15 min, 4°C) to remove debris, the supernatant was spun twice at 9000 × g (15 min, 4°C). The resulting mitochondrial pellet was resuspended in 100 μL IMBc for PDH assays or 300 μL MIR05 for respirometry (Sladowska et al. 2021).

HFF‐1 mitochondria were isolated in a similar manner. Cells were detached with 0.05% trypsin–EDTA, resuspended in 1 mL IMBc, and homogenized using the Isobiotec cell homogenizer with 6 μm clearance.

A commercially available activity kit (ab109902; Abcam, US) was used to determine the PDH activity of isolated mitochondria from nematode samples. PDH was immunocaptured within the wells of a microplate, and the activity was determined by the reduction of NAD⁺ to NADH + H⁺, coupled to a reporter dye, which was measured at an absorbance of 450 nm. The protein concentration was determined by a BCA protein assay. Each sample was adjusted to 100 μg of protein per well. The assay is reflecting in a kinetic mode for 1 h. The rate (ΔmOD/min) was then calculated by using the following formula: Rate (mOD/min) = (Absorbance 2‐ Absorbance 1) / time (min) (Shimada et al. 2022).

Isolated mitochondria (50–100 μg) from nematodes and HFF‐1 cells were resuspended in 300 μL MIR05 respiration medium and added to an Oxygraph‐2 k respirometer chamber (Oroboros, Austria), prefilled with MIR05 at 20°C. Respiration was assessed using the SUIT‐008 protocol. Complex I activity was stimulated with 5 mM pyruvate, 2 mM malate, 2.5 mM ADP, and Mg²⁺. Membrane integrity was tested with 10 μM cytochrome c. Complex II was assessed after 0.05 μM rotenone inhibition using 10 mM glutamate and 10 mM succinate. Maximal respiration was induced with 0.5 μM CCCP. Complex IV activity was analyzed after inhibiting complexes I and III (0.5 μM rotenone, 2.5 μM antimycin A), using 2 mM ascorbate and 0.5 mM tetramethyl phenylenediamine (TMPD), followed by inhibition with 50 mM NaN₃. O₂ flux was normalized to protein content and analyzed using DatLab 7 (Oroboros) (Sladowska et al. 2021).

Data were processed in Excel 2010 and analyzed using GraphPad Prism 9.3.1. Results are shown as mean ± SEM from at least three independent experiments. Two‐tailed Student's t‐tests were used for pairwise comparisons and one‐way ANOVA for multiple groups. For comparing significant distributions between different groups in the lifespan assays, statistical calculations were carried out using the log‐rank test. Nematode sample sizes are listed in Table 1. Cell sample sizes from live‐cell imaging are noted in the respective Figure's legend, providing the number of total cells measured and the number of independent experiments. Significance was set at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.