Calcium (Ca2+) fluxes at mitochondria-ER contact sites (MERCS) are a new target of senolysis in therapy-induced senescence (TIS)

Reagents

From Thermo Fisher Scientific (Waltham, Massachusetts, USA): Dulbecco´s modified Eagle medium-high glucose (DMEM-HG) (12100-046), DMEM-HG no glutamine and calcium (21068028), trypsin (0.25%)-EDTA phenol red (25200072), antibiotic-antimycotic (100×) (15240062), trypan Blue solution 0.4% (15250061), Opti-MEM™ GlutaMAX™ supplemented (51985034), DMEM FluoroBrite™ (A1896701), Fluo-4/AM (F-14201), Rhod2/AM (R-1245MP), Lipofectamine™ 3000 (L3000015), Lipofectamine™ RNAiMAX (13778150), TMRE (T669), Hoechst 33342 (H3570), Fluoromount-G mounting medium (00-4958-02), Pierce™ Bradford plus protein assay (23236), SuperSignal™ West Pico PLUS chemiluminescent substrate (34580), PageRuler™ Plus prestained protein ladder (26619), Image-iT™ fixative solution (FB002), X-Gal (B-1690). From Cytiva (Chicago, Illinois, USA): Fetal bovine serum (HC.SV30160.03), Amersham Hybond P 0.45 PVDF blotting membrane (10600023). From Sigma-Aldrich (St. Louis, Missouri, USA): Histamine dihydrochloride (H7250-5G), PLA Duolink® (DUO92014-100RXN), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (C2920), protease Inhibitor Cocktail (P2714), PhosSTOP™ (4906837001). From Tocris Bioscience (Bristol, United Kingdom): From Merck (Darmstadt, Alemania): Acrylamide bis-acrylamide 29:1, 40% solution (1690-OP), triton® X-100 (112298), methanol (106009), 10× PBS (6505-OP), dimethyl sulfoxide (317275), hydrochloric acid fuming 37% (100317), sodium hydroxide (106498), TWEEN® 20 (655205), RIPA buffer (20–188). From Santa Cruz Biotechnology (Dallas, Texas, EE. UU.): BSA (sc-2323), Z-VAD-FMK (Z-VAD) (sc-3067). From Winkler (Santiago, RM, Chile): TRIS buffer (BM-0585), glycerol (BM-0800), 2-mercaptoethanol (BM-1200), SDS (BM-1750), bromophenol blue (AZ-0395), glycine (BM-0820), methanol (AL-0210), glutaraldehyde (WK- 106), NaCl (SO-1455), KCl (PO-1260), Tween-20 (016520).

Plasmids

From Addgene (Watertown, MA, USA.): pCAG mito-GCaMP5G (105009), CMV-mito-GEM-GECO1 (32461), BFP-KDEL (49150).

Antibodies

From Cell Signaling Technology (Danvers, MA, USA): anti-IP₃R1 (8568), anti MCU (14997), anti-p21 Waf1/Cip1 (12D1) (2947), anti-MCU (D2Z3B), anti-MICU1 (D4P8Q). GRP75 (D13H4) (3593S), PDI (C81H6) (3501S). From Abcam (Cambridge, UK): anti-VDAC1 (AB10527), anti-VDAC1 (AB14734), anti-TOMM20 (AB56783), anti-cleaved caspasa-3 (AB32042), p16: CDKN2A/P16INK4a (ab108349). From Becton & Dickinson (Franklin Lakes, NJ, USA.): anti IP₃R3 (610313). From Thermo Fisher Scientific (Waltham, Massachusetts, USA): Anti-IP₃R2 (PA1-904), anti β-tubulin (32–2600), anti-mouse secondary antibody Alexa Fluor™ 350 (A11045), anti-rabbit secondary antibody Alexa Fluor™ 488 (A11008), anti-mouse secondary antibody Alexa Fluor™ 488 (A11001), anti-rabbit secondary antibody Alexa Fluor™ 568 (A11011), anti-mouse secondary antibody Alexa Fluor™ 568 (A11004), anti-rabbit secondary antibody Alexa Fluor™ 647 (A21244), anti-mouse secondary antibody Alexa Fluor™ 647 (A21235), anti-rabbit secondary antibody HRP (31460), anti-mouse secondary antibody HRP (31430). From Santa Cruz Biotechnology (Dallas, Texas, EE. UU.): anti-β-actin (sc-47778), anti-HSP90 (13119sc). From Millipore (Burlington, Massachusetts, EE. UU): Anti-phospho-Histone H2A.X (Ser139) (05-636).

Cell culture and treatment

IMR90 (ATCC® CCL186™) cell line was maintained in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum (FBS; Biological Industries), 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 µg/ml amphotericin B (antibiotic-antimycotic solution), at 37 °C, 10% CO₂ and 3% O₂. To induce senescence experiments, cells were incubated with 250 nm Doxorubicin or 10 µM Etoposide for 48 h, and the culture medium was refreshed every 2 days. All cell counts were performed manually by using ImageJ and performed in at least three independent replicates. From each replicate, 100 cells were counted. All experiments, both control and cells at the start of doxorubicin treatments, were conducted using between 5000 and 10,000 cells per cm².

Detection of SA-β-Gal

SA-β-Gal activity was evaluated as previously described in ref. ¹⁸.

Multiplex Luminex assay of cytokines

Serum-free media from 24 h were collected and centrifuged at 1000 × g for 10 minutes to eliminate cell debris. The conditioned media were stored at −80 °C until use. A Human Luminex Discovery Assay of IL1α, Ilβ, IL6, CXCL1 and MMP3 available in the kit were performed using the Customization Tool. Serial dilution techniques were used to quantify the concentrations of cytokines, following the manufacturers’ recommendations using Luminex MAGPIX Analyzer, and the results were expressed as pg/mL.

Live imaging

For ER labeling, 1 μg of ER-BFP expression vector (mBFP fused to KDEL) was transfected in transfection medium and Lipofectamine 3000, according to the manufacturer’s instructions. For mitochondrial labeling, TMRE was used at a concentration of 10 nM, incubated for 20 min at RT (room temperature). Images were acquired using a TCS SP8 confocal microscope (Leica, Wetzlar, Germany) and were taken in different channels, then merged to evaluate the points of greatest proximity between ER-Mitochondria and their respective correlation coefficients known as Manders’ coefficients. Finally, they were processed and analyzed using ImageJ software (NIH Image).

Transmission electron microscopy

Cells were fixed using a specialized fixative mixture for electron microscopy (3% glutaraldehyde, 0.05% picric acid, and 50 mM sodium cacodylate buffer) for 10 minutes. Subsequently, the cells were mechanically detached from the plate and centrifuged at 3000 rpm for 5 minutes. The resulting pellet was incubated overnight in the electron microscopy fixative solution.

The samples were then washed multiple times with sodium cacodylate buffer and post-fixed with 1% osmium tetroxide in cacodylate buffer for 2 hours. Dehydration was carried out using a graded ethanol series, progressing from lower to higher concentrations, and concluded with an acetone bath. The samples were then infiltrated with acetone and Epon resin in increasing concentrations, ultimately embedding the samples in concentrated Epon resin.

Block processing, ultrathin sectioning, grid mounting, and imaging were conducted at the laboratory of Dr. José Manuel Villalba, from the Department of Cell Biology, Physiology, and Immunology at the University of Córdoba, Spain.

Images of mitochondria were taken at ×60,000 magnification. From these images, those showing interactions with the endoplasmic reticulum (ER) at distances between 10 and 50 nm were selected. In these selected images, the distance between the reticular membrane and the outer mitochondrial membrane was measured, as well as the percentage of mitochondrial surface covered by the ER. Image analysis was performed using ImageJ software (NIH Image).

Plasmid transfection

Plasmid transfection was performed using Lipofectamine™ 3000 (Thermo Fisher Scientific) on cells plated in 6-well plate. Transfection mixture was prepared considering 2 µg/well of plasmid DNA, 5 µL P3000 and 10 µL Lipofectamine™ 3000 diluted in Opti-MEM. After 12 h the culture media were replaced with fresh medium and cells were incubated at 37 °C in a 5% CO₂ and 3% O₂ incubator. The experiments were performed 24–48 h post-transfection.

Cell viability

Viability assays was determined using LIVE/DEAD Cell Imaging Kit (R37601) according to manufacturer’s instructions. Images were obtained using confocal microscopy (TCS SP8 Spectral Confocal Microscope; Leica™) and subsequently processed and analyzed using ImageJ software (NIH).

Measurement of cytoplasmic and mitochondrial Ca²⁺ signals

Cells were grown on 18 mm Ø glass coverslips in 6-well plates and cytoplasmic and mitochondrial Ca²⁺ signals were evaluated by timelapse in confocal microscopy, using Nikon C²⁺ Confocal Microscope System (Nikon™), or Leica TCS SP8 Confocal Laser Scanning Microscope (Leica™) with control of temperature (37 °C), humidity and CO₂ (5%). Measurements were performed in calcium-free medium, using DMEM no calcium (Gibco™ #21068) supplemented with 100 µM EGTA.

Cytoplasmic Ca²⁺ signals

IP₃R activity was evaluated using the Ca²⁺ sensitive cytoplasmic probe Fluo-4/AM (Max. ex/em: 494/506 nm). IMR90 cells growth in coverslips were loaded with 5 µM Fluo-4/AM (30 min) in complete medium and mounted in round chamber for microscopy imaging. The cells were washed twice with calcium-free medium and imaged in presence of the corresponding compounds. After a period of baseline recording, the cells were stimulated with a pulse of 0,3 mM histamine to induce IP₃R-mediated Ca²⁺ release. A total of 80 to 100 frames were recorded every 1.5 seconds approximately, at 488 nm excitation and using a ×63 objective. Images were analyzed and quantified using ImageJ (NIH).

Mitochondrial Ca²⁺ signals

Mitochondrial Ca²⁺ uptake from IP₃R-mediated Ca²⁺ release after histamine stimuli was determined initially using the mitochondria-targeted genetically encoded fluorescent Ca²⁺ indicator mito-GCaMP5G t (K_d = 160 nM; Max. ex/em: 488/500 and 550 nm). For these experiments, cells were transfected with plasmid DNA (2 µg/well on 6-well plates) using lipofectamine 3000. After 24 h pos-transfection, cells were trypsinized, seeded on 25 mm Ø glass coverslips and finally cultured for 24 h. Confocal image recording was performed as described above, at 488 nm excitation laser. In addition, we used Rhod-2/AM (Max. ex/em: 552/581 nm) to measure mitochondrial Ca²⁺ signals, co-localized with the mitotracker green FM (Max. ex/em: 490/516 nm) labeling. For this approach, cells seeded on 25 mm Ø glass coverslips were loaded with 5 µM Rhod-2/AM and 100 nM mitotracker green FM (30 min) in complete medium after completion of the experimental treatments. For the confocal timelapse, cells were mounted in the microscope chambers, washed twice with calcium-free medium, and finally recorded before and after the stimulation with 0,3 mM histamine, using a ×63 objective, at 488 nm and 561 nm excitation lasers for mitotracker green FM and Rhod-2/AM respectively. In addition, we used an FCCP stimuli to uncouple mitochondrial membrane potential, after a period of baseline recording. This resulted in the release of mitochondrial Ca²⁺, which was measured in the cytoplasm using the Fluo-4/AM probe. IMR90 cells were loaded with 5 µM Fluo-4/AM for 30 minutes in complete medium, as previously described. A total of 140 frames were recorded every 1.5 seconds approximately. Images were analyzed and quantified using ImageJ (NIH).

Finally, basal mitochondrial Ca²⁺ levels were measured as described in ref. ¹⁹.

Protein extraction and quantification

Cells seeded in 6-well plates were cultured for 24 h and then subjected to corresponding experimental treatments. Once treatments were finished, cells were placed on ice, washed twice with ice-cold PBS, and finally scraped and lysed using CytoBuster™ protein extraction reagent supplemented with protease and phosphatase inhibitors. The obtained homogenates were sonicated, incubated 15 minutes on ice, centrifugated at 12,500 × rpm for 20 minutes at 4 °C, and the supernatant was collected. Protein quantification was performed using the Bradford method in 96-well plates, and OD₅₉₅ nm was determined using the Infinite 200 PRO plate reader (Tecan™).

SDS-PAGE and western blotting

Protein extracts were mixed with sample buffer 5× (250 mM Tris-HCl; 40% v/v glycerol; 8% v/v 2-mercaptoethanol; 10% w/v SDS; 0,5% w/v bromophenol blue, pH 6.8) and then boiled for 5 minutes at 100 °C. Once cooled, 30 µg of protein extract from each sample was separated electrophoretically in either 5%, 10% or 15% SDS-polyacrylamide gels using a Mini-PROTEAN Tetra Cell (Bio-Rad™) with running buffer containing: 25 mM Tris; 192 mM glycine; 0,1% w/v SDS at pH 8.3, and subsequently transferred to PDVF membranes by Trans-Blot™ SD Semi-Dry Transfer Cell (Bio-Rad ®). Membranes were subsequently blocked 60 minutes with 5% w/v bovine serum albumin (BSA) prepared in Tris buffered saline-tween (TBS-T) containing: 20 mM Tris-HCl, 150 mM NaCl and 0.05% w/v Tween 20, at pH 7.5. Corresponding primary antibodies (dilution 1:1000 or 1:3000) were incubated overnight at 4 °C, then washed with TBS-T and subsequently incubated with HRP-conjugated secondary antibody (dilution 1:2000) for 1 h at room temperature with shaking. Protein signal was visualized using SuperSignal™ West Pico PLUS chemiluminescent substrate and documented with ChemiDoc™ Imaging System (Bio-Rad). Images were analyzed and quantified using ImageJ (NIH).

PLA

For Immunofluorescence and PLA assays, 5000 cells per cm² were seeded on 12 mm Ø glass coverslips. Once the corresponding experimental treatments were completed, cells were fixed with 4% w/v paraformaldehyde (Image-iT™ fixative solution; Thermo Fisher Scientific), washed in PBS, then permeabilized in 0.1% v/v triton X-100 in PBS and blocked for 1 h in 10% w/v BSA in PBS at room temperature. After blocking, cells were incubated with the indicated antibodies); overnight at 4 °C followed by Duolink manufacturer’s instructions (Duolink, Sigma-Aldrich). Once incubation was completed, cells were washed 3 times in PBS and Hoechst 33342 was applied during the last wash. The coverslips were mounted on slides using the mounting medium Fluoromount-G™. Images were obtained using confocal microscopy (TCS SP8 Spectral Confocal Microscope; Leica™) and subsequently processed and analyzed using ImageJ software (NIH).

Mice and doxorubicin-induced in vivo senescence

This study was carried out following the strict recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Committee on the Ethics of Animal Experiments of Universidad Mayor (number:05/2020). Mice were provided standard chow ad libitum and maintained under a 12:12-hour light/dark cycle. Systemic cellular senescence was introduced by treating mice with doxorubicin, as described. Briefly, a single dose of doxorubicin (10 mg/kg) was injected intraperitoneally and/or ABT-263 inhibitor was administered by gavage. Organs (lung, kidney, and liver) were collected.

Fluorescence images of organs were captured by In-Vivo FX PRO (Bruker) imaging system. To obtain a white-black image, organs were exposed to the white light for 0.175 seconds and to obtain a fluorescent image, they were exposed to 530 nm excitation wavelength for 10 seconds, capturing the obtained fluorescence using a filter of 600 nm. Auto-fluorescence was corrected for each organ, respectively to the non-treated control auto-fluorescence. Subsequently, a merge of both images, white-black and fluorescence, was created, and a representative image was constructed. Graphics of fluorescence quantification were created using the obtained mean intensity by each organ n = 4.

Statistical analysis

Depending on the type of experiment, the results are shown as representative images or mean ± SEM of at least 3 independent experiments. Statistical analyses were performed using Prism 8 software (GraphPad software). Significance of differences was assessed using unpaired t-tests. In the case of data that exhibited a normal distribution, the data were analyzed using a T-Test and one-way ANOVA with comparisons among the different experimental groups using Sidak correction. Additionally, comparisons were made with the control group using Dunnett’s test correction. Differences with a p value < 0.05 were considered statistically significant.