Intracellular calcium (Ca²⁺) homeostasis is crucial for various cellular functions, including survival, growth, and apoptosis. Annexin A5 (AnxA5), a Ca²⁺-dependent phospholipid-binding protein, is involved in regulating intracellular Ca²⁺ signaling and apoptosis. AnxA5's ability to facilitate Ca²⁺ influx has been demonstrated in various systems, including large unilamellar vesicles and liposomes, with its function dependent on lipid composition. In mitochondria, AnxA5 binds to cardiolipin and influences mitochondria-mediated apoptosis; AnxA5 depletion enhances resistance to Ca²⁺-induced apoptosis. Voltage-Dependent Anion Channel 1 (VDAC1), a Ca²⁺-permeable channel in the outer mitochondrial membrane (OMM), plays a critical role in Ca²⁺ influx into mitochondria upon inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release from the endoplasmic reticulum (ER). VDAC1's Ca²⁺ permeability is modulated by proteins such as α-synuclein and Bcl-xL. Within the intermembrane space (IMS), Ca²⁺ must exceed a threshold to cross the inner mitochondrial membrane (IMM) via the mitochondrial Ca²⁺ uniporter complex, counteracted by the mitochondrial Na⁺/Ca²⁺ exchanger. This study hypothesized that AnxA5 modulates mitochondrial functions by influencing mitochondrial Ca²⁺ signaling, particularly the trans-OMM Ca²⁺ fluxes and IMS Ca²⁺ signaling upon ER Ca²⁺ release, potentially via interaction with VDAC1.

Existing literature established AnxA5's role in intracellular Ca²⁺ signaling and apoptosis, but its precise function in mitochondrial Ca²⁺ homeostasis remained unclear. Previous research showed AnxA5's Ca²⁺-dependent binding to cardiolipin in mitochondria and its impact on apoptosis. AnxA5 depletion increased resistance to Ca²⁺-induced apoptosis, suggesting its involvement in apoptosis-inducing pathways. Studies also highlighted VDAC1's role as a Ca²⁺-permeable channel, whose permeability is influenced by other proteins. The interplay between ER Ca²⁺ release, VDAC1, and mitochondrial Ca²⁺ uptake via the uniporter complex, and efflux via the Na⁺/Ca²⁺ exchanger, has been extensively characterized. However, a direct link between AnxA5 and mitochondrial Ca²⁺ handling, particularly its interaction with VDAC1, was lacking prior to this study.

This study used a multi-faceted approach to investigate AnxA5's role in mitochondrial Ca²⁺ homeostasis. AnxA5 knockout (AnxA5-KO) HeLa cells and human umbilical vein-derived endothelial cells (EA.hy926) were generated using the CRISPR/Cas9 system. Mitochondrial matrix Ca²⁺ levels ([Ca²⁺]_Matrix), cytosolic Ca²⁺ levels ([Ca²⁺]_Cyto), and ER Ca²⁺ levels ([Ca²⁺]_ER) were measured using genetically encoded Ca²⁺ sensors (4mtD3cpv, Fura2-AM, and a genetically encoded ER-targeted Ca²⁺ sensor, respectively). IMS Ca²⁺ levels ([Ca²⁺]_IMS) were monitored using the MICU1-140-GEMGECO1 sensor. Mitochondrial membrane potential (Ψ_mito) was assessed using TMRM. The expression levels of proteins involved in mitochondrial Ca²⁺ uptake (VDAC1, MICU1, MICU2, UCP2, MCU, EMRE) were analyzed using immunoblotting. ER-mitochondria contact sites (MERCs) were examined using 3D-confocal microscopy and SPLICS technology. Mitochondrial ultrastructure was analyzed using transmission electron microscopy (TEM). AnxA5 localization was determined using subcellular fractionation, proteinase K treatment, and immunogold labeling with TEM. Proximity ligation assays (PLA) were used to assess the proximity of AnxA5 and VDAC1. Single-channel currents in isolated mitochondria were measured using patch-clamp techniques. The effect of cisplatin and selenite on mitochondrial Ca²⁺ levels, cell viability, and apoptosis was assessed using flow cytometry and live-cell imaging. VDAC1 oligomerization was examined using cross-linking and immunoblotting. Statistical analyses included one-way ANOVA with Tukey's multiple comparison test, Student's t-test, and Kruskal-Wallis test.

The study found that AnxA5-KO cells exhibited significantly reduced mitochondrial Ca²⁺ elevation upon IP₃-induced ER Ca²⁺ release, without affecting cytosolic or ER Ca²⁺ signaling. This effect was not due to changes in mitochondrial membrane potential, expression of Ca²⁺ uniporter components, or MERC integrity. AnxA5-KO cells showed increased mitochondrial volume and branching but unchanged cristae density. Immunogold labeling revealed AnxA5 localization in the OMM and within mitochondria, with accumulation at the OMM upon ER Ca²⁺ release. PLA showed AnxA5's proximity to VDAC1, indicating functional interaction rather than direct binding. AnxA5-KO cells showed reduced IP₃-mediated Ca²⁺ transfer from the ER into the IMS, and this reduction occurred across various histamine concentrations. The Ca²⁺ binding of AnxA5, but not its self-assembly, was critical for regulating IMS Ca²⁺ signaling. AnxA5-mediated IMS Ca²⁺ signaling influenced MICU1 dimerization and cristae membrane dynamics in MERCs. Patch-clamp experiments revealed a 35 pS Ca²⁺-permeable channel in the OMM, whose occurrence and open probability were reduced in AnxA5-KO mitochondria and rescued by recombinant AnxA5. AnxA5-KO cells showed increased susceptibility to cisplatin-induced apoptosis due to enhanced cisplatin-induced VDAC1 dimerization; similar results were observed with selenite. Treatment with VBIT-4, a VDAC1 oligomerization inhibitor, reduced cell death and VDAC1 dimerization in both WT and AnxA5-KO cells. VDAC2, but not VDAC3, could rescue the reduced mitochondrial Ca²⁺ signaling in VDAC1-depleted cells, and this rescue was AnxA5-dependent.

This study demonstrates AnxA5's crucial role in regulating mitochondrial Ca²⁺ homeostasis by modulating VDAC1's Ca²⁺ permeability. AnxA5's proximity to VDAC1, revealed by PLA, suggests a regulatory mechanism rather than a direct physical interaction. The Ca²⁺-dependent binding of AnxA5 to negatively charged phospholipids in the OMM likely stabilizes VDAC1's monomeric, Ca²⁺-permeable state. The isoform-specific rescue of mitochondrial Ca²⁺ signaling by VDAC2 highlights the importance of the microenvironment created by AnxA5 and specific VDAC isoforms. The increased susceptibility of AnxA5-KO cells to cisplatin and selenite-induced apoptosis, driven by enhanced VDAC1 dimerization, confirms AnxA5's protective role. The observed differences in Ca²⁺ conductance in intact mitochondria versus artificial bilayers may reflect the complexity of the OMM environment. Overall, this study highlights AnxA5's function as a critical regulator of VDAC1-dependent mitochondrial Ca²⁺ homeostasis, affecting not only Ca²⁺ transport, but also mitochondrial structure and cell survival.

This study identified a novel role for AnxA5 as a key regulator of VDAC1-dependent mitochondrial calcium signaling. AnxA5, by localizing near VDAC1, modulates its calcium permeability, impacting IMS calcium levels, mitochondrial architecture, and ultimately cellular susceptibility to apoptosis induced by agents like cisplatin and selenite. Future research could investigate the specific phospholipids AnxA5 interacts with in the OMM and explore the therapeutic potential of targeting the AnxA5-VDAC1 interaction to modulate apoptotic pathways.

The study primarily focused on HeLa cells and EA.hy926 cells. While findings were validated in perivascular cells from AnxA5-KO mice, further investigation in other cell types and organisms is needed to confirm the generalizability of the results. The modest knockdown efficiency of VDAC1 in some experiments may have limited the interpretation of specific results. The exact mechanism by which AnxA5 modulates VDAC1's Ca²⁺ permeability remains to be fully elucidated.