Maintaining intracellular calcium (Ca²⁺) homeostasis is crucial for various cellular processes, including cell survival, growth, and apoptosis. Annexin A5 (AnxA5), a Ca²⁺-dependent phospholipid-binding protein, plays a role in regulating intracellular Ca²⁺ signaling and apoptosis. While AnxA5's cytosolic localization is well-established, its precise involvement in mitochondrial Ca²⁺ signaling remains unclear. Previous studies have shown AnxA5's ability to facilitate Ca²⁺ influx in vesicles and liposomes through Ca²⁺-dependent membrane insertion and its impact on mitochondria-mediated apoptosis. AnxA5 depletion increases resistance to Ca²⁺-induced apoptosis, suggesting its role in apoptotic pathways. VDAC1, a crucial protein in the outer mitochondrial membrane (OMM), acts as a Ca²⁺-permeable channel. Upon stimulation, inositol 1,4,5-trisphosphate (IP₃) triggers Ca²⁺ release from the endoplasmic reticulum (ER), and Ca²⁺ ions then traverse the OMM via VDAC1. VDAC1's Ca²⁺ permeability is influenced by proteins like α-synuclein and Bcl-xL. In the intermembrane space (IMS), Ca²⁺ concentrations need to reach a threshold to pass through the inner mitochondrial membrane (IMM) via the mitochondrial Ca²⁺ uniporter complex. This intricate system of Ca²⁺ handling is further balanced by mitochondrial Na⁺/Ca²⁺ exchanger for Ca²⁺ efflux. This study hypothesized that AnxA5 modulates mitochondrial function by influencing mitochondrial Ca²⁺ signaling, investigating AnxA5's role in intracellular Ca²⁺ signaling across various cellular and mitochondrial compartments using genetically encoded Ca²⁺ sensors and electrophysiology.

The existing literature supports a role for AnxA5 in intracellular Ca²⁺ homeostasis and apoptosis. Studies have shown AnxA5's Ca²⁺-dependent binding to negatively charged phospholipids, its influence on Ca²⁺ influx across the plasma membrane, and its localization within cardiolipin-rich regions of the mitochondrial membrane. Depletion of AnxA5 leads to increased resistance to various apoptosis-inducing agents, highlighting its involvement in cell death pathways. VDAC1's role as a Ca²⁺-permeable channel and its modulation by other proteins has been established. However, the interplay between AnxA5, VDAC1, and mitochondrial Ca²⁺ homeostasis remained poorly understood before this study. The research aimed to clarify AnxA5's precise role in mitochondrial Ca²⁺ regulation, particularly in the context of ER Ca²⁺ release and apoptosis.

This study employed a multi-faceted approach to investigate AnxA5's role in mitochondrial Ca²⁺ homeostasis. CRISPR/Cas9 technology was used to generate AnxA5 knockout (AnxA5-KO) HeLa cells and EA.hy926 cells, allowing for the comparison of mitochondrial Ca²⁺ dynamics between wild-type (WT) and AnxA5-KO cells. Genetically encoded Ca²⁺ sensors, specifically targeted to the mitochondrial matrix, IMS, and ER, were utilized to perform dynamic Ca²⁺ measurements in various cellular compartments. Electrophysiological techniques, including patch clamp of isolated mitochondria, provided direct assessment of Ca²⁺ currents across the OMM. Immunoblotting, immunogold labeling with transmission electron microscopy (TEM), and proximity ligation assays (PLA) were used to determine AnxA5's subcellular localization, particularly its proximity to VDAC1. Confocal and structured illumination microscopy (SIM) techniques allowed for the morphological analysis of mitochondria and the dynamics of cristae membranes. Functional studies included assessment of mitochondrial membrane potential, the expression of Ca²⁺ uniporter complex components, ER-mitochondria contact site (MERCs) integrity, and the effects of apoptotic stimuli (cisplatin and selenite) on cell viability and VDAC1 oligomerization. AnxA5 mutants with disrupted Ca²⁺ binding or self-assembly functions were also generated and tested to further explore the mechanistic role of AnxA5. Statistical analyses, including Student's t-tests, ANOVA, and Kruskal-Wallis tests, were used to determine the significance of the results.

This research demonstrated that AnxA5 is crucial for mitochondrial Ca²⁺ homeostasis, particularly during ER Ca²⁺ release. AnxA5-KO cells showed significantly reduced mitochondrial Ca²⁺ uptake in response to IP₃-generating agonists, while cytosolic Ca²⁺ levels remained unchanged. AnxA5 was found to localize at the OMM and within mitochondria. Immunogold labeling with TEM revealed that AnxA5 accumulates in the OMM's vicinity upon ER Ca²⁺ release. PLA showed close proximity between AnxA5 and VDAC1, suggesting that AnxA5 influences VDAC1's activity without direct binding. AnxA5 depletion did not affect the mitochondrial membrane potential or MERCs but increased mitochondrial volume and branching. The study also found that AnxA5 regulates the Ca²⁺ permeability of VDAC1 and VDAC2 but not VDAC3. Electrophysiological recordings demonstrated that AnxA5 is essential for the occurrence and open probability of a 35 pS Ca²⁺-permeable channel in the OMM. AnxA5's presence near VDAC1 protected against cisplatin and selenite-induced VDAC1 dimerization, thereby reducing apoptosis. In contrast, AnxA5-KO cells showed enhanced VDAC1 oligomerization and increased susceptibility to cisplatin- and selenite-induced apoptosis. Mutational studies revealed that AnxA5's Ca²⁺ binding, but not self-assembly, is critical for its function in regulating mitochondrial Ca²⁺ signaling.

This study provides compelling evidence for AnxA5's critical role in regulating mitochondrial Ca²⁺ uptake, particularly its modulation of VDAC1 activity. The observed reduction in mitochondrial Ca²⁺ uptake in AnxA5-KO cells following ER Ca²⁺ release highlights AnxA5's importance in this process. AnxA5's close proximity to VDAC1, as shown by PLA, suggests a functional interaction that influences VDAC1's Ca²⁺ permeability without direct binding. The findings regarding AnxA5's effects on mitochondrial morphology and cristae dynamics further emphasize its multifaceted role in mitochondrial function. The isoform-specific effects on VDACs and the rescue experiments using AnxA5 mutants highlight the mechanistic importance of AnxA5's Ca²⁺ binding in this regulation. The protective effect of AnxA5 against cisplatin- and selenite-induced apoptosis by preventing VDAC1 oligomerization highlights its cytoprotective role.

This research uncovered AnxA5 as a key regulator of mitochondrial Ca²⁺ homeostasis and apoptosis. AnxA5 modulates VDAC1's Ca²⁺ permeability, affecting IMS Ca²⁺ signaling and subsequently mitochondrial structure and dynamics. Its ability to prevent excessive VDAC1 oligomerization under apoptotic stress highlights its cytoprotective function. Future research could explore the precise molecular mechanism of AnxA5-VDAC1 interaction, identify the specific phospholipids AnxA5 binds to on the OMM, and investigate AnxA5's potential therapeutic applications in preventing apoptosis in diseases involving mitochondrial dysfunction.

The study primarily focused on HeLa cells and EA.hy926 cells. Although perivascular cells from AnxA5-KO mice were also studied, the findings might not be fully generalizable across all cell types. The modest knockdown efficiency of VDAC1 in some experiments might have limited the interpretation of certain findings. The study concentrated on ER Ca²⁺ release as a primary source of Ca²⁺; thus, additional investigation could explore the role of AnxA5 in other Ca²⁺ influx mechanisms. Further research is needed to fully elucidate the complex interplay between AnxA5 and other OMM proteins.