Coping with the calcium overload caused by cell injury: ER to the rescue

It was nearly a century ago that Lewis Victor Heilbrunn identified the requirement of extracellular Ca²⁺ for what he termed as ‘surface precipitation reaction‘, a process that allows the cytoplasm streaming out from an injured cell to coagulate. Studies since then have identified that entry of extracellular Ca²⁺ into the injured cells trigger vesicle fusion, membrane remodeling, and cytoskeletal reorganization, all of which are required to repair the PM of the injured cell. While it is well recognized that excess cytosolic Ca²⁺ causes cell death, there is little understanding of how cells handle the large amount of extracellular Ca²⁺ that enters the injured cell. Being the largest membrane bound organelle and the major site for storing intracellular Ca²⁺, the ER is likely to aid in coping with the cytosolic Ca²⁺ overload. In a recent study, we used an ER-targeted Ca²⁺ sensor and found that ER responds to PM injury by rapidly sequestering the extracellular Ca²⁺ that enters the injured cell. This uptake of Ca²⁺ by the ER was mediated by the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) – the ER resident pump required for cytosolic Ca²⁺ uptake into the ER. Inhibition of SERCA activity in injured cells through a pharmacological inhibitor (thapsigargin), prevented ER Ca²⁺ uptake. Concomitantly, this also impaired the ability of the injured cells to sequester the cytosolic Ca²⁺, and impaired PMR. While this identified the role of ER in the injured cell’s ability to cope with Ca²⁺ influx, it raised two additional questions:

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(1) What is the mechanism by which ER senses and buffers increased cytosolic Ca²⁺?

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(2) Why does failure to buffer cytosolic Ca²⁺ result in poor repair of the injured cell?

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The answer to the first question is confounded by the fact that SERCA activity is not regulated by the cytosolic Ca²⁺ level. Instead, sustaining SERCA-mediated ER Ca²⁺ uptake requires the lumen of the ER to be kept electroneutral through the export of H⁺ and import of negatively charged ions such as Chloride (Cl⁻). Previously, we had observed that cells from Limb Girdle Muscular Dystrophy 2L (LGMD2L) patients, repair poorly due to lack of an ER-localized putative Ca²⁺ activated chloride channel (CaCC) protein – Anoctamin 5 (ANO5) also called Transmembrane 16E (TMEM16E). If injury-triggered increase in cytosolic Ca²⁺ activates ANO5-mediated pumping of Cl^– ions into the ER, then this could be the mechanism for the activation of Ca²⁺ uptake by the ER in healthy cells. In our recent study we found that similar to thapsigargin-treated healthy cells, LGMD2L patient cells are also poor at coping with injury-triggered increase in the cytosolic Ca²⁺. Through the use of a ER-targeted anion sensor, our study identified that PM injury led to anion uptake into the ER, which is compromised by NPPB (5-nitro-2-(3-phenylpropyl-amino) benzoic acid) – an inhibitor of CaCC. Similar to NPPB treated healthy cells, anion uptake into the ER was compromised in the injured LGMD2L patient cells. Concomitantly, the ER of the NPPB treated healthy cells and of the LGMD2L patient cells were both compromised in their ability to uptake injury-triggered increase in cytosolic Ca²⁺. By using a ANO5 knockout mouse, we examined Ca²⁺ homeostasis in injured ANO5-deficient muscle fibers, which identified that ANO5-deficient fibers are compromised in their ability to cope with the injury-triggered Ca²⁺ overload and in repairing PM damaged by focal injury or mechanical activity. Together, above results identify that cytosolic Ca²⁺ increase in injured cells triggers ER-resident CaCC activity, which supports clearance of cytosolic Ca²⁺ by the ER (Figure 1). This mechanism is the basis for the ER Ca²⁺ uptake in the injured cells and ANO5/TMEM16E is the CaCC that supports ER-mediated clearance of cytosolic Ca²⁺ overload in injured muscle cells.

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FIGURE 1: ER-mediated buffering of injury-triggered cytosolic Ca2+ increase. Plasma membrane acts as a barrier to maintain cytosolic ion balance, which is disrupted by cell injury leading to influx of ions such as Ca2+ (1). This increase in cytosolic Ca2+ activates opening of the ER-resident ion channel ANO5 and increases influx of the Cl- ions into the ER lumen (2). This Cl- ion serves as the counterion to maintain ER electroneutrality as the excess cytosolic Ca2+ is pumped into the ER lumen by the ER-resident Ca2+ pump SERCA (3). Together, these transporters enable buffering the cytosolic Ca2+ overload caused by PM injury, and their inhibition prevents clearance of cytosolic Ca2+ increase in the injured cell, compromising repair of the injured cell.

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Given the requirement of Ca²⁺ increase for PMR, the next question was why does failure to rapidly buffer cytosolic Ca²⁺ compromise the ability of the injured cell to repair. The answer came from examining (A) Annexin-mediated PMR, and (B) Mitochondria-mediated PMR. Based on the Ca²⁺ affinity of the different annexins, PM injury causes sequential recruitment and clearance of annexins at the injury site. This injury-triggered dynamics of annexins is required for remodeling and repair of the injured PM. ANO5 deficit in the LGMD2L patient cells altered the injury-triggered dynamics of two of the annexin proteins we examined – Annexin A1 and Annexin A2. While both these annexins were recruited to the site of injury in patient cells at the kinetics similar to the healthy cells, prolonged cytosolic Ca²⁺ increase in patient cells correlated with the failure of annexins to clear from the injury site, interfering with annexin-mediated PMR. Another PMR pathway is mediated by the injury-proximal mitochondria, which helps clear some of the cytosolic Ca²⁺, but also get depolarized and increases reactive oxygen species (ROS) production. ROS from these mitochondria then activates local buildup of actin cytoskeleton that helps close the PM wound. Injury of patient cells lacking ANO5 increased Ca²⁺ uptake and caused extensive depolarization of their mitochondria, impairing mitochondria-mediated PMR. Based on these findings we concluded that failure of ER to sequester injury-triggered increase in cytosolic Ca²⁺ impairs spatial and temporal regulation of PMR response, which compromises the ability of these cells to repair. This indicated that approaches to restore cytosolic Ca²⁺ balance in the injured ANO5 deficient cells could improve their repair ability. Accordingly, our study identified improved repair of ANO5 knockout muscle fibers treated with a cell permeant Ca²⁺ chelator – BAPTA-AM (1,2-bis(o-aminophenoxy) ethane-N,N,N9,N9-tetraacetic acid-acetoxy-methyl ester).

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Together, these results highlight the Goldilocks-like nature of injury-triggered Ca²⁺ increase, such that too little or too much increase both compromise PMR. It shows that ER-mediated cytosolic Ca²⁺ uptake allows the control over the amplitude and time of cytosolic Ca²⁺ increase in the injured cells. It highlights that Ca²⁺ uptake by the ER in injured cell relies upon Ca²⁺-activated anion transport and failure of anion or Ca²⁺ uptake by the ER disrupts the fine balance of multiple PMR repair responses mounted by the cell, compromising the cell’s repair ability. Identification of this ER-mediated Ca²⁺ and PMR regulation not only identifies a new role for this organelle, but also offer insights into how defect in a putative anion channel can cause poor muscle repair in LGMD2L patients and point to future avenues for its therapy.