Significance. Acute lung injury (ALI) is major cause of mortality and morbidity. Our goal is to define mechanisms that might lead to a cure. We will focus on alveolar mitochondrial mechanisms, which are poorly understood. In ALI, mitochondrial dysfunction might underlie alveolar dysfunction, leading to lung injury. The dysfunction might follow loss of mitochondrial Ca2+ buffering in which Ca2+ diffuses from cytosol to mitochondrial matrix across the mitochondrial Ca2+ uniporter (MCU), preventing proinflammatory increase of the cytosolic Ca2+ (cytCa2+), hence protecting against injury. Better understanding of these mechanisms might lead to restoration of mitochondrial Ca2+ buffering by MCU as a therapy for LPS-ALI. Proposal. We propose the novel hypothesis that mitochondrial Ca2+ (mitCa2+) determine surfactant secretion, because the mitochondrial Ca2+ uptake increases ATP and H2O2 production. In LPS-ALI, sustained cytCa2+ increase causes failure of mitochondrial Ca2+ buffering leading to the activation of the Ca2+-dependent phosphatase, calcineurin, which in turn, dephosphorylates and activates the mitochondrial fission protein, DRP1. The resulting mitochondrial fission leads to abrogation of MCU. Calcineurin also promotes actin depolymerization. Together these effects induce alveolar dysfunction as reflected in loss of surfactant secretion.
Specific Aims. The specific aims are to determine the role of the MCU in alveolar surfactant secretion (Aim 1) and to determine the extent to which loss of MCU exacerbates lung injury in LPS-induced ALI (Aim 2). We will establish the role of the MCU as a specific Ca2+ channel, as well as its role in surfactant secretion. We will evaluate molecular mechanisms underlying the loss of mitochondrial Ca2+ buffering in LPS-ALI and we will consider bone-marrow-derived mesenchymal stromal cell (BMSC)-based therapeutic approaches aimed at reinstating Ca2+ buffering by mitochondria damaged alveoli as a strategy for lung repair. Approach. We will achieve these aims by live confocal and two-photon microscopy of isolated perfused mouse lungs and by studies in isolated alveolar type 2 (AT2) cells. Our determinations will include: (1) cytCa2+ and mitCa2+ in intact alveoli and in isolated AT2 cells; (2) regulation of surfactant secretion by single cell imaging through determinations of Ca2+, ATP, H2O2 and the F-actin fence; (3) MCU and Ca2+ buffering in endotoxin ALI through lung and AT2 cell expressions of specific mutants and siRNA; (4) Expression of MCU mutants in BMSCs to test the hypothesis that MCU restitution re-instates mitochondrial Ca2+ buffering; (5) MCU overexpression by mitochondrial transfer as a means to protect against LPS-ALI. Preliminary data. We show (1) MCU regulates cytCa2+ and mitCa2+ and surfactant secretion, (2) endotoxin ALI blocks mitochondrial Ca2+ buffering by downregulating MCU through activation of DRP1, and that (3) while alveolar transfection of a mutant MCU worsens mouse survival in endotoxin ALI, expression of a fill length MCU increases mouse survival. These and other preliminary data on our measurement strategies support feasibility of the project. Impact. This project will provide the first systematic understanding of the role of the MCU as a determinant of alveolar function. The extent to which loss of the MCU exacerbates ALI, and the extent to which MCU reinstatement promotes lung repair will be understood for the first time. Outstandingly new understanding of the pathogenesis of ALI will be achieved.
This project aims to determine fundamental mitochondrial mechanisms regulating alveolar functions in normal and in endotoxin-treated lungs. Specifically, the proposal aims to determine that loss of mitochondrial calcium uniporter (MCU) leading to a loss of mitochondrial Ca2+ buffering, exacerbates endotoxin-induced acute lung injury (ALI) and re-instatement of MCU, hence mitochondrial Ca2+ buffering is protective in ALI. The findings of this research are likely to impact understanding of mechanisms of acute lung injury and the development of relevant therapy
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| Bhattacharya, Jahar; Westphalen, Kristin (2016) Macrophage-epithelial interactions in pulmonary alveoli. Semin Immunopathol 38:461-9 |
