Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA313

Poster Communications

Endoplasmic reticulum/ mitochondrial stress in human airway smooth muscle

G. C. Sieck1, P. Delmotte1

1. Physiology & Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, United States.

Inflammation is key to a number of diseases that affect airway smooth muscle (ASM) function. Inflammatory cytokines (e.g., TNFα) enhance cytosolic Ca2+ ([Ca2+]cyt) responses to agonist stimulation triggering ASM hyperreactivity. Inflammation also induces ASM cell proliferation and remodeling. We hypothesized that TNFα induces a vicious cycle including reactive oxidant species (ROS) formation, endoplasmic reticulum (ER) stress, mitochondrial fragmentation via increased dynamin-related protein (Drp1) and reduced mitofusin (Mfn2) expression, and uncoupling of mitochondria and ER resulting in dysregulation of mitochondrial and cytosolic Ca2+ responses to agonist stimulation. Human ASM cells were isolated from lung specimens incidental to patient surgery and exposed to TNFα for 24 h. Expression of ER stress markers were evaluated by Western blot including GRP78, PERK, IRE1, ATF6, PDI, ERO-1, and calnexin. Expression of the spliced isoform of XBP1 was also measured by quantitative RT-PCR. Mitochondrial fission/fusion proteins, Drp1 and Mfn2 were measured by Western blot, and mitochondrial morphology was analyzed in ASM cells loaded with Mitotracker Green. hASM cell proliferation was evaluated using CyQUANT assay. Isolated ASM cells were loaded with Fluo3 and Rhod2 to measure [Ca2+]cyt and mitochondrial ([Ca2+]mito) responses to 1 μM ACh stimulation using high-speed real time confocal imaging. We found that 24-h exposure to TNFα increased ROS formation (MitoSOX fluorescence) and increased and expression of ER stress protein markers, an effect reversed by the ROS scavenger, tempol. Associated with ER stress, we found that Drp1 expression increased and Mfn2 expression decreased and this was associated with increased mitochondrial fragmentation. Exposure to TNFα also resulted in the uncoupling of [Ca2+]cyt and [Ca2+]mito responses to ACh stimulation with a net increase in [Ca2+]cyt and reduced [Ca2+]mito. Exposure to TNFα increased hASM cell proliferation. Together these results indicate that inflammation induces ROS formation leading to ER stress in human ASM cells with downstream changes in Mfn2 and Drp1 expression, mitochondrial fragmentation, uncoupling of mitochondria and ER, and altered cytosolic and mitochondrial Ca2+ regulation - all characteristic of ASM hyperreactivity and remodeling in asthma.

Where applicable, experiments conform with Society ethical requirements