Effect of TNFα on Mitochondrial Function and Mitochondrial Biogenesis in Human Airway Smooth Muscle

Abstract

Airway inflammation is a key aspect of asthma and is associated with airway smooth muscle (ASM) hyperreactivity as well as airway remodeling. Proinflammatory cytokines, such as TNFα mediate this airway inflammatory response. Previously, we found that TNFα induces an increase airway smooth muscle (ASM) force and ATP hydrolysis. In response, TNFα may also induce an increase in ATP production in ASM. This could be achieved by either an increase in mitochondrial function and/or mitochondrial biogenesis. We previously found that TNFα decreases mitochondrial Ca2+ influx. Since dehydrogenase enzymes in the TCA cycle are Ca2+ dependent, this would tend to reduce mitochondrial function in response to agonist stimulation. We also found that TNFα increases mitochondrial fragmentation, an initial step in mitochondrial biogenesis. We hypothesized that TNFα increases mitochondrial biogenesis and decreases mitochondrial function in human ASM.Human ASM cells were isolated from lung specimens incidental to patient surgery. The patients were normal as evaluated by a clinical pathologist (no history of asthma or COPD). hASM cells were exposed to TNFα (20 ng/ml) for 24 h. To examine mitochondrial biogenesis, protein expression of porin (an indicator of mitochondrial density) and PGC1α (a regulator of mitochondria biogenesis) were determined by Western blot analysis. In addition, the relative number of copies of human mitochondrial DNA (mtDNA) was determined by PCR and normalized to nuclear DNA. Mitochondrial function was assessed by measuring O2 Consumption Rate (OCR – using a Seahorse Bioscience XF24 Analyzer), succinate dehydrogenase (SDH) activity (using a quantitative histochemical technique) and mitochondrial membrane potential – (DYm – using TMRM and confocal microscopy). OCR and SDH activity measurements were normalized to mitochondrial volume density. Results were analyzed by one‐way ANOVA with post hoc analysis when justified.We found that TNFα increases mitochondrial biogenesis as indicated by an increase in porin and PGC1α protein expression in hASM and an increase in mtDNA copy numbers. We also found that TNFα decreases mitochondrial membrane potential in hASM cells, and also decreases OCR and SDH activity when normalized for the increase in mitochondrial volume density induced by TNFα. In a normal homeostatic response, an increase in hASM force and ATP hydrolysis (energy demand) would be met by increased mitochondrial ATP production. Our results indicate that TNFα disrupts this normal homeostatic response by reducing mitochondrial Ca2+ influx and thereby reducing mitochondrial TCA activity. However, TNFα increase the number of mitochondria to compensate the decrease in mitochondrial TCA activity.Support or Funding InformationSupported by NIH grant HL126451This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.