Doğan, Murat

Inflammation and pro‐inflammatory cytokines (e.g., TNFα, IL‐13) induce airway smooth muscle (ASM) hyper‐reactivity and remodeling characteristic of asthma. We hypothesized that exposure to TNFα (and IL‐13) increases isometric force generation in porcine ASM via several underlying mechanisms: 1) increasing sensitivity to ACh stimulation; 2) enhancing the cytosolic Ca 2+ ([Ca 2+ ] cyt ) response to ACh; 3) increasing the Ca 2+ sensitivity of force generation; 4) enhancing the Ca 2+ ‐dependent phosphorylation of myosin regulator light chain (rMLC); 5) promoting expression of non‐muscle rMLC; 6) increasing actin and myosin heavy chain (MyHC) content (i.e., the number of contractile units); and/or 7) promoting cytoskeletal remodeling (actin polymerization). Studies were performed on intact ASM strips (~1 mm wide × ~5 mm long) dissected from porcine tracheas obtained from a local abattoir. The ASM strips were placed in PSS with and without TNFα (100 ng/ml) for 24 h. The ACh concentration dependence of force generation ASM was determined (ACh range from 10 nM to 1mM), and the EC50 (i.e., the ACh concentration that produces 50% of maximal ASM force) was determined. In control, untreated ASM, the EC50 for ACh was 2.6 μM compared to 1.3 μM following TNFα treatment. We found that across all ACh concentrations, ASM force increased after TNFα treatment with an ~50% increase in maximum ASM force. The [Ca 2+ ] cyt response induced by the EC50 [ACh] was similar in control and TNFα treated ASM. Thus, with an increase in force and no effect on [Ca 2+ ] cyt , there was an apparent increase in Ca 2+ sensitivity of force after TNFα treatment. However, in both control and TNFα treated ASM, the extent of rMLC phosphorylation induced by ACh stimulation was not affected by TNFα treatment. Importantly, the extent of rMLC phosphorylation at the ACh EC50 was comparable between control and TNFα treatment indicating no change in Ca 2+ sensitivity. The expression of non‐muscle rMLC (determined by 2D gel electrophoresis) was also unaffected by TNFα treatment. Total actin and MyHC concentrations increased by ~45% following TNFα treatment; thus, the number of contractile units contributing to force increased. This was reflected by a marked increase in the Ca 2+ activation of ATP consumption (cross‐bridge cycling) of ASM following TNFα treatment. Cytoskeletal remodeling was evaluated by ACh‐induced actin polymerization (F‐/G‐actin ratio) but was found to be unaffected by TNFa treatment. Together, our results show that two primary mechanisms underlie the marked increase in ASM force generation induced by 24‐h TNFα exposure: 1) an increase in ACh sensitivity, and 2) an increase in actin and MyHC concentrations (number of contractile units). Support or Funding Information Supported by NIH grant HL126451 (GCS) and HL150890 (GCS)

Airway inflammation is a hallmark of asthma, triggering airway smooth muscle (ASM) hyperreactivity and airway remodeling. TNFα increases both agonist-induced cytosolic Ca2+ concentration ([Ca2+]cyt) and force in ASM. The effects of TNFα on ASM force may also be due to an increase in Ca2+ sensitivity, cytoskeletal remodeling, and/or changes in contractile protein content. We hypothesized that 24 h of exposure to TNFα increases ASM force by changing actin and myosin heavy chain (MyHC) content and/or polymerization. Porcine ASM strips were permeabilized with 10% Triton X-100, and force was measured in response to increasing concentrations of Ca2+ (pCa 9.0 to 4.0) in control and TNFα-treated groups. Relative phosphorylation of the regulatory myosin light chain (p-MLC) and total actin, MLC, and MyHC concentrations were quantified at pCa 9.0, 6.1, and 4.0. Actin polymerization was quantified by the ratio of filamentous to globular actin at pCa 9.0 and 4.0. For determination of total cross-bridge formation, isometric ATP hydrolysis rate at pCa 4.0 was measured using an enzyme-coupled NADH-linked fluorometric technique. Exposure to TNFα significantly increased force across the range of Ca2+ activation but did not affect the intrinsic Ca2+ sensitivity of force generation. The TNFα-induced increase in ASM force was associated with an increase in total actin, MLC, and MyHC content, as well as an increase in actin polymerization and an increase in maximum isometric ATP hydrolysis rate. The results of this study support our hypothesis that TNFα increases force generation in ASM by increasing the number of contractile units (actin-myosin content) contributing to force generation.