Kafaji, Mustafa

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)

Cytoskeletal remodeling in smooth muscle occurs with activation and involves actin polymerization. Remodeling of the cortical cytoskeleton allows tethering of contractile proteins and force transmission to the extracellular matrix. Smooth muscle contraction involves cross‐bridge cycling and ATP hydrolysis, which are both sensitive to loading. During isometric activation, the rate of ATP hydrolysis is initially high but declines with time reaching a steady‐state, while peak force is maintained. Thus, tension cost, i.e., the rate of ATP hydrolysis per unit of force, decreases over time, reflecting increased efficiency of smooth muscle activation. We hypothesize that the time‐dependent decrease in ATP hydrolysis rate and tension cost is due to cytoskeletal remodeling and an increase in internal loading on cross‐bridges. In permeabilized porcine airway smooth muscle (ASM) strips, isometric force and ATP hydrolysis rate were simultaneously measured after maximum Ca 2+ activation (pCa 4.0). From each animal, two ASM strips were tested: 1) Inhibition of actin polymerization and cytoskeletal remodeling using Cytochalasin‐D (Cyto‐D; treated (1 μM for 30 min); and 2) Untreated controls. Compared to untreated controls, Cyto‐D‐induced inhibition of actin polymerization reduced both peak and steady‐state isometric force by ~40%, while peak and steady‐state ATP hydrolysis rate increased by ~30% and ~20%, respectively. As a result, at peak and steady‐state, tension cost increased by ~100% and ~60%, respectively, following Cyto‐D treatment. These results support our hypothesis that the time‐dependent decrease in ATP hydrolysis rate reflects an increase in internal load on cross‐bridges via cytoskeletal remodeling. Cyto‐D inhibition of cytoskeletal remodeling impairs contractile protein tethering and thus reduces internal loading, resulting in faster cross‐bridge cycling and increased ATP hydrolysis rate. Support or Funding Information Supported by NIH grant HL126451(GCS), HL150890 (GCS) and the Mayo Clinic