Mechanisms underlying TNFα-induced enhancement of force generation in airway smooth muscle

Abstract

Airway diseases such as asthma are triggered by inflammation and mediated by proinflammatory cytokines such as tumor necrosis factor alpha (TNFα). Our goal was to systematically examine the potential mechanisms underlying the effect of TNFα on airway smooth muscle (ASM) contractility. Porcine ASM strips were incubated for 24 h with and without TNFα. Exposure to TNFα increased maximum ASM force in response to acetylcholine (Ach), with an increase in ACh sensitivity (hyperreactivity), as reflected by a leftward shift in the dose-response curve (EC₅₀ ). At the EC₅₀ , the [Ca²⁺ ]_cyt response to ACh was similar between TNFα and control ASM, while force increased; thus, Ca²⁺ sensitivity appeared to increase. Exposure to TNFα increased the basal level of regulatory myosin light chain (rMLC) phosphorylation in ASM; however, the ACh-dependent increase in rMLC phosphorylation was blunted by TNFα with no difference in the extent of rMLC phosphorylation at the EC₅₀ ACh concentration. In TNFα-treated ASM, total actin and myosin heavy chain concentrations increased. TNFα exposure also enhanced the ACh-dependent polymerization of G- to F-actin. The results of this study confirm TNFα-induced hyperreactivity to ACh in porcine ASM. We conclude that the TNFα-induced increase in ASM force, cannot be attributed to an enhanced [Ca²⁺ ]_cyt response or to an increase in rMLC phosphorylation. Instead, TNFα increases Ca²⁺ sensitivity of ASM force generation due to increased contractile protein content (greater number of contractile units) and enhanced cytoskeletal remodeling (actin polymerization) resulting in increased tethering of contractile elements to the cortical cytoskeleton and force translation to the extracellular matrix.

Keywords: Actin polymerization; inflammation; myosin heavy chain; myosin light chain phosphorylation.

Figure 1

Representative tracings of specific force (force per muscle cross‐sectional area) generated by stimulating with different concentrations of ACh (ranging from 10 nmol/L to 1 mmol/L) in porcine ASM strips. Note that 24‐h exposure to TNFα increased ASM force generation across all ACh concentrations compared to control (P < 0.01). ACh, acetylcholine; ASM, airway smooth muscle; TNFα, tumor necrosis factor alpha.

Figure 2

Summary results showing a dependency of porcine ASM‐specific force on ACh concentration (A). After 24‐h TNFα (100 ng/mL) exposure, ASM force increased across all ACh concentrations (A). The sensitivity of force generation to ACh concentrations was assessed by normalizing force to the maximum force (F _max) for each ASM strip (B). The ACh concentration that induced 50% F _max (EC₅₀) was determined as an index of ACh sensitivity. Note that after 24‐h TNFα exposure the EC₅₀ shifted leftward compared to control ASM strips. Values are means ± SD. *Significant difference (P < 0.05) from control (n = 6 animals). ASM, airway smooth muscle; ACh, acetylcholine; TNFα, tumor necrosis factor alpha; SD, standard deviation.

Figure 3

Summary of results showing that 24‐h TNFα (100 ng/mL) exposure increased maximum specific force (F _max) evoked by ACh stimulation (A). Exposure to TNFα (100 ng/mL for 24 h) increased sensitivity to ACh as reflected by a decrease in the ACh concentration that induced 50% F _max (EC₅₀) (B). Values are medians – IQR *Significant difference (P < 0.05) from control (n = 6 animals). TNFα, tumor necrosis factor alpha; ACh, acetylcholine; IQR, interquartile range.

Figure 4

Representative tracings of ACh‐induced elevation of [Ca²⁺]_cyt and increased specific force in control and TNFα‐exposed (100 ng/mL, 24 h) ASM strips at three different ACh concentrations. (A) 1 μmol/L ACh; (B) 1.3 μmol/L ACh for control (EC₅₀ ) and 2.6 μmol/L ACh for TNFα (EC₅₀ ); (C) ACh 10 μmol/L. ACh, acetylcholine; TNFα, tumor necrosis factor alpha.

Figure 5

Summary of the specific force (A) and peak [Ca²⁺]_cyt (B) responses induced by ACh at three different concentrations (control: 1, 2.6 [EC₅₀], and 10 μmol/L; TNF: 1, 1.3 [EC₅₀], 10 and 10 μmol/L). Exposure to TNFα (100 ng/mL, 24 h) significantly increased ASM‐specific force across all ACh concentrations (A). TNFα exposure significantly increased the peak amplitude of the ACh‐induced [Ca²⁺]_cyt response except at the EC₅₀ concentration (B). Due to the disproportionate increase in specific force induced by TNFα exposure, Ca²⁺ sensitivity (force per [Ca²⁺]_cyt) significantly increased (C). Values are medians – IQR. *Significant difference (P < 0.05) versus initial 1 μmol/L ACh for each group (n = 6 animals). **Significant interaction between ACh concentration dependence and treatment group. ACh, acetylcholine; TNFα, tumor necrosis factor alpha; ASM, airway smooth muscle; IQR, interquartile range.

Figure 6

Representative western blots in which phosphorylated (mono‐ and di‐ p‐rMLC) and unphosphorylated (un‐p‐rMLC) rMLC were separated using a modified Phos‐tag™ gels. In control ASM, ACh stimulation increased rMLC phosphorylation (p‐rMLC relative to total rMLC) up to the EC₅₀ concentration (2.6 μmol/L) but with no change at 10 μmol/L. Exposure to TNFα (100 ng/mL, 24 h) significantly increased the basal level of rMLC phosphorylation but blunted the ACh‐dependent increase in pMLC. The ratio of p‐rMLC to total rMLC increased with 1 and 2.6 μmol/L ACh stimulation in control ASM strips and phosphorylation of rMLC did not increase at the 10 μmol/L ACh stimulation. In the TNFα‐treated group, basal p‐rMLC was increased in unstimulated ASM strips and there was no significant difference at the other doses of ACh stimulation. Values are medians – IQR. *Significant difference (P < 0.05) compared to unstimulated ASM strip within the control group (n = 6 animals). **Significant interaction between ACh concentration dependence and treatment group. rMLC, regulatory myosin light chain; ASM, airway smooth muscle; ACh, acetylcholine; TNFα, tumor necrosis factor alpha.

Figure 7

Representative silver‐stained 2‐D gel used for quantifying phosphorylated (dots 1 and 3) and nonphosphorylated (dots 2 and 4) NM and SM rMLC, respectively, in control and TNFα‐treated (100 ng/mL, 24 h) porcine ASM. Exposure to TNFα significantly increased the ratio of NM myosin to total rMLC at the baseline. After ACh stimulation at the EC₅₀ concentration (2.6 μmol/L for control and 1.3 for TNFα‐treated ASM), the ratio of phosphorylated NM rMLC to phosphorylated SM rMLC was reduced in TNFα‐treated ASM strips. Values for each of the four animals are presented. NM, nonmuscle; SM, smooth muscle; rMLC, regulatory myosin light chain; TNFα, tumor necrosis factor alpha; ASM, airway smooth muscle; ACh, acetylcholine.

Figure 8

Representative Coomassie‐stained gels used for quantifying MyHC (A) and actin (B) protein concentrations in control and TNFα‐treated (100 ng/mL, 24 h) porcine ASM. Exposure to TNFα significantly increased both MyHC and actin concentration in ASM compared to controls. Values are medians – IQR. *Significant difference (P < 0.05) compared to control (n = 6 animals). MyHC, myosin heavy chain; TNFα, tumor necrosis factor alpha; ASM, airway smooth muscle; IQR, interquartile range.

Figure 9

Representative western blots for F‐ and G‐ actin content in ASM strips that were either unstimulated or stimulated by ACh at three different concentrations (control: 1, 2.6 [EC₅₀], and 10 μmol/L; TNF: 1, 1.3 [EC₅₀], 10 and 10 μmol/L). In both control and TNFα‐treated (100 ng/mL, 24 h) groups, the extent of actin polymerization (increase in F‐ to G‐actin ratio) increased with ACh stimulation. However, exposure to TNFα significantly increased actin polymerization across all ACh concentrations. Values are medians – IQR. *Significant difference (P < 0.05) compared to unstimulated ASM strip (n = 6 animals). **Significant interaction between ACh concentration dependence and treatment group. ASM, airway smooth muscle; ACh, acetylcholine; TNFα, tumor necrosis factor alpha.