LIMK2 Is Required For F-actin Cytoskeleton Organization And Contributes To Mechanical Property Of Contracting Airway Smooth Muscle

Airway smooth muscle is a major structural component of the walls of airway.It is responsible for the contraction of airway and is a key contributor to airway narrowing.The mechanical contractile properties of airway smooth muscle function essentially for airway physio-and pathological processes.During normal breathing,the airway smooth muscle is exposed to continuously changing mechanical conditions.To adapt mechanical forces from patho-physiological breathing,airway smooth muscle has developed unique contractile properties concerning hyper-responsive and lengthsensitive contractility and force transduction.Many studies have shown that these properties essentially function in diverse pathological processes such as asthma and chronic obstructive pulmonary disease(COPD).Smooth muscle cytoskeleton reorganization has been considered as a fundamental process for the genesis of these properties,but the regulatory mechanism underlying remains unclear.It is well documented that actin filament cytoskeletal remodeling is essentially regulated by LIM-kinases/cofilin signaling.Activated cofilin can depolymerize actin filaments into actin monomers,while LIM-kinases(LIMKs,including LIMK1 and LIMK2)can phosphorylate cofilin and inhibiting the ability of cofilin to sever actin filaments,thereby regulating the remodeling of actin cytoskeleton.LIMKs participate in multiple physiological processes,such as cell morphology and cell migration,but the significance of LIMKs-mediated cytoskeletal organization on smooth muscle mechanical contractility is still unclear.We found that LIMKs are predominantly expressed in airway smooth muscle compare with other smooth muscle tissues,and upregulated in the airway smooth muscle from cigarette smoking-induced COPD animal model.Therefore,we speculated an essential role of LIMKs in the cytoskeleton reorganization of contracting airway smooth muscle as well as mechanical properties.To address this hypothesis,we established two mouse lines with the deletions of LIMK1 and LIMK2,then systematically evaluated their role in cytoskeleton and contractility of smooth muscle.In contrast to control mice,the airways from LIMK1 deficient mice displayed normal contractile responses to various stimuli including KCl depolarization,methacholine(MCh)and 5-hydroxytryptamine(5-HT),and normal relaxant response to chloroquine.These observations showed that LIMK1 is not involved in the contractile properties of airway smooth muscle.However,the LIMK2 mutant airways showed about 30% inhibition of maximal force development in response to various stimuli comparing with control muscle,but the sensitivity of airway smooth muscle did not change,and ablation of LIMK2 did not affect relaxant property of airway.These observations indicated that LIMK2 deletion significantly inhibits the contraction of airway smooth muscle,and LIMK2 plays an important role in airway smooth muscle contractile process.Moreover,the LIMK1 and LIMK2 double knockout muscle showed a comparable contractile response to LIMK2 mutant muscle,indicating LIMK1 could not compensate for the effect of LIMK2 in the contractile process of airway smooth muscle,these two kinases have different functions in smooth muscles.To verify how LIMK2 participates in the contraction process of smooth muscle cells,we used live-cell imaging and found that the contractile stimulus-induced submembranous actin filament polymerization was abolished in LIMK2 mutant airway smooth muscle cells in contrast to control cells.The biochemical assay also showed that compared with control,the deletion of LIMK2 suppressed the stimuli-induced actin filament polymerization.Meanwhile,the deletion of LIMK2 leads to an abolished cofilin phosphorylation triggered by contractile stimulations.Taken together,our findings provide direct evidence to corroborate the importance of LIMK2/cofilin mediated submembranous actin filament reorganization in airway smooth muscle contractile properties.Remarkably,the deletion of LIMK2 also suppressed the length sensitivity of airway smooth muscle.Under physiological conditions,mechanical stretching can significantly inhibit the smooth muscle contractile force caused by various stimuli,and the dilation effects of mechanical stretching absent in pathological conditions.Our results showed that when the LIMK2 mutant smooth muscle was stimulated with inflammatory mediator 5-HT,the length sensitive effect was abolished.We found that mechanical stretching influences intracellular Ca2+ signaling by inactivating TMEM16 A in the 5-HT/GPCR/Gq/IP3/TMEM16A/VDCC pathway,thereby sequentially inhibiting LIMK2 phosphorylation and smooth muscle contractility.We thus suggested that LIMK2-mediated contraction contributes to the length sensitive effect of airway smooth muscle.In summary,taking the advantage of genetic approach,we firstly provide the in vivo evidence supporting that LIMK2,rather than LIMK1,was necessary for the maximal force development and actin filament cytoskeleton organization of contracting airway smooth muscle.Moreover,LIMK2-mediated force partially contributed to the mechanical properties of airway smooth muscle in terms of length-sensitive contraction.