Regulation Of Passive Stiffness And Force Maintenance In Airway Smooth Muscle

Asthma has become one of the most common chronic airway diseases in the world.Nowadyas, it was actually being regonized as one of the four biggest public health issuearound the world, but unfortunately the mechanism underlying the development ofasthma remain unknown. Asthma is characterized by excessive airway narrowing,termed airway hyperresponsiveness (AHR). Since airway smooth muscle (ASM) is thekey-end factor for airway narrowing, mechanical property and contractility of ASM likeforce production, stiffness and force-velocity relationship are critical for airwaymechanical property and asthma research. Huge gap still remain in understanding of asmechanical property and ASM contraction; previous research from our groupdemonstrated the abnormal ability of asthmatic ASM to maintain force againstmechanical perturbation comparing to health subjects. The initial phase of smoothmuscle contraction is largely cross-bridges dependent; this regulation and signalingpathway of this process has been well documented during the last decades of smoothmuscle research, but the mechanism underlying the sustain phase of ASM contraction isstill unclear. Understanding the regulation of ASM contraction, especially themaintenance of smooth muscle force and stiffness maintenance contribute to illustratethe physiopathological mechanism of asthma. Thus, tracheal smooth muscle was used inthis research to demonstrate the regulation of passive stiffness, force maintenanceagainst oscillatory strain and signaling pathway involved in these processes during ASMactivation. In short,the results are:1) Passive stiffness is regulatable independent to cross bridge activation; thisstiffness change is regulated via Rho kinase (ROCK) pathway. In order todemonstrated cytoskeleton stiffness change, separate the effect from activation fromcross bridges cycling,1%Trition-x100were used to skin ASM. After permiblization,calcium and Ach weer used to stimuli the stiffness of muscle, interestingly, even whenpCa=10-9, Ach can still activated cytoskeleton stiffness, significant increase in passivestiffness happened without any change from resting tension; more importantly, westernblot suggeste that there is no change from MLC20phosphorylation comparing tocontrol. Further investigation illustrated that the increase in stiffness is completedblocked by ROCK inhibitor H1152(1μM) whereas myosin light chain kinase (MLCK)inhibitor ML-7(1μM) does not affect this process. 2) The ability of ASM to maintain contraction both statically and againstosclitory stain is regulated by ROCK. To gain insight of the ability of ASM tomaintain force and its signaling pathway, we employed4different inhibitors: Proteinkinase C (PKC) inhibitor GF109023x (15μM), MLCK inhibitor ML-7(5μM) and ROCKinhibitors Y27632(3μM) and H1152(3μM), these inhibitor are known to abolish threemost important signaling pathway in ASM contraction. To tease out the decline in forcedue to incubation of inhibitors, all experiment group match peak force production to50%of Fmax,100s isometric force was recorded after the muscle reached the plateau.Data suggest that GF109023x and ML-7didn’t affect the force maintenance whereasboth Rho kinase inhitors decrease the force maintenance of force significantly.100s25%Lref oscillatory strain was then applied in the presents of different inhibitor toexamine the inbitors effect on force maintenance over mechanical perturbation. Similarto previous finding, the force maintenance against length oscillation was attenuated btboth ROCK inhibitors. Force recovery data after length oscillation showed that onlyML-7does not affect the recovery, inhibition of ROCK and PKC leads to significantdecrease in velocity and level in force recovery.3) Myosin filament assembly regulatesforce maintenance in ASM. In order toinvestigate the force maintenance process and the ultrastructure changes happenedduring activation. We employed the same protocol as previously described, ASM werethen fixed and sectioned for electronic microscopy to gain ultra thin cross-sectional EMimages, Y27632(3μM) and PKC (10μM) were used in this experiment. Analsisy of theEM images demonstrate a significant decrease of myosin filament density from Y27632treated group, however PKC inhibition didn’t affect the myosin density comparing tocontrol.Take together, current research suggested that passive stiffness is seprated fromcross-brdige activation and could be activated by Ach; myosin filament assembly inASM facilitied the force maintenance of airway smooth muscle. ROCK kinase plays animportant in all these process. Understanding the regulation of ASM passive stiffnessand force maintenance were enhanced by this work, at the same time, Rho-kinase iscritical to ASM contraction, further investigation may provide new target for clinicaltherapy for asthma by targeting ROCK and asscociated pathway.