Using polyacrylamide hydrogels to model physiological aortic stiffness reveals that microtubules are critical regulators of isolated smooth muscle cell morphology and contractility.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aortic wall and normally exist in a quiescent, contractile phenotype where actomyosin-derived contractile forces maintain vascular tone. However, VSMCs are not terminally differentiated and can dedifferentiate into a proliferative, synthetic phenotype. Actomyosin force generation is essential for the function of both phenotypes. Whilst much is already known about the mechanisms of VSMC actomyosin force generation, existing assays are either low throughput and time consuming, or qualitative and inconsistent. In this study, we use polyacrylamide hydrogels, tuned to mimic the physiological stiffness of the aortic wall, in a VSMC contractility assay. Isolated VSMC area decreases following stimulation with the contractile agonists angiotensin II or carbachol. Importantly, the angiotensin II induced reduction in cell area correlated with increased traction stress generation. Inhibition of actomyosin activity using blebbistatin or Y 27632 prevented angiotensin II mediated changes in VSMC morphology, suggesting that changes in VSMC morphology and actomyosin activity are core components of the contractile response. Furthermore, we show that microtubule stability is an essential regulator of isolated VSMC contractility. Treatment with either colchicine or paclitaxel uncoupled the morphological and/or traction stress responses of angiotensin II stimulated VSMCs. Our findings support the tensegrity model and we demonstrate that microtubules act to balance the actomyosin-derived traction stress generation and regulate the morphological responses of VSMCs.

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Effect of maturational changes in myosin content and morphometry on airway smooth muscle contraction

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1991.260.6.l471 ◽

1991 ◽

Vol 260 (6) ◽

pp. L471-L480 ◽

Cited By ~ 12

Author(s):

T. M. Murphy ◽

R. W. Mitchell ◽

A. Halayko ◽

J. Roach ◽

L. Roy ◽

...

Keyword(s):

Smooth Muscle ◽

Muscle Mass ◽

Morphological Changes ◽

Rank Order ◽

Force Generation ◽

Electron Microscopic ◽

Cross Sectional ◽

Tissue Content ◽

Electron Microscopic Morphometry ◽

Contractile Forces

We studied the relationship of airway morphometry, the content of myosin heavy-chain and isoform stoichiometry, and the distribution of bronchoconstrictor responses in the airways of maturing swine. Lungs were excised in 2-wk-old farm swine (2ws; n = 13) and 10-wk-old swine (10ws; n = 13), and tracheal smooth muscle strips and bronchial rings from generations 2–5 were fixed for in vitro isometric measurement of force generation. Split samples were placed in formaldehyde solution or glutaraldehyde for light- or electron-microscopic morphometry or frozen for analysis of tissue myosin content. The rank order of force generation elicited by both receptor- and nonreceptor-dependent mechanisms for both 2ws and 10ws was generation 4 greater than 3 greater than or equal to 2. For all matched airway generations, contractile force was 257#x2013;100% greater in 2ws than 10ws. Differences in force generation were not related to morphometric differences in smooth muscle mass content among airways. The relative cross-sectional area of smooth muscle derived by computerized morphometry was 5.5–7% for each airway generation and did not change with age. Electron-microscopic morphometry demonstrated comparable myocyte content within muscle bundles for all airways in both age groups. In generation 4 airways, myocyte size in 2ws (27.3 +/- 0.8 nuclei/2,500 microns2) hypertrophied approximately 15% in 10ws (20.4 +/- 0.6 nuclei/2,500 microns2; P less than 0.01). Tissue content of myosin measured by computerized laser densitometry of gel electrophoresis of homogenates was greater in trachea from 2ws than 10ws (135 +/- 10 vs. 90 +/- 4 micrograms/g tissue; P less than 0.01); homology of 200- and 205-kDa isoforms was confirmed by Western blot against polyclonal myosin antibody and Cleveland digest analysis of each band. Differences in contractile forces between generations in 2ws and 10ws were not correlated to functional myosin isoform content. We demonstrate a maturational downregulation of contractile forces in maturing swine. This response is independent of smooth muscle receptor distribution and is not related to morphological changes in airways muscle mass, cellularity, changes in content of nonmyocyte tissues, or tissue content of functional myosin isoform.

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Different ontogeny of rate of force generation and shortening velocity in guinea pig trachealis

Journal of Applied Physiology ◽

10.1152/jappl.2000.88.4.1338 ◽

2000 ◽

Vol 88 (4) ◽

pp. 1338-1345 ◽

Cited By ~ 25

Author(s):

Pasquale Chitano ◽

Jizhong Wang ◽

Carrie M. Cox ◽

Newman L. Stephens ◽

Thomas M. Murphy

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Force Production ◽

Internal Resistance ◽

Force Generation ◽

Stress Generation ◽

Shortening Velocity ◽

Smooth Muscle Function ◽

Old Adult ◽

Force Velocity

Juveniles of many species, including humans, display greater airway responsiveness than do adults. This may involve changes in airway smooth muscle function. In the present work we studied force production and shortening velocity in trachealis from 1-wk-old (1 wk), 3-wk-old (3 wk), and 3-mo-old (adult) guinea pigs. Strips were electrically stimulated (60 Hz, 18 V) at their optimal length ( l o) to obtain maximum active stress (Po) and rate of stress generation. Then, force-velocity curves were elicited at 2.5 s from the onset of the stimulus. By applying a recently developed modification of Hill's equation for airway smooth muscle, the maximum shortening velocity at zero load ( V o) and the value α ⋅ γ/β, an index of internal resistance to shortening (Rsi), were calculated (α, β, and γ are the constants of the equation). Poincreased little with maturation, whereas the rate of stress generation increased significantly (0.40 ± 0.03, 0.45 ± 0.03, 0.51 ± 0.03 P o/s for 1 wk, 3 wk, and adult animals). V o slightly increased early with maturation to decrease significantly later (1.79 ± 0.67, 2.45 ± 0.92, and 0.55 ± 0.09 l o/s for 1 wk, 3 wk, and adult animals), whereas the Rsi showed an opposite trend (14.98 ± 5.19, 8.99 ± 3.01, and 32.07 ± 5.54 mN ⋅ mm−2 ⋅ l o −1 ⋅ s for 1 wk, 3 wk, and adult animals). This early increase of force generation in combination with late increase of Rsi may explain the changes of V o with age. An elevated V o may contribute to the incidence of airway hyperresponsiveness in healthy juveniles.

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