Differences In Force Generation Research Articles

Substrate Interactions and Free-Swimming Dynamics in the Crayfish Escape Response.

The caridoid or "tail flip" escape behavior of decapod crustaceans is a model system in neurobiology, but many aspects of its biomechanics are not well understood. To understand how the freshwater virile crayfish Faxonius virilis interacts with the substrate during the tail flip, we studied tail-flip hydrodynamics and force generation for free-moving animals standing on substrate, as well as tethered animals held at different distances from the substrate. We found no significant differences in force generation when distance from substrate was varied. Particle image velocimetry revealed that vortex formation was similar at all distances, but there were notable differences in interactions between shed vortices and substrate at different distances. Negative vorticity (clockwise flow of water) was observed in tethered animals interacting with the substrate but was largely absent in free-swimming animals. We found no evidence of ground effects enhancing tail flip performance in either tethered or free-swimming individuals, as peak force generation occurred before vortex shedding. This study contributes to our understanding of the crayfish escape response and highlights the need for more work that incorporates free-swimming animals and complex environments in the study of crustacean biomechanics.

Measuring Single-Cell Platelet Forces via Microcontact-Printed, Reference-Free Traction Force Microscopy Reveals Relationships between Cell Shape, F-Actin Localization, and Force

Platelets bind, spread, and contract to stop bleeding and reinforce hemostatic plugs. Platelet contraction occurs when interactions between actin and myosin create cytoskeletal tension and force generation. In this study, single-cell platelet forces were measured using a novel microcontact-printed, reference-free traction force microscopy approach. This approach facilitates single-cell force measurement on a contiguous topology that enables unrestricted platelet spreading without necessitating platelet removal to calculate force. Microcontact printing is used to deposit a grid of fluorescent bovine serum albumin onto a flexible polydimethylsiloxane substrate. Platelets from healthy donors were washed and seeded onto von Willebrand Factor-coated substrates. Platelets were fixed, stained, and imaged via confocal microscopy to simultaneously visualize fluorescently labeled platelets and grid displacement (used to calculate single-cell force.) Because microcontact-printing facilitates printing the grid on large, scalable surfaces, platelet forces were able to be measured on >100 single-cells per donor (N = 6 donors). Donor-to-donor platelet force variability was examined, and some donors produce significantly more force per platelet than other donors (p<0.01). Even within each donor, ∼15-fold variability in single platelet forces exists. A well-established area-force relationship (more spread cells produce more force) accounts for some variability in force generation, but for cells spread equally, there is still ∼5-fold differences in force generation. After examining the relationship between cell shape and force, a relationship was observed wherein platelets that are more circular produce more force. Additionally, amongst spread platelets, platelets with more spread F-actin produce more force. This versatile method that facilitates measurement of single-cell forces over large areas without restricting cell spreading or requiring cell removal reveals donor variability, cell shape, and F-actin localization as contributors to the large variability of single-platelet forces.

Analysis of the ACTN3 heterozygous genotype suggests that α-actinin-3 controls sarcomeric composition and muscle function in a dose-dependent fashion.

A common null polymorphism (R577X) in ACTN3 causes α-actinin-3 deficiency in ∼ 18% of the global population. There is no associated disease phenotype, but α-actinin-3 deficiency is detrimental to sprint and power performance in both elite athletes and the general population. However, despite considerable investigation to date, the functional consequences of heterozygosity for ACTN3 are unclear. A subset of studies have shown an intermediate phenotype in 577RX individuals, suggesting dose-dependency of α-actinin-3, while others have shown no difference between 577RR and RX genotypes. Here, we investigate the effects of α-actinin-3 expression level by comparing the muscle phenotypes of Actn3(+/-) (HET) mice to Actn3(+/+) [wild-type (WT)] and Actn3(-/-) [knockout (KO)] littermates. We show reduction in α-actinin-3 mRNA and protein in HET muscle compared with WT, which is associated with dose-dependent up-regulation of α-actinin-2, z-band alternatively spliced PDZ-motif and myotilin at the Z-line, and an incremental shift towards oxidative metabolism. While there is no difference in force generation, HET mice have an intermediate endurance capacity compared with WT and KO. The R577X polymorphism is associated with changes in ACTN3 expression consistent with an additive model in the human genotype-tissue expression cohort, but does not influence any other muscle transcripts, including ACTN2. Overall, ACTN3 influences sarcomeric composition in a dose-dependent fashion in mouse skeletal muscle, which translates directly to function. Variance in fibre type between biopsies likely masks this phenomenon in human skeletal muscle, but we suggest that an additive model is the most appropriate for use in testing ACTN3 genotype associations.

The myosin converter region influences cross‐bridge stiffness and muscle kinetics

The converter has been proposed to be the myosin elastic element that sets cross‐bridge stiffness. According to most cross‐bridge models, the elastic element's stiffness should directly influence muscle fiber type force and power generation levels. We tested these hypotheses by using the natural variation in converter regions found in Drosophila muscle types. In Drosophila, myosin isoforms are generated by alternative splicing from a single myosin heavy chain gene. The converter region is encoded by five alternative exon versions. We expressed four non‐native versions in Drosophila indirect flight muscle (IFM) and found that two of the three versions, 11b and 11e (found in larval and adult body wall muscles) decreased IFM rigor stiffness by 63% and 43%, respectively. This supports the hypothesis that the converter is the critical elastic element of myosin. The converters also influenced IFM fiber and myosin kinetics. 11b, 11d, and 11e decreased the frequency at which maximum power was generated (fmax) by 28%, 40%, and 53%, respectively. All three decreased myosin attachment kinetics, however, 11d and 11e also increased detachment kinetics. Differences in force generation are minimal, likely due to myosin stiffness changes being offset by changes in myosin duty ratio. With little to no force differences, changes in muscle power almost directly correlated with fmax. All three lines displayed reduced wing beat frequency, 11 to 15% less than control lines, with flight performance better correlating with fiber fmax than cross‐bridge stiffness. Thus, the converter is critical for muscle fiber type diversity as it helps set myosin stiffness, cross‐bridge kinetics and muscle power generation. Supported by NIH grant R01 AR055611 to D.S.

Myosin Stiffness and Cross-Bridge Kinetics are Influenced by Five Drosophila Alternative Converters

The converter has been proposed to be the myosin elastic element that sets cross-bridge stiffness. According to most cross-bridge models, the elastic element's stiffness should influence muscle fiber type force and power generation levels. We tested these hypotheses by exploiting the natural variation in converter regions found in Drosophila muscle types. In Drosophila, myosin isoforms are generated by alternative splicing from a single myosin heavy chain gene. The converter region is encoded by five alternative versions. We forced the four non-native versions to be expressed in Drosophila indirect flight muscle (IFM) and found that two of the three versions tested to date, 11b and 11e (found in larval and adult body wall muscles) significantly decreased IFM rigor stiffness by 63% and 43%, respectively. This supports the hypothesis that the converter is at least part of the elastic element of myosin. The converters influenced more than myosin stiffness, as all three converters slowed the super fast IFM fiber kinetics. 11b, 11d, and 11e decreased the frequency at which maximum power was generated (fmax) by 28%, 40%, and 53%, respectively. All three decreased apparent rate constant 2πb (set by myosin attachment and Pi release kinetics). However, 11d and 11e increased 2πc (set by detachment kinetics). Differences in force generation are minimal, likely due to myosin stiffness changes being offset by changes in duty ratio. With little to no force differences, power almost directly correlated with fmax, and the optimal percent ML amplitude for power inversely correlated with fmax. All three lines showed reduced wing beat frequency, with flight performance better correlating with fiber fmax than rigor stiffness. Thus, the converter is critical for muscle fiber type diversity as it helps set myosin stiffness, cross-bridge kinetics and power generation.

Fluid transport and coherent structures of translating and flapping wings

The Lagrangian coherent structures (LCSs) of simple wing cross sections in various low Reynolds number motions are extracted from high-fidelity numerical simulation data and examined in detail. The entrainment process in the wake of a translating ellipse is revealed by studying the relationship between attracting structures in the wake and upstream repelling structures, with the help of blocks of tracer particles. It is shown that a series of slender lobes in the repelling LCS project upstream from the front of the ellipse and "pull" fluid into the wake. Each lobe is paired with a corresponding wake vortex, into which the constituent fluid particles are folded. Flexible and rigid foils in flapping motion are studied, and the resulting differences in coherent structures are used to elucidate their differences in force generation. The clarity with which these flow structures are revealed, compared to the vorticity or velocity fields, provides new insight into the vortex shedding mechanisms that play an important role in unsteady aerodynamics.

Contractile forces generated by striae distensae fibroblasts embedded in collagen lattices

Striae distensae are characterized by linear, smooth bands of atrophic-appearing skin that are reddish at first and finally white. They are due to stretching of the skin, as in rapid weight gain, or mechanical stress, as in weight lifting. The pathogenesis of striae distensae is unknown but probably relates to changes in the fibroblast phenotype. In order to characterize striae distensae fibroblasts, alpha-smooth muscle actin expression and contractile forces were studied. Five healthy women with early erythematous striae and five healthy women with older striae were selected. Paired biopsies were taken from the center of lesional striae and adjacent normal skin. Fibroblasts were obtained by an explant technique and expanded in vitro in Dulbecco's modified Eagle's medium. Contractile forces generated by fibroblasts in collagen lattices were measured with the Glasbox device developed in our laboratory. Alpha-smooth muscle actin expression was studied by immunofluorescence labeling of cells and by flow cytometry. Fibroblasts from early striae distensae were the richest cells in alpha-smooth muscle actin filaments and generated the highest contractile forces. Their peak contractile force was 26% greater than normal fibroblasts. There was a 150% higher level of alpha-smooth muscle actin content in fibroblasts from early striae distensae compared with fibroblasts from normal skin. In contrast, there was no significant difference in force generation between old striae fibroblasts and normal fibroblasts with cells expressing no alpha-smooth muscle actin. The contractile properties of fibroblasts from striae distensae varies depending on the stage of the disease. In early striae distensae, fibroblasts acquire a more contractile phenotype, corresponding to that of myofibroblasts.

3-D in vitro model of early skeletal muscle development.

An understanding of the mechanical and mechano-molecular responses that occur during the differentiation of mouse C2C12 [corrected] myoblasts in 3-D culture is critical for understanding growth, which is important for progress towards producing a tissue-engineered muscle construct. We have established the main differences in force generation between skeletal myoblasts, dermal fibroblasts, and smooth muscle cells in a 3-D culture model in which cells contract a collagen gel construct. This model was developed to provide a reproducible 3-D muscle organoid in which differences in force generation could be measured, as the skeletal myoblasts fused to form myotubes within a collagen gel. Maintenance of the 3-D culture under sustained uni-axial tension, was found to promote fusion of myoblasts to form aligned multi-nucleate myotubes. Gene expression of both Insulin Like Growth Factor (IGF-1 Ea) and an isoform of IGF-1 Ea, Mechano-growth factor (IGF-1 Eb, also termed MGF), was monitored in this differentiating collagen construct over the time course of fusion and maturation (0-7 days). This identified a transient surge in both IGF-1 and MGF expression on day 3 of the developing construct. This peak of IGF-1 and MGF expression, just prior to differentiation, was consistent with the idea that IGF-1 stimulates differentiation through a Myogenin pathway [Florini et al., 1991: Mol. Endocrinol. 5:718-724]. MGF gene expression was increased 77-fold on day 3, compared to a 36-fold increase with IGF-1 on day 3. This indicates an important role for MGF in either differentiation or, more likely, a response to mechanical or tensional cues.

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

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 25-100% 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.