Rebecca E. Taylor

The phenomenon of twisted growth: humeral torsion in dominant arms of high performance tennis players.

This manuscript is driven by the need to understand the fundamental mechanisms that cause twisted bone growth and shoulder pain in high performance tennis players. Our ultimate goal is to predict bone mass density in the humerus through computational analysis. The underlying study spans a unique four level complete analysis consisting of a high-speed video analysis, a musculoskeletal analysis, a finite element based density growth analysis and an X-ray based bone mass density analysis. For high performance tennis players, critical loads are postulated to occur during the serve. From high-speed video analyses, the serve phases of maximum external shoulder rotation and ball impact are identified as most critical loading situations for the humerus. The corresponding posts from the video analysis are reproduced with a musculoskeletal analysis tool to determine muscle attachment points, muscle force vectors and overall forces of relevant muscle groups. Collective representative muscle forces of the deltoid, latissimus dorsi, pectoralis major and triceps are then applied as external loads in a fully 3D finite element analysis. A problem specific nonlinear finite element based density analysis tool is developed to predict functional adaptation over time. The density profiles in response to the identified critical muscle forces during serve are qualitatively compared to X-ray based bone mass density analyses.

Multidimensional structure-function relationships in human β-cardiac myosin from population-scale genetic variation

Significance Genetic variants in human β-cardiac myosin, which causes muscle contraction in the heart, can lead to hypertrophic cardiomyopathy (HCM), an inherited heart disease that can cause sudden death. New technologies have generated sequence data for large numbers of patients with HCM and unaffected individuals. In this study, we compare the protein structural locations of genetic variants of patients with HCM and the general population to identify spatial regions of the myosin that have a higher than expected proportion of genetic variants associated with HCM and earlier age at diagnosis. In addition, we develop new methods to interrogate the localization of genetic changes in protein structures. Our study demonstrates the power of combining clinical, genetic, and structural data to gain insight into Mendelian disease. Myosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human β-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from two population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 patients with HCM to identify regions of disease enrichment within β-cardiac myosin. We first developed computational models of the human β-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein 3D space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified a larger region significantly enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as the myosin mesa. Notably, patients with HCM with variants in the enriched regions have earlier disease onset than patients who have HCM with variants elsewhere. Our study provides a model for integrating protein structure, large-scale genetic sequencing, and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease.

Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments

The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes.

Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin.

ABSTRACT Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles. Summary: The underlying molecular basis of genetic-based cardiomyopathy diseases is largely unknown. This review describes recent molecular studies that have used human cardiac proteins to begin to elucidate the mechanisms whereby mutations cause disease.

Computational modeling of bone density profiles in response to gait: a subject-specific approach

The goal of this study is to explore the potential of computational growth models to predict bone density profiles in the proximal tibia in response to gait-induced loading. From a modeling point of view, we design a finite element-based computational algorithm using the theory of open system thermodynamics. In this algorithm, the biological problem, the balance of mass, is solved locally on the integration point level, while the mechanical problem, the balance of linear momentum, is solved globally on the node point level. Specifically, the local bone mineral density is treated as an internal variable, which is allowed to change in response to mechanical loading. From an experimental point of view, we perform a subject-specific gait analysis to identify the relevant forces during walking using an inverse dynamics approach. These forces are directly applied as loads in the finite element simulation. To validate the model, we take a Dual-Energy X-ray Absorptiometry scan of the subject’s right knee from which we create a geometric model of the proximal tibia. For qualitative validation, we compare the computationally predicted density profiles to the bone mineral density extracted from this scan. For quantitative validation, we adopt the region of interest method and determine the density values at fourteen discrete locations using standard and custom-designed image analysis tools. Qualitatively, our two- and three-dimensional density predictions are in excellent agreement with the experimental measurements. Quantitatively, errors are less than 3% for the two-dimensional analysis and less than 10% for the three-dimensional analysis. The proposed approach has the potential to ultimately improve the long-term success of possible treatment options for chronic diseases such as osteoarthritis on a patient-specific basis by accurately addressing the complex interactions between ambulatory loads and tissue changes.

Stretchable microelectrode array using room-temperature liquid alloy interconnects

In this paper, we present a stretchable microelectrode array for studying cell behavior under mechanical strain. The electrode array consists of gold-plated nail-head pins (250 µm tip diameter) or tungsten micro-wires (25.4 µm in diameter) inserted into a polydimethylsiloxane (PDMS) platform (25.4 × 25.4 mm2). Stretchable interconnects to the outside were provided by fusible indium-alloy-filled microchannels. The alloy is liquid at room temperature, thus providing the necessary stretchability and electrical conductivity. The electrode platform can withstand strains of up to 40% and repeated (100 times) strains of up to 35% did not cause any failure in the electrodes or the PDMS substrate. We confirmed biocompatibility of short-term culture, and using the gold pin device, we demonstrated electric field pacing of adult murine heart cells. Further, using the tungsten microelectrode device, we successfully measured depolarizations of differentiated murine heart cells from embryoid body clusters.

Planar patterned stretchable electrode arrays based on flexible printed circuits

For stretchable electronics to achieve broad industrial application, they must be reliable to manufacture and must perform robustly while undergoing large deformations. We present a new strategy for creating planar stretchable electronics and demonstrate one such device, a stretchable microelectrode array based on flex circuit technology. Stretchability is achieved through novel, rationally designed perforations that provide islands of low strain and continuous low-strain pathways for conductive traces. This approach enables the device to maintain constant electrical properties and planarity while undergoing applied strains up to 15%. Materials selection is not limited to polyimide composite devices and can potentially be implemented with either soft or hard substrates and can incorporate standard metals or new nano-engineered conductors. By using standard flex circuit technology, our planar microelectrode device achieved constant resistances for strains up to 20% with less than a 4% resistance offset over 120,000 cycles at 10% strain.

A Stretchable Cell Culture Platform with Embedded Electrode Array

In this paper, we present a stretchable electrode array for studying cell behavior subjected to mechanical strain. The electrode array consists of four gold nail-head pins (250¿m tip diameter and 1.75mm spacing) inserted into a polydimethylsiloxane (PDMS) platform (25.4×25.4mm2). Fusible indium alloy (liquid at room temperature) filled microchannels are used to connect the electrodes to the outside, thus providing the required stretchability. The electrode platform is biocompatible and can withstand strains of up to 40%. We tested these electrodes by repeatedly (100 times) subjecting them to 35% strain and did not notice any failure. We also successfully cultured mice cardiomyocytes onto the platform and performed electrical pacing.

Tools for Studying Biomechanical Interactions in Cells

Cells interact with their environment through forces that are generated and sensed by the cell. Forces generated by cells are in the few nanoNewton to several microNewton range and can change spatially over subcellular size scales. Transducing forces at such size and force scales requires development of platforms that can mechanically interface with cells. We describe several techniques that have been developed to study the role of mechanical forces in cellular processes. The measurement tools include those to measure the forces exerted by the cell on the extracellular environment, internal forces of contraction and the cytoskeletal properties.

Controlling all variables in an experiment

Recently, our students were attempting to experimentally measure a sphere’s moment of inertia by releasing a steel sphere from rest on a straight inclined channel. Why was there such disparity in results?