Skeletal muscle is the engine that produces force to power movement in humans and animals alike. To date the invasive nature of obtaining muscle-tendon forces in humans has limited our understanding of muscle function during coordinated locomotor tasks. Phenomenological, Hill-type models of skeletal muscle are often used, providing estimates of a muscle’s force as a function of its activation state, force-length, and force-velocity properties. However, few studies have examined the accuracy of whole muscle-tendon forces obtained from such models during in vivo motor tasks. The goal of my thesis was to develop, test, and refine methods to better quantify muscle mechanical output in humans, using ultrasound and electromyographic recordings, together with advanced Hill-type models. My first study developed techniques to non-invasively estimate in vivo Achilles tendon forces. I used ultrasound-based measures of tendon length and tendon mechanical properties to determine forces during cycling. In my second study, I compared gastrocnemii forces, predicted from a traditional Hill-type model with one contractile element, to force estimates derived from ultrasound-based tendon length changes. Because the traditional Hill-type model fails to account for variable activation states of different fibre types, I additionally tested a two-element model that includes both slow and fast contractile elements. I found that Hill-type models predicted 31-85% of the cyclists’ gastrocnemii forces across a range of conditions elicited, producing results comparable to those reported in animal models. Further, at higher cadences, the two-element model better estimated forces because it accounted for the increased recruitment of fast fibres. Traditional Hill-type models also neglect dynamic shape changes in contracting muscles, which may be important in modulating the velocities at which fascicles operate. My third study compared predictions of muscle architecture (fascicle lengths and pennation angles) generated from a 1D Hill-type model and additionally from 2D and 3D geometric models that allowed dynamic shape changes to occur. I found that the 1D model provided predictions of muscle architecture that were similar to the predictions of 2D and 3D models and that muscle shape changes and fascicle velocities were more closely linked to force than activation. Taken together, this research provides a non-invasive approach for studying in vivo muscle-tendon mechanics and testing the predictions of Hill-type models.
Cardiac Rehabilitation Programs (CRP) are effective behavioural interventions that reduce morbidity and mortality in patients with cardiovascular disease. Despite the myriad of benefits, participation remains sub-optimal with drop-out rates as high as 60%. Patients who discontinue CRP are under-treated and consequently, are at greater risk for further cardiac events. It is imperative to find alternative strategies to support to this high-risk population. The objectives of the present thesis were three fold: i) to identify baseline characteristics of participants who previously dropped-out of a CRP (chapter 2); ii) to assess self-efficacy among patients who complete a CRP versus those who drop-out (chapter 3); iii) to test the feasibility of an Internet-mediated VC intervention to provide ongoing psycho-social support among patients who previously dropped-out of a CRP (chapter 4).
Modeling a disease “in a dish,” a new research tool to study human heart disease mechanisms, is becoming as popular as more established techniques such as the use of transgenic mice. The primary practical challenge of this “disease in a dish” method is efficiently directing human induced pluripotent stem cell (hiPSC) differentiation into the desired lineages, with the major concern being the variability within hiPSC clones.To generate a reliable in vitro model for inherited cardiac diseases and address the variability problem, characteristics of hiPSCs derived from the blood of four human donors using both the episomal and Sendai virus reprogramming systems were examined. The hiPSC-cardiomyocytes (hiPSC-CMs) generated were then characterized according to their cardiac-specific gene expression properties. No differences were observed on the effect of the reprogramming system on expression of pluripotent genes in iPSCs but differences were observed in expression of cardiac specific genes in cardiomyocytes derived from those iPSCs despite a high variance in the analysis.
The palatine tonsils are a collection of lymph nodes overlaid by stratified non-keratinized epithelium that invaginates deep into the tissue, forming tonsillar “crypts” where ingested and inhaled pathogens are collected and initiate an immune response followed by transepithelial lymphocyte infiltration. The dynamic nature of this site suggests the existence of primitive cells responsible for the constant tissue repair and regeneration; however, such cells in the tonsils have not been characterized. Human Papillomavirus (HPV)-associated cancer of oropharynx is a global health concern that is on the rise, with HPV16 oncoproteins E6/E7 frequently detected in the epithelium of tonsillar crypts. It is hypothesized that the long-term self-renewing progenitor cells are the target of HPV-induced malignancy, but the lack of a method to specifically study these critical cells has been a barrier to further investigation. In this study, I have developed and optimized the methodology to identify, isolate and quantitate epithelial progenitors from human palatine tonsil. I show that tonsillar progenitors that form colonies in vitro in 2D colony assays and differentiate into multilayered epithelial tissues in a 3D culture system are CD44+NGFR+ and present in both surface and crypt regions. Transcriptome analysis indicates a high similarity between CD44+NGFR+ cells in both regions, although those isolated from the crypt contained a higher proportion of the most primitive (holo)clonogenic cells. The method was then applied to study effects of HPV infection on purified CD44+NGFR+ cells from both regions. Lentiviral transduction of CD44+NGFR+ cells with HPV16 E6/E7 oncogenes prolonged their growth in 2D cultures and caused aberrant differentiation in 3D cultures. Interestingly, in the presence of the normal cells, the E6/E7-transduced cells proliferated more slowly in 2D cultures and formed uniquely heterogeneous epithelial structures displaying varying degrees of perturbation in 3D cultures suggesting possible inhibitory effects of the cocultured normal cells. The system developed and presented in this thesis allows for a targeted approach to study a specific subset of epithelial cells purified from the tonsillar crypts and their response to E6/E7 infection, setting the stage for addressing many unanswered questions pertaining to the early stages of tonsillar oncogenesis.
The cardiac human ether-a-go-go related gene (hERG) channel is a voltage-gated potassium (Kv) channel that plays a fundamental role in cardiac repolarization. The importance of the hERG channel derives from its unusually slow activation (opening) and deactivation (closing) processes. Like other Kv channels, structural reconfigurations of the hERG voltage sensor upon membrane depolarization and repolarization underlie channel activation and deactivation, respectively. However, specific rearrangements of the voltage sensor that may dictate the unique slow activation and deactivation in hERG channels remain unclear. Lanthanide-based resonance transfer (LRET) is a spectroscopic technique that has previously demonstrated an ability to provide quantitative description of voltage sensor dynamics in an archetypal Kv channel. In this report, we outline a rationalized approach to applying LRET to examine hERG channel voltage sensor dynamics that may be of physiological significance. As a result, we report the first distance measurement from voltage sensors across the hERG channel pore.
Post-operative arrhythmias, such as Junctional Ectopic Tachycardia (JET) and atrioventricular (AV) block, are serious post-operative complications for children with congenital heart disease 1. We hypothesize that these arrhythmias arise within the AV node because of an ischemia-reperfusion (I/R) insult in the setting of immature myocytes, exacerbated by post-operative inotropy. Rabbit whole heart models of post-operative arrhythmias were generated, focusing on three primary risk factors: age, I/R exposure and the application of dopamine, an inotropic agent. Using optical mapping technology, neonatal rabbit hearts were found to experience persistent post-ischemia arrhythmias of differing severity while mature hearts exhibited no arrhythmias or transient ones, when the three risk factors were varied.Compared to rabbit mature hearts, rabbit neonatal whole hearts demonstrated a susceptibility to I/R insults resulting in alterations in automaticity. An analogous susceptibility may predispose human neonates to post-operative arrhythmias such as JET and AV block.
Forty-two percent of concussions in ice hockey are caused by hits involving shoulder-to-head contact. The goal of this project was to determine how shoulder pad stiffness affects head impact severity when players delivered checks to an instrumented dummy. Fifteen participants administered “the hardest shoulder checks they were comfortable delivering” to the head of an instrumented dummy. Trials were conducted with participants wearing two common types of shoulder pads, with and without a 2 cm thick layer of polyurethane foam over the shoulder pad cap. The study found that a 2 cm thick foam layer overlying the shoulder cap reduced peak linear accelerations to the head by 21.6-27.7%, peak rotational velocities by 10.5-13.8%, while causing no significant increase in shoulder impact velocity. Therefore, integration of foam padding on top of plastic caps warrants further examination as a method for preventing brain injuries in ice hockey.
Gene duplication results in extra copies of genes that can be sub-functionalized on structural and/or regulatory levels. Multiple paralogs are expressed in teleosts for troponin (Tn) components of the contractile unit (TnC, TnI and TnT), likely to maximize survival in different environmental conditions. The evolution of Tn subunits can be used as a model for understanding the variation in contractile function in ectotherms. The studies in this dissertation integrate evolutionary analysis with structural information to expand upon the knowledge of Tn function across phylogeny. Multiple parameters of cardiac structure and function were determined in vivo in the adult zebrafish, using high-resolution echocardiography, to accurately characterize the responses of this teleost model to both acute and chronic temperature perturbations. Cardiac output was modulated primarily by heart rate in response to acute temperature changes. With cold acclimation, a decreased E/A ratio suggests an increased reliance on atrial contraction for ventricular filling.The evolutionary history and sub-functionalization of the cardiac-specific TnC1 genes were characterized on both regulatory and structural levels. Three paralogs of TnC exist in fish, two of which are homologous to mammalian TnC1/cTnC. The TnC1 paralogs are likely the result of a tandem gene duplication that occurred in the common ancestor of the teleosts. In both zebrafish and trout hearts, TnC1 paralogs display temperature and chamber specific patterns in their usage of mRNA transcripts. While the zebrafish TnC1 paralogs have minimal variation in structure based on homology models, TnC1b has a higher Ca2+ affinity relative to TnC1a as measured by isothermal titration calorimetry. Variation in the apparent affinity of zebrafish TnC1 paralogs for Ca2+ results from dynamic conformational flexibility changes rather than from the direct interaction of site II with Ca2+. Finally, the inter-related roles of regulatory and structural sub-functionalization that guide the co-evolution of interacting proteins in the Tn complex were explored. Transcriptional expression patterns predict various TnC/TnI complexes exist in the zebrafish heart with differential interaction strengths between the N-TnC and TnI switch region. Domain-specific divergent selection pressures and interaction energies suggest that substitutions in the TnI switch region are crucial to modifying TnI/TnC function to maintain cardiac contraction with temperature changes. Through these studies, the interacting proteins of the Tn complex have been established as an important model of the functional divergence of paralogs in the adaptive evolution of the teleost cardiac contractile element.
Over 90% of hip fractures in older adults are due to falls. Wearable hip protectors have been shown in clinical trials to reduce the risk for hip fracture by up to 80% when worn, but user compliance with conventional garment-based hip protectors averages less than 50%. Improvements in product design may lead to enhanced compliance. This thesis describes the development and preliminary evaluation of usability in the acute care environment of a “stick-on” hip protector (secured over the hip with a skin-friendly adhesive). Through biomechanical testing, I developed a prototype that attenuates impact force by over 30% (higher than protectors currently used in Fraser Health). In a feasibility pilot trial, five of six patients wore the device for seven days. Additional input from 43 acute care providers during a Feedback Fair resulted in a 20 mm thick donut- shaped prototype of surface area 19x15.5 cm, that provided 36% force attenuation.
Groups of muscles are recruited in various combinations to perform smooth, controlled limb movements. Within these muscle groups, the excitation of a single muscle is commonly the focus of manipulation when attempting to influence limb movement, but it is the coordinated excitation of multiple muscles (muscle coordination) that ultimately determines the limb movement and mechanics. Despite successes with single muscles, the capabilities of manipulating muscle coordination are unknown. Therefore the goal of this research was to develop a biofeedback tool to purposefully manipulate muscle coordination.This research is comprised of four studies, three studies facilitated the development of a biofeedback tool and a fourth study finalized the development and validated the capabilities of the tool to purposefully manipulate muscle coordination in real-time during movement. The first study established a physiologically relevant outcome to be used by the biofeedback tool. The study showed that muscle excitation provides good predictions of changes in metabolic power and could therefore be used to determine the relative mechanical efficiency of different muscle coordination strategies.The second and third studies established a muscle coordination reference frame, specific to the relative efficiency outcome ascertained in the first study, used to characterize the current and desired end states of muscle coordination. Specifically, muscle coordination patterns, and their associated relative efficiencies, were determined across a range of mechanical demands to distinguish the key features responsible for differences in relative efficiency that were subsequently used to guide the biofeedback tool.In the final study, a novel biofeedback tool for manipulating muscle coordination was developed and validated. The underlying algorithm used principal component decomposition of muscle excitation to characterize the changes in coordination between the muscles at different mechanical demands. The algorithm was modified to render it feasible for implementation in real-time and the tool was validated by having subjects cycle while receiving feedback comparing their muscle coordination to the reference frame. The results showed that the subjects were successfully able to manipulate muscle coordination to improve the relative efficiency of the movement. Taken together, this research provides a valuable tool for research into motor learning and could be applied to improve rehabilitation and sport performance.