Development of microfluidic platforms for muscle strength and aging investigations in the model organism C. elegans
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Maintenance of physical fitness is essential for an individual’s health and well-being. While there are many aspects of physical fitness, a decline in muscle strength correlates with poor physical performance. Loss of muscle strength alone is a prognostic indicator for a variety of disorders including dynapenia, cancer, cardiovascular and neuromuscular diseases. Although it is straightforward to record muscle strength in people, it is not feasible to conduct whole life studies looking at the impact of genetics on muscle health. Promisingly, prospective life-long studies can be accomplished in the millimeter-sized roundworm C. elegans that is easy to manipulate genetically and has a short lifespan of ~3 weeks. The nematode body wall muscles have similarities to human muscle and strikingly deteriorate with age similar to humans, which makes C. elegans a premier genetic model for muscle strength and aging investigations. In this study, a soft deformable micropillar based microfluidic device, NemaFlex, is reported for quantifying muscle strength in C. elegans. Animals crawl through the pillars pushing them, allowing extraction of local forces from pillar displacements. It is expected that the forces fluctuate depending on animal’s behavior, velocity, body shape and position with respect to pillar, making quantitation of animal strength thus far elusive. Driven by the need to anchor NemaFlex for high throughput muscle strength assays, a robust experimental protocol and analysis workflow has been developed for quantifying maximum strength in C. elegans and its genetic mutants. It is found that forced contractions, such as induced by an acetylcholine agonist, show the same maximum strength as the untreated animals suggesting NemaFlex quantitates true strength. Mutants with neuronal defects (unc-17) and impaired sarcomeres (unc-52 and unc-112) have been tested and observed changes that verify the neuromuscular origin of strength. Also, it is found that the strength of C. elegans is a cubic function of its body diameter during its developmental period. The knowledge of how animal size affects muscle strength allows accurate comparisons among worms at different developmental stages or among mutants with size variations. NemaFlex has been configured into a simple and high-throughput lifespan measuring device called NemaLife, which is free from the limitations of using progeny blocking drugs, tedious worm picking, and high rate of matricide. The multifunctional capabilities of the device have been tested by conducting a pilot screen that includes wild-type, daf-2, daf-16, age-1 and eat-2 animals. Also, the lifespan curves obtained in a targeted RNAi screen are consistent with those of the genetic mutants. The device is equipped with unique capability of washing, delivering, and changing reagent at any time. This has been shown by inducing dietary restriction (DR) on wild-type worms and observing individual animals longitudinally and tracking physiological changes over age. The efficacy of DR has been demonstrated through the extension of worm lifespan and development at a single animal resolution with precise control of food. Thus, NemaLife will enable highly parallelized cross-sectional and longitudinal aging experiments especially when precise control of stimuli delivery is required. In addition to lifespan, NemaLife can make standard healthspan readouts such as locomotory prowess and pharyngeal pumping. Using NemaLife novel measures of healthspan such as muscle strength and agility can be recorded virtually from ‘womb to tomb’ providing insights into how muscle strength changes during development and declines with age. Profiling strength across the lifespan for individual worms show that strength increases 5-fold from the young adult followed by a sharp late-age decline (~45%)—providing the first direct evidence of muscle strength loss due to aging, similar to dynapenia in humans. In summary, NemaFlex and NemLife are powerful tools to conduct prospective life-long investigations of muscle strength and aging in C. elegans.