David P. Ferguson

Strain screen and haplotype association mapping of wheel running in inbred mouse strains.

Previous genetic association studies of physical activity, in both animal and human models, have been limited in number of subjects and genetically homozygous strains used as well as number of genomic markers available for analysis. Expansion of the available mouse physical activity strain screens and the recently published dense single-nucleotide polymorphism (SNP) map of the mouse genome (approximately 8.3 million SNPs) and associated statistical methods allowed us to construct a more generalizable map of the quantitative trait loci (QTL) associated with physical activity. Specifically, we measured wheel running activity in male and female mice (average age 9 wk) in 41 inbred strains and used activity data from 38 of these strains in a haplotype association mapping analysis to determine QTL associated with activity. As seen previously, there was a large range of activity patterns among the strains, with the highest and lowest strains differing significantly in daily distance run (27.4-fold), duration of activity (23.6-fold), and speed (2.9-fold). On a daily basis, female mice ran further (24%), longer (13%), and faster (11%). Twelve QTL were identified, with three (on Chr. 12, 18, and 19) in both male and female mice, five specific to males, and four specific to females. Eight of the 12 QTL, including the 3 general QTL found for both sexes, fell into intergenic areas. The results of this study further support the findings of a moderate to high heritability of physical activity and add general genomic areas applicable to a large number of mouse strains that can be further mined for candidate genes associated with regulation of physical activity. Additionally, results suggest that potential genetic mechanisms arising from traditional noncoding regions of the genome may be involved in regulation of physical activity.

Lessons learned from vivo-morpholinos: How to avoid vivo-morpholino toxicity.

Vivo-morpholinos are a promising tool for gene silencing. These oligonucleotide analogs transiently silence genes by blocking either translation or pre-mRNA splicing. Little to no toxicity has been reported for vivo-morpholino treatment. However, in a recent study conducted in our lab, treatment of mice with vivo-morpholinos resulted in high mortality rates. We hypothesized that the deaths were the result of oligonucleotide hybridization, causing an increased cationic charge associated with the dendrimer delivery moiety of the vivo-morpholino. The cationic charge increased blood clot formation in whole blood treated with vivo-morpholinos, suggesting that clotting could have caused cardiac arrest in the deceased mice. Therefore, we investigate the mechanism by which some vivo-morpholinos increase mortality rates and propose techniques to alleviate vivo-morpholino toxicity.

Vivo-Morpholinos Induced Transient Knockdown of Physical Activity Related Proteins

Physical activity is associated with disease prevention and overall wellbeing. Additionally there has been evidence that physical activity level is a result of genetic influence. However, there has not been a reliable method to silence candidate genes in vivo to determine causal mechanisms of physical activity regulation. Vivo-morpholinos are a potential method to transiently silence specific genes. Thus, the aim of this study was to validate the use of Vivo-morpholinos in a mouse model for voluntary physical activity with several sub-objectives. We observed that Vivo-morpholinos achieved between 60–97% knockdown of Drd1-, Vmat2-, and Glut4-protein in skeletal muscle, the delivery moiety of Vivo-morpholinos (scramble) did not influence physical activity and that a cocktail of multiple Vivo-morpholinos can be given in a single treatment to achieve protein knockdown of two different targeted proteins in skeletal muscle simultaneously. Knocking down Drd1, Vmat2, or Glut4 protein in skeletal muscle did not affect physical activity. Vivo-morpholinos injected intravenously alone did not significantly knockdown Vmat2-protein expression in the brain (p = 0.28). However, the use of a bradykinin analog to increase blood-brain-barrier permeability in conjunction with the Vivo-morpholinos significantly (p = 0.0001) decreased Vmat2-protein in the brain with a corresponding later over-expression of Vmat2 coincident with a significant (p = 0.0016) increase in physical activity. We conclude that Vivo-morpholinos can be a valuable tool in determining causal gene-phenotype relationships in whole animal models.

Effects of Aromatase Inhibition on the Physical Activity Levels of Male Mice.

Increasing activity levels in an inactive population can lead to associative increases in health and well-being. Both biologic and genetic factors have been identified that alter physical activity levels in humans and rodents with an extensive early literature regarding sex steroid effects on physical activity. Currently, it is suggested that the androgens require conversion to estrogens prior to eliciting any effects on activity patterns. Recent data contradicts this assertion; thus, the purpose of this study was to evaluate the necessity of the aromatase complex in activity regulation. Wheel running was assessed in male C57BL/6J mice under various sex steroid-disrupted and aromatase-inhibited conditions. Inhibition of the aromatase complex was achieved through administration of two different aromatase inhibiting substances-letrozole and exemestane. Wheel running was unaffected by aromatase inhibition in reproductively intact and sex steroid supplemented mice. Orchidectomy significantly reduced wheel running activity. Steroid replacement recovered wheel running to pre-surgical levels; however, aromatase inhibition did not further affect wheel running levels. The recovery of wheel running in mice with androgen supplementation and the further persistence of wheel running in mice with compromised aromatase function suggests that the androgens-testosterone in particular-may directly affect wheel running patterns in male mice.

Differential skeletal muscle proteome of high- and low-active mice.

Physical inactivity contributes to cardiovascular disease, type II diabetes, obesity, and some types of cancer. While the literature is clear that there is genetic regulation of physical activity with existing gene knockout data suggesting that skeletal muscle mechanisms contribute to the regulation of activity, actual differences in end-protein expression between high- and low-active mice have not been investigated. This study used two-dimensional differential gel electrophoresis coupled with mass spectrometry to evaluate the proteomic differences between high-active (C57L/J) and low-active (C3H/HeJ) mice in the soleus and extensor digitorum longus (EDL). Furthermore, vivo-morpholinos were used to transiently knockdown candidate proteins to confirm their involvement in physical activity regulation. Proteins with higher expression patterns generally fell into the calcium-regulating and Krebs (TCA) cycle pathways in the high-active mice (e.g., annexin A6, P = 0.0031; calsequestrin 1; P = 0.000025), while the overexpressed proteins in the low-active mice generally fell into cytoskeletal structure- and electron transport chain-related pathways (e.g., ATPase, P = 0.031; NADH dehydrogenase, P = 0.027). Transient knockdown of annexin A6 and calsequestrin 1 protein of high-active mice with vivo-morpholinos resulted in decreased physical activity levels (P = 0.001). These data suggest that high- and low-active mice have unique protein expression patterns and that each pattern contributes to the peripheral capability to be either high- or low-active, suggesting that different specific mechanisms regulate activity leading to the high- or low-activity status of the animal.

Differential protein expression in the nucleus accumbens of high and low active mice.

Physical inactivity is associated with the development of a variety of chronic illnesses. Literature has shown that physical activity is genetically regulated; however there is limited information on the mechanisms that influence this process with existing studies primarily focused on genomic and/or transcription association studies. There have been no studies to determine differential protein expression in the nucleus accumbens, the brain site thought to be involved in activity regulation, between high and low active animals. We compared the global nucleus accumbens proteome signature from known high- and low-active mice and identified seven differentially expressed proteins. Low active mice generally over expressed proteins associated with neural stress (Stress 70 protein and V type proton ATPase catalytic subunit A), and the high-active mice over expressed proteins associated with metabolism (creatine kinase B, succinyl-CoA ligase). Previously suggested mechanisms associated with activity regulation in the nucleus accumbens have centered on dopamine receptor 1 and endocannabinoid receptor 1. However, these proteins and the associated pathways were not differentially expressed between high and low active mice. In conclusion, protein expression must be determined as part of the effort to identify involved mechanisms in regulating activity and there appears to be separate nucleus accumbens proteome signatures associated with high- and low-active mice.

Optimizing the Physical Conditioning of the NASCAR Sprint Cup Pit Crew Athlete

Abstract Ferguson, DP, Davis, AM, and Lightfoot, JT. Optimizing the physical conditioning of the NASCAR Sprint Cup Pit Crew athlete. J Strength Cond Res 29(3): 567–577, 2015—Stock car racing is the largest spectator sport in the United States. As a result, National Association for Stock Car Automobile Racing (NASCAR) Sprint Cup teams have begun to invest in strength and conditioning programs for their pit crew athletes. However, there is limited knowledge regarding the physical characteristics of elite NASCAR pit crew athletes, how the NASCAR Sprint Cup season affects basic physiological parameters such as body composition, and what is the most appropriate physical training program that meets the needs of a pit crew athlete. We conducted 3 experiments involving Sprint Cup motorsport athletes to determine predictors of success at the elite level, seasonal physiological changes, and appropriate physical training programs. Our results showed that hamstring flexibility (p = 0.015) and the score on the 2-tire front run test (p = 0.012) were significant predictors of NASCAR Sprint Cup Pit Crew athlete performance. Additionally, during the off season, pit crew athletes lost lean body mass, which did not return until the middle of the season. Therefore, a strength and conditioning program was developed to optimize pit crew athlete performance throughout the season. Implementation of this strength and conditioning program in 1 NASCAR Sprint Cup team demonstrated that pit crew athletes were able to prevent lean body mass loss and have increased muscle power output from the start of the season to the end of the season.