Abstract
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.
Highlights
Physical inactivity has been correlated with cardiovascular disease, obesity, type II diabetes and some types of cancers [1]
Of the candidate genes identified, Dopamine Receptor 1 (Drd1; [1,6,7,8,9]), Glucose Transporter 4 [Glut4, aka: Slc2a4; 11], and Vesicular Monoamine Transporter 2 (Vmat2; [4,10,12,13]), have been the most widely studied in association with voluntary physical activity
Treatment with a Vivo-morpholino targeting Vmat2 did not result in a significant knockdown of Vmat2 expression in the nucleus accumbens of the brain four days after the last morpholino injection as compared to saline or scramble treatment (Figure 1, Panel A)
Summary
Physical inactivity has been correlated with cardiovascular disease, obesity, type II diabetes and some types of cancers [1]. Several potential candidate genes associated with the genetic influence on voluntary physical activity have been identified [1,4,5,6,7,8,9,10,11] with potential regulatory effects both in the brain and skeletal muscle. A loss of Vmat has been associated with a decrease in physical activity and development of Parkinson’s disease [10,13] Another central factor contributing the regulation of physical activity is Drd; high active mice have an under expression of Drd which results in a decrease in dopamine turnover in the nucleus accumbens [6] and a suggested increase in reward driven behavior and voluntary physical activity [6,7]. Glut transports glucose into skeletal muscle [14] and it has been shown that an over expression of Glut is associated with a fourfold increase in voluntary physical activity [11]
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