Duchenne Muscular Dystrophy (DMD) is an X-linked degenerative muscle disease that affects approximately 1 in 3500 male births worldwide. While substantial evidence exists for an enhancement in mechanical stress dependent activation of reactive oxygen signaling (ROS) and calcium influx pathways in the mdx, little mechanistic insight has been revealed. In this proposal, new technology developed by the PI, enables mechano- signaling to be assayed in single enzymatically isolated adult skeletal myofibers. With this approach, we have discovered that in the mdx, stretch induced a burst of ROS release from NADPH Oxidase 2 (NOX2) a pathway termed X-ROS signaling. In mdx skeletal muscle we show that stretched-induced X-ROS activates sarcolemmal stretch sensitive Ca2+ channels to elicit excessive Ca2+ influx across the sarcolemma. Furthermore, we show that the activation of X-ROS in the mdx is dependent on a significant microtubule densification. In support of this, we demonstrate an increase in near membrane cytoskeletal stiffness in mdx as measured by Atomic Force Microscopy (AFM). Finally, in new studies since the previous submission, we have used an established contraction injury assay to show that in vivo pharmacological targeting of X- ROS components (microtubule network or NOX2) prevents contraction-induced injury in the mdx muscle from adult mice. This finding supports the X-ROS signaling components as valid targets for therapeutic opportunity.
Recent work has identified that sarcolemmal Ca2+ influx and ROS (reactive oxygen species) generation is essential for this enhanced damage susceptibility in mdx skeletal muscle. Here we reveal our surprising and novel finding that the microtubule network is responsible for transmitting the mechanical stress of stretch to the respective ROS producing elements and mechano-sensitive stretch activated Ca2+ channels in the sarcolemmal membrane of dystrophic mdx muscle. Further, we show an increased expression microtubule network of proteins in the mdx and that disruption of this network abrogates the gain in stretch induced ROS and Ca2+ signaling in the mdx. We believe that the results of these studies will lead to important mechanistic findings that will be valuable as we seek to identify therapeutic targets for slowing or ameliorating the pathogenic progression of DMD.
|Lyons, James S; Iyer, Shama R; Lovering, Richard M et al. (2016) Novel multi-functional fluid flow device for studying cellular mechanotransduction. J Biomech 49:4173-4179|
|Ackermann, Maegen A; Kerr, Jaclyn P; King, Brendan et al. (2015) The Phosphorylation Profile of Myosin Binding Protein-C Slow is Dynamically Regulated in Slow-Twitch Muscles in Health and Disease. Sci Rep 5:12637|
|Kerr, Jaclyn P; Robison, Patrick; Shi, Guoli et al. (2015) Detyrosinated microtubules modulate mechanotransduction in heart and skeletal muscle. Nat Commun 6:8526|
|Ackermann, Maegen A; Ward, Christopher W; Gurnett, Christina et al. (2015) Myosin Binding Protein-C Slow Phosphorylation is Altered in Duchenne Dystrophy and Arthrogryposis Myopathy in Fast-Twitch Skeletal Muscles. Sci Rep 5:13235|
|Ward, Christopher W; Prosser, Benjamin L; Lederer, W Jonathan (2014) Mechanical stretch-induced activation of ROS/RNS signaling in striated muscle. Antioxid Redox Signal 20:929-36|
|Prosser, Benjamin L; Ward, Christopher W; Lederer, W Jonathan (2013) X-ROS signalling is enhanced and graded by cyclic cardiomyocyte stretch. Cardiovasc Res 98:307-14|
|Prosser, Benjamin L; Khairallah, Ramzi J; Ziman, Andrew P et al. (2013) X-ROS signaling in the heart and skeletal muscle: stretch-dependent local ROS regulates [Ca²?]i. J Mol Cell Cardiol 58:172-81|
|Khairallah, Ramzi J; Shi, Guoli; Sbrana, Francesca et al. (2012) Microtubules underlie dysfunction in duchenne muscular dystrophy. Sci Signal 5:ra56|