Microtubule Network Density Tunes Both Stretch and Contraction Activated X-ROS

Abstract

We recently identified the microtubule (MT) cytoskeleton (Khairallah et al, 2012) as the critical mechano-transduction element for stretch activated NADPH Oxidase 2 (NoX2) dependent ROS generation (i.e., X-ROS; Prosser et al, 2011) which activates mechano-sensitive Ca2+ influx. In WT skeletal muscle, X-ROS was minimal to undetectable with acute mechanical stretch but dramatically amplified by increasing MT density. In dystrophic myofibers (mdx), elevated X-ROS was coincident with increased MT density and ameliorated by depolymerizing MT (Khairallah et al, 2012, Prosser et al, 2013). Our data support the hypothesis that MT density tunes X-ROS activity and Ca2+ influx. These novel insights were possible because of our use of brief, acute stretch in single, enzymatically isolated skeletal myofibers - a model where baseline ROS generation and cytosolic [Ca2+] is low, allowing the identification of stretch-induced X-ROS and its subsequent Ca2+ influx. In vivo studies targeted MT density in mdx and blunted contraction-induced injury. We now report a similar effect in WT mice, indicating that contraction-activated X-ROS is operant in WT. However, our initial single cell approach could not support electrically-elicited contraction, leading us to develop novel technology and methodology that allows the reliable measurement of dynamic stretch and isometric contractility. We find that X-ROS and Ca2+ influx in WT are modulated by the frequency of passive stretch and electrically-elicited contraction. We are extending these studies with new genetic models and pharmacologic and molecular approaches that alter MT density to evaluate its contribution to mechano-activation of X-ROS and Ca2+ during dynamic stretch or contraction. We also show the contribution of MT density to the mechanical properties of the myofiber with measures of near-membrane stiffness by axial deformation (AFM) and passive stiffness during longitudinal stretch.Work funded by R01-AR062554, R01-HL106056.