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Dysregulated mechano-activated calcium (Ca2+) and reactive oxygen species (ROS) signaling pathways underscore a growing number of diseases; however, a lack of mechanistic detail has limited the discovery of therapeutic targets. The cytoskeleton has garnered recent interest as it integrates and focuses mechanical stress on mechano-activated enzymes, ion channels, and proteins. In striated muscle, we recently discovered the microtubule network as the critical cytoskeletal element which activates X-ROS, a novel pathway in which the mechanical stress of stretch/contraction drives NADPH Oxidase 2 (NoX2) ROS production. In healthy muscle, X-ROS sensitizes the activation of ligand or stretch-activated Ca2+ channels and pharmacologic modulation of MT density ‘tunes’ X-ROS. In disparate models of muscular dystrophy, we find elevated MT network density drives excess X-ROS and Ca2+ signaling dysfunction. Acute pharmacologic ablation of MT network structure in diseased muscle revealed the MT network as a target with translational potential, however MT structure is critical to normal cellular function and significant MT ablation may limit this approach. We identified elevated levels of α-tubulin de-tyrosination - a post-translational modification (PTM) associated with increased mechanical properties of the MT network - in muscle exhibiting both enhanced MT density and X-ROS. In dystrophic mdx muscle, the pharmacologic reduction of α-tubulin de-tyrosination had no effect on the overall density or structure of the MT cytoskeleton, yet significantly reduced X-ROS and Ca2+ dysregulation in vitro and limited contraction-induced muscle injury in vivo. Measures of sarcomere and sub-sarcolemmal cytoskeletal mechanics revealed that a reduction in α-tubulin de-tyrosination decreased the mechanical stiffness of the MT cytoskeletal network. We conclude that α-tubulin de-tyrosination is a PTM that regulates the mechanosensitivity of striated muscle and propose that pharmacological targeting of this PTM will have broad therapeutic potential for the muscular dystrophies.

In striated muscle, we discovered that force transduced through the microtubule (MT) network activates NADPH Oxidase 2 (NoX2) ROS signals (i.e., X-ROS) that target calcium (Ca2+) channels. The significance of our discovery was revealed in dystrophin-deficient heart and skeletal muscle, where MT proliferation leads to an increase in both the cytoskeletal stiffness and the mechano-activated X-ROS and Ca2+ signaling that underscores disease progression and enhanced contraction-induced injury. Our most recent work (Kerr, J.P., et. al., Nat. Comm. http://dx.doi.org/10.1038/ncomms9526) implicated the abundance of detyrosinated MT filaments, rather than MT network proliferation per se, in the amplification of X-ROS mechanotransduction and depressed contractile mechanics through the alteration of cytoskeletal stiffness. Further, reducing MT detyrosination in vivo protected against contraction-induced injury in the mdx mouse, highlighting the significance of X-ROS and detyrosinated MT in the disease process. Here we extend our discovery by examining the passive mechanical properties of single, intact skeletal myofibers using novel technology and techniques. We show that the longitudinal stress-strain relationship and shear stiffness of the muscle fiber is regulated by the abundance of detyrosinated MTs. These results are consistent with the MT network acting as a compression-resistant element both within the myofilament lattice and beneath the sarcolemmal membrane, and that detyrosination of the MT network ‘tunes’ this behavior. Our ongoing studies will inform an integrated understanding and computational modeling of the cytoskeleton's role in cell mechanics and mechanotransduction, forwarding our goal of defining novel therapeutic targets for disease.

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.

In dystrophic muscle, an increase in reactive oxygen species (ROS) production and sarcolemmal calcium (Ca2+) influx contributes to stretch-induced muscle damage however mechanistic insights into the activation of these pathways is lacking. In mdx myofibers (murine Duchenne muscular dystrophy), we have demonstrated that with mechanical stretch, the microtubule (MT) cytoskeleton is a critical mechano-transduction element for the activation of NADPH oxidase2 (Nox2) derived ROS production; a pathway we term X-ROS signaling[1]. Downstream, we showed that X-ROS sensitized stretch activated channels (SACs) to increase sarcolemmal Ca2+ influx during stretch. The significance of the MT cytoskeleton activation of X-ROS in mdx was revealed when the acute targeting of MT density proffered protection from contraction induced damage. In mammalian cells, the MT network is a dynamic structure in which MT density is determined by the stability of MT filaments. Our initial studies used acute pharmacological stabilization (taxol) or destabilization (colchicine) to establish MT network density as critical for the mechano-activation of X-ROS. We now interrogate critical upstream pathways and use new pharmacological and molecular approaches to explore the role of endogenous modulators of MT stability and how they may contribute to the enhanced X-ROS in dystrophic skeletal muscle.1. Khairallah RJ, Shi G, Sbrana F, Prosser BL, Borroto C, Mazaitis MJ, Hoffman EP, Mahurkar A, Sachs F, Sun Y, Chen YW, Raiteri R, Lederer WJ, Dorsey SG, Ward CW (2012) Microtubules underlie dysfunction in Duchenne muscular dystrophy. Sci Signal 5: ra56.

Duchenne muscular dystrophy (DMD) is a common X-linked progressive muscle wasting disease characterized by an increased susceptibility to stretch-induced damage. While an abnormal regulation of reactive oxygen species (ROS) and calcium (Ca2+) signaling cascades are established contributors, little mechanistic insight has been revealed for how mechanical stress induces the dysregulation of these signaling pathways. With novel tools to mechanically manipulate enzymatically isolated skeletal muscle fibers, we show for the first time that a small physiologic stretch of a single mdx myofiber produces an almost instantaneous increase in ROS. The inhibition of NADPH oxidase 2 (NOX2) abrogated the stretch-induced ROS consistent with the recently reported X-ROS signaling in the heart (Prosser et al Science, 2011); however X-ROS was not detectable in wild-type fibers. Our group and others have demonstrated that the microtubule cytoskeleton is an important mechano-transduction element in muscle. Our investigation shows increased microtubule density in mdx muscle that displays X-ROS signaling. In this regard, microtubule depolymerization with colchicine was sufficient to inhibit X-ROS signaling in mdx myocytes implicating microtubules as critical proximate mechano-transducers in this pathway. Accordingly, proteins necessary for X-ROS (tubulin subunits and the NOX2 subunits gp91phox and rac1) are increased in dystrophic muscle. An important consequence of X-ROS signaling in mdx is increased sarcolemmal Ca2+ influx which is abrogated by the stretch-activated channel blocker GsMTx4 or microtubule destabilization. Lastly, we find increased surface membrane stiffness in mdx (measured by atomic force microscopy) suggesting that the abundance of microtubules contributes to important functional changes in skeletal muscle properties including stiffness which impacts X-ROS signaling. Our discoveries suggest that X-ROS may underscore the pathogenic stretch-dependent signaling in DMD and thus may provide novel therapeutic targets. Funding: RJK- T32AR007592, CWW- RC2 NR011968, WJL- R01 HL106059, R01 HL36974

Generation of reactive oxygen species (ROS) under physiological conditions is required for normal force production in skeletal muscle. However, high levels of ROS promote contractile dysfunction, resulting in muscle weakness and fatigue. Our recent studies suggest that sub-cellular site-specific ROS production governs the beneficial vs. damaging effects of ROS. NADPH oxidase (Nox2) is an enzyme complex that generates ROS. In this study we investigated the role of Nox2 in skeletal muscle EC coupling. Using sub-cellular site-specific redox probes we show that cytosolic glutathione redox potential becomes more oxidized during contractile activity while mitochondrial redox potential does not change. We show that increased contractile activity promotes Nox2 dependent ROS production. Interestingly, we have found that the non-tyrosine kinase Src is activated in response to increased contractile activity. These data support a hypothesis in which Src acts as a redox switch to activate Nox2. Increased ROS production during strenuous exercise would decrease sarcolemmal Ca2+ influx and decrease sarcoplasmic reticulum refilling, which could contribute to the development of fatigue. As trials of general antioxidant therapy to combat increased ROS production have not been convincingly beneficial, understanding the sub-cellular signaling pathways by which oxidants influence muscle function will allow for the development of targeted therapeutic interventions to combat the deleterious effects of sustained contractile activity as well as skeletal muscle diseases.

An acute physiologic stretch of a cardiomyocyte triggers an increase in the production of reactive oxygen species (ROS) by the membrane-localized enzyme complex NADPH oxidase 2 (Nox2, X-ROS signaling). The ROS act locally to sensitize nearby Ca2+ release channels in the sarcoplasmic reticulum, the ryanodine receptors type 2 (RyR2), resulting in a brief increase in the frequency of calcium sparks. During sustained, cyclical stretch the rate of ROS production remains elevated and is graded by both the degree and frequency of cyclic stretch. The elevated ROS production results in a sustained increase of calcium spark rate, thus coupling the mechanical load on the heart cell to its calcium signaling sensitivity. However, an increase in RyR2 sensitivity alone is insufficient to explain a persistent increase in Ca2+ sparks, implicating additional stretch-dependent players in the sustained elevation of calcium signaling sensitivity with increased mechanical stress. We initially investigated three potential players based on their previous links to mechanical stress and modulation by ROS signaling. Here we report on the contribution of nitric oxide (NO), Ca2+/Calmodulin-dependent kinase II (CaMKII), and mechano-sensitive channels (MSC) on stretch-dependent regulation of calcium signaling in ventricular myocytes. We find that both NO and CaMKII have little to no effect on the rapid acute increase in the Ca2+ spark rate with stretch (≤10s), but each contribute significantly to maintaining elevated calcium signaling sensitivity with prolonged cyclic stretch (≥1min). We also explore the role of ROS signaling in the stretch-dependent activation of these signaling pathways, and how this affects both diastolic calcium sparks as well as systolic calcium transients and contractility in electrically paced myocytes.