We report that in heart cells, physiologic stretch rapidly activates reduced-form nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) to produce reactive oxygen species (ROS) in a process dependent on microtubules (X-ROS signaling). ROS production occurs in the sarcolemmal and t-tubule membranes where NOX2 is located and sensitizes nearby ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR). This triggers a burst of Ca(2+) sparks, the elementary Ca(2+) release events in heart. Although this stretch-dependent "tuning" of RyRs increases Ca(2+) signaling sensitivity in healthy cardiomyocytes, in disease it enables Ca(2+) sparks to trigger arrhythmogenic Ca(2+) waves. In the mouse model of Duchenne muscular dystrophy, hyperactive X-ROS signaling contributes to cardiomyopathy through aberrant Ca(2+) release from the SR. X-ROS signaling thus provides a mechanistic explanation for the mechanotransduction of Ca(2+) release in the heart and offers fresh therapeutic possibilities.

INTRODUCTION-Along with its well-documented role as a track for cargo transport, the microtubule (MT) cytoskeleton is linked to diverse structural and signaling roles in the cardiac myocyte. MTs can facilitate the rapid transmission of mechanical signals to intracellular effectors, a process termed mechanotransduction. A proliferated MT network may also provide a mechanical resistance to cardiac contraction in certain disease states. Yet our understanding of how MTs resist compression and transmit mechanical signals has been impaired by a lack of direct observation and by the unpredictable effects of blunt pharmacological tools.

Duchenne muscular dystrophy (DMD) is a fatal X-linked degenerative muscle disease caused by the absence of the microtubule-associated protein dystrophin, which results in a disorganized and denser microtubule cytoskeleton. In addition, mechanotransduction-dependent activation of calcium (Ca2+) and reactive oxygen species (ROS) signaling underpins muscle degeneration in DMD. We show that in muscle from adult mdx mice, a model of DMD, a brief physiologic stretch elicited microtubule-dependent activation of NADPH (reduced-form nicotinamide adenine dinucleotide phosphate) oxidase–dependent production of ROS, termed X-ROS. Further, X-ROS amplified Ca2+ influx through stretch-activated channels in mdx muscle. Consistent with the importance of the microtubules to the dysfunction in mdx muscle, muscle cells with dense microtubule structure, such as those from adult mdx mice or from young wild-type mice treated with Taxol, showed increased X-ROS production and Ca2+ influx, whereas cells with a less dense microtubule network, such as young mdx or adult mdx muscle treated with colchicine or nocodazole, showed little ROS production or Ca2+ influx. In vivo treatments that disrupted the microtubule network or inhibited NADPH oxidase 2 reduced contraction-induced injury in adult mdx mice. Furthermore, transcriptome analysis identified increased expression of X-ROS–related genes in human DMD skeletal muscle. Together, these data show that microtubules are the proximate element responsible for the dysfunction in Ca2+ and ROS signaling in DMD and could be effective therapeutic targets for intervention.

Detyrosinated microtubules (MTs) provide mechanical resistance that can impede the motion of contracting cardiomyocytes. However, the functional effects of MT detyrosination in heart failure or in human hearts have not previously been studied. Here we utilize mass spectrometry and single-myocyte mechanical assays to characterize changes to the cardiomyocyte cytoskeleton and their functional consequences in human heart failure. Proteomic analysis of left ventricle tissue reveals a consistent upregulation and stabilization of intermediate filaments and MTs in failing human hearts. As revealed by super-resolution imaging, failing cardiomyocytes are characterized by a dense, heavily detyrosinated MT network, which is associated with increased myocyte stiffness and impaired contractility. Pharmacological suppression of detyrosinated MTs lowers the viscoelasticity of failing myocytes and restores 40–50% of lost contractile function; reduction of MT detyrosination using a genetic approach also softens cardiomyocytes and improves contractile kinetics. Together, these data demonstrate that a modified cytoskeletal network impedes contractile function in cardiomyocytes from failing human hearts and that targeting detyrosinated MTs could represent a new inotropic strategy for improving cardiac function.

In striated muscle, X-ROS is the mechanotransduction pathway by which mechanical stress transduced by the microtubule network elicits reactive oxygen species. X-ROS tunes Ca2+ signalling in healthy muscle, but in diseases such as Duchenne muscular dystrophy (DMD), microtubule alterations drive elevated X-ROS, disrupting Ca2+ homeostasis and impairing function. Here we show that detyrosination, a post-translational modification of α-tubulin, influences X-ROS signalling, contraction speed and cytoskeletal mechanics. In the mdx mouse model of DMD, the pharmacological reduction of detyrosination in vitro ablates aberrant X-ROS and Ca2+ signalling, and in vivo it protects against hallmarks of DMD, including workload-induced arrhythmias and contraction-induced injury in skeletal muscle. We conclude that detyrosinated microtubules increase cytoskeletal stiffness and mechanotransduction in striated muscle and that targeting this post-translational modification may have broad therapeutic potential in muscular dystrophies.