Patrick Robison

Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes

A close-up view of cardiac cell mechanics Heart cells contain a very well-organized array of cytoskeletal elements, including actin and microtubules that help them to perform their mechanical functions. Robison et al. used an advanced imaging approach to study the inner workings of mouse cardiac myocytes in real time. They observed microtubule “buckling” under contractile force in beating cardiomyocytes. This buckling was regulated by interaction with desmin and by the tubulin tyrosination state. The findings suggest a role for stable detyrosinated microtubules whose buckling under tension contributes to cardiac muscle strength. Science, this issue p. 10.1126/science.aaf0659 Posttranslational detyrosination of the microtubule network influences the mechanical properties of heart cells. 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. RATIONALE Direct observation of MT mechanical behavior during contraction is the most straightforward way to elucidate the mechanisms underlying MT contributions to heart function. Advances in imaging have made this possible at temporal and spatial resolutions that permit quantification of MT geometry during the contraction cycle. Furthermore, recent evidence suggests that posttranslational modification of the microtubule network, specifically “detyrosination,” regulates cardiac mechano-transduction. This raises the question of whether detyrosination alters how microtubules respond to the changing mechanical loads inherent to each cardiac cycle. To answer these questions, we used advanced imaging techniques to explore MT behavior in beating murine cardiomyocytes. RESULTS During contraction, MTs must somehow accommodate the changing geometry of the myocyte. In a typical myocyte, this was accomplished by deforming into a sinusoidal buckled configuration that returned to an identical resting configuration after each beat. The periodic nature of these buckles coincided with the repeating contractile units of the cardiomyocyte known as sarcomeres, which suggested a direct interaction. Desmin intermediate filaments were identified as a key component of an anchoring complex that links MTs to the sarcomere and imparts structural organization to the MT network. The physical link between microtubules and the sarcomere was highly dependent on detyrosination. In myocytes where detyrosination was suppressed, MTs often accommodated the contraction by sliding past each other rather than buckling as the sarcomere shortened. Disrupting the MT-sarcomere interaction allowed the sarcomere to shorten farther and faster, as well as decreased overall stiffness. Conversely, promoting detyrosination was sufficient to increase myocyte stiffness and impede the contraction of the myocyte. Consistently, clinical data showed a direct correlation between excess detyrosination and functional decline in patients with hypertrophic cardiomyopathy. CONCLUSION Thus, microtubules can provide mechanical resistance to the myocyte through interactions with the sarcomere, forming load-bearing spring elements in parallel with the contractile apparatus. These interactions are mediated by a detyrosination-dependent association with desmin that regulates myocyte stiffness and contractility. Excess detyrosination promotes the interaction between MTs and the sarcomere, which increases resistance to contraction and may contribute to reductions in cardiac function in certain disease states. MTs in the beating heart. When a cardiomyocyte (A) is compressed (B), as occurs during systolic contraction, MTs buckle under load. In a typical myocyte (C), detyrosinated MTs are mechanically coupled to the sarcomere and buckle during contraction (D). When detyrosination is reduced (E), this interaction is disrupted and MTs buckle less, which allows sarcomeres to shorten and stretch with less resistance. The microtubule (MT) cytoskeleton can transmit mechanical signals and resist compression in contracting cardiomyocytes. How MTs perform these roles remains unclear because of difficulties in observing MTs during the rapid contractile cycle. Here, we used high spatial and temporal resolution imaging to characterize MT behavior in beating mouse myocytes. MTs deformed under contractile load into sinusoidal buckles, a behavior dependent on posttranslational “detyrosination” of α-tubulin. Detyrosinated MTs associated with desmin at force-generating sarcomeres. When detyrosination was reduced, MTs uncoupled from sarcomeres and buckled less during contraction, which allowed sarcomeres to shorten and stretch with less resistance. Conversely, increased detyrosination promoted MT buckling, stiffened the myocyte, and correlated with impaired function in cardiomyopathy. Thus, detyrosinated MTs represent tunable, compression-resistant elements that may impair cardiac function in disease.

Detyrosinated microtubules modulate mechanotransduction in heart and skeletal muscle

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.

Mechanical signaling coordinates the embryonic heartbeat

Significance There is a mounting body of evidence that physical forces induce biochemical changes. Here, we suggest that the early embryonic heart provides a striking illustration of the importance of mechanics in living matter. Whereas adult hearts use electrical signaling to coordinate the heartbeat, we propose that embryonic hearts use mechanical signaling. We model the embryonic heart as mechanically excitable tissue, with cardiac myocytes that are triggered to contract under strain. Such contractions exert strains on nearby cells and induce further contraction, thus propagating the signal through the heart. This simple model captures key features observed in the heartbeat of stiffness-modified embryonic hearts that cannot be explained by standard electrochemical signaling and yields predictions that we confirm with experiments. In the beating heart, cardiac myocytes (CMs) contract in a coordinated fashion, generating contractile wave fronts that propagate through the heart with each beat. Coordinating this wave front requires fast and robust signaling mechanisms between CMs. The primary signaling mechanism has long been identified as electrical: gap junctions conduct ions between CMs, triggering membrane depolarization, intracellular calcium release, and actomyosin contraction. In contrast, we propose here that, in the early embryonic heart tube, the signaling mechanism coordinating beats is mechanical rather than electrical. We present a simple biophysical model in which CMs are mechanically excitable inclusions embedded within the extracellular matrix (ECM), modeled as an elastic-fluid biphasic material. Our model predicts strong stiffness dependence in both the heartbeat velocity and strain in isolated hearts, as well as the strain for a hydrogel-cultured CM, in quantitative agreement with recent experiments. We challenge our model with experiments disrupting electrical conduction by perfusing intact adult and embryonic hearts with a gap junction blocker, β-glycyrrhetinic acid (BGA). We find this treatment causes rapid failure in adult hearts but not embryonic hearts—consistent with our hypothesis. Last, our model predicts a minimum matrix stiffness necessary to propagate a mechanically coordinated wave front. The predicted value is in accord with our stiffness measurements at the onset of beating, suggesting that mechanical signaling may initiate the very first heartbeats.

Microtubule mechanics in the working myocyte

The mechanical role of cardiac microtubules (MTs) has been a topic of some controversy. Early studies, which relied largely on pharmacological interventions that altered the MT cytoskeleton as a whole, presented no consistent role. Recent advances in the ability to observe and manipulate specific properties of the cytoskeleton have strengthened our understanding. Direct observation of MTs in working myocytes suggests a spring‐like function, one that is surprisingly tunable by post‐translational modification (PTM). Specifically, detyrosination of MTs facilitates an interaction with intermediate filaments that complex with the sarcomere, altering myocyte stiffness, contractility, and mechanosignalling. Such results support a paradigm of cytoskeletal regulation based on not only polymerization, but also associations with binding partners and PTMs that divide the MT cytoskeleton into functionally distinct subsets. The evolutionary costs and benefits of tuning cytoskeletal mechanics remain an open question, one that we discuss herein. Nevertheless, mechanically distinct MT subsets provide a rich new source of therapeutic targets for a variety of phenomena in the heart.

Suppression of detyrosinated microtubules improves cardiomyocyte function in human heart failure

Detyrosinated microtubules provide mechanical resistance that can impede the motion of contracting cardiomyocytes. However, the functional effects of microtubule 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 microtubules in failing human hearts. As revealed by super-resolution imaging, failing cardiomyocytes are characterized by a dense, heavily detyrosinated microtubule network, which is associated with increased myocyte stiffness and impaired contractility. Pharmacological suppression of detyrosinated microtubules lowers the viscoelasticity of failing myocytes and restores 40–50% of lost contractile function; reduction of microtubule 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 microtubules could represent a new inotropic strategy for improving cardiac function.Post-translational modification of microtubules by detyrosination is prevalent in failing human cardiomyocytes and inhibits cardiomyocyte contraction, suggesting a new therapeutic strategy for improving heart function.

Impaired calcium signaling in muscle fibers from intercostal and foot skeletal muscle in a cigarette smoke‐induced mouse model of COPD

Respiratory and locomotor skeletal muscle dysfunction are common findings in chronic obstructive pulmonary disease (COPD); however, the mechanisms that cause muscle impairment in COPD are unclear. Because Ca2+ signaling in excitation–contraction (E‐C) coupling is important for muscle activity, we hypothesized that Ca2+ dysregulation could contribute to muscle dysfunction in COPD.