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.
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.
Rationale: Mechanical forces are transduced to nuclear responses via the linkers of the nucleoskeleton and cytoskeleton (LINC) complex, which couples the cytoskeleton to the nuclear lamina and associated chromatin. While disruption of the LINC complex can cause cardiomyopathy, the relevant interactions that bridge the nucleoskeleton to cytoskeleton are poorly understood in the cardiomyocyte, where cytoskeletal organization is unique. Furthermore, while microtubules and desmin intermediate filaments associate closely with cardiomyocyte nuclei, the importance of these interactions is unknown. Objective: Here, we sought to determine how cytoskeletal interactions with the LINC complex regulate nuclear homeostasis in the cardiomyocyte. Methods and Results: To this end, we acutely disrupted the LINC complex, microtubules, actin, and intermediate filaments and assessed the consequences on nuclear morphology and genome organization in rat ventricular cardiomyocytes via a combination of super-resolution imaging, biophysical, and genomic approaches. We find that a balance of dynamic microtubules and desmin intermediate filaments is required to maintain nuclear shape and the fidelity of the nuclear envelope and lamina. Upon depletion of desmin (or nesprin [nuclear envelope spectrin repeat protein]-3, its binding partner in the LINC complex), polymerizing microtubules collapse the nucleus and drive infolding of the nuclear membrane. This results in DNA damage, a loss of genome organization, and broad transcriptional changes. The collapse in nuclear integrity is concomitant with compromised contractile function and may contribute to the pathophysiological changes observed in desmin-related myopathies. Conclusions: Disrupting the tethering of desmin to the nucleus results in a loss of nuclear homeostasis and rapid alterations to cardiomyocyte function. Our data suggest that a balance of forces imposed by intermediate filaments and microtubules is required to maintain nuclear structure and genome organization in the cardiomyocyte.
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. mechanotransduction | excitable media | cardiac development | heartbeat | reaction-diffusion
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