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Background: Microtubules (MTs) buckle and bear load during myocyte contraction, a behavior enhanced by post-translational detyrosination. This buckling suggests a spring-like resistance against myocyte shortening, which could store energy and aid myocyte relaxation. Despite this visual suggestion of elastic behavior, the precise mechanical contribution of the cardiac MT network remains to be defined. Methods: Here we experimentally and computationally probe the mechanical contribution of stable MTs and their influence on myocyte function. We use multiple approaches to interrogate viscoelasticity and cell shortening in primary murine myocytes in which either MTs are depolymerized or detyrosination is suppressed and use the results to inform a mathematical model of myocyte viscoelasticity. Results: MT ablation by colchicine concurrently enhances both the degree of shortening and speed of relaxation, a finding inconsistent with simple spring-like MT behavior and suggestive of a viscoelastic mechanism. Axial stretch and transverse indentation confirm that MTs increase myocyte viscoelasticity. Specifically, increasing the rate of strain amplifies the MT contribution to myocyte stiffness. Suppressing MT detyrosination with parthenolide or via overexpression of tubulin tyrosine ligase has mechanical consequences that closely resemble colchicine, suggesting that the mechanical impact of MTs relies on a detyrosination-dependent linkage with the myocyte cytoskeleton. Mathematical modeling affirms that alterations in cell shortening conferred by either MT destabilization or tyrosination can be attributed to internal changes in myocyte viscoelasticity. Conclusions: The results suggest that the cardiac MT network regulates contractile amplitudes and kinetics by acting as a cytoskeletal shock-absorber, whereby MTs provide breakable cross-links between the sarcomeric and nonsarcomeric cytoskeleton that resist rapid length changes during both shortening and stretch.

The heart is the first organ to develop in vertebrate embryos, and coordinated beating of cardiomyocytes is likely facilitated by structural proteins of the extracellular matrix, cytoskeleton, and even the nucleus. We first show that the nucleus beats in the intact embryonic heart and that nuclear beating in vitro is regulated by matrix stiffness. However, it has been unclear when the nuclear structural protein Lamin-A turns on, which seems important because Lmna knockout mice are known to exhibit ‘developmental defects of the heart’ and die shortly after birth. We show that nuclear lamin levels parallel the development of mechanical stiffness and stress in normal embryos. While early embryos are well-known to be uniformly soft, our measurements using a chick embryo model show that the heart begins to stiffen almost immediately following initial differentiation, with tissue stiffness E of 1-2 kPa at embryonic day 4 (E4) increasing significantly by E6. This is accompanied by an increase in Lamin-A levels and comparatively constant Lamin-B expression, with Lamin-A:B stoichiometry already known to function as a mechanosensor in adult tissue [Swift, Science 2013]. Proteomic analyses of lamins from E4 to E10 indeed show that Lamin-A:B stoichiometry scales with stiffness as measured for adult tissue. Inhibition of actomyosin with blebbistatin reversibly reduces beating and contractility of isolated E4 hearts, leading to lower cytoskeletal tension and changes in lamin levels. Isolated cardiomyocytes beat optimally on gels that match the stiffness of embryonic hearts and nuclear beating follows the same trend, with ongoing studies focused on lamin responsiveness including phosphorylation and transcriptional regulation. Nuclear lamins are thus embryonic stress sensors that mechanically link to the cytoskeleton and the matrix, potentially feeding back to cytoskeletal maturation in differentiation.