The adaptation of the skeleton to its mechanical environment is orchestrated by mechanosensitive osteocytes, largely by regulating the abundance of sclerostin, a secreted inhibitor of bone formation. We defined a microtubule-dependent mechanotransduction pathway that linked fluid shear stress to reactive oxygen species (ROS) and calcium (Ca²⁺) signals that led to a reduction in sclerostin abundance in cultured osteocytes. We demonstrated that microtubules stabilized by detyrosination, a reversible posttranslational modification of polymerized α-tubulin, determined the stiffness of the cytoskeleton, which set the mechanoresponsive range of cultured osteocytes to fluid shear stress. We showed that fluid shear stress through the microtubule network activated NADPH oxidase 2 (NOX2)–generated ROS that target the Ca²⁺ channel TRPV4 to elicit Ca²⁺ influx. Furthermore, tuning the abundance of detyrosinated tubulin affected cytoskeletal stiffness to define the mechanoresponsive range of cultured osteocytes to fluid shear stress. Finally, we demonstrated that NOX2-ROS elicited Ca²⁺ signals that activated the kinase CaMKII to decrease the abundance of sclerostin protein. Together, these discoveries may identify potentially druggable targets for regulating osteocyte mechanotransduction to affect bone quality.

Microtubules tune cytoskeletal stiffness, which affects cytoskeletal mechanics and mechanotransduction of striated muscle. While recent evidence suggests that microtubules enriched in detyrosinated α-tubulin regulate these processes in healthy muscle and increase them in disease, the possible contribution from several other α-tubulin modifications has not been investigated. Here, we used genetic and pharmacologic strategies in isolated cardiomyocytes and skeletal myofibers to increase the level of acetylated α-tubulin without altering the level of detyrosinated α-tubulin. We show that microtubules enriched in acetylated α-tubulin increase cytoskeletal stiffness and viscoelastic resistance. These changes slow rates of contraction and relaxation during unloaded contraction and increased activation of NADPH oxidase 2 (Nox2) by mechanotransduction. Together, these findings add to growing evidence that microtubules contribute to the mechanobiology of striated muscle in health and disease.

The downregulation of sclerostin in osteocytes mediates bone formation in response to mechanical cues and parathyroid hormone (PTH). To date, the regulation of sclerostin has been attributed exclusively to the transcriptional downregulation of the Sost gene hours after stimulation. Using mouse models and rodent cell lines, we describe the rapid, minute-scale post-translational degradation of sclerostin protein by the lysosome following mechanical load and PTH. We present a model, integrating both new and established mechanically and hormonally activated effectors into the regulated degradation of sclerostin by lysosomes. Using a mouse forelimb mechanical loading model, we find transient inhibition of lysosomal degradation or the upstream mechano-signaling pathway controlling sclerostin abundance impairs subsequent load-induced bone formation by preventing sclerostin degradation. We also link dysfunctional lysosomes to aberrant sclerostin regulation using human Gaucher disease iPSCs. These results reveal how bone anabolic cues post-translationally regulate sclerostin abundance in osteocytes to regulate bone formation.

Mechanistically driven therapies for atrial fibrillation (AF), the most common cardiac arrhythmia, are urgently needed, the development of which requires improved understanding of the cellular signaling pathways that facilitate the structural and electrophysiological remodeling that occurs in the atria. Similar to humans, increased persistent Na ⁺ current leads to the development of an atrial myopathy and spontaneous and long-lasting episodes of AF in mice. How increased persistent Na ⁺ current causes both structural and electrophysiological remodeling in the atria is unknown. We crossbred mice expressing human F1759A-Na _V 1.5 channels with mice expressing human mitochondrial catalase (mCAT). Increased expression of mCAT attenuated mitochondrial and cellular reactive oxygen species (ROS) and the structural remodeling that was induced by persistent F1759A-Na ⁺ current. Despite the heterogeneously prolonged atrial action potential, which was unaffected by the reduction in ROS, the incidences of spontaneous AF, pacing-induced after-depolarizations, and AF were substantially reduced. Expression of mCAT markedly reduced persistent Na ⁺ current–induced ryanodine receptor oxidation and dysfunction. In summary, increased persistent Na ⁺ current in atrial cardiomyocytes, which is observed in patients with AF, induced atrial enlargement, fibrosis, mitochondrial dysmorphology, early after-depolarizations, and AF, all of which can be attenuated by resolving mitochondrial oxidative stress.

Stromal interaction molecule 1 (STIM1), the sarcoplasmic reticulum (SR) transmembrane protein, activates store-operated Ca ²⁺ entry (SOCE) in skeletal muscle and, thereby, coordinates Ca ²⁺ homeostasis, Ca ²⁺ -dependent gene expression, and contractility. STIM1 occupies space in the junctional SR membrane of the triads and the longitudinal SR at the Z-line. How STIM1 is organized and is retained in these specific subdomains of the SR is unclear. Here, we identified desmin, the major type III intermediate filament protein in muscle, as a binding partner for STIM1 based on a yeast 2-hybrid screen. Validation of the desmin-STIM1 interaction by immunoprecipitation and immunolocalization confirmed that the CC1-SOAR domains of STIM1 interact with desmin to enhance STIM1 oligomerization yet limit SOCE. Based on our studies of desmin-KO mice, we developed a model wherein desmin connected STIM1 at the Z-line in order to regulate the efficiency of Ca ²⁺ refilling of the SR. Taken together, these studies showed that desmin-STIM1 assembles a cytoskeletal-SR connection that is important for Ca ²⁺ signaling in skeletal muscle.

Significance: In breast cancer, the chronically stiffening mechanical microenvironment promotes tumor growth/invasion. However, the molecular details and integration of cancer mechanotransduction signaling pathways are not well understood. We find that nontumorigenic breast epithelial cells are mechanically sensitive and respond to acute mechanical stimuli with the generation of ROS-stimulated calcium signaling in a microtubule-dependent manner. These results establish that epithelial cells conserve a mechanotransduction pathway from muscle and bone (X-ROS). Using common breast cancer genetic mutations, we further report that oncogene activation reduces X-ROS pathway mechanoresponsiveness at the level of ROS sensitivity. This definition of how oncogene activation disrupts highly conserved mechanotransduction signaling mechanisms could improve the understanding of altered tumor cell responses to the mechanical microenvironment and reveal new therapeutic opportunities.