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In response to mechanical loading of bone, osteocytes produce nitric oxide (NO•) and decrease sclerostin protein expression, leading to an increase in bone mass. However, it is unclear whether NO• production and sclerostin protein loss are mechanistically linked, and, if so, the nature of their hierarchical relationship within an established mechano-transduction pathway. Prior work showed that following fluid-shear stress (FSS), osteocytes produce NOX2-derived reactive oxygen species, inducing calcium (Ca2+) influx. Increased intracellular Ca2+ results in calcium-calmodulin dependent protein kinase II (CaMKII) activation, which regulates the lysosomal degradation of sclerostin protein. Here, we extend our discoveries, identifying NO• as a regulator of sclerostin degradation downstream of mechano-activated CaMKII. Pharmacological inhibition of nitric oxide synthase (NOS) activity in Ocy454 osteocyte-like cells prevented FSS-induced sclerostin protein loss. Conversely, short-term treatment with a NO• donor in Ocy454 cells or isolated murine long bones was sufficient to induce the rapid decrease in sclerostin protein abundance, independent of changes in Sost gene expression. Ocy454 cells express all three NOS genes, and transfection with siRNAs targeting eNOS/Nos3 was sufficient to prevent FSS-induced loss of sclerostin protein, while siRNAs targeting iNOS/Nos2 mildly blunted the loss of sclerostin but did not reach statistical significance. Similarly, siRNAs targeting both eNOS/Nos3 and iNOS/Nos2 prevented FSS-induced NO• production. Together, these data show iNOS/Nos2 and eNOS/Nos3 are the primary producers of FSS-dependent NO•, and that NO• is necessary and sufficient for sclerostin protein control. Further, selective inhibition of elements within this sclerostin-controlling mechano-transduction pathway indicated that NO• production occurs downstream of CaMKII activation. Targeting Camk2d and Camk2g with siRNA in Ocy454 cells prevented NO• production following FSS, indicating that CaMKII is needed for NO• production. However, NO• donation (1min) resulted in a significant increase in CaMKII activation, suggesting that NO• may have the ability to tune CaMKII response. Together, these data support that CaMKII is necessary for, and may be modulated by NO•, and that the interaction of these two signals is involved in the control of sclerostin protein abundance, consistent with a role in bone anabolic responses.

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Cellular mechanical properties influence cellular functions across pathological and physiological systems. The observation of these mechanical properties is limited in part by methods with a low throughput of acquisition or with low accessibility. To overcome these limitations, we have designed, developed, validated, and optimized a microfluidic cellular deformation system (MCDS) capable of mechanotyping suspended cells on a population level at a high throughput rate of ∼300 cells pers second. The MCDS provides researchers with a viable method for efficiently quantifying cellular mechanical properties towards defining prognostic implications of mechanical changes in pathology or screening drugs to modulate cytoskeletal integrity.

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Low bone mass is a pervasive global health concern, with implications for osteoporosis, frailty, disability, and mortality. Lifestyle factors, including sedentary habits, metabolic dysfunction, and an aging population, contribute to the escalating prevalence of osteopenia and osteoporosis. The application of mechanical load to bone through physical activity and exercise prevents bone loss, while sufficient mechanical load stimulates new bone mass acquisition. Osteocytes, cells embedded within the bone, receive mechanical signals and translate these mechanical cues into biological signals, termed mechano-transduction. Mechano-transduction signals regulate other bone resident cells, such as osteoblasts and osteoclasts, to orchestrate changes in bone mass. This review explores the mechanisms through which osteocyte-mediated response to mechanical loading regulates osteoblast differentiation and bone formation. An overview of bone cell biology and the impact of mechanical load will be provided, with emphasis on the mechanical cues, mechano-transduction pathways, and factors that direct progenitor cells toward the osteoblast lineage. While there are a wide range of clinically available treatments for osteoporosis, the majority act through manipulation of the osteoclast and may have significant disadvantages. Despite the central role of osteoblasts to the deposition of new bone, few therapies directly target osteoblasts for the preservation of bone mass. Improved understanding of the mechanisms leading to osteoblastogenesis may reveal novel targets for translational investigation.

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3D bioprinting holds great promise for meeting the increasing need for transplantable tissues and organs. However, slow printing, interlayer mixing, and the extended exposure of cells to non-physiological conditions in thick structures still hinder clinical applications. Here the DeepFreeze-3D (DF-3D) procedure and bioink for creating multilayered human-scale tissue mimetics is presented for the first time. The bioink is tailored to support stem cell viability, throughout the rapid freeform DF-3D biofabrication process. While the printer nozzle is warmed to room temperature, each layer solidifies at contact with the stage (-80 °C), or the subsequent layers, ensuring precise separation. After thawing, the encapsulated stem cells remain viable without interlayer mixing or delamination. The composed cell-laden constructs can be cryogenically stored and thawed when needed. Moreover, it is shown that under inductive conditions the stem cells differentiate into bone-like cells and grow for months after thawing, to form large tissue-mimetics in the scale of centimeters. This is important, as this approach allows the generation and storage of tissue mimetics in the size and thickness of human tissues. Therefore, DF-3D biofabrication opens new avenues for generating off-the-shelf human tissue analogs. It further holds the potential for regenerative treatments and for studying tissue pathologies caused by disease, tumor, or trauma.

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Bone regulates its mass and quality in response to diverse mechanical, hormonal, and local signals. The bone anabolic or catabolic responses to these signals are often received by osteocytes, which then coordinate the activity of osteoblasts and osteoclasts on bone surfaces. We previously established that calcium/calmodulin-dependent kinase 2 (CaMKII) is required for osteocytes to respond to some bone anabolic cues in vitro. However, a role for CaMKII in bone physiology in vivo is largely undescribed. Here, we show that conditional codeletion of the most abundant isoforms of CaMKII (delta and gamma) in mature osteoblasts and osteocytes [Ocn-cre:Camk2d/Camk2g double-knockout (dCKO)] caused severe osteopenia in both cortical and trabecular compartments by 8 wk of age. In addition to having less bone mass, dCKO bones are of worse quality, with significant deficits in mechanical properties, and a propensity to fracture. This striking skeletal phenotype is multifactorial, including diminished osteoblast activity, increased osteoclast activity, and altered phosphate homeostasis both systemically and locally. These dCKO mice exhibited decreased circulating phosphate (hypophosphatemia) and increased expression of the phosphate-regulating hormone fibroblast growth factor 23. Additionally, dCKO mice expressed less bone-derived tissue nonspecific alkaline phosphatase protein than control mice. Consistent with altered phosphate homeostasis, we observed that dCKO bones were hypo-mineralized with prominent osteoid seams, analogous to the phenotypes of mice with hypophosphatemia. Altogether, these data reveal a fundamental role for osteocyte CaMKIIδ and CaMKIIγ in the maintenance of bone mass and bone quality and link osteoblast/osteocyte CaMKII to phosphate homeostasis.

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The nuclear receptor peroxisome proliferator activated receptor-γ (PPARγ) is a key contributor to metabolic function via its adipogenic and insulin-sensitizing functions, but it has negative effects on skeletal homeostasis. Here, we questioned whether the skeletal and metabolic actions of PPARγ are linked. Ablating Pparg expression in osteoblasts and osteocytes produced a high bone mass phenotype, secondary to increased osteoblast activity, and a reduction in subcutaneous fat mass because of reduced fatty acid synthesis and increased fat oxidation. The skeletal and metabolic phenotypes in Pparg mutants proceed from the regulation of sclerostin production by PPARγ. Mutants exhibited reductions in skeletal Sost expression and serum sclerostin levels while increasing production normalized both phenotypes. Importantly, disrupting the production of sclerostin synergized with the insulin-sensitizing actions of a PPARγ agonist while preventing bone loss. These data suggest that modulating sclerostin action may prevent bone loss associated with anti-diabetic therapies and augment their metabolic actions.

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Skeletal muscle adapts to different exercise training modalities with age; however, the impact of both variables at the systemic and tissue levels is not fully understood. Here, adult and old C57BL/6 male mice were assigned to one of three groups: sedentary, daily high-intensity intermittent training (HIIT), or moderate intensity continuous training (MICT) for 4 weeks, compatible with the older group's exercise capacity. Improvements in body composition, fasting blood glucose, and muscle strength were mostly observed in the MICT old group, while effects of HIIT training in adult and old animals was less clear. Skeletal muscle exhibited structural and functional adaptations to exercise training, as revealed by electron microscopy, OXPHOS assays, respirometry, and muscle protein biomarkers. Transcriptomics analysis of gastrocnemius muscle combined with liver and serum metabolomics unveiled an age-dependent metabolic remodeling in response to exercise training. These results support a tailored exercise prescription approach aimed at improving health and ameliorating age-associated loss of muscle strength and function in the elderly.

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Methylsulfonylmethane (MSM) is a naturally occurring anti-inflammatory compound that effectively treats multiple degenerative diseases such as osteoarthritis and acute pancreatitis. Our previous studies have demonstrated the ability of MSM to differentiate stem cells from human exfoliated deciduous (SHED) teeth into osteoblast-like cells. This study examined the systemic effect of MSM in 36-week-old aging C57BL/6 female mice in vivo by injecting MSM for 13 weeks. Serum analyses showed an increase in expression levels of bone formation markers [osteocalcin (OCN) and procollagen type 1 intact N-terminal propeptide (P1NP)] and a reduction in bone resorption markers [tartrate-resistant acid phosphatase (TRAP) and C-terminal telopeptide of type I collag (CTX-I)] in MSM-injected animals. Micro-computed tomographic images demonstrated an increase in trabecular bone density in mandibles. The trabecular bone density tended to be higher in the femur, although the increase was not significantly different between the MSM- and phosphate-buffered saline (PBS)-injected mice. In mandibles, an increase in bone density with a corresponding decrease in the marrow cavity was observed in the MSM-injected mice. Furthermore, immunohistochemical analyses of the mandibles for the osteoblast-specific marker - OCN, and the mesenchymal stem cell-specific marker - CD105 showed a significant increase and decrease in OCN and CD105 positive cells, respectively. Areas of bone loss were observed in the inter-radicular region of mandibles in control mice. However, this loss was considerably decreased due to stimulation of bone formation in response to MSM injection. In conclusion, our study has demonstrated the ability of MSM to induce osteoblast formation and function in vivo, resulting in increased bone formation in the mandible. Hence, the application of MSM and stem cells of interest may be the right combination in alveolar bone regeneration under periodontal or other related diseases that demonstrate bone loss.

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Myosin binding protein-C slow (sMyBP-C) comprises a subfamily of cytoskeletal proteins encoded by MYBPC1 that is expressed in skeletal muscles where it contributes to myosin thick filament stabilization and actomyosin cross-bridge regulation. Recently, our group described the causal association of dominant missense pathogenic variants in MYBPC1 with an early-onset myopathy characterized by generalized muscle weakness, hypotonia, dysmorphia, skeletal deformities, and myogenic tremor, occurring in the absence of neuropathy. To mechanistically interrogate the etiologies of this MYBPC1-associated myopathy in vivo, we generated a knock-in mouse model carrying the E248K pathogenic variant. Using a battery of phenotypic, behavioral, and physiological measurements spanning neonatal to young adult life, we found that heterozygous E248K mice faithfully recapitulated the onset and progression of generalized myopathy, tremor occurrence, and skeletal deformities seen in human carriers. Moreover, using a combination of biochemical, ultrastructural, and contractile assessments at the level of the tissue, cell, and myofilaments, we show that the loss-of-function phenotype observed in mutant muscles is primarily driven by disordered and misaligned sarcomeres containing fragmented and out-of-register internal membranes that result in reduced force production and tremor initiation. Collectively, our findings provide mechanistic insights underscoring the E248K-disease pathogenesis and offer a relevant preclinical model for therapeutic discovery.

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L-plastin (LPL) was identified as a potential regulator of the actin-bundling process involved in forming nascent sealing zones (NSZs), which are precursor zones for mature sealing zones. TAT-fused cell-penetrating small molecular weight LPL peptide (TAT- MARGSVSDEE, denoted as an inhibitory LPL peptide) attenuated the formation of NSZs and impaired bone resorption in vitro in osteoclasts. Also, the genetic deletion of LPL in mice demonstrated decreased eroded perimeters and increased trabecular bone density. In the present study, we hypothesized that targeting LPL with the inhibitory LPL peptide in vivo could reduce osteoclast function and increase bone density in a mice model of low bone mass. We injected aging C57BL/6 female mice (36 weeks old) subcutaneously with the inhibitory and scrambled peptides of LPL for 14 weeks. Micro-CT and histomorphometry analyses demonstrated an increase in trabecular bone density of femoral and tibial bones with no change in cortical thickness in mice injected with the inhibitory LPL peptide. A reduction in the serum levels of CTX-1 peptide suggests that the increase in bone density is associated with a decrease in osteoclast function. No changes in bone formation rate and mineral apposition rate, and the serum levels of P1NP indicate that the inhibitory LPL peptide does not affect osteoblast function. Our study shows that the inhibitory LPL peptide can block osteoclast function without impairing the function of osteoblasts. LPL peptide could be developed as a prospective therapeutic agent to treat osteoporosis.

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Bone is a mechano-responsive tissue that adapts to changes in its mechanical environment. Increases in strain lead to increased bone mass acquisition, whereas decreases in strain lead to a loss of bone mass. Given that mechanical stress is a regulator of bone mass and quality, it is important to understand how bone cells sense and transduce these mechanical cues into biological changes to identify druggable targets that can be exploited to restore bone cell mechano-sensitivity or to mimic mechanical load. Many studies have identified individual cytoskeletal components - microtubules, actin, and intermediate filaments - as mechano-sensors in bone. However, given the high interconnectedness and interaction between individual cytoskeletal components, and that they can assemble into multiple discreet cellular structures, it is likely that the cytoskeleton as a whole, rather than one specific component, is necessary for proper bone cell mechano-transduction. This review will examine the role of each cytoskeletal element in bone cell mechano-transduction and will present a unified view of how these elements interact and work together to create a mechano-sensor that is necessary to control bone formation following mechanical stress.

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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.

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The decline in the mass and function of bone and muscle is an inevitable consequence of healthy aging with early onset and accelerated decline in those with chronic disease. Termed osteo-sarcopenia, this condition predisposes the decreased activity, falls, low-energy fractures, and increased risk of co-morbid disease that leads to musculoskeletal frailty. The biology of osteo-sarcopenia is most understood in the context of systemic neuro-endocrine and immune/inflammatory alterations that drive inflammation, oxidative stress, reduced autophagy, and cellular senescence in the bone and muscle. Here we integrate these concepts to our growing understanding of how bone and muscle senses, responds and adapts to mechanical load. We propose that age-related alterations in cytoskeletal mechanics alter load-sensing and mechano-transduction in bone osteocytes and muscle fibers which underscores osteo-sarcopenia. Lastly, we examine the evidence for exercise as an effective countermeasure to osteo-sarcopenia.

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OBJECTIVES: To evaluate the efficacy of intraoperative tobramycin powder in preventing surgical site infection (SSI) and implant colonization with Enterobacter cloacae in a rabbit fixation model. Gram-negative rods, particularly Enterobacter species, comprise an increasing percentage of SSI at our institution. METHODS: Eighteen New Zealand White rabbits underwent surgical fixation of the left tibia with implantation of a plate and screws. The surgical site and implant were inoculated with 1 × 107 CFUs E. cloacae. The selected E. cloacae isolate was resistant to tobramycin and capable of forming biofilms. Nine rabbits received 125 mg tobramycin powder directly into the surgical site, overlying the implant. The control group was untreated. Fourteen days postinfection, the tibiae and implants were explanted. Radiographs were taken with and without the implants in place. One tibia from each group was examined after hematoxylin and eosin staining. The remaining tibiae and implants were morselized or sonicated, respectively, and plated on agar to determine infection burden. Data were analyzed with Fisher exact tests and Mann-Whitney U tests. RESULTS: No bone infection or implant colonization occurred in the tobramycin-treated group. In the control group, 7 of 8 rabbits developed bone infections (P = 0.001), and 4 of 8 implants were colonized (P = 0.07). No gross disruption of the normal bone architecture was observed in either group. CONCLUSIONS: Intraoperative tobramycin powder applied at the time of contamination prevented bone infection with E. cloacae in this rabbit fixation model. The results are encouraging because the E. cloacae isolate was tobramycin-resistant, demonstrating the utility of intraoperative powdered antibiotics.

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Bone is a dynamic tissue that adapts to changes in its mechanical environment. Mechanical stimuli pressurize interstitial fluid in the lacunar-canalicular system within the bone matrix, causing fluid shear stress (FSS) across bone embedded, mechano-sensitive osteocytes. Therefore, modeling this mechanical stimulus in vitro is vital for identifying mechano-transduction cascades that contribute to the regulation of mechano-responsive proteins, such as the Wnt/ß-catenin antagonist, sclerostin, which is reduced in response to FSS. Recently, we reported the rapid post-translational degradation of sclerostin protein in bone cells following FSS. Given the fundamental nature of sclerostin to bone physiology and the nuances of studying its rapid post-translational control, here, we detail our FSS protocol, and adaptations that can be made, to stimulate Ocy454 osteocyte-like cells to study sclerostin protein in vitro. While this protocol is optimized for detecting sclerostin degradation by western blot, this protocol can be adapted to examine transcriptional changes with RT-qPCR, cellular dynamics with live cell imaging, or secreted factors in the FSS buffer. This protocol utilizes 3D-printed FSS tips that are compatible with commercially available 96-well plates, allowing for high experimental accessibility, versatility, and throughput. However, this protocol can be adapted for any FSS chamber. It can also be combined with pharmacological inhibitors or genetic manipulations to interrogate the role of specific cellular components. In all, this experimental set-up and protocol is highly adaptable to allow for many experimental outcomes to examine many aspects of cell mechano-transduction.

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Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

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Skeletal remodeling is driven in part by the osteocyte's ability to respond to its mechanical environment by regulating the abundance of sclerostin, a negative regulator of bone mass. We have recently shown that the osteocyte responds to fluid shear stress via the microtubule network-dependent activation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species and subsequent opening of TRPV4 cation channels, leading to calcium influx, activation of CaMKII, and rapid sclerostin protein downregulation. In addition to the initial calcium influx, purinergic receptor signaling and calcium oscillations occur in response to mechanical load and prior to rapid sclerostin protein loss. However, the independent contributions of TRPV4-mediated calcium influx and purinergic calcium oscillations to the rapid sclerostin protein downregulation remain unclear. Here, we showed that NOX2 and TRPV4-dependent calcium influx is required for calcium oscillations, and that TRPV4 activation is both necessary and sufficient for sclerostin degradation. In contrast, calcium oscillations are neither necessary nor sufficient to acutely decrease sclerostin protein abundance. However, blocking oscillations with apyrase prevented fluid shear stress induced changes in osterix (Sp7), osteoprotegerin (Tnfrsf11b), and sclerostin (Sost) gene expression. In total, these data provide key mechanistic insights into the way bone cells translate mechanical cues to target a key effector of bone formation, sclerostin.

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Bone resorption requires the formation of complex, actin-rich cytoskeletal structures. During the early phase of sealing ring formation by osteoclasts, L-plastin regulates actin-bundling to form the nascent sealing zones (NSZ). Here, we show that L-plastin knockout mice produce osteoclasts that are deficient in the formation of NSZs, are hyporesorptive, and make superficial resorption pits in vitro. Transduction of TAT-fused full-length L-plastin peptide into osteoclasts from L-plastin knockout mice rescued the formation of nascent sealing zones and sealing rings in a time-dependent manner. This response was not observed with mutated full-length L-plastin (Ser-5 and -7 to Ala-5 and -7) peptide. In contrast to the observed defect in the NSZ, L-plastin deficiency did not affect podosome formation or adhesion of osteoclasts in vitro or in vivo. Histomorphometry analyses in 8- and 12-week-old female L-plastin knockout mice demonstrated a decrease in eroded perimeters and an increase in trabecular bone density, without a change in bone formation by osteoblasts. This decrease in eroded perimeters supports that osteoclast function is attenuated in L-plastin knockouts. Micro-CT analyses confirmed a marked increase in trabecular bone mass. In conclusion, female L-plastin knockout mice had increased trabecular bone density due to impaired bone resorption by osteoclasts. L-plastin could be a potential target for therapeutic interventions to treat trabecular bone loss.

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Connexin43 is an important modulator of many signaling pathways in bone. ß-Catenin, a key regulator of the osteoblast differentiation and function, is among the pathways downstream of connexin43-dependent intercellular communication. There are striking overlaps between the functions of these two proteins in bone cells. However, differential effects of connexin43 on ß-catenin activity have been reported. Here, we examined how connexin43 influenced both Wnt-dependent and Wnt-independent activation of ß-catenin in osteoblasts in vitro. Our data show that loss of connexin43 in primary osteoblasts or connexin43 overexpression in UMR106 cells regulated active ß-catenin and phospho-Akt levels, with loss of connexin43 inhibiting and connexin43 overexpression increasing the levels of active ß-catenin and phospho-Akt. Increasing connexin43 expression synergistically enhanced Wnt3a-dependent activation of ß-catenin protein and ß-catenin transcriptional activity, as well as Wnt-independent activation of ß-catenin by prostaglandin E2 (PGE2). Finally, we show that the activation of ß-catenin by PGE2 required signaling through the phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3ß) pathway, as the PI3K inhibitor, LY-294002, disrupted the synergy between connexin43 and PGE2. These data show that connexin43 regulates Akt and ß-catenin activity and synergistically enhances both Wnt-dependent and Wnt-independent ß-catenin signaling in osteoblasts.

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Mechanical forces regulate muscle development, hypertrophy, and homeostasis. Force-transmitting structures allow mechanotransduction at the sarcolemma, cytoskeleton, and nuclear envelope. There is growing evidence that Yes-associated protein (YAP) serves as a nuclear relay of mechanical signals and can induce a range of downstream signaling cascades. Dystrophin is a sarcolemma-associated protein, and its absence underlies the pathology in Duchenne muscular dystrophy. We tested the hypothesis that the absence of dystrophin in muscle would result in reduced YAP signaling in response to loading. Following in vivo contractile loading in muscles of healthy (wild-type; WT) mice and mice lacking dystrophin (mdx), we performed Western blots of whole and fractionated muscle homogenates to examine the ratio of phospho (cytoplasmic) YAP to total YAP and nuclear YAP, respectively. We show that in vivo contractile loading induced a robust increase in YAP expression and its nuclear localization in WT muscles. Surprisingly, in mdx muscles, active YAP expression was constitutively elevated and unresponsive to load. Results from qRT-PCR analysis support the hyperactivation of YAP in vivo in mdx muscles, as evidenced by increased gene expression of YAP downstream targets. In vitro assays of isolated myofibers plated on substrates with high stiffness showed YAP nuclear labeling for both genotypes, indicating functional YAP signaling in mdx muscles. We conclude that while YAP signaling can occur in the absence of dystrophin, dystrophic muscles have altered mechanotransduction, whereby constitutively active YAP results in a failure to respond to load, which could be attributed to the increased state of "pre-stress" with increased cytoskeletal and extracellular matrix stiffness.

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