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Receptors for activated C kinases (RACKs) have been shown to coordinate PKC-mediated hypertrophic signalling in mice. However, little information is available on its participation in embryonic gene expression. This study investigated the involvement of RACK1 in the expression of embryonic genes in a zebrafish (ZF) ex vivo heart culture model by using phenylephrine (PE) or a growth factors cocktail (GFs) as a prohypertrophic/regeneration stimulus. Blebbistatin (BL) inhibition has also been studied for its ability to block the signal transduction actions of some PEs. qRT‒PCR and immunoblot analyses confirmed the upregulation of RACK1 in the PE- and GFs-treated groups. BL administration counteracted PE-induced hypertrophy and downregulated RACK1 expression. Immunohistochemical analyses of the heart revealed the colocalization of RACK1 and embryonic genes, namely, Gata4, Wt1, and Nfat2, under stimulation, whereas these genes were expressed at lower levels in the BL treatment group. Culturing ZF heart cells activated via GFs treatment increased the expression of RACK1. The overexpression of RACK1 induced by the transfection of recombinant RACK1 cDNA in ZF heart cells increased the expression of embryonic genes, especially after one week of GFs treatment. In summary, these results support the involvement of RACK1 in the induction of embryonic genes during cardiac hypertrophy/GFs stimulation in a fish heart model, which can be used as an alternative study model for mammals. [ABSTRACT FROM AUTHOR]

High-resolution respirometry in human permeabilized muscle fibers is extensively used for analysis of mitochondrial adaptions to nutrition and exercise interventions, and is linked to athletic performance. However, the lack of standardization of experimental conditions limits quantitative inter- and intra-laboratory comparisons. In our study, an international team of investigators measured mitochondrial respiration of permeabilized muscle fibers obtained from three biopsies (vastus lateralis) from the same healthy volunteer to avoid inter-individual variability. High-resolution respirometry assays were performed together at the same laboratory to assess whether the heterogenity in published results are due to the effects of respiration media (MiR05 versus Z) with or without the myosin inhibitor blebbistatin at low- and high-oxygen regimes. Our findings reveal significant differences between respiration media for OXPHOS and ETcapacities supported by NADH&succinate-linked substrates at different oxygen concentrations. Respiratory capacities were approximately 1.5-fold higher in MiR05 at high-oxygen regimes compared to medium Z near air saturation. The presence or absence of blebbistatin in human permeabilized muscle fiber preparations was without effect on oxygen flux. Our study constitutes a basis to harmonize and establish optimum experimental conditions for respirometric studies of permeabilized human skeletal muscle fibers to improve reproducibility. [Display omitted] • Understanding muscle energetics' link to athletic performance is crucial. • Harmonizing mitochondrial bioenergetics protocols is critical for inter-study data analysis. • Medium composition and oxygen concentration influence muscle mitochondria respiratory capacity in permeabilized fibers. • Optimizing experimental protocols is pivotal for precise athletic performance evaluation. [ABSTRACT FROM AUTHOR]

Introduction: Equid herpesvirus type 8 (EqHV-8) poses a significant threat to equine health, leading to miscarriages and respiratory diseases in horses and donkeys, and results in substantial economic losses in the donkey industry. Currently, there are no effective drugs or vaccines available for EqHV-8 infection control. Methods: In this study, we investigated the in vitro and in vivo antiviral efficacy of Blebbistatin, a myosin II ATPase inhibitor, against EqHV-8. Results: Our results demonstrated that Blebbistatin significantly inhibited EqHV-8 infection in Rabbit kidney (RK-13) and Madin-Darby Bovine Kidney (MDBK) cells in a concentration-dependent manner. Notably, Blebbistatin was found to disrupt EqHV-8 infection at the entry stage by modulating myosin II ATPase activity. Moreover, in vivo experiments revealed that Blebbistatin effectively reduced EqHV-8 replication and mitigated lung pathology in a mouse model. Conclusion: Collectively, these findings suggest that Blebbistatin holds considerable potential as an antiviral agent for the control of EqHV-8 infection, presenting a novel approach to addressing this veterinary challenge. [ABSTRACT FROM AUTHOR]

Zebrafish larvae have emerged as a valuable model for studying heart physiology and pathophysiology, as well as for drug discovery, in part thanks to its transparency, which simplifies microscopy. However, in fluorescence-based optical mapping, the beating of the heart results in motion artifacts. Two approaches have been employed to eliminate heart motion during calcium or voltage mapping in zebrafish larvae: the knockdown of cardiac troponin T2A and the use of myosin inhibitors. However, these methods disrupt the mechano-electric and mechano-mechanic coupling mechanisms. We have used ratiometric genetically encoded biosensors to image calcium in the beating heart of intact zebrafish larvae because ratiometric quantification corrects for motion artifacts. In this study, we found that halting heart motion by genetic means (injection of tnnt2a morpholino) or chemical tools (incubation with para-aminoblebbistatin) leads to bradycardia, and increases calcium levels and the size of the calcium transients, likely by abolishing a feedback mechanism that connects contraction with calcium regulation. These outcomes were not influenced by the calcium-binding domain of the gene-encoded biosensors employed, as biosensors with a modified troponin C (Twitch-4), calmodulin (mCyRFP1-GCaMP6f), or the photoprotein aequorin (GFP-aequorin) all yielded similar results. Cardiac contraction appears to be an important regulator of systolic and diastolic Ca2+ levels, and of the heart rate. [ABSTRACT FROM AUTHOR]

Zebrafish larvae have emerged as a valuable model for studying heart physiology and pathophysiology, as well as for drug discovery, in part thanks to its transparency, which simplifies microscopy. However, in fluorescence-based optical mapping, the beating of the heart results in motion artifacts. Two approaches have been employed to eliminate heart motion during calcium or voltage mapping in zebrafish larvae: the knockdown of cardiac troponin T2A and the use of myosin inhibitors. However, these methods disrupt the mechano-electric and mechano-mechanic coupling mechanisms. We have used ratiometric genetically encoded biosensors to image calcium in the beating heart of intact zebrafish larvae because ratiometric quantification corrects for motion artifacts. In this study, we found that halting heart motion by genetic means (injection of tnnt2a morpholino) or chemical tools (incubation with para-aminoblebbistatin) leads to bradycardia, and increases calcium levels and the size of the calcium transients, likely by abolishing a feedback mechanism that connects contraction with calcium regulation. These outcomes were not influenced by the calcium-binding domain of the gene-encoded biosensors employed, as biosensors with a modified troponin C (Twitch-4), calmodulin (mCyRFP1-GCaMP6f), or the photoprotein aequorin (GFP-aequorin) all yielded similar results. Cardiac contraction appears to be an important regulator of systolic and diastolic Ca2+ levels, and of the heart rate.

Optical mapping is a high-resolution fluorescence imaging technique, that uses voltage- or calcium-sensitive dyes to visualize electrical excitation waves on the heart surface. However, optical mapping is very susceptible to the motion of cardiac tissue, which results in so-called motion artifacts in the fluorescence signal. To avoid motion artifacts, contractions of the heart muscle are typically suppressed using pharmacological excitation-contraction uncoupling agents, such as Blebbistatin. The use of pharmacological agents, however, may influence cardiac electrophysiology. Recently, it has been shown that numerical motion tracking can significantly reduce motion-related artifacts in optical mapping, enabling the simultaneous optical measurement of cardiac electrophysiology and mechanics. Here, we combine ratiometric optical mapping with numerical motion tracking to further enhance the robustness and accuracy of these measurements. We evaluate the methods performance by imaging and comparing cardiac restitution and ventricular fibrillation (VF) dynamics in contracting, non-working vs. Blebbistatin-arrested Langendorff-perfused rabbit hearts (N = 10). We found action potential durations (APD) to be, on average, 25 ± 5% shorter in contracting hearts compared to hearts uncoupled with Blebbistatin. The relative shortening of the APD was found to be larger at higher frequencies. VF was found to be significantly accelerated in contracting hearts, i.e., 9 ± 2Hz with Blebbistatin and 15 ± 4Hz without Blebbistatin, and maintained a broader frequency spectrum. In contracting hearts, the average number of phase singularities was N PS = 11 ± 4 compared to N PS = 6 ± 3 with Blebbistatin during VF on the anterior ventricular surface. VF inducibility was reduced with Blebbistatin. We found the effect of Blebbistatin to be concentration-dependent and reversible by washout. Aside from the electrophysiological characterization, we also measured and analyzed cardiac motion. Our findings may have implications for the interpretation of optical mapping data, and highlight that physiological conditions, such as oxygenation and metabolic demand, must be carefully considered in ex vivo imaging experiments.

Objective To investigate the protective effect of blebbistatin, a non-muscle myosin Ⅱ (NMⅡ) inhibitor, on inflammatory injury of lung tissue after chronic common bile duct ligation (CBDL) in rats. Methods A total of 55 SPF male SD rats (200~220 g) were randomly divided into 3 groups, namely control group (Sham, n=11), high CBDL group (model group, n=22) and blebbistatin treatment after high CBDL group (treatment group, n=22). The treatment group was intraperitoneally injected with blebbistatin at a dose of 5 mg/kg once a day for a week, while the model group was only given the solvent (PEG-300∶saline=1∶1). The changes of lung tissue were observed with HE staining. Western blotting was performed to detect the expression of myeloperoxidase (MPO), IL-6, TNF-α, and superoxide dismutase (SOD3) in the lung tissues. The expression levels of MPO, IL-6, IL-1β, TNF-α, LY6B, CD163, and vascular endothelium related factor CD31 were also determined by immunohistochemical assay. Results The lung injury at the 3rd week after CBDL presented a large number of inflammatory cells infiltration, accompanied by local alveolar space and pulmonary interstitial hemorrhage. Immunohistochemical results demonstrated that the numbers of MPO positive cells, LY6B positive cells and CD163 positive cells were all increased significantly in the model group as compared with the Sham group (P < 0.01), with enhanced expression of IL-1β and TNF-α (P < 0.01). Western blotting results confirmed the up-regulated levels of MPO, IL-6 and SOD3 in the model group than the sham group (P < 0.05). In comparison with the model group, the treatment group showed decreased numbers of MPO positive cells, LY6B positive cells, and CD163 positive cells (P < 0.01), and declined levels of inflammatory factors IL-1β, IL-6, and TNF-α (P < 0.01). Western blotting indicated as well that the expression of MPO, IL-6 and TNF-α in the treatment group was reduced greatly (P < 0.05). Moreover, at the 5th week after surgery, the lung injury mainly manifested as pulmonary microvascular dilation, which was more severe in the model group than the Sham group (P < 0.01), while the severity of pulmonary microvascular dilatation was remarkably milder after treatment (P < 0.01). Conclusion Blebbistatin can effectively inhibit the infiltration of inflammatory cells in lung tissue, reduce pneumonia injury and alleviate pulmonary microvascular dilatation after CBDL.

Blebbistatin potently inhibits actin-myosin interaction, preventing contractile activity of excitable cells including cardiac myocytes, despite electrical excitation of an action potential (AP). We collected intracellular microelectrode recordings of pacemaker cells located in the sinoatrial region (SAR) of the zebrafish heart at room temperature and during acute warming to investigate whether or not blebbistatin inhibition of contraction significantly alters pacemaker cell electrophysiology. Changes were evaluated based on 16 variables that characterized the AP waveform. None of these AP variables nor the spontaneous heart rate were significantly modified with the application of 10 μM blebbistatin when recordings were made at room temperature. Compared with the control group, the blebbistatin-treated group showed minor changes in the rate of spontaneous diastolic depolarization (P = 0.027) and the 50% and 80% repolarization (P = 0.008 and 0.010, respectively) in the 26°C–29°C temperature bin, but not at higher temperatures. These findings suggest that blebbistatin is an effective excitation-contraction uncoupler that does not appreciably affect APs generated in pacemaking cells of the SAR and can, therefore, be used in zebrafish cardiac studies.

In mammals, prolonged mechanical unloading results in a significant decrease in passive stiffness of postural muscles. The nature of this phenomenon remains unclear. The aim of the present study was to investigate possible causes for a reduction in rat soleus passive stiffness after 7 and 14 days of unloading (hindlimb suspension, HS). We hypothesized that HS-induced decrease in passive stiffness would be associated with calpain-dependent degradation of cytoskeletal proteins or a decrease in actomyosin interaction. Wistar rats were subjected to HS for 7 and 14 days with or without PD150606 (calpain inhibitor) treatment. Soleus muscles were subjected to biochemical analysis and ex vivo measurements of passive tension with or without blebbistatin treatment (an inhibitor of actomyosin interactions). Passive tension of isolated soleus muscle was significantly reduced after 7- and 14-day HS compared to the control values. PD150606 treatment during 7- and 14-day HS induced an increase in alpha-actinin-2 and -3, desmin contents compared to control, partly prevented a decrease in intact titin (T1) content, and prevented a decrease in soleus passive tension. Incubation of soleus muscle with blebbistatin did not affect HS-induced reductions in specific passive tension in soleus muscle. Our study suggests that calpain-dependent breakdown of cytoskeletal proteins, but not a change in actomyosin interaction, significantly contributes to unloading-induced reductions in intrinsic passive stiffness of rat soleus muscle. [ABSTRACT FROM AUTHOR]

Right‐sided myocardial mechanical efficiency (work output/metabolic energy input) in pulmonary hypertension can be severely reduced. We determined the contribution of intrinsic myocardial determinants of efficiency using papillary muscle preparations from monocrotaline‐induced pulmonary hypertensive (MCT‐PH) rats. The hypothesis tested was that efficiency is reduced by mitochondrial dysfunction in addition to increased activation heat reported previously. Right ventricular muscle preparations were subjected to 5 Hz sinusoidal length changes at 37°C. Work and suprabasal oxygen consumption (V̇O2${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$) were measured before and after cross‐bridge inhibition by blebbistatin. Cytosolic cytochrome c concentration, myocyte cross‐sectional area, proton permeability of the inner mitochondrial membrane and monoamine oxidase and glucose 6‐phosphate dehydrogenase activities and phosphatidylglycerol/cardiolipin contents were determined. Mechanical efficiency ranged from 23% to 11% in control (n = 6) and from 22% to 1% in MCT‐PH (n = 15) and correlated with work (r2 = 0.68, P < 0.0001) but not with V̇O2${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ (r2 = 0.004, P = 0.7919). V̇O2${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ for cross‐bridge cycling was proportional to work (r2 = 0.56, P = 0.0005). Blebbistatin‐resistant V̇O2${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ (r2 = 0.32, P = 0.0167) and proton permeability of the mitochondrial inner membrane (r2 = 0.36, P = 0.0110) correlated inversely with efficiency. Together, these variables explained the variance of efficiency (coefficient of multiple determination r2 = 0.79, P = 0.0001). Cytosolic cytochrome c correlated inversely with work (r2 = 0.28, P = 0.0391), but not with efficiency (r2 = 0.20, P = 0.0867). Glucose 6‐phosphate dehydrogenase, monoamine oxidase and phosphatidylglycerol/cardiolipin increased in the right ventricular wall of MCT‐PH but did not correlate with efficiency. Reduced myocardial efficiency in MCT‐PH is a result of activation processes and mitochondrial dysfunction. The variance of work and the ratio of activation heat reported previously and blebbistatin‐resistant V̇O2${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ are discussed. Key points: Mechanical efficiency of right ventricular myocardium is reduced in pulmonary hypertension. Increased energy use for activation processes has been demonstrated previously, but the contribution of mitochondrial dysfunction is unknown.Work and oxygen consumption are determined during work loops. Oxygen consumption for activation and cross‐bridge cycling confirm the previous heat measurements.Cytosolic cytochrome c concentration, proton permeability of the mitochondrial inner membrane and phosphatidylglycerol/cardiolipin are increased in experimental pulmonary hypertension.Reduced work and mechanical efficiency are related to mitochondrial dysfunction.Upregulation of the pentose phosphate pathway and a potential gap in the energy balance suggest mitochondrial dysfunction in right ventricular overload is a resiult of the excessive production of reactive oxygen species. [ABSTRACT FROM AUTHOR]

Objective: To study the clinical characteristics, causative organism, treatment, and clinical outcomes of bleb-related infection. Materials and Methods: The medical charts of patients who were diagnosed with bleb-related infection, including blebitis and bleb-related endophthalmitis (BRE), from September 2001 to December 2019 at Siriraj Hospital were reviewed. The patients' demographic data, clinical characteristics, microorganisms found, treatment, and clinical outcomes were explored. Results: We found a total of 42 eyes from 41 patients had been diagnosed with blebitis (11 eyes) and BRE (31 eyes) over the 18-year study period. More than 80% of the patients had experienced pain and redness as the presenting symptoms. The most common bleb characteristic in BRE was purulent bleb (74.2%), while in blebitis it was bleb injection (45.5%). Bleb leakage was documented in 27.3% and 22.6% of patients with blebitis and BRE, respectively. Among the 41 patients, 10 had a history of minor trauma before the onset of infection, such as a rubbed eye, foreign body entering into the eye, water splashed into the eyes, or the eyes had been washed with soap. The yield of vitreous culture in bleb-related endophthalmitis was 48.3%. The most common microorganisms were Streptococcus spp., Enterococcus spp., and Haemophilus Influenzae. Generally, the treatment for blebitis at our institute is broad spectrum topical and systemic antibiotics, while intravitreal broad spectrum antibiotics are added to the treatment regimen for BRE patients. For BRE in our cohort, 11 eyes required vitrectomy and 7 eyes had undergone bleb excision. Treatment for blebitis tended to have a good visual outcome, with a stable visual prognosis for 9 out of the 11 eyes diagnosed with blebitis. However, most of the BRE eyes had a worsened visual outcome. Enterococcus spp. and Haemophilus Influenzae resulted in poor visual outcomes. Conclusion: The clinical characteristics of bleb-related infection in our patients were pain and redness. One fourth of patients had a history of minor trauma to the eye. To prevent bleb-related infection, the importance of patient education after trabeculectomy should be highlighted. Patients with presenting symptoms and unwanted behavior that could result in bleb infection should be identified and receive treatment alongside education. [ABSTRACT FROM AUTHOR]

Elbow trauma can lead to joint contracture and reduced range of motion (ROM). Nonsurgical interventions can improve ROM, but in some cases capsule release surgery is required. Although surgery can improve ROM, it often does not restore full ROM. Thus, alternatives are needed. One approach is to target activated myofibroblasts, which are commonly associated with fibrotic tissue. Mechanical and biochemical cues drive a feedback loop that can result in normal or pathological healing. We hypothesize that this feedback loop exists in joint contracture and can be manipulated so that myofibroblast activity is reduced, normal healing is achieved, and ROM is improved. We previously demonstrated that blebbistatin can inhibit myofibroblast contractile forces and reduce collagen synthesis in vitro. Thus, the purpose of this study was to assess the use of blebbistatin in an animal model of elbow contracture, which was induced in 7 groups of 4 rats each (n = 28). All elbows were mechanically and histologically tested. The uninjured contralateral elbows of each rat were used as a control group. Capsule release surgery significantly improved (p < 0.01) outcomes 1 week after surgery compared to injury alone and was not significantly different from uninjured elbows. Three weeks after surgery, outcomes worsened, indicating joint stiffening consistent with what is observed clinically. The addition of blebbistatin did not significantly improve outcomes. Future work will investigate relationships among treatment, fibrotic tissue deposition, myofibroblast activity, and biomechanics to determine if blebbistatin is a useful adjunctive therapy for treating joint contracture., (© 2024 The Author(s). Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Stretch-shortening cycles (SSCs) involve muscle lengthening (eccentric contractions) instantly followed by shortening (concentric contractions). This combination enhances force, work and power output compared with pure shortening contractions, which is known as the SSC effect. Recent evidence indicates both cross-bridge (XB)-based and non-XB-based (e.g. titin) structures contribute to this effect. This study analysed force re-development following SSCs and pure shortening contractions to gain further insight into the roles of XB and non-XB structures regarding the SSC effect. Experiments were conducted on rat soleus muscle fibres (n=16) with different SSC velocities (30%, 60% and 85% of maximum shortening velocity) and constant stretch-shortening magnitudes (18% of optimum length). The XB inhibitor blebbistatin was used to distinguish between XB and non-XB contributions to force generation. The results showed SSCs led to significantly greater [mean±s.d. 1.02±0.15 versus 0.68±0.09 (ΔF/Δt); t62=8.61, P<0.001, d=2.79) and faster (75 ms versus 205 ms; t62=-6.37, P<0.001, d=-1.48) force re-development compared with pure shortening contractions in the control treatment. In the blebbistatin treatment, SSCs still resulted in greater [0.11±0.03 versus 0.06±0.01 (ΔF/Δt); t62=8.00, P<0.001, d=2.24) and faster (3010±1631 versus 7916±3230 ms; t62=-8.00, P<0.001, d=-1.92) force re-development compared with pure shortening contractions. These findings deepen our understanding of the SSC effect, underscoring the involvement of non-XB structures such as titin in modulating force production. This modulation is likely to involve complex mechanosensory coupling from stretch to signal transmission during muscle contraction., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Cardiomyocyte cultures exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. In such preparations, beat rate variability exhibits features similar to those of heart rate variability in vivo. Mechanical deformations and forces feed back on the electrical properties of cardiomyocytes, but it is not fully elucidated how this mechano‐electrical interplay affects beating variability in such preparations. Using stretchable microelectrode arrays, we assessed the effects of the myosin inhibitor blebbistatin and the non‐selective stretch‐activated channel blocker streptomycin on beating variability and on the response of neonatal or fetal murine ventricular cell cultures against deformation. Spontaneous electrical activity was recorded without stretch and upon predefined deformation protocols (5% uniaxial and 2% equibiaxial strain, applied repeatedly for 1 min every 3 min). Without stretch, spontaneous activity originated from the edge of the preparations, and its site of origin switched frequently in a complex manner across the cultures. Blebbistatin did not change mean beat rate, but it decreased the spatial complexity of spontaneous activity. In contrast, streptomycin did not exert any manifest effects. During the deformation protocols, beat rate increased transiently upon stretch but, paradoxically, also upon release. Blebbistatin attenuated the response to stretch, whereas this response was not affected by streptomycin. Therefore, our data support the notion that in a spontaneously firing network of cardiomyocytes, active force generation, rather than stretch‐activated channels, is involved mechanistically in the complexity of the spatiotemporal patterns of spontaneous activity and in the stretch‐induced acceleration of beating. Key points: Monolayer cultures of cardiac cells exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. Beating variability in these preparations recapitulates the power‐law behaviour of heart rate variability in vivo. However, the effects of mechano‐electrical feedback on beating variability are not yet fully understood.Using stretchable microelectrode arrays, we examined the effects of the contraction uncoupler blebbistatin and the non‐specific stretch‐activated channel blocker streptomycin on beating variability and on stretch‐induced changes of beat rate.Without stretch, blebbistatin decreased the spatial complexity of beating variability, whereas streptomycin had no effects.Both stretch and release increased beat rate transiently; blebbistatin attenuated the increase of beat rate upon stretch, whereas streptomycin had no effects.Active force generation contributes to the complexity of spatiotemporal patterns of beating variability and to the increase of beat rate upon mechanical deformation. Our study contributes to the understanding of how mechano‐electrical feedback influences heart rate variability. [ABSTRACT FROM AUTHOR]

When a constraint is removed, confluent cells migrate directionally into the available space. How the migration directionality and speed increase are initiated at the leading edge and propagate into neighboring cells are not well understood. Using a quantitative visualization technique-Particle Image Velocimetry (PIV)-we revealed that migration directionality and speed had strikingly different dynamics. Migration directionality increases as a wave propagating from the leading edge into the cell sheet, while the increase in cell migration speed is maintained only at the leading edge. The overall directionality steadily increases with time as cells migrate into the cell-free space, but migration speed remains largely the same. A particle-based compass (PBC) model suggests cellular interplay (which depends on cell-cell distance) and migration speed are sufficient to capture the dynamics of migration directionality revealed experimentally. Extracellular Ca2+ regulated both migration speed and directionality, but in a significantly different way, suggested by the correlation between directionality and speed only in some dynamic ranges. Our experimental and modeling results reveal distinct directionality and speed dynamics in collective migration, and these factors can be regulated by extracellular Ca2+ through cellular interplay. Quantitative visualization using PIV and our PBC model thus provide a powerful approach to dissect the mechanisms of collective cell migration.

Tissue fibrosis is a major health issue that impacts millions of people and is costly to treat. However, few effective anti-fibrotic treatments are available. Due to their central role in fibrotic tissue deposition, fibroblasts and myofibroblasts are the target of many therapeutic strategies centered primarily on either inducing apoptosis or blocking mechanical or biochemical stimulation that leads to excessive collagen production. Part of the development of these drugs for clinical use involves in vitro prescreening. 2D screens, however, are not ideal for discovering mechanobiologically significant compounds that impact functions like force generation and other cell activities related to tissue remodeling that are highly dependent on the conditions of the microenvironment. Thus, higher fidelity models are needed to better simulate in vivo conditions and relate drug activity to quantifiable functional outcomes. To provide guidance on effective drug dosing strategies for mechanoresponsive drugs, we describe a custom force-bioreactor that uses a fibroblast-seeded fibrin gels as a relatively simple mimic of the provisional matrix of a healing wound. As cells generate traction forces, the volume of the gel reduces, and a calibrated and embedded Nitinol wire deflects in proportion to the generated forces over the course of 6 days while overhead images of the gel are acquired hourly. This system is a useful in vitro tool for quantifying myofibroblast dose-dependent responses to candidate biomolecules, such as blebbistatin. Administration of 50 μM blebbistatin reliably reduced fibroblast force generation approximately 40% and lasted at least 40 h, which in turn resulted in qualitatively less collagen production as determined via fluorescent labeling of collagen.

CDK5 belongs to the cyclin-dependent kinase family. CDK5 is multifunctional and plays an important role in neural differentiation. However, the role of CDK5 in osteoblastic differentiation remains unclear. The present study investigated functions and molecular mechanism of CDK5 in osteoblastic differentiation. It was found that, CDK5 inhibition promoted the expression of Runx2, ALP, OCN and OPN of MSCs and the mineralization of MC-3T3E1 cells and MSCs. CDK5 inhibition enhanced the development of F-actin, nuclear localization of ß-catenin and YAP, as well as the expression of RMRP RNA. When F-actin was suppressed by Blebbistatin, the nuclear localization of YAP and ß-catenin, and expression of RMRP RNA as well as Runx2 and ALP were decreased. These indicate Seliciclib promotes osteoblastic differentiation mainly by F-actin. Moreover, Seliciclib also suppressed the migration of MG-63, suggesting a potential application for Seliciclib in bone defect repair and inhibition of the migration of osteosarcoma cells after osteosarcoma surgical resection. [ABSTRACT FROM AUTHOR]

Cardiac excitation originates in the sinoatrial node (SAN), due to the automaticity of this distinct region of the heart. SAN automaticity is the result of a gradual depolarisation of the membrane potential in diastole, driven by a coupled system of transarcolemmal ion currents and intracellular Ca2+ cycling. The frequency of SAN excitation determines heart rate and is under the control of extra- and intracardiac (extrinsic and intrinsic) factors, including neural inputs and responses to tissue stretch. While the structure, function, and control of the SAN have been extensively studied in mammals, and some critical aspects have been shown to be similar in zebrafish, the specific drivers of zebrafish SAN automaticity and the response of its excitation to vagal nerve stimulation and mechanical preload remain incompletely understood. As the zebrafish represents an important alternative experimental model for the study of cardiac (patho-) physiology, we sought to determine its drivers of SAN automaticity and the response to nerve stimulation and baseline stretch. Using a pharmacological approach mirroring classic mammalian experiments, along with electrical stimulation of intact cardiac vagal nerves and the application of mechanical preload to the SAN, we demonstrate that the principal components of the coupled membrane- Ca2+ pacemaker system that drives automaticity in mammals are also active in the zebrafish, and that the effects of extra- and intracardiac control of heart rate seen in mammals are also present. Overall, these results, combined with previously published work, support the utility of the zebrafish as a novel experimental model for studies of SAN (patho-) physiological function. [ABSTRACT FROM AUTHOR]

Optical mapping is an imaging technique that is extensively used in cardiovascular research, wherein parameter-sensitive fluorescent indicators are used to study the electrophysiology and excitation-contraction coupling of cardiac tissues. Despite many benefits of optical mapping, eliminating motion artifacts within the optical signals is a major challenge, as myocardial contraction interferes with the faithful acquisition of action potentials and intracellular calcium transients. As such, excitation-contraction uncoupling agents are frequently used to reduce signal distortion by suppressing contraction. When compared with other uncoupling agents, blebbistatin is the most frequently used, as it offers increased potency with minimal direct effects on cardiac electrophysiology. Nevertheless, blebbistatin may exert secondary effects on electrical activity, metabolism, and coronary flow, and the incorrect administration of blebbistatin to cardiac tissue can prove detrimental, resulting in erroneous interpretation of optical mapping results. In this "Getting It Right" perspective, we briefly review the literature regarding the use of blebbistatin in cardiac optical mapping experiments, highlight potential secondary effects of blebbistatin on cardiac electrical activity and metabolic demand, and conclude with the consensus of the authors on best practices for effectively using blebbistatin in optical mapping studies of cardiac tissue. [ABSTRACT FROM AUTHOR]

Introduction: Equid herpesvirus type 8 (EqHV-8) poses a significant threat to equine health, leading to miscarriages and respiratory diseases in horses and donkeys, and results in substantial economic losses in the donkey industry. Currently, there are no effective drugs or vaccines available for EqHV-8 infection control., Methods: In this study, we investigated the in vitro and in vivo antiviral efficacy of Blebbistatin, a myosin II ATPase inhibitor, against EqHV-8., Results: Our results demonstrated that Blebbistatin significantly inhibited EqHV-8 infection in Rabbit kidney (RK-13) and Madin-Darby Bovine Kidney (MDBK) cells in a concentration-dependent manner. Notably, Blebbistatin was found to disrupt EqHV-8 infection at the entry stage by modulating myosin II ATPase activity. Moreover, in vivo experiments revealed that Blebbistatin effectively reduced EqHV-8 replication and mitigated lung pathology in a mouse model., Conclusion: Collectively, these findings suggest that Blebbistatin holds considerable potential as an antiviral agent for the control of EqHV-8 infection, presenting a novel approach to addressing this veterinary challenge., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Cui, Yu, Sun, Li, Li, Li, Chen, Khan, Wang and Wang.)

Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of the manual patch-clamp (MPC) technique, which is labor intensive and gives low output. However, the application of the APC platform is still limited in iPSC-CM based research, due to the difficulty in maintaining the high quality of single iPSC-CMs during dissociation and recording. In this study, we improved the method for single iPSC-CM preparation by applying 2.5 µM blebbistatin (BB, an excitation–contraction coupling uncoupler) throughout APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology. However, BB-supplement led to significant improvements in morphology and INa recording, and we even obtained several CMs that showed spontaneous action potentials with typical morphology. Furthermore, APC faithfully recapitulated the single-cell electrophysiological phenotypes of iPSC-CMs derived from Brugada syndrome patients, as detected with MPC. Our study indicates that APC is capable of replacing MPC in the modeling of cardiac channelopathies using human iPSC-CMs by providing high-quality data with higher throughput.

Studies on neural development and neuronal regeneration after injury are mainly based on animal models. The establishment of pluripotent stem cell (PSC) technology, however, opened new perspectives for better understanding these processes in human models by providing unlimited cell source for hard-to-obtain human tissues. Here, we aimed at identifying the molecular factors that confine and modulate an early step of neural regeneration, the formation of neurites in human neural progenitor cells (NPCs). Enhanced green fluorescent protein (eGFP) was stably expressed in NPCs differentiated from human embryonic and induced PSC lines, and the neurite outgrowth was investigated under normal and injury-related conditions using a high-content screening system. We found that inhibitors of the non-muscle myosin II (NMII), blebbistatin and its novel, non-toxic derivatives, initiated extensive neurite outgrowth in human NPCs. The extracellular matrix components strongly influenced the rate of neurite formation but NMII inhibitors were able to override the inhibitory effect of a restrictive environment. Non-additive stimulatory effect on neurite generation was also detected by the inhibition of Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), the upstream regulator of NMII. In contrast, inhibition of c-Jun N-terminal kinases (JNKs) had only a negligible effect, suggesting that the ROCK1 signal is dominantly manifested by actomyosin activity. In addition to providing a reliable cell-based in vitro model for identifying intrinsic mechanisms and environmental factors responsible for impeded axonal regeneration in humans, our results demonstrate that NMII and ROCK1 are important pharmacological targets for the augmentation of neural regeneration at the progenitor level. These studies may open novel perspectives for development of more effective pharmacological treatments and cell therapies for various neurodegenerative disorders.

Osteocytes differentiated from osteoblasts play significant roles as mechanosensors in modulating the bone remodeling process. While the well-aligned osteocyte network along the trabeculae with slender cell processes perpendicular to the trabeculae surface is known to facilitate the sensing of mechanical stimuli by cells and the intracellular communication in the bone matrix, the mechanisms underlying osteocyte network formation remains unclear. Here, we developed a novel in vitro collagen matrix system exerting a uniaxially-fixed mechanical boundary condition on which mouse osteoblast-like MC3T3-E1 cells were subcultured, evoking cellular alignment along the uniaxial boundary condition. Using a myosin II inhibitor, blebbistatin, we showed that the intracellular tension via contraction of actin fibers contributed to the cellular alignment under the influence of isometric matrix condition along the uniaxially-fixed mechanical boundary condition. Furthermore, the cells actively migrated inside the collagen matrix and promoted the expression of osteoblast and osteocyte genes with their orientations aligned along the uniaxially-fixed boundary condition. Collectively, our results suggest that the intracellular tension of osteoblasts under a uniaxially-fixed mechanical boundary condition is one of the factors that determines the osteocyte alignment inside the bone matrix. [ABSTRACT FROM AUTHOR]

Cultured cardiomyocytes have been shown to possess significant potential as a model for characterization of mechano-Ca2+, mechano-electric, and mechano-metabolic feedbacks in the heart. However, the majority of cultured cardiomyocytes exhibit impaired electrical, mechanical, biochemical, and metabolic functions. More specifically, the cells do not beat spontaneously (pacemaker cells) or beat at a rate far lower than their physiological counterparts and self-oscillate (atrial and ventricular cells) in culture. Thus, efforts are being invested in ensuring that cultured cardiomyocytes maintain the shape and function of freshly isolated cells. Elimination of contraction during culture has been shown to preserve the mechano-Ca2+, mechano-electric, and mechano-metabolic feedback loops of cultured cells. This review focuses on pacemaker cells, which reside in the sinoatrial node (SAN) and generate regular heartbeat through the initiation of the heart’s electrical, metabolic, and biochemical activities. In parallel, it places emphasis on atrial cells, which are responsible for bridging the electrical conductance from the SAN to the ventricle. The review provides a summary of the main mechanisms responsible for mechano-electrical, Ca2+, and metabolic feedback in pacemaker and atrial cells and of culture methods existing for both cell types. The work concludes with an explanation of how the elimination of mechano-electrical, mechano-Ca2+, and mechano-metabolic feedbacks during culture results in sustained cultured cell function.

Arterial thrombosis (AT) causes various ischemia-related diseases, which impose a serious medical burden worldwide. As an inhibitor of myosin II, blebbistatin has an important role in thrombosis development. We investigated the effect of blebbistatin on carotid artery ligation (CAL)-induced carotid AT and its potential underlying mechanism. A model of carotid AT in mice was generated by CAL. Mice were divided into three groups: CAL model, blebbistatin-treated, and sham-operation. After 7 days, blood vessels were harvested from mice in each group. The procoagulant activity of tissue factor (TF) was tested by a chromogenic assay, and thrombus severity assessed by histopathology scores. Expression of non-muscle myosin heavy chain II A (NMMHCIIA), TF, glycogen synthase kinase 3β (GSK3β), and nuclear factor-kappa B (NF-κB) was detected by immunohistochemical and immunofluorescence staining. mRNA expression was measured by quantitative polymerase chain reaction. Blebbistatin (1 mg/kg) inhibited development of carotid AT, reduced infiltration of inflammatory cells, and prevented vascular-tissue damage, relative to the model group. Furthermore, blebbistatin also reduced the procoagulant activity of TF. Immunohistochemical and immunofluorescence data demonstrated that, compared with the model group, blebbistatin intervention reduced expression of NMMHCIIA, TF, GSK3β, p65, and p-p65 in carotid-artery endothelia in the CAL-induced AT model, but it increased levels of p-GSK3β. Blebbistatin could inhibit expression of NMMHCIIA mRNA in the CAL model. Overall, our data demonstrated that blebbistatin could inhibit TF expression and AT development in arterial endothelia (at least in part) via GSK3β/NF-κB signaling.

Aging, noise, and ototoxic drug-induced hair cell (HC) loss are the major causes of sensorineural hearing loss. Aminoglycoside antibiotics are commonly used in the clinic, but these often have ototoxic side effects due to the accumulation of oxygen-free radicals and the subsequent induction of HC apoptosis. Blebbistatin is a myosin II inhibitor that regulates microtubule assembly and myosin–actin interactions, and most research has focused on its ability to modulate cardiac or urinary bladder contractility. By regulating the cytoskeletal structure and reducing the accumulation of reactive oxygen species (ROS), blebbistatin can prevent apoptosis in many different types of cells. However, there are no reports on the effect of blebbistatin in HC apoptosis. In this study, we found that the presence of blebbistatin significantly inhibited neomycin-induced apoptosis in HC-like HEI-OC-1 cells. We also found that blebbistatin treatment significantly increased the mitochondrial membrane potential (MMP), decreased ROS accumulation, and inhibited pro-apoptotic gene expression in both HC-like HEI-OC-1 cells and explant-cultured cochlear HCs after neomycin exposure. Meanwhile, blebbistatin can protect the synaptic connections between HCs and cochlear spiral ganglion neurons. This study showed that blebbistatin could maintain mitochondrial function and reduce the ROS level and thus could maintain the viability of HCs after neomycin exposure and the neural function in the inner ear, suggesting that blebbistatin has potential clinic application in protecting against ototoxic drug-induced HC loss.

Fibrosis is a complication of tendon injury where excessive scar tissue accumulates in and around the injured tissue, leading to painful and restricted joint motion. Unfortunately, fibrosis tends to recur after surgery, creating a need for alternative approaches to disrupt scar tissue. We posited a strategy founded on mechanobiological principles that collagen under tension generated by fibroblasts is resistant to degradation by collagenases. In this study, we tested the hypothesis that blebbistatin, a drug that inhibits cellular contractile forces, would increase the susceptibility of scar tissue to collagenase degradation. Decellularized tendon scaffolds (DTS) were treated with bacterial collagenase with or without external or cell-mediated internal tension. External tension producing strains of 2–4% significantly reduced collagen degradation compared with non-tensioned controls. Internal tension exerted by human fibroblasts seeded on DTS significantly reduced the area of the scaffolds compared to acellular controls and inhibited collagen degradation compared to free-floating DTS. Treatment of cell-seeded DTS with 50 mM blebbistatin restored susceptibility to collagenase degradation, which was significantly greater than in untreated controls (p < 0.01). These findings suggest that therapies combining collagenases with drugs that reduce cell force generation should be considered in cases of tendon fibrosis that do not respond to physiotherapy. [ABSTRACT FROM AUTHOR]

Aging, noise, and ototoxic drug-induced hair cell (HC) loss are the major causes of sensorineural hearing loss. Aminoglycoside antibiotics are commonly used in the clinic, but these often have ototoxic side effects due to the accumulation of oxygen-free radicals and the subsequent induction of HC apoptosis. Blebbistatin is a myosin II inhibitor that regulates microtubule assembly and myosin–actin interactions, and most research has focused on its ability to modulate cardiac or urinary bladder contractility. By regulating the cytoskeletal structure and reducing the accumulation of reactive oxygen species (ROS), blebbistatin can prevent apoptosis in many different types of cells. However, there are no reports on the effect of blebbistatin in HC apoptosis. In this study, we found that the presence of blebbistatin significantly inhibited neomycin-induced apoptosis in HC-like HEI-OC-1 cells. We also found that blebbistatin treatment significantly increased the mitochondrial membrane potential (MMP), decreased ROS accumulation, and inhibited pro-apoptotic gene expression in both HC-like HEI-OC-1 cells and explant-cultured cochlear HCs after neomycin exposure. Meanwhile, blebbistatin can protect the synaptic connections between HCs and cochlear spiral ganglion neurons. This study showed that blebbistatin could maintain mitochondrial function and reduce the ROS level and thus could maintain the viability of HCs after neomycin exposure and the neural function in the inner ear, suggesting that blebbistatin has potential clinic application in protecting against ototoxic drug-induced HC loss. [ABSTRACT FROM AUTHOR]

Phosphorylation of skeletal myosin regulatory light chain (RLC) occurs in fatigue and may play a role in the inhibition of shortening velocities observed in vivo. Forces and shortening velocities were measured in permeabilized rabbit psoas fibers with either phosphorylated or dephosphorylated RLCs and in the presence or absence of the myosin inhibitor blebbistatin. Addition of 20 μM blebbistatin decreased tensions by ~80% in fibers, independent of phosphorylation. In blebbistatin maximal shortening velocities (V max) at 30°C, were decreased by 45% (3.2 ± 0.34 vs. 5.8 ± 0.18 lengths/s) in phosphorylated fibers but were not inhibited in dephosphorylated fibers (6.0 ± 0.30 vs. 5.4 ± 0.30). In the presence of 20 μM blebbistatin, K m for V max as a function of [ATP] was lower for phosphorylated fibers than for dephosphorylated fibers (50 ± 20 vs. 330 ± 84 μM) indicating that the apparent binding of ATP is stronger in these fibers. Phosphorylation of RLC in situ during fiber preparation or by addition of myosin light chain kinase yielded similar data. RLC phosphorylation inhibited velocity in blebbistatin at both 30 and 10°C, unlike previous reports where RLC phosphorylation only affected shortening velocities at higher temperatures.

Proliferative vitreoretinopathy (PVR) is a common complication of rhegmatogenous retinal detachment, eventually leading to vision loss. To date, there are no effective drugs for the treatment of this disease. In this study, we investigated the effect of blebbistatin, a non-muscle myosin II inhibitor, on the ARPE-19 cell line and in a rabbit model of proliferative vitreoretinopathy. In vitro , we found that blebbistatin inhibited the epithelial-mesenchymal transition of retinal pigment epithelial (RPE) cells and inhibited the ability of RPE cells to migrate, proliferate, generate extracellular matrix, and affect contractility. In vivo the PVR model showed that blebbistatin significantly delayed PVR progression. It also partially prevents the loss of retinal function caused by PVR. Our results suggest that blebbistatin is a potential drug with clinical applications for the treatment of PVR. [Display omitted] • Blebbistatin was able to significantly inhibit the epithelial-mesenchymal transformation of ARPE-19 in vitro. • Blebbistatin could significantly slow down the progression of PVR and improve some visual dysfunction caused by PVR in the eye of the rabbit. • Cell mechanical contraction may be an important target for the treatment of PVR. [ABSTRACT FROM AUTHOR]

Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca++ probe as a small and mobile tracer in live T cells. Ca++, highlighted by Fluo-4, is localized in intracellular organelles. Inhibiting intracellular mechanical work by myosin II through blebbistatin treatment increased cellular dis-homogeneity of Ca++-rich features in length scale < 1.1 μm. We detected a similar effect in cells imaged by label-free bright-field (BF) microscopy, in mitochondria-highlighted cells and in ATP-depleted cells. Blebbistatin treatment also reduced the dynamics of the Ca++-rich features and generated prominent negative temporal correlations in their signals. Following Guggenberger et al. and numerical simulations, we suggest that diffusion in the viscoelastic and confined medium of intracellular organelles may promote spatial dis-homogeneity and stability of their content. This may be revealed only after inhibiting intracellular mechanical work and related cell vibrations. Our described mechanisms may allow the cell to control its organization via balancing its viscoelasticity and mechanical activity, with implications to cell physiology in health and disease.

Currently available data on the energetics of isolated muscle preparations are based on bouts of less than 10 muscle contractions, whereas metabolic energy consumption is mostly relevant during steady state tasks such as locomotion. In this study we quantified the energetics of small fiber bundles of mouse soleus muscle during prolonged (2 min) series of contractions. Bundles (N = 9) were subjected to sinusoidal length changes, while measuring force and oxygen consumption. Stimulation (five pulses at 100 Hz) occurred either during shortening or during lengthening. Movement frequency (2–3 Hz) and amplitude (0.25–0.50 mm; corresponding to ± 4–8% muscle fiber strain) were close to that reported for mouse soleus muscle during locomotion. The experiments were performed at 32°C. The contributions of cross-bridge cycling and muscle activation to total metabolic energy expenditure were separated using blebbistatin. The mechanical work per contraction cycle decreased sharply during the first 10 cycles, emphasizing the importance of prolonged series of contractions. The mean ± SD fraction of metabolic energy required for activation was 0.37 ± 0.07 and 0.56 ± 0.17 for concentric and eccentric contractions, respectively (both 0.25 mm, 2 Hz). The mechanical efficiency during concentric contractions increased with contraction velocity from 0.12 ± 0.03 (0.25 mm 2 Hz) to 0.15 ± 0.03 (0.25 mm, 3 Hz) and 0.16 ± 0.02 (0.50 mm, 2 Hz) and was -0.22 ± 0.08 during eccentric contractions (0.25 mm, 2 Hz). The percentage of type I fibers correlated positively with mechanical efficiency during concentric contractions, but did not correlate with the fraction of metabolic energy required for activation.

Background: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests., Objectives: To determine how diminished myosin activity affects platelet functions, including and beyond clot contraction., Methods: Using the myosin IIA-specific pharmacologic inhibitor blebbistatin, we modulated myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition by interrogating distinct platelet functions at each stage of thrombus formation using a range of complementary assays., Results: Partial myosin IIA inhibition neither affected platelet von Willebrand factor interactions under arterial shear nor platelet spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting platelet spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion, and platelet aggregation downstream of glycoprotein (GP)VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by adenosine diphosphate or arachidonic acid was unaffected., Conclusion: Our findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling., (© 2024 The Author(s).)

The stretching of a cardiomyocyte leads to the increased production of reactive oxygen species that increases ryanodine receptor open probability through a process termed X-ROS signaling. The stretching of the myocyte also increases the calcium affinity of myofilament Troponin C, which increases its calcium buffering capacity. Here, an integrative experimental and modeling study is pursued to explain the interplay of length-dependent changes in calcium buffering by troponin and stretch-activated X-ROS calcium signaling. Using this combination, we show that the troponin C-dependent increase in myoplasmic calcium buffering during myocyte stretching largely offsets the X-ROS-dependent increase in calcium release from the sarcoplasmic reticulum. The combination of modeling and experiment are further informed by the elimination of length-dependent changes to troponin C calcium binding in the presence of blebbistatin. Here, the model suggests that it is the X-ROS signaling-dependent Ca2+ release increase that serves to maintain free myoplasmic calcium concentrations during a change in myocyte length. Together, our experimental and modeling approaches have further defined the relative contributions of X-ROS signaling and the length-dependent calcium buffering by troponin in shaping the myoplasmic calcium transient.

Background: Aquatic species in several clades possess cement glands producing adhesive secretions of various strengths. In vertebrates, transient adhesive organs have been extensively studied in Xenopus laevis, other anurans, and in several fish species. However, the development of these structures is not fully understood. Results: Here, we report on the development and functional morphology of the adhesive gland of a giant danio species, Devario malabaricus. We found that the gland is localized on the larval head, is composed of goblet‐like secretory cells framed by basal, bordering, and intercalated apical epithelial cells, and is innervated by the trigeminal ganglion. The gland allows nonswimming larvae to adhere to various substrates. Its secretory cells differentiate by 12 hours postfertilization and begin to disappear in the second week of life. Exogenous retinoic acid disrupts the gland's patterning. More importantly, the single mature gland emerges from fusion of two differentiated secretory cells fields; this fusion is dependent on nonmuscle myosin II function. Conclusions: Taken together, our studies provide the first documentation of the embryonic development, structure, and function of the adhesive apparatus of a danioninae. To our knowledge, this is also the first report of a cement gland arising from convergence of two bilateral fields. Key Findings: The giant danio (Devario malabaricus) possesses a functional cement gland.Developmental and functional morphology of the cement gland.First evidence of a single mature cement gland emerging from the convergence and fusion of two differentiated secretory cells fields.Cement gland field fusion requires non-muscle myosin II function. [ABSTRACT FROM AUTHOR]

The aims of the study were to determine the effectiveness of blebbistatin (BLEB) on detrusor overactivity (DO) in an animal model induced by retinyl acetate (RA) and, because of potential urothelial permeability, to evaluate the degenerative impact of BLEB on the urothelium. Three days after RA instillation into the urinary bladder, BLEB was administered into the bladder and immediately after cystometric assessment was performed. Furthermore, Evans Blue extravasation into bladder tissue and urothelium thickness were measured. Sixty female Wistar rats were used and randomly assigned to one of four groups (n = 15 in each group): (1) control, (2) RA, (3) BLEB, and (4) RA + BLEB. RA administration induced changes in cystometric parameters reflecting DO, as previously reported. Treatment with BLEB did not significantly alter cystometric parameters in rats which did not receive RA. Administration of BLEB to rats pretreated with RA reversed changes in cystometric parameters induced by RA in basal pressure, threshold pressure, detrusor overactivity index, amplitude of nonvoiding contractions, frequency of nonvoiding contractions, voided volume, volume threshold, intercontraction interval, bladder compliance, and volume threshold to elicit nonvoiding contractions. There were no significant differences in Evans Blue extravasation into bladder tissue or urothelium thickness between the groups. The current research provides new data on the possible utility of blebbistatin in the pharmacotherapy of DO, which is an important feature of overactive bladder (OAB). Further studies in human patients with DO/OAB are warranted to confirm these preclinical results. [ABSTRACT FROM AUTHOR]

Currently available data on the energetics of isolated muscle preparations are based on bouts of less than 10 muscle contractions, whereas metabolic energy consumption is mostly relevant during steady state tasks such as locomotion. In this study we quantified the energetics of small fiber bundles of mouse soleus muscle during prolonged (2 min) series of contractions. Bundles (N = 9) were subjected to sinusoidal length changes, while measuring force and oxygen consumption. Stimulation (five pulses at 100 Hz) occurred either during shortening or during lengthening. Movement frequency (2–3 Hz) and amplitude (0.25–0.50 mm; corresponding to ± 4–8% muscle fiber strain) were close to that reported for mouse soleus muscle during locomotion. The experiments were performed at 32°C. The contributions of cross-bridge cycling and muscle activation to total metabolic energy expenditure were separated using blebbistatin. The mechanical work per contraction cycle decreased sharply during the first 10 cycles, emphasizing the importance of prolonged series of contractions. The mean ± SD fraction of metabolic energy required for activation was 0.37 ± 0.07 and 0.56 ± 0.17 for concentric and eccentric contractions, respectively (both 0.25 mm, 2 Hz). The mechanical efficiency during concentric contractions increased with contraction velocity from 0.12 ± 0.03 (0.25 mm 2 Hz) to 0.15 ± 0.03 (0.25 mm, 3 Hz) and 0.16 ± 0.02 (0.50 mm, 2 Hz) and was -0.22 ± 0.08 during eccentric contractions (0.25 mm, 2 Hz). The percentage of type I fibers correlated positively with mechanical efficiency during concentric contractions, but did not correlate with the fraction of metabolic energy required for activation. [ABSTRACT FROM AUTHOR]

Small intestinal organoids have become an important tool to study crypt homeostasis, cell fate dynamics and tissue biomechanics. Yet, the mechanisms that drive the budding of crypts from the smooth organoid epithelium remain incompletely understood. Locally enhanced proliferation has been suggested to induce tissue buckling and crypt initiation. Here we report that changes in cell morphology play a crucial role in crypt formation. Crypt formation is preceded by local epithelial thickening, apicobasal elongation, and apical narrowing, resulting in a wedge-like cell-shape, followed by apical evagination and crypt outgrowth. Myosin II activity is found to coincide with apical constriction of cells, while inhibition of myosin suppresses apical constriction and bud formation. The data suggest that myosin-driven apical constriction is a key driving force of bud initiation in small intestinal organoids. • Budding crypts in intestinal organoids show cellular elongation, apical narrowing, and tilt. • Myosin inhibition stalls, but does not revert apical constriction and crypt initiation. • Data suggests long-term role of myosin in crypt maintenance. [ABSTRACT FROM AUTHOR]

Force enhancement (FE) is a phenomenon that is present in skeletal muscle. It is characterized by progressive forces upon active stretching—distinguished by a linear rise in force—and enhanced isometric force following stretching (residual FE (RFE)). In skeletal muscle, non-cross-bridge (XB) structures may account for this behaviour. So far, it is unknown whether differences between non-XB structures within the heart and skeletal muscle result in deviating contractile behaviour during and after eccentric contractions. Thus, we investigated the force response of intact cardiac trabeculae during and after isokinetic eccentric muscle contractions (10% of maximum shortening velocity) with extensive magnitudes of stretch (25% of optimum muscle length). The different contributions of XB and non-XB structures to the total muscle force were revealed by using an actomyosin inhibitor. For cardiac trabeculae, we found that the force–length dynamics during long stretch were similar to the total isometric force–length relation. This indicates that no (R)FE is present in cardiac muscle while stretching the muscle from 0.75 to 1.0 optimum muscle length. This finding is in contrast with the results obtained for skeletal muscle, in which (R)FE is present. Our data support the hypothesis that titin stiffness does not increase with activation in cardiac muscle. [ABSTRACT FROM AUTHOR]

Aims: The main goal of our study was to investigate whether blebbistatin would prevent the cyclophosphamide (CYP)‐induced changes in cystometric and inflammatory parameters indicating the development of bladder inflammation and bladder overactivity. As the nature of CYP‐induced urotoxicity is inflammatory, we assume that agents presenting an anti‐inflammatory potential, such as blebbistatin, are worth special attention. Materials and Methods: The experiments were carried out in female Wistar rats. Surgical procedures, cystometric investigations, measurements of bladder edema and urothelium thickness as well as biochemical analyses were performed according to the published literature. Results: As expected, an acute administration of CYP (200 mg/kg, intraperitoneally) induced changes in the cystometric parameters and the levels of the tested biomarkers (ie, interleukin 1‐β, interleukin 6, interleukin 10, tumor necrosis factor‐α, nerve growth factor, brain‐derived neurotrophic factor, heparin‐binding epidermal growth factor‐like growth factor, insulin‐like growth factor‐binding protein 3, C‐X‐C motif chemokine 10, orosomucoid‐1, Tamm‐Horsfall protein, hemopexin, and occludin), indicating the development of bladder overactivity and bladder inflammation, respectively. These changes were accompanied by bladder edema and increased urothelium thickness. Intravesical infusion of blebbistatin for 7 days (125 nmol/day) prevented all symptoms of the CYP‐induced urotoxicity. Conclusions: Blebbistatin might be a promising novel agent for the treatment of bladder dysfunctions, like CYP‐induced hemorrhage cystitis or bladder overactivity, since it diminished the increased urinary bladder levels of proinflammatory markers and normalized the concentrations of the anti‐inflammatory ones. This effect was accompanied by amelioration of bladder edema and permeability, and normalization of both urothelium thickness and values of the cystometric parameters. [ABSTRACT FROM AUTHOR]

Overactive bladder (OAB) coexists with depression in women. Here, we assessed the effects of a 1-week treatment with blebbistatin, a myosin II inhibitor, on changes in behavior and detrusor overactivity (DO) symptoms induced by a 6-week administration of 13-cis-retinoic acid (13-cis-RA), with the aid of the forced swim test (FST), spontaneous locomotor activity test, and in vivo cystometric investigations in female Wistar rats. 13-cis-RA-induced depressive-like behavior and DO symptoms were associated with increased corticotropin-releasing factor (CRF) level in the plasma, prefrontal cortex (PFC), hippocampus (Hp), Barrington's nucleus (BN), and urinary bladder. Moreover, 13-cis-RA decreased brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) levels in plasma, PFC, Hp, and BN, while it increased BDNF and NGF levels in urinary bladder. Blebbistatin exerted antidepressant-like effect and attenuated changes in the cystometric parameters as well as the central and peripheral levels of CRF, BDNF, and NGF that were induced by 13-cis-RA, while it did not affect urine production, mean, systolic or diastolic blood pressure, or heart rate. The results point to blebbistatin as a potential treatment option for OAB coexisting with depression. [ABSTRACT FROM AUTHOR]

Cell contractility is mainly imagined as a force dipole-like interaction based on actin stress fibers that pull on cellular adhesion sites. Here, we present a different type of contractility based on isotropic contractions within the actomyosin cortex. Measuring mechanosensitive cortical contractility of suspended cells among various cell lines allowed us to exclude effects caused by stress fibers. We found that epithelial cells display a higher cortical tension than mesenchymal cells, directly contrasting to stress fiber-mediated contractility. These two types of contractility can even be used to distinguish epithelial from mesenchymal cells. These findings from a single cell level correlate to the rearrangement effects of actomyosin cortices within cells assembled in multicellular aggregates. Epithelial cells form a collective contractile actin cortex surrounding multicellular aggregates and further generate a high surface tension reminiscent of tissue boundaries. Hence, we suggest this intercellular structure as to be crucial for epithelial tissue integrity. In contrast, mesenchymal cells do not form collective actomyosin cortices reducing multicellular cohesion and enabling cell escape from the aggregates.

The motor protein myosin drives a wide range of cellular and muscular functions by generating directed movement and force, fueled through adenosine triphosphate (ATP) hydrolysis. Release of the hydrolysis product adenosine diphosphate (ADP) is a fundamental and regulatory process during force production. However, details about the molecular mechanism accompanying ADP release are scarce due to the lack of representative structures. Here we solved a novel blebbistatin-bound myosin conformation with critical structural elements in positions between the myosin pre-power stroke and rigor states. ADP in this structure is repositioned towards the surface by the phosphate-sensing P-loop, and stabilized in a partially unbound conformation via a salt-bridge between Arg131 and Glu187. A 5 Å rotation separates the mechanical converter in this conformation from the rigor position. The crystallized myosin structure thus resembles a conformation towards the end of the two-step power stroke, associated with ADP release. Computationally reconstructing ADP release from myosin by means of molecular dynamics simulations further supported the existence of an equivalent conformation along the power stroke that shows the same major characteristics in the myosin motor domain as the resolved blebbistatin-bound myosin-II·ADP crystal structure, and identified a communication hub centered on Arg232 that mediates chemomechanical energy transduction.

In multitarget drug design, it is critical to identify active and inactive compounds against a variety of targets and antitargets. Multitarget strategies thus test the limits of available technology, be that in screening large databases of compounds vs. a large number of targets, or in using in silico methods for understanding and reliably predicting these pharmacological outcomes. In this paper, we have evaluated the potential of several in silico approaches to predict the target, antitarget and physicochemical profile of (S)-blebbistatin, the best-known myosin II ATPase inhibitor, and a series of analogs thereof. Standard and augmented structure-based design techniques could not recover the observed activity profiles. A ligand-based method using molecular fingerprints was, however, able to select actives for myosin II inhibition. Using further ligand- and structure-based methods, we also evaluated toxicity through androgen receptor binding, affinity for an array of antitargets and the ADME profile (including assay-interfering compounds) of the series. In conclusion, in the search for (S)-blebbistatin analogs, the dissimilarity distance of molecular fingerprints to known actives and the computed antitarget and physicochemical profile of the molecules can be used for compound design for molecules with potential as tools for modulating myosin II and motility-related diseases.

Abstract Background Mitochondrial fission is the essential mechanisms of myocardial ischemia/reperfusion (MI/R)-induced cardiomyocytes apoptosis. Myosin II plays a key role in fission due to the recruitment and actomyosin constriction at the fission site in U2OS cells. However, the role of myosin IIA–actin interaction in regulating MI/R-induced cardiomyocytes mitochondrial fission and apoptosis remains to be fully elucidated. Methods and results When cardiomyocytes are exposed to simulated I/R injury, the myosin IIA protein translocated from the juxtamembrane to the cytoplasm, interacted with actin filaments, formed stress fibers and generated contractile forces. Treatment with the myosin II inhibitor blebbistatin attenuated the myosin IIA–actin complex induced actomyosin contractility and prevented cardiomyocytes apoptosis as reflected by inhibition of cleaved caspase-3 expression, normalization of Bcl-2/Bax levels and decreased apoptotic cells. Meanwhile, blebbistatin inhibited the activation of PINK1/Parkin pathway and ameliorated mitochondrial fission as evidenced by improvement of mitochondrial morphology, inhibition of Drp1 phosphorylation at Ser616 and translocation. Furthermore, CRISPR/Cas9 knockout of myosin IIA blocked I/R-induced apoptosis, suppressed PINK1/Parkin pathway and reduced mitochondrial fission. Importantly, blebbistatin attenuated myocardial apoptosis, inhibited myosin IIA–actin interaction and PINK1/Parkin pathway, suppressed myocardial ultrastructure abnormalities and mitochondrial fission in a mouse MI/R injury model. Conclusions Inhibition of actomyosin contractility induced by myosin IIA–actin interaction could impede myocardial apoptosis and MI/R injury via PINK1/Parkin pathway and mitochondrial fission modulation both in vitro and in vivo , which may be applicable for the development of therapies for cardiovascular diseases. Highlights • Nonmuscle myosin IIA–actin regulates MI/R-induced cardiomyocyte apoptosis. • Nonmuscle myosin IIA–actin modulates MI/R-induced mitochondrial fission. • Myosin IIA-related actomyosin contractility modulates PINK1/Parkin pathway. • Inhibition of myosin IIA–actin interaction attenuates myocardial injury. • Provide new insights on MI/R-induced myocardial apoptosis [ABSTRACT FROM AUTHOR]

Cytoplasmic flows are an ubiquitous feature of biological systems, in particular in large cells, such as oocytes and eggs in early animal development. Here we show that cytoplasmic flows in starfish oocytes, which can be imaged well with transmission light microscopy, are fully determined by the cortical dynamics during surface contraction waves. We first show that the dynamics of the oocyte surface is highly symmetric around the animal-vegetal axis. We then mathematically solve the Stokes equation for flows inside a deforming sphere using the measured surface displacements as boundary conditions. Our theoretical predictions agree very well with the intracellular flows quantified by particle image velocimetry, proving that during this stage the starfish cytoplasm behaves as a simple Newtonian fluid on the micrometer scale. We calculate the pressure field inside the oocyte and find that its gradient is too small as to explain polar body extrusion, in contrast to earlier suggestions. Myosin II inhibition by blebbistatin confirms this conclusion, because it diminishes cell shape changes and hydrodynamic flow, but does not abolish polar body formation. [ABSTRACT FROM AUTHOR]

FRET-based biosensor experiments in adult cardiomyocytes are a powerful way of dissecting the spatiotemporal dynamics of the complicated signaling networks that regulate cardiac health and disease. However, although much information has been gleaned from FRET studies on cardiomyocytes from larger species, experiments on adult cardiomyocytes from mice have been difficult at best. Thus the large variety of genetic mouse models cannot be easily used for this type of study. Here we develop cell culture conditions for adult mouse cardiomyocytes that permit robust expression of adenoviral FRET biosensors and reproducible FRET experimentation. We find that addition of 6.25 μM blebbistatin or 20 μM (S)-nitroblebbistatin to a minimal essential medium containing 10 mM HEP ES and 0.2% BSA maintains morphology of cardiomyocytes from physiological, pathological, and transgenic mouse models for up to 50 h after adenoviral infection. This provides a 10-15-h time window to perform reproducible FRET readings using a variety of CFP/YFP sensors between 30 and 50 h postinfection. The culture is applicable to cardiomyocytes isolated from transgenic mouse models as well as models with cardiac diseases. Therefore, this study helps scientists to disentangle complicated signaling networks important in health and disease of cardiomyocytes. [ABSTRACT FROM AUTHOR]