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Chronic diabetic wounds are a growing threat globally. Many aspects contribute to its deterioration, including bacterial infection, unbalanced microenvironment, dysfunction of cell repair, etc. In this work, we designed a multipronged micelles-hydrogel platform loaded with curcumin and rifampicin (CRMs-hydrogel) for bacteria-infected chronic wound treatment. The curcumin- and rifampicin-loaded micelles (CRMs) exhibited both MMP9-responsive and epidermal growth factor receptor (EGFR)-targeting abilities. On the one hand, drugs could be released from micelles due to responsive disassembly by MMP9, a matrix metalloproteinase overexpressed in a chronic wound environment; on the other hand, CRMs showed specific targeting to EGFR on epithelial cells and fibroblasts and therefore increased intracellular drug delivery. The thermosensitive CRMs-hydrogel could form strong adhesion with the wound area and served as a suitable matrix for sustained release of CRMs directly at the wound bed, with excellent intracellular and extracellular bacterial elimination efficiency and wound healing promotion capability. We found that a single dose of CRMs-hydrogel achieved 99% antibacterial rate at the MRSA-infected diabetic wound, which effectively reduced inflammatory response and promoted the neovascularization and re-epithelialization process, with nearly half reduction of the skin barrier regeneration period. Collectively, our thermosensitive, MMP9-responsive, and targeted micelles-hydrogel nanoplatform is promising for chronic wound treatment.

Biocompatible cationic graphene flakes efficiently complex and deliver the enzyme to the lysosomes of the fibroblasts derived from the patients with Mucopolysaccharidosis VI, leading to enhanced degradation of the accumulated lysosomal substrate.

Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC50 values of 12.9 and 8.6 ng/mL, with a linear range of 6.9–24.0 and 4.7–16.0 ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC50 of 3.5 ng/mL for LAS and 4.1 ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2–92.7%(CV

An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on immunomagnetic bead clean-up for detection of Maduramicin (MAD) was developed using a single-chain antibody (scFv). The single-chain antibody synthesized by genetic engineering technology was immobilized on the surface of carboxylic acid magnetic beads with a diameter of 2.8 ��m. After simple extraction, the residue in the sample extract was specifically adsorbed, and the supernatant was removed by magnetic separation. The half-maximal inhibitory concentration (IC50) of 15.43 ng��ml���1 with a limit of detection of 6.31 ng��ml���1 for MAD in chicken muscle. The recovery ranged from 72.93 to 89.51% with variation coefficients in intraday and interday less than 15%. We improved the inhibition efficiency and sensitivity of this method compared with traditional hydrophilic-lipophilic balance column clean up icELISA. The convenience and repeatability of the immunomagnetic clean-up render the new method for the analysis of drug residues in complex matrices.

Stabilisers, such as surfactants, polymers and polyaromatic molecules, offer an effective way to produce graphene dispersions in water by Liquid Phase Exfoliation (LPE) without degrading the properties of graphene. In particular, pyrene derivatives provide better exfoliation efficiency than traditional surfactants and polymers. A stabiliser is expected to be relatively soluble in order to disperse hydrophobic graphene in water. Here, we show that exfoliation can also be achieved with insoluble pyrene stabilisers if appropriately designed. In particular, bis-pyrene stabilisers (BPSs) functionalised with pyrrolidine provide a higher exfoliation efficiency and percentage of single layers compared to traditional pyrene derivatives under the same experimental conditions. This is attributed to the enhanced interactions between BPS and graphene, provided by the presence of two pyrene binding groups. This approach is therefore attractive not only to produce highly concentrated graphene, but also to use graphene to disperse insoluble molecules in water. The enhanced adsorption of BPS on graphene, however, is reflected in higher toxicity towards human epithelial bronchial immortalized cells, limiting the use of this material for biomedical applications.

Carbon nanomaterials codoped with transition metal and nitrogen have appeared as one of the most potential candidates to replace noble metal-based oxygen reduction catalyst. However, it remains a c...

Background: Diastolic dysfunction is a prevalent and therapeutically intractable feature of heart failure (HF). Increasing ventricular compliance can improve diastolic performance, but the viscoelastic forces that resist diastolic filling and become elevated in human HF are poorly defined. Having recently identified posttranslationally detyrosinated microtubules as a source of viscoelasticity in cardiomyocytes, we sought to test whether microtubules contribute meaningful viscoelastic resistance to diastolic stretch in human myocardium. Methods: Experiments were conducted in isolated human cardiomyocytes and trabeculae. First, slow and rapid (diastolic) stretch was applied to intact cardiomyocytes from nonfailing and HF hearts and viscoelasticity was characterized after interventions targeting microtubules. Next, intact left ventricular trabeculae from HF patient hearts were incubated with colchicine or vehicle and subject to pre- and posttreatment mechanical testing, which consisted of a staircase protocol and rapid stretches from slack length to increasing strains. Results: Viscoelasticity was increased during diastolic stretch of HF cardiomyocytes compared with nonfailing counterparts. Reducing either microtubule density or detyrosination reduced myocyte stiffness, particularly at diastolic strain rates, indicating reduced viscous forces. In myocardial tissue, we found microtubule depolymerization reduced myocardial viscoelasticity, with an effect that decreased with increasing strain. Colchicine reduced viscoelasticity at strains below, but not above, 15%, with a 2-fold reduction in energy dissipation upon microtubule depolymerization. Post hoc subgroup analysis revealed that myocardium from patients with HF with reduced ejection fraction were more fibrotic and elastic than myocardium from patients with HF with preserved ejection fraction, which were relatively more viscous. Colchicine reduced viscoelasticity in both HF with preserved ejection fraction and HF with reduced ejection fraction myocardium. Conclusions: Failing cardiomyocytes exhibit elevated viscosity and reducing microtubule density or detyrosination lowers viscoelastic resistance to diastolic stretch in human myocytes and myocardium. In failing myocardium, microtubules elevate stiffness over the typical working range of strains and strain rates, but exhibited diminishing effects with increasing length, consistent with an increasing contribution of the extracellular matrix or myofilament proteins at larger excursions. These studies indicate that a stabilized microtubule network provides a viscous impediment to diastolic stretch, particularly in HF.

Exchange protein directly activated by cAMP (Epac) mediates cAMP-mediated cell signal independent of protein kinase A (PKA). Mice lacking Epac1 displayed metabolic defect suggesting possible functional involvement of skeletal muscle and exercise capacity. Epac1 was highly expressed, but not Epac 2, in the extensor digitorum longus (EDL) and soleus muscles. The exercise significantly increased protein expression of Epac 1 in EDL and soleus muscle of wild-type (WT) mice. A global proteomics and pathway analyses revealed that Epac 1 deficiency mainly affected "the energy production and utilization" process in the skeletal muscle. We have tested their forced treadmill exercise tolerance. Epac1-/- mice exhibited significantly reduced exercise capacity in the forced treadmill exercise and lower number of type 1 fibers than WT mice. The basal protein level of proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) was reduced in the Epac1-/- mice. Furthermore, increasing expression of PGC-1α by exercise was also significantly attenuated in the skeletal muscle of Epac1-/- mice. The expressions of downstream target genes of PGC-1α, which involved in uptake and oxidation of fatty acids, ERRα and PPARδ, and fatty acid content were lower in muscles of Epac1-/-, suggesting a role of Epac1 in forced treadmill exercise capacity by regulating PGC-1α pathway and lipid metabolism in skeletal muscle. Taken together, Epac1 plays an important role in exercise capacity by regulating PGC-1α and fatty acid metabolism in the skeletal muscle.

Truncating variants in TTN (TTNtvs) are the most common known cause of nonischemic dilated cardiomyopathy (DCM), but how TTNtvs cause disease has remained controversial. Efforts to detect truncated titin proteins in affected human DCM hearts have failed, suggesting that disease is caused by haploinsufficiency, but reduced amounts of titin protein have not yet been demonstrated. Here, we leveraged a collection of 184 explanted posttransplant DCM hearts to show, using specialized electrophoretic gels, Western blotting, allelic phasing, and unbiased proteomics, that truncated titin proteins can quantitatively be detected in human DCM hearts. The sizes of truncated proteins corresponded to that predicted by their respective TTNtvs; the truncated proteins were encoded by the TTNtv-bearing allele; and no degradation fragments from protein encoded by either allele were detectable. In parallel, full-length titin was less abundant in TTNtv(+) than in TTNtv(−) DCM hearts. Disease severity or need for transplantation did not correlate with TTNtv location. Transcriptomic profiling revealed few differences in splicing or allelic imbalance of the TTN transcript between TTNtv(+) and TTNtv(−) DCM hearts. Studies with isolated human adult cardiomyocytes revealed no defects in contractility in cells from TTNtv(+) compared to TTNtv(−) DCM hearts. Together, these data demonstrate the presence of truncated titin protein in human TTNtv(+) DCM, show reduced amounts of full-length titin protein in TTNtv(+) DCM hearts, and support combined dominant-negative and haploinsufficiency contributions to disease.

Methicillin-resistant Staphylococcus aureus (MRSA) infection at impaired wound is associated with high risks of developing to persistent bacterial infections since bacterial biofilm is easy to form in MRSA infected wounds. An advanced therapeutic approach to effectively penetrate and eliminate bacterial biofilm and to accelerate cell proliferation and migration at the wound is crucial.The poly(ε-caprolactone)-monomethoxyl poly (ethylene glycol) (PCL-mPEG) micelles loaded with Quercetin and Rifampicin (QRMs) were prepared. Bacterial biofilm proliferation and elimination effect of QRMs were evaluated with confocal laser scanning microscopy. Antibacterial assay was further performed to detect antibacterial activity and mechanism. The cell scratch assay and cellular uptake were performed in HaCaT skin epithelial cells.Our results showed that the small sized QRMs could penetrate the interior of MRSA biofilm to disperse and eradicate biofilm. Then, antibiotics are released and accumulated in the acidic biofilm environment. QRMs could kill bacteria through increasing bacterial membrane permeability and altering membrane potential and membrane fluidity. Moreover, QRMs improved intracellular and cytoplasmic delivery efficiency of drugs to epithelial cells, and in the scratch test, presented a stronger ability to promote migration and proliferation of HaCaT cells compared with free drugs. Hemolysis test further proved good biocompatibility of QRMs.QRMs could potentially be used as a novel dual-functional nanotherapeutic for anti-bacterial infection by eradicating biofilm and accelerating cells proliferation at MRSA infected wound.

Graphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is essential for successful clinical translation. In this study, we exploit intrinsic fluorescent properties of thin GO sheets to establish the relationship between lateral dimensions of the material, its cellular uptake mechanisms and intracellular fate over time. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO) were thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was initially interrogated using a range of pharmaceutical inhibitors and validated using polystyrene beads of different diameters (0.1 and 1 μm). Subsequently, RNA-Seq was used to follow the changes in the uptake mechanism used to internalize s-GO flakes over time. Regardless of lateral dimensions, both types of GO were found to interact with the plasma membrane and to be internalized by a panel of cell lines studied. However, s-GO was internalized mainly via macropinocytosis while us-GO was mainly internalized via clathrin- and caveolae-mediated endocytosis. Importantly, we report the shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of s-GO at 24 h, mediated by upregulation of mTORC1/2 pathway. Finally, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results offer an insight into the mechanism of interaction of GO with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems.

Diffuse axonal injury (DAI) accounts for 30-40% of total neurotrauma,majority among them manifest with consciousness disturbance.At present, the understanding of the treatment of coma and awakening in patients with DAIs is still limited.This study is characterized by the use of electroacupuncture along with conventional Western medicine to promote consciousness more effectively in comatose patients with DAIs, shorten their time spent in a coma, and gain time for more favorable treatments during follow-up rehabilitation in order to improve the cure rate, reduce the morbidity rate, and achieve better therapeutic effects.In this randomized controlled study, 145 comatose patients with DAIs (type III) were divided into the treatment group (n = 71) and control group (n = 74). The patients in the control group were treated with conventional Western medicine, while those in the treatment group were treated with both electroacupuncture and conventional treatment. The Glasgow Coma Scale (GCS) scores and consciousness-promotion rates of both groups were observed before treatment as well as 10, 20, and 30 days after treatment. Meanwhile, serum acetylcholinesterase E (AchE) concentrations in both groups were measured with ELISA, while AchE activity was determined with the rate method. Correlations between GCS score, AchE concentration, and AchE activity in the treatment group were analyzed by using the stepwise multiple regression method.The GCS scores in the treatment group showed significant increases after the first, second, and third courses of treatment when compared to the pre-treatment scores (P0.05). After 1 course of treatment, the GCS scores in the control group were not statistically significantly different compared to the pre-treatment scores(P0.05), whereas after 2 and 3 courses of treatment, the differences were of greater statistical significance (P0.05). Statistically significant differences between the 2 groups were found in GCS scores in the same course of treatment (P0.05). The consciousness-promotion rates between the 2 groups after the same treatment course were statistically significantly different (P0.05). Both the standardized regression coefficients and partial correlation coefficients showed that AchE concentration had a certain influence on GCS score (|Beta| = 0.3601; r Y2.1 = 0.726).Conventional Western medicine combined with electroacupuncture treatment may promote the consciousness of patients with DAIs and shorten the amount of time they spend comatose. Furthermore, the neurotransmitter AchE may play a role in the pathophysiological mechanism of consciousness promotion.

Its anatomical localization, a highly heterogeneous and drug-resistant tumor cell population and a “cold” immune microenvironment, all challenge the treatment of glioblastoma. Nanoscale drug delivery systems, including graphene oxide (GO) flakes, may circumvent some of these issues bypassing biological barriers, delivering multiple cargoes to impact several pathways simultaneously, or targeting the immune compartment. Here, the interactions of GO flakes with in vitro (U-87 MG three-dimensional spheroids, without stromal or immune compartments) and in vivo (U-87 MG orthotopic xenograft) models of glioblastoma are investigated. In vitro, GO flakes translocated deeply into the spheroids with little internalization in tumor cells. In vivo, intracranially administered GO also show extensive distribution throughout the tumor and demonstrate no impact on tumor growth and progression for the duration of the study. Internalization within tumor cells is also scarce, with the majority of flakes preferentially taken up by microglia/macrophages. The results indicate that GO flakes could offer deep and homogenous distribution throughout glioblastoma tumors and a means to target their myeloid compartment. Further studies are warranted to investigate the mechanisms of GO flakes transport within the tumor mass and their capacity to deliver bioactive cargoes but, ultimately, this information could inform the development of immunotherapies against glioblastoma., K.K., P.S., S.V., and M.S. would like to acknowledge the United Kingdom Research and Innovation (UKRI) Engineering and Physical Sciences Research Council (EPSRC) for funding most of this work under the 2D-Health Programme Grant (EP/P00119X/1). K.K. and I.d.L. would also like to acknowledge the UKRI (EPSRC) for an International Centre-to-Centre grant (EP/S030719/1) between the University of Manchester and Harvard University that funded part of this work. T.K. would like to thank the UKRI (EPSRC and Medical Research Council, MRC) Center for Doctoral Training (CDT) in Regenerative Medicine (EP/L014904/1) for a funded PhD studentship. The authors would like to thank Dr. Marta Quintanilla and Dr. David Spiller for help with LSFM. The equipment at the University of Manchester Bioimaging and Genomic Technologies facilities used in this study were purchased with grants from the Biotechnology and Biological Sciences Research Council (BBSRC), Wellcome Trust, and the University of Manchester Strategic Fund. A.Y., C.G., and H.T. would like to thank the project “G-IMMUNOMICS” funded under the Joint Transnational Call (JTC) Graphene 2015. A.Y. is thankful to the Turkish Academy of Sciences (TUBA) for the financial support.

Background Glioma is a common type of brain tumor and is classified as low and high grades according to morphology and molecules. Growing evidence has proved that long non-coding RNAs (lncRNAs) play pivotal roles in numerous tumors or diseases including glioma. Proteasome 20S subunit alpha 3 antisense RNA 1 (PSMA3-AS1), as a member of lncRNAs, has been disclosed to play a tumor-promoting role in cancer progression. However, the role of PSMA3-AS1 in glioma remains unknown. Therefore, we concentrated on researching the regulatory mechanism of PSMA3-AS1 in glioma. Methods PSMA3-AS1 expression was detected using RT-qPCR. Functional assays were performed to measure the effects of PSMA3-AS1 on glioma progression. After that, ENCORI (http://starbase.sysu.edu.cn/) database was used to predict potential genes that could bind to PSMA3-AS1, and miR-411-3p was chosen for further studies. The interaction among PSMA3-AS1, miR-411-3p and homeobox A10 (HOXA10) were confirmed through mechanism assays. Results PSMA3-AS1 was verified to be up-regulated in glioma cells and promote glioma progression. Furthermore, PSMA3-AS1 could act as a competitive endogenous RNA (ceRNA) for miR-411-3p to regulate HOXA10 and thus affecting glioma progression. Conclusion PSMA3-AS1 stimulated glioma progression via the miR-411-3p/HOXA10 pathway, which might offer a novel insight for the therapy and treatment of glioma.

Rationale: Impaired myocardial relaxation is an intractable feature of several heart failure (HF) causes. In human HF, detyrosinated microtubules stiffen cardiomyocytes and impair relaxation. Yet the identity of detyrosinating enzymes have remained ambiguous, hindering mechanistic study and therapeutic development. Objective: We aimed to determine if the recently identified complex of VASH1/2 (vasohibin 1/2) and SVBP (small vasohibin binding protein) is an active detyrosinase in cardiomyocytes and if genetic inhibition of VASH-SVBP is sufficient to lower stiffness and improve contractility in HF. Methods and Results: Transcriptional profiling revealed that VASH1 transcript is >10-fold more abundant than VASH2 in human hearts. Using short hairpin RNAs (shRNAs) against VASH1 , VASH2 , and SVBP , we showed that both VASH1- and VASH2-SVBP complexes function as tubulin carboxypeptidases in cardiomyocytes, with a predominant role for VASH1. We also generated a catalytically dead version of the tyrosinating enzyme TTL (TTL-E331Q) to separate the microtubule depolymerizing effects of TTL from its enzymatic activity. Assays of microtubule stability revealed that both TTL and TTL-E331Q depolymerize microtubules, while VASH1 and SVBP depletion reduce detyrosination independent of depolymerization. We next probed effects on human cardiomyocyte contractility. Contractile kinetics were slowed in HF, with dramatically slowed relaxation in cardiomyocytes from patients with HF with preserved ejection fraction. Knockdown of VASH1 conferred subtle kinetic improvements in nonfailing cardiomyocytes, while markedly improving kinetics in failing cardiomyocytes. Further, TTL, but not TTL-E331Q, robustly sped relaxation. Simultaneous measurements of calcium transients and contractility demonstrated that VASH1 depletion speeds kinetics independent from alterations to calcium cycling. Finally, atomic force microscopy confirmed that VASH1 depletion reduces the stiffness of failing human cardiomyocytes. Conclusions: VASH-SVBP complexes are active tubulin carboxypeptidases in cardiomyocytes. Inhibition of VASH1 or activation of TTL is sufficient to lower stiffness and speed relaxation in cardiomyocytes from patients with HF, supporting further pursuit of detyrosination as a therapeutic target for diastolic dysfunction.

The outstanding properties of graphene offer high potential for biomedical applications. In this framework, positively charged nanomaterials show better interactions with the biological environment, hence there is strong interest in the production of positively charged graphene nanosheets. Currently, production of cationic graphene is either time consuming or producing dispersions with poor stability, which strongly limit their use in the biomedical field. In this study, we made a family of new cationic pyrenes, and have used them to successfully produce water-based, highly concentrated, stable, and defect-free graphene dispersions with positive charge. The use of different pyrene derivatives as well as molecular dynamics simulations allowed us to get insights on the nanoscale interactions required to achieve efficient exfoliation and stabilisation. The cationic graphene dispersions show outstanding biocompatibility and cellular uptake as well as exceptional colloidal stability in the biological medium, making this material extremely attractive for biomedical applications., This work has been supported by the EPSRC in the framework of the project 2D Health (EP/P00119X/1) and the M-ERA.NET project MODIGLIANI (project2110). Computer time for this study was provided by the Consortium des Equipements de Calcul Intensif (CECI, http://www.ceci-hpc.be), and particularly those of the Technological Platform on High-Performance Computing, for which we gratefully acknowledge the financial support of the FNRS-FRFC (Conventions No. 2.4.617.07.F and 2.5020.11).

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

Cardiomyocytes are large (∼40,000 µm3), rod-shaped muscle cells that provide the working force behind each heartbeat. These highly structured cells are packed with dense cytoskeletal networks that can be divided into two groups—the contractile (i.e. sarcomeric) cytoskeleton that consists of filamentous actin-myosin arrays organized into myofibrils, and the non-sarcomeric cytoskeleton, which is composed of β- and γ-actin, microtubules, and intermediate filaments. Together, microtubules and intermediate filaments form a cross-linked scaffold, and these networks are responsible for the delivery of intracellular cargo, the transmission of mechanical signals, the shaping of membrane systems, and the organization of myofibrils and organelles. Microtubules are extensively altered as part of both adaptive and pathological cardiac remodeling, which has diverse ramifications for the structure and function of the cardiomyocyte. In heart failure, the proliferation and post-translational modification of the microtubule network is linked to a number of maladaptive processes, including the mechanical impediment of cardiomyocyte contraction and relaxation. This raises the possibility that reversing microtubule alterations could improve cardiac performance, yet therapeutic efforts will strongly benefit from a deeper understanding of basic microtubule biology in the heart. The aim of this review is to summarize the known physiological roles of the cardiomyocyte microtubule network, the consequences of its pathological remodeling, and to highlight the open and intriguing questions regarding cardiac microtubules.Impact statementAdvancements in cell biological and biophysical approaches and super-resolution imaging have greatly broadened our view of tubulin biology over the last decade. In the heart, microtubules and microtubule-based transport help to organize and maintain key structures within the cardiomyocyte, including the sarcomere, intercalated disc, protein clearance machinery and transverse-tubule and sarcoplasmic reticulum membranes. It has become increasingly clear that post translational regulation of microtubules is a key determinant of their sub-cellular functionality. Alterations in microtubule network density, stability, and post-translational modifications are hallmarks of pathological cardiac remodeling, and modified microtubules can directly impede cardiomyocyte contractile function in various forms of heart disease. This review summarizes the functional roles and multi-leveled regulation of the cardiac microtubule cytoskeleton and highlights how refined experimental techniques are shedding mechanistic clarity on the regionally specified roles of microtubules in cardiac physiology and pathophysiology.

Detyrosinated microtubules (dTyr MTs) provide a viscoelastic resistance to myocyte motion and are proliferated in end-stage heart failure. In isolated myocytes, proliferation of the MT network leads to reduced shortening and relaxation, consistent with an underlying viscoelastic resistance, yet a clear contribution of MTs to the passive mechanical properties of myocytes or myocardium in heart failure remains to be determined. To this end, intact human myocytes were isolated from failing and non-failing hearts and their viscoelasticity was measured using a physiological stretch assay where strain-rates were consistent with diastolic filling. In myocytes from heart failure patients, we find an increase in myocyte viscoelasticity and slowing of passive relaxation during cell stretch compared to myocytes from non-failing hearts. Depolymerizing MTs with colchicine, or suppressing dTyr MTs either pharmacologically with parthenolide or genetically by overexpression of tubulin tyrosine ligase reduces myocyte passive viscoelasticity, accelerates passive relaxation time and leads to consistent improvements in contractile and relaxation dynamics in failing cardiomyocytes. Manipulation of dTyr MT levels does not significantly alter the calcium transient during unloaded contraction, suggesting that the observed gain in function is mechanical in nature. We additionally have begun to assay the MT contribution to mechanics at the tissue level. In intact trabeculae from failing hearts, colchicine treatment to depolymerize MTs reduces passive myocardial viscoelasticity upon diastolic stretch. The results indicate that microtubules contribute to myocardial mechanical properties in the context of end-stage heart failure.

In the beating heart, cardiac myocytes (CMs) contract in a coordinated fashion, generating contractile wave fronts that propagate through the heart with each beat. Coordinating this wave front requires fast and robust signaling mechanisms between CMs. The primary signaling mechanism has long been identified as electrical: gap junctions conduct ions between CMs, triggering membrane depolarization, intracellular calcium release, and actomyosin contraction. In contrast, we propose here that, in the early embryonic heart tube, the signaling mechanism coordinating beats is mechanical rather than electrical. We present a simple biophysical model in which CMs are mechanically excitable inclusions embedded within the extracellular matrix (ECM), modeled as an elastic-fluid biphasic material. Our model predicts strong stiffness dependence in both the heartbeat velocity and strain in isolated hearts, as well as the strain for a hydrogel-cultured CM, in quantitative agreement with recent experiments. We challenge our model with experiments disrupting electrical conduction by perfusing intact adult and embryonic hearts with a gap junction blocker, β-glycyrrhetinic acid (BGA). We find this treatment causes rapid failure in adult hearts but not embryonic hearts—consistent with our hypothesis. Last, our model predicts a minimum matrix stiffness necessary to propagate a mechanically coordinated wave front. The predicted value is in accord with our stiffness measurements at the onset of beating, suggesting that mechanical signaling may initiate the very first heartbeats.

Detyrosinated microtubules provide mechanical resistance that can impede the motion of contracting cardiomyocytes. However, the functional effects of microtubule detyrosination in heart failure or in human hearts have not previously been studied. Here, we utilize mass spectrometry and single-myocyte mechanical assays to characterize changes to the cardiomyocyte cytoskeleton and their functional consequences in human heart failure. Proteomic analysis of left ventricle tissue reveals a consistent upregulation and stabilization of intermediate filaments and microtubules in failing human hearts. As revealed by super-resolution imaging, failing cardiomyocytes are characterized by a dense, heavily detyrosinated microtubule network, which is associated with increased myocyte stiffness and impaired contractility. Pharmacological suppression of detyrosinated microtubules lowers the viscoelasticity of failing myocytes and restores 40-50% of lost contractile function; reduction of microtubule detyrosination using a genetic approach also softens cardiomyocytes and improves contractile kinetics. Together, these data demonstrate that a modified cytoskeletal network impedes contractile function in cardiomyocytes from failing human hearts and that targeting detyrosinated microtubules could represent a new inotropic strategy for improving cardiac function.

"Pure shift" methods can greatly improve the resolution of proton NMR spectra. However, current pure shift spectra show small periodic artefacts that prevent their use for studying dilute mixture components. A new technique, compatible with all current pure shift methods, is presented that suppresses such sidebands to arbitrary order, allowing ultraclean spectra to be obtained.