Antimyosin Antibody Research Articles

Postcardiac Injury Syndrome After Cardiac Surgery: An Evidence-Based Review.

Postcardiac injury syndrome (PCIS) serves as a comprehensive term encompassing a spectrum of conditions, namely postpericardiotomy syndrome, postmyocardial infarction (MI) related pericarditis (Dressler syndrome), and post-traumatic pericarditis stemming from procedures like percutaneous coronary intervention or cardiac implantable electronic device placement. These conditions collectively give rise to PCIS, triggered by cardiac injury affecting pericardial or pleural mesothelial cells, leading to subsequent inflammation syndromes spanning from uncomplicated pericarditis to substantial pleural effusion. A thorough literature search conducted on MEDLINE/PubMed utilizing search terms including "postacute cardiac injury syndrome," "postcardiac injury syndrome," "postcardiotomy syndrome," "postpericardiotomy syndrome," and "post-MI pericarditis" was instrumental in collating pertinent studies. To encapsulate the amassed evidence, relevant full-text materials were meticulously selected and amalgamated narratively. The pathophysiology of PCIS is proposed to manifest through an autoimmune-mediated process, particularly in predisposed individuals. This process involves the development of anti-actin and antimyosin antibodies after a cascade of cardiac injuries in diverse forms. Treatment strategies aimed at preventing recurrent PCIS episodes have shown efficacy, with colchicine and nonsteroidal anti-inflammatory drugs, including ibuprofen, demonstrating positive outcomes. Conversely, corticosteroids have exhibited no discernible benefit concerning prognosis or recurrence rates for this ailment. In summary, PCIS serves as a unifying term encompassing a spectrum of cardiac injury-related syndromes. A comprehensive review of relevant literature underscores the autoimmune-mediated pathophysiology in susceptible individuals. The therapeutic landscape involves the proficient use of colchicine and Nonsteroidal anti-inflammatory drugs to deter recurrent PCIS episodes, while corticosteroids do not appear to contribute to improved prognosis or reduced recurrence rates. This nuanced understanding contributes to an enhanced comprehension of PCIS and its multifaceted clinical manifestations, potentially refining its diagnosis and management.

Cooperative & competitive binding of anti-myosin tail antibodies revealed by super-resolution microscopy

The MF30 monoclonal antibody, which binds to the myosin subfragment-2 (S2), was found to increase the extent of myofibril shortening. Yet, previous observations found no effect of this antibody on actin sliding over myosin during in vitro motility assays with purified proteins in which myosin binding protein C (MyBPC) was absent. MF30 is hypothesized to enhance the availability of myosin heads (subfragment-1 or S1) to bind actin by destabilizing the myosin S2 coiled-coil and sterically blocking S2 from binding S1. The mechanism of action likely includes MF30's substantial size, thereby inhibiting S1 heads and MyBPC from binding S2. Hypothetically, MF30 should enhance the ON state of myosin, thereby increasing muscle contraction. Our findings indicate that MF30 binds preferentially to the unfolded heavy chains of S2, displaying positive cooperativity. However, the dose-response curve of MF30's enhancement of myofibril shortening did not suggest complex interactions with S2. Single, double, and triple-stained myofibrils with increasing amounts of antibodies against myosin rods indicate a possible competition with MyBPC. Additional assays revealed decreased fluorescence intensity at the C-zone (central zone in the sarcomere, where MyBPC is located), where MyBPC may inhibit MF30 binding. Another monoclonal antibody named MF20, which binds to the light meromyosin (LMM) without affecting myofibril contraction, showed less reduction in fluorescence intensity at the C-zone in expansion microscopy than MF30. Expansion microscopy images of myofibrils labeled with MF20 revealed labeling of the A-band (anisotropic band) and a slight reduction in the labeling at the C-zone. The staining pattern obtained from the expansion microscopy image was consistent with images from photolocalization microscopy which required the synthesis of unique photoactivatable quantum dots, and Zeiss Airyscan imaging as well as alternative expansion microscopy digestion methods. Consistent with the hypothesis that MF30 competes with MyBPC binding to S2, cardiac tissue from MyBPC knockout mice was stained more intensely, especially in the C-zone, by MF30 compared to the wild type.

(008) Chronic High Fat Diet Decreases Vaginal Smooth Muscle Tone but does not Affect Pelvic Floor Skeletal Muscle in Mice

Abstract Introduction Pelvic organ prolapse (POP) is a disease that affects the physical and mental health of women, as well as their quality of life. POP, regardless of severity, can be a direct cause of female sexual dysfunction (FSD) by producing pain during intercourse or a general reluctance to engage in sexual activity. The largest risk factors for POP include parity, obesity, and advanced age. Obesity, a modifiable risk factor, is believed to increase intra-abdominal pressure and weaken the pelvic floor muscles and fascia. Studies have shown that obesity and a high fat diet (HFD) can weaken skeletal muscle by increasing collagen deposition and inducing muscle apoptosis. Objectives There is paucity in the literature of animal studies that have specifically investigated the effect of obesity on the support structures of the pelvic floor and how it contributes to POP. The objective of this study is to examine the structure and function of the vagina and pelvic floor in female mice fed a chronic HFD. Methods Adult female C57/Blk6N mice (10 weeks, n=32) were fed a control (10% kcal fat) or HFD (45% kcal fat) for 24 weeks. Ex vivo external vaginal sphincter contraction to increasing concentrations of adrenergic agonist norepinephrine and relaxation to nitric oxide donor DEA NONOate and electric field stimulation (EFS) was measured. Iliococcygeus (IC) muscles of the pelvic floor were isolated and set to optimal length and tetanic contractions were performed at increasing frequencies to asses absolute and specific force. Fatigue was measured following repetitive stimulations over 5 minutes. IC muscle fiber types were characterized with anti-myosin antibodies and cross-sectional area (CSA) of each fiber type was measured. Results Vaginal sphincter adrenergic-mediated contraction was a significantly decreased in HFD-fed mice (Fig 1A; p<0.05). The vaginal sphincter smooth muscle relaxation in response to a nitric oxide donor was greater in the HFD mice (Fig 1B; p<0.05). Neurogenic-mediated EFS vaginal relaxations were also enhanced in the HFD mice (p<0.05). Surprisingly, chronic HFD did not impact iliococcygeus muscle specific force, absolute force, or muscle fatigue (Fig 1C). Furthermore, HFD did not affect the CSA or population of type I, IIa, IIX, or IIb iliococcygeus muscle fibers. Conclusions HFD-induced obesity in mice leads to pelvic floor weakness by enhancing relaxation and decreasing tone of the vaginal smooth muscle. Interestingly, the function and composition of pelvic skeletal muscle was unaffected by HFD. As the vagina serves as a primary support structure to the pelvic organs, lack in muscle contractility and increase in relaxation following HFD suggests that obesity-induced POP may play a direct role in FSD. Disclosure

Fusion of Wild-Type Mesoangioblasts with Myotubes of mtDNA Mutation Carriers Leads to a Proportional Reduction in mtDNA Mutation Load.

In 25% of patients with mitochondrial myopathies, pathogenic mitochondrial DNA (mtDNA) mutation are the cause. For heteroplasmic mtDNA mutations, symptoms manifest when the mutation load exceeds a tissue-specific threshold. Therefore, lowering the mutation load is expected to ameliorate disease manifestations. This can be achieved by fusing wild-type mesoangioblasts with mtDNA mutant myotubes. We have tested this in vitro for female carriers of the m.3271T>C or m.3291T>C mutation (mutation load >90%) using wild-type male mesoangioblasts. Individual fused myotubes were collected by a newly-developed laser capture microdissection (LCM) protocol, visualized by immunostaining using an anti-myosin antibody. Fusion rates were determined based on male-female nuclei ratios by fluorescently labelling the Y-chromosome. Using combined 'wet' and 'air dried' LCM imaging improved fluorescence imaging quality and cell yield. Wild-type mesoangioblasts fused in different ratios with myotubes containing either the m.3271T>C or the m.3291T>C mutation. This resulted in the reduction of the mtDNA mutation load proportional to the number of fused wild-type mesoangioblasts for both mtDNA mutations. The proportional reduction in mtDNA mutation load in vitro after fusion is promising in the context of muscle stem cell therapy for mtDNA mutation carriers in vivo, in which we propose the same strategy using autologous wild-type mesoangioblasts.

Astrocytes control the spiking of mouse visual cortex layer 5 pyramidal neurons.

Astrocytes control the spiking of mouse visual cortex layer 5 pyramidal neurons.

PD36-03 CHRONIC HIGH FAT DIET INCREASES VAGINAL SPHINCTER LAXITY BUT NOT PELVIC FLOOR MUSCLE FUNCTION IN MICE

You have accessJournal of UrologySexual Function/Dysfunction: Female (PD36)1 Apr 2020PD36-03 CHRONIC HIGH FAT DIET INCREASES VAGINAL SPHINCTER LAXITY BUT NOT PELVIC FLOOR MUSCLE FUNCTION IN MICE Lindsey Burleson*, Adam Amorese, Michelle Oyen, Espen Spengenburg, and Johanna Hannan Lindsey Burleson*Lindsey Burleson* More articles by this author , Adam AmoreseAdam Amorese More articles by this author , Michelle OyenMichelle Oyen More articles by this author , Espen SpengenburgEspen Spengenburg More articles by this author , and Johanna HannanJohanna Hannan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000907.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Pelvic organ prolapse (POP) affects 41% women and is a direct cause of female sexual dysfunction (FSD) by producing pain during intercourse or a reluctance to engage in sexual activity. The largest risk factors for POP include parity, obesity, and advanced age. Obesity, a modifiable risk factor, is believed to increase intra-abdominal pressure and weaken the pelvic floor muscles and fascia. The impact of obesity on vaginal smooth muscle versus pelvic floor skeletal muscle has not been studied in animal models of POP. We will examine the structure and function of the vagina and pelvic floor in female mice fed a chronic high fat diet (HFD). METHODS: Adult female C57/Blk6N mice (10 weeks, n=32) were fed a control (10% kcal fat) or HFD (45% kcal fat) for 24 weeks. Ex vivo external vaginal sphincter contraction to increasing concentrations of adrenergic agonist norepinephrine and relaxation to nitric oxide donor DEA NONOate and electric field stimulation (EFS) was measured. Iliococcygeus muscles of the pelvic floor were isolated and set to optimal length and tetanic contractions were performed at increasing frequencies to asses absolute and specific force. Fatigue was measured following repetitive stimulations over 5 minutes. Iliococcygeus muscle fiber types were characterized with anti-myosin antibodies and cross-sectional area (CSA) of each fiber type was measured. RESULTS: Vaginal sphincter adrenergic-mediated contraction was a significantly decreased in HFD-fed mice (Fig 1A; p<0.05). The vaginal sphincter smooth muscle relaxation in response to a nitric oxide donor was greater in the HFD mice (Fig 1B; p<0.05). Neurogenic-mediated EFS vaginal relaxations were also enhanced in the HFD mice (p<0.05). Surprisingly, chronic HFD did not impact iliococcygeus muscle specific force, absolute force, or muscle fatigue (Fig 1C). Furthermore, HFD did not affect the CSA or population of type I, IIa, IIX, or IIb iliococcygeus muscle fibers. CONCLUSIONS: HFD-induced obesity in mice leads to pelvic floor weakness by enhancing relaxation and decreasing tone of the external vaginal sphincter. Interestingly, the function or composition of pelvic skeletal muscle was unaffected. As the vagina provides significant support to pelvic organs, obesity-induced decreased vaginal sphincter tone and structure may lead to POP and FSD. Source of Funding: International Society for the Study of Women’s Sexual Health, Alpha Omega Alpha Medical Society © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e723-e724 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lindsey Burleson* More articles by this author Adam Amorese More articles by this author Michelle Oyen More articles by this author Espen Spengenburg More articles by this author Johanna Hannan More articles by this author Expand All Advertisement PDF downloadLoading ...

Early Detection of Localized Immunity in Experimental Autoimmune Myocarditis Using [99mTc]Fucoidan SPECT.

The aim of the study was to evaluate the ability of technetium-99m-fucoidan ([99mTc]fucoidan), a molecular imaging agent specific for selectins, in the assessment of early localized immunity in a rat model of experimental autoimmune myocarditis (EAM). EAM was induced in Lewis rats and troponin T; brain natriuretic peptide (BNP) and anti-myosin antibodies were measured in plasma. Separately, [99mTc]fucoidan single-photon emission computed tomography (SPECT)/x-ray computed tomography (CT) was performed in the very early phase of myocarditis at 10, 15, and 21days after immunization. Then, hearts were collected and used for autoradiography, well counting, histology, and flow cytometry analysis. The EAM acute phase is characterized by extensive myocardial necrosis, release of troponin and BNP, and pericardial effusion. [99mTc]Fucoidan uptake was significantly increased in EAM compared with controls starting from D15. There was a close relationship between uptake of the tracer and myocardial content in CD45+, CD8+, CD11b+, and CD31+ cells. [99mTc]Fucoidan SPECT/CT accurately diagnosed the autoimmune attack in the early steps of EAM and could be used to monitor disease evolution and therapy efficiency.

Immune-Mediated Myocarditis in Fabry Disease Cardiomyopathy.

BackgroundGlycosphingolipid accumulation in Fabry cells generates a proinflammatory response that may influence disease evolution and responsiveness to enzyme replacement therapy. This study evaluated incidence, mechanism, and impact of myocarditis in Fabry disease cardiomyopathy (FDCM).Methods and ResultsMyocarditis, defined as CD3+ T lymphocytes >7/mm2 associated with necrosis of glycolipid‐laden myocardiocytes, was retrospectively evaluated in endomyocardial biopsies from 78 patients with FDCM: 13 with maximal wall thickness (MWT) <11 mm (group 1), 17 with MWT 11 to 15 mm (group 2), 30 with MWT 16 to 20 mm (group 3), and 18 with MWT >20 mm (group 4). Myocarditis was investigated by polymerase chain reaction for cardiotropic viruses, by serum antiheart and antimyosin antibodies, and by cardiac magnetic resonance. Myocarditis was recognized at histology in 48 of 78 patients with FDCM (38% of group 1, 41% of group 2, 66% of group 3, and 72% of group 4). Myocarditis was characterized by positive antiheart and antimyosin antibodies and negative polymerase chain reaction for viral genomes. CD3+ cells/mm2 correlated with myocyte necrosis, antimyosin autoantibody titer, and MWT (P<0.001,r=0.79; P<0.001, r=0.84; P<0.001, r=0.61, respectively). Cardiac magnetic resonance showed myocardial edema in 24 of 78 patients (31%): 0% of group 1, 23% of group 2, 37% of group 3, and 50% of group 4.ConclusionsMyocarditis is detectable at histology in up to 56% of patients with FDCM. It is immune mediated and correlates with disease severity. It can be disclosed by antiheart/antimyosin autoantibodies and in the advanced phase by cardiac magnetic resonance. It may contribute to progression of FDCM and resistance to enzyme replacement therapy.

Autoimmunity to Tropomyosin-Specific Peptides Induced by Mycobacterium leprae in Leprosy Patients: Identification of Mimicking Proteins.

It has been shown earlier that there is a rise in the levels of autoantibodies and T cell response to cytoskeletal proteins in leprosy. Our group recently demonstrated a rise in both T and B cell responses to keratin and myelin basic protein in all types of leprosy patients and their associations in type 1 reaction (T1R) group of leprosy. In this study, we investigated the association of levels of autoantibodies and lymphoproliferation against myosin in leprosy patients across the spectrum and tried to find out the mimicking proteins or epitopes between host protein and protein/s of Mycobacterium leprae. One hundred and sixty-nine leprosy patients and 55 healthy controls (HC) were enrolled in the present study. Levels of anti-myosin antibodies and T-cell responses against myosin were measured by ELISA and lymphoproliferation assay, respectively. Using 2-D gel electrophoresis, western blot and MALDI-TOF/TOF antibody-reactive spots were identified. Three-dimensional structure of mimicking proteins was modeled by online server. B cell epitopes of the proteins were predicted by BCPREDS server 1.0 followed by identification of mimicking epitopes. Mice of inbred BALB/c strain were hyperimmunized with M. leprae soluble antigen (MLSA) and splenocytes and lymph node cells of these animals were adoptively transferred to naïve mice. Highest level of anti-myosin antibodies was noted in sera of T1R leprosy patients. We observed significantly higher levels of lymphoproliferative response (p < 0.05) with myosin in all types of leprosy patients compared to HC. Further, hyperimmunization of inbred BALB/c strain of female mice and rabbit with MLSA revealed that both hyperimmunized rabbit and mice evoked heightened levels of antibodies against myosin and this autoimmune response could be adoptively transferred from hyperimmunized to naïve mice. Tropomyosin was found to be mimicking with ATP-dependent Clp protease ATP-binding subunit of M. leprae. We found four mimicking epitopes between these sequences. These data suggest that these mimicking proteins tropomyosin and ATP-dependent Clp protease ATP-binding subunit of M. leprae or more precisely mimicking epitopes (four B cell epitopes) might be responsible for extensive tissue damage during type1 reaction in leprosy.

Significance of Anti-Myosin Antibody Formation in Patients With Myocardial Infarction: A Prospective Observational Study

Anti-myosin antibodies (AMAs) are often formed in response to myocardial infarction (MI) and have been implicated in maladaptive cardiac remodelling. We aimed to: (1) compare AMA formation in patients with Non-ST-Elevation MI (NSTEMI) and ST-Elevation MI (STEMI); (2) evaluate factors predicting autoantibody formation; and, (3) explore their functional significance. Immunoglobulin M (IgM) and Immunoglobulin G (IgG) AMA titres were determined in serum samples collected at admission, 3 and 6 months post MI. The relationship between demographic and clinical data, and antibody formation, was investigated to determine factors predicting antibody formation and functional significance. Forty-three (43) patients were consecutively recruited; 74.4% were positive for IgM at admission, compared with 23.3% for IgG. Mean IgG levels increased by 1.24% (±0.28) at 3 months, and 13.55% (±0.13) at 6 months post MI. Mean antibody levels were significantly higher in the NSTEMI cohort at both follow-up time points for IgG (p<0.001, p<0.0001), but not IgM (p=0.910, p=0.066). A moderately positive correlation between infarct size and increase in mean IgM concentration was observed at 3 months (r(98)=0.455; p=0.015). Anti-myosin antibody formation was not associated with an unfavourable outcome at follow-up. Anti-myosin antibodies are formed in a significant proportion of patients following MI, particularly among those with NSTEMI. While IgM levels fall after infarction, IgG levels increase and persist beyond 6 months of follow-up. This raises the possibility that they may contribute to long-term myocardial damage and dysfunction. Future research should focus on the specific epitopes that are targeted by these antibodies, and their functional significance. This may result in the emergence of novel therapies to attenuate cardiac dysfunction in MI patients.

Morphological classification and comparison of suboccipital muscle fiber characteristics.

In an attempt to clarify the function of the suboccipital muscles, we performed morphological observation of the suboccipital muscles for variations in the muscle belly and compared the morphology of their muscle fibers in terms of cross-sectional area by immunostaining with anti-myosin heavy chain antibodies. The cadavers of 25 Japanese individuals were used: 22 for morphological examinations and three for histological examinations. Among samples of the rectus capitis posterior major muscle (RCPma) and rectus capitis posterior minor muscle (RCPmi), 86.4% had a typical muscle appearance with a single belly, and 13.6% had an anomalous morphology. None of the samples of the obliquus capitis superior (OCS) or obliquus capitis inferior (OCI) muscles had an anomalous appearance. Measurement of cross-sectional area revealed that fast-twitch muscle fibers in the RCPma and OCI had a significantly greater cross-sectional area than those of the RCPmi and OCS. The cross-sectional area of intermediate muscle fibers was also significantly greater in the OCS than in the RCPma, RCPmi, and OCI. The cross-sectional area of slow-twitch muscle fibers was significantly greater in the OCS than in the RCPma, RCPmi, and OCI, and the RCPmi showed a significantly greater cross-sectional area for slow-twitch muscle fibers than did the RCPma, and OCI. Our findings indicate that the RCPmi and OCS exert a greater force than the RCPma and OCI, and act as anti-gravity agonist muscles of the head. Prolonged head extension in individuals with anomalous suboccipital muscle groups could result in dysfunction due to undue stress.

Prothrombotic skeletal muscle myosin directly enhances prothrombin activation by binding factors Xa and Va

AbstractTo test the hypothesis that skeletal muscle myosins can directly influence blood coagulation and thrombosis, ex vivo studies of the effects of myosin on thrombogenesis in fresh human blood were conducted. Addition of myosin to blood augmented the thrombotic responses of human blood flowing over collagen-coated surfaces (300 s−1 shear rate). Perfusion of human blood over myosin-coated surfaces also caused fibrin and platelet deposition, evidencing myosin's thrombogenicity. Myosin markedly enhanced thrombin generation in both platelet-rich plasma and platelet-poor plasma, indicating that myosin promoted thrombin generation in plasma primarily independent of platelets. In purified reaction mixtures composed only of factor Xa, factor Va, prothrombin, and calcium ions, myosin greatly enhanced prothrombinase activity. The Gla domain of factor Xa was not required for myosin's prothrombinase enhancement. When binding of purified clotting factors to immobilized myosin was monitored using biolayer interferometry, factors Xa and Va each showed favorable binding interactions. Factor Va reduced by 100-fold the apparent Kd of myosin for factor Xa (Kd ∼0.48 nM), primarily by reducing koff, indicating formation of a stable ternary complex of myosin:Xa:Va. In studies to assess possible clinical relevance for this discovery, we found that antimyosin antibodies inhibited thrombin generation in acute trauma patient plasmas more than in control plasmas (P = .0004), implying myosin might contribute to acute trauma coagulopathy. We posit that myosin enhancement of thrombin generation could contribute either to promote hemostasis or to augment thrombosis risk with consequent implications for myosin's possible contributions to pathophysiology in the setting of acute injuries.

Anti-Myosin Antibodies Following Myocardial Infarction: A Prospective Observational Study

Anti-Myosin Antibodies Following Myocardial Infarction: A Prospective Observational Study

FTY720 (Gilenya) treatment prevents spontaneous autoimmune myocarditis and dilated cardiomyopathy in transgenic HLA-DQ8-BALB/c mice.

Although dilated cardiomyopathy (DCM) is often caused by viral infections, it frequently involves autoimmune mechanisms associated with particular HLA-DR and DQ alleles. Our homozygous HLA-DQ8Ab(0) transgenic mice in the BALB/c background (HLA-DQ8(BALB/c)-Tg) developed early and progressive fatal heart failure from 4 to 5 weeks of age. Clinical signs of the disease included cyanotic eyes, tachycardia with dyspnea (from pale to cyanotic limbs), and terminal whole body edema. Sick mice had extremely dilated hearts, enlarged liver and spleen, and pleural/peritoneal effusion. Histology of the heart showed extensive heart muscle destruction with signs of fibrosis. The autoimmune nature of the disease was shown by high titers of antimyosin antibodies in the sera and IgG deposits in sick heart muscles, as well as focal neutrophil, T cell, and macrophage infiltration of the heart muscle. The sera of the sick mice showed a granular staining pattern on sections of healthy heart muscle. Quantitative analyses of DCM-specific gene expression studies revealed that sets of genes are involved in inflammation, hypoxia, and fibrosis. Treatment with FTY720 (Fingolimod/Gilenya) protected animals from the development of cardiomyopathy. HLA-DQ8(BALB/c)-Tg mice represent a spontaneous autoimmune myocarditis model that may provide a useful tool for studying the autoimmune mechanism of DCM and testing immunosuppressive drugs.

Exome Genotyping Links Venous Thrombosis Risk with the Skeletal Muscle Myosin Gene Cluster and Leads to Discovery of New Family of Procoagulant Factors

Venous thromboembolism (VTE) exacts a severe toll, and its toll persists even after the acute thrombotic event is resolved. Although several low frequency, racially-specific, genetic variants have been linked to VTE (e.g., factor V Leiden, prothrombin nt20210A, protein S Tokushima, Protein C R147W and del-Lys150), these polymorphisms fail to explain the majority of VTE risk. These variants are causally linked to increased VTE risk and, based on their racial-specific occurrence, they likely provided evolutionary benefits. Thus, to discover new rare missense functional variants linked to VTE, we used the new Affymetrix Axiom array to determine ~ 300,000 mostly exomic rare variant polymorphisms in the Scripps venous thrombosis registry (N=214 controls and 107 VTE cases) and gained multiple novel insights. When recurrent VTE cases were analyzed, 34 SNPs, including two F5 SNPs (rs6025 and rs6687813), were significant (p<0.05) after FDR correction. Analysis of clustering for these SNPs revealed associations of VTE recurrence risk with skeletal muscle myosin (MYH) rare SNPs (chr 17p13.1) in a highly conserved region with clustered myosin heavy chain genes (MYH1, 2, 4, 8). Among 11 rare MYH SNPs, rs111567318 was significantly linked to VTE (p=8.55x10-6, FDR p<0.05). Notably, 16 of 107 (15. %) VTE subjects had ≥ one SNP in the MYH chr17 cluster compared to only 1 of 212 (0.5 %) controls.Studies were initiated to define procoagulant or anticoagulant properties of skeletal muscle myosins which heretofore had no known function in blood clotting and which are broadly found in the body, even in plasma (e.g., at 20 nM). Remarkably, purified rabbit skeletal muscle myosin enhanced TF-induced thrombin generation in plasma. In neat plasma, anti-myosin antibodies reduced TF-induced thrombin generation, suggesting the contribution of endogenous plasma myosin to plasma thrombin generation. Skeletal muscle myosin also enhanced recalcification-induced thrombin generation in plasma. Studies to tests if prothrombinase activity is promoted by skeletal muscle myosin in purified prothrombinase (IIase) assays (factor Xa, factor Va, and II plus Ca++, with or without phospholipid added) showed that skeletal muscle myosin greatly enhanced IIase activity. When factor (F) Va was absent, myosin did not detectably enhance IIase activity, showing FVa was required. The myosin heavy chain can be split into 1 light meromyosin (LMM) and 1 heavy meromyosin (HMM) domains which can be further split into 2 globular subfragments (S1) and 1 rod-shaped subfragment (S2). HMM enhanced IIase activity whereas the myosin S1 subfragment did not, suggesting the S2 subfragment and LMM were required for the procoagulant activity. When prothrombin cleavage kinetics in the presence of skeletal muscle myosin were studied using PAGE, negligible amounts of meizothrombin were observed while fragment 1.2 and prothrombin 2 (or a-thrombin) bands were observed. This implies that skeletal muscle myosin promotes the prothrombin cleavage pathway similar to that enhanced by platelets, but not similar to that enhanced by phospholipid vesicles. The IIase activity of plasma-derived, purified extra cellular vesicles (ECVs) was markedly inhibited (> 60 %) by anti-skeletal muscle myosin antibodies, suggesting that a major fraction of procoagulant extracellular vesicle procoagulant activity involves skeletal muscle myosin. In purified systems, the apparent Kd of skeletal myosin in the presence of FVa for FXa was 9 nM.In summary, we found that MYH SNPs may be linked to VTE risk which led to the discovery of a new family of procoagulant proteins, skeletal myosins, that bind FXa, stimulate thrombin generation in plasma and in purified systems, thus paving the way for future genetic and functional VTE research. DisclosuresNo relevant conflicts of interest to declare.

Immuno-modification of enhancing stem cells targeting for myocardial repair.

Despite the controversy in mechanism, rodent and clinical studies have demonstrated beneficial effects of stem/progenitor cell therapy after myocardial infarction (MI). In a rat ischaemic reperfusion MI model, we investigated the effects of immunomodification of CD 34+ cells on heart function and myocardial conduction. Bispecific antibody (BiAb), consisting of an anti-myosin light chain antibody and anti-CD45 antibody, injected intravenously was used to direct human CD34+ cells to injured myocardium. Results were compared to echocardiography guided intramyocardial (IM) injection of CD34+ cells and PBS injected intravenously. Treatment was administered 2 days post MI. Echocardiography was performed at 5 weeks and 3 months which demonstrated LV dilatation prevention and fractional shortening improvement in both the BiAb and IM injection approaches, with BiAb achieving better results. Histological analyses demonstrated a decrease in infarct size and increase in arteriogenesis in both BiAb and IM injection. Electrophysiological properties were studied 5 weeks after treatments by optical mapping. Conduction velocity (CV), action potential duration (APD) and rise time were significantly altered in the MI area. The BiAb treated group demonstrated a more normalized activation pattern of conduction and normalization of CV at shorter pacing cycle lengths. The ventricular tachycardia inducibility was lowest in the BiAb treatment group. Intravenous administration of BiAb offers an effective means of stem cell delivery for myocardial repair post-acute MI. Such non-invasive approach was shown to offer a distinct advantage to more invasive direct IM delivery.

Specific Sorting and Post-Golgi Trafficking of Dendritic Potassium Channels in Living Neurons

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.

Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age

Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.

Development of the Novel PEG-PE-Based Polymer for the Reversible Attachment of Specific Ligands to Liposomes: Synthesis and in Vitro Characterization

Surface grafting of liposomes with the wide variety of ligands including antibodies and other proteins is a promising approach for targeted delivery of therapeutics. In this paper, we describe a simple method of synthesizing a hydrazine-functionalized poly(ethylene glycol)-phosphatidylethanolamine (PEG-PE)-based amphiphilic polymer which can conjugate a variety of ligands via a reversible, pH-cleavable bond. In this method, the targeting ligand is attached to the distal end of the PEG chain, which facilitates its easy access to the targeted site of interaction. The reversible attachment of targeting ligands is useful especially in multifunctional liposomal systems, whereafter successfully performing the function of targeting to the specific site, the bulky ligands, such as proteins or antibodies, are cleaved off in response to an environmental stimulus to expose some other functionalities such as ligands for intracellular penetration or organelle-specific targeting. To investigate the applicability of the protocol, the model ligands monoclonal antinucleosome antibody 2C5 and antimyosin antibody 2G4, and glycoproteins concanavalin A (Con-A) and avidin were conjugated to the synthesized polymer and incorporated into liposomes. In vitro assays including biochemical, enzyme-linked immunosorbent, fluorescence microscopy, and flow cytometry were used to confirm three key characteristics of the modified and/or liposome-attached proteins: successful conjugation of the targeting ligands to the polymer, preservation of specific activity of the ligands after the conjugation and liposome attachment, and the facile pH-sensitive ligand detachment. Monoclonal antibody 2C5 and 2G4, immobilized on the liposome surface, retained their binding affinity to corresponding antigens as confirmed by ELISA. The Con A-bearing liposomes showed significantly higher agglutination in the presence of its substrate mannan compared to plain liposomes (PL) and avidin-functionalized liposomes bound specifically with biotin-agarose. The study on the pH-dependence showed that almost 80% of the hydrazone bond was cleaved after rather brief preincubation of the immunoliposomes at pH 5 for 0.5 to 1 h. Fluorescence microscopy and flow cytometry analysis of cancer cells (HeLa and MCF-7) treated with cancer cell-specific targeting ligand mAb 2C5-bearing liposomes showed enhanced cellular binding. Studies at low pH clearly confirmed the easy cleavability of the targeting ligand from the liposomes resulting in significantly less or virtually no cellular association.

Bone marrow mesenchymal stromal cells with support of bispecific antibody and ultrasound-mediated microbubbles prevent myocardial fibrosis via the signal transducer and activators of transcription signaling pathway

This study was initiated to investigate the efficacy of myocardial fibrosis intervention via signal transducer and activators of transcription (STAT) signaling using bone marrow (BM) mesenchymal stromal cells (MSC) with the aid of bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB). BiAb (anti-CD29 × anti-myosin light chain antibody; AMLCA) was prepared and combined with isolated MSC from male mice and transfused into female mice with isoproterenol-induced myocardial fibrosis via the tail vein, followed by MB (MSC + BiAb + MB). This study included seven groups: MSC + BiAb + MB; MSC; BiAb; MB; MSC + BiAb; untreated; and control. Five weeks after treatment, expression levels of the sex-determining region of Y-chromosome (SRY), matrix metalloproteinases (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1 and vascular endothelial growth factor (VEGF) in myocardium were detected by fluorescent quantitative real-time polymerase chain reaction (qRT-PCR). Collagen distribution was observed using Sirius Red staining. The protein expression of signal transducer and activators of transcription (STAT)1 and STAT3 was detected by Western blot. The highest homing number of MSC was in the MSC + BiAb + MB group, second highest in the MSC + BiAb group, and lowest in MSC alone. Compared with the untreated group, MSC + BiAb + MB, MSC + BiAb and MSC groups had decreased levels of MMP-9, TIMP-1, STAT1 and collagen deposition, and increased levels of STAT3. Upregulated STAT3 and downregulated TIMP-1 were significantly different in MSC + BiAb + MB compared with MSC alone or MSC + BiAb. The homing rate and repairing efficacy of MSC improved with treatment utilizing a combination of BiAb and MB. MSC can improve MMP-TIMP expression in injured myocardium and interfere with myocardial fibrosis after homing, a mechanism that may be related to the STAT-mediated signaling pathway.