Non-muscle Tissue Research Articles

Integrating muscle fiber orientation from visible human data into radiotherapy target volumes.

Objective.A major challenge in treatment of tumors near skeletal muscle is defining the target volume for suspected tumor invasion into the muscle. This study develops a framework that generates radiation target volumes with muscle fiber orientation directly integrated into their definition. The framework is applied to nineteen sacral tumor patients with suspected infiltration into surrounding muscles.Approach.To compensate for the poor soft-tissue contrast of CT images, muscle fiber orientation is derived from cryo-images of two cadavers from the human visible project (VHP). The approach consists of (a) detecting image gradients in the cadaver images representative of muscle fibers, (b) mapping this information onto the patient image, and (c) embedding the muscle fiber orientation into an expansion method to generate patient-specific clinical target volumes (CTV). The validation tested the consistency of image gradient orientation across VHP subjects for the piriformis, gluteus maximus, paraspinal, gluteus medius, and gluteus minimus muscles. The model robustness was analyzed by comparing CTVs generated using different VHP subjects. The difference in shape between the new CTVs and standard CTV was analyzed for clinical impact.Main results.Good agreement was found between the image gradient orientation across VHP subjects, as the voxel-wise median cosine similarity was at least 0.86 (for the gluteus minimus) and up to 0.98 for the piriformis. The volume and surface similarity between the CTVs generating from different VHP subjects was on average at least 0.95 and 5.13 mm for the Dice Similarity Coefficient and the Hausdorff 95% Percentile Index, showing excellent robustness. Finally, compared to the standard CTV with different margins in muscle and non-muscle tissue, the new CTV margins are reduced in muscle tissue depending on the chosen clinical margins.Significance.This study implements a method to integrate muscle fiber orientation into the target volume without the need for additional imaging.

Nerve pathology is prevented by linker proteins in mouse models for LAMA2-related muscular dystrophy.

LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is a devastating congenital muscular dystrophy that is caused by mutations in the LAMA2 gene encoding laminin-α2, the long chain of several heterotrimeric laminins. Laminins are essential components of the extracellular matrix that interface with underlying cells. The pathology of LAMA2 MD patients is dominated by an early-onset, severe muscular dystrophy that ultimately leads to death by respiratory insufficiency. However, pathology in nonmuscle tissues has been described. Prior work in the dyW /dyW mouse model for LAMA2 MD has shown that two linker proteins, mini-agrin and αLNNd, when expressed in skeletal muscle fibers, greatly increase survival from a few months up to more than 2 years. However, the restoration of skeletal muscle function accentuates the pathology in nonmuscle tissue in dyW /dyW mice, first and foremost in the peripheral nerve resulting in paralysis of the hind limbs. We now show that the expression of the two linker proteins in all tissues ameliorates the muscular dystrophy and prevents the appearance of the hind limb paralysis. Importantly, the same ameliorating effect of the linker proteins was seen in dy3K /dy3K mice, which represent the most severe mouse model of LAMA2 MD. In summary, these data show that the two linker proteins can compensate the loss of laminin-α2 in muscle and peripheral nerve, which are the two organs most affected in LAMA2 MD. These results are of key importance for designing appropriate expression constructs for mini-agrin and αLNNd to develop a gene therapy for LAMA2 MD patients.

P.58 MRI muscle segmentation in Duchenne muscular dystrophy (DMD): Stepwise region of interest (ROI) contractions to minimize fat fraction variability

MRI proton density fat fraction (MRI-PDFF) to quantify fatty infiltration is a key efficacy endpoint in DMD and other neuromuscular clinical trials. We have previously shown that fat fraction variability can be reduced by contracting the ROI to eliminate noise from non-muscle tissue due to small segmentation errors. Multiple muscles are of interest due to progressive involvement in DMD, and the ideal contraction may vary depending on the muscle size or location. We tested a strategy for optimizing the segmentation of muscles of varying sizes and depths, designed to minimize the variability of fat fraction measurements while maximizing the volume of the contracted ROI. The vastus lateralis (VL) and soleus muscle bellies were segmented by two technologists from T1-weighted images and the center 3 slices were computed for the MRI-PDFF ROI (VL: N=13; soleus: N=14). Uniform axial contractions between 0 mm to 2 mm (in steps of 0.2 mm) were applied to the ROI and the resulting segmentations were registered to PDFF maps to obtain the fat fraction at every step. The minimal detectable change (MDC) for each contracted ROI was computed from the appropriate intraclass correlation coefficient (2-way or 1-way, random-effects, single-rating, absolute agreement) and pooled standard deviation. The Dice coefficient was used to record how much of the original volume is represented at each contraction. The variability approached a minimum as the Dice coefficient neared 0.9 for both muscles. The fat fraction MDC was reduced by 75% in the VL when a 1.2 mm contraction was applied (MDC=1.0%; Dice 0.91) and by 29% in the soleus when a 1.0 mm contraction was applied (MDC=1.3%; Dice 0.90). Further contracting the muscles had minimal effects on MDC. Therefore, we conclude that an axial contraction of the central ROI that represents 90% of the segmented muscle area is optimal for maximizing the interrogated muscle volume and the ability to demonstrate efficacy of disease-modifying therapeutics.

Estrogen promotes fetal skeletal muscle myofiber development important for insulin sensitivity in offspring.

Using our nonhuman primate baboon model, we showed that offspring born to mothers deprived of estrogen during the second half of gestation exhibited insulin resistance and a deficit in first phase insulin release. Although insulin resistance was not due to an impairment of fetal or offspring growth, nor to an alteration in adipose or hepatic sensitivity to insulin, skeletal muscle microvacularization critical for delivery of nutrients/insulin was significantly reduced in fetuses and offspring deprived of estrogen in utero. Skeletal muscle myofiber maturation occurs in utero and estrogen modulates myofiber growth in adults. Therefore, the current study determined whether fetal skeletal muscle development was altered in baboons in which estradiol levels were suppressed/restored during the second half of gestation by maternal treatment with letrozole ± estradiol benzoate. In estrogen-suppressed animals, fetal skeletal muscle fascicles were structurally less organized, smaller, and comprised of slow type I and fast type II fibers, the size, but not the number of which were smaller than in untreated baboons. Moreover, the proportion of non-muscle fiber tissue was greater and that of muscle fibers lower in estrogen-deprived fetuses. Thus, the maintenance of fetal body weight in estrogen-deprived animals was maintained at the expense of muscle fibers and likely reflected increased deposition of non-muscle proteins. Importantly, fetal skeletal muscle development, including fascicle organization, myofiber size and composition was normal in baboons treated with letrozole and estradiol benzoate. Collectively, these and our previous findings support our proposal that exposure of the fetus to estrogen is important for fetal skeletal muscle development and glucose homeostasis in adulthood.

Characterization and analysis of myosin gene family in the whitefly (<i>Bemisia tabaci</i>)

<abstract> <p>Myosin is an actin-based motor protein that widely exists in muscle tissue and non-muscle tissue, and myosin of a diverse subfamily has obvious differences in structure and cell function. Many eukaryotes and even some unicellular organisms possess a variety of myosins. They have been well characterized in human, fungi and other organisms. However, the myosin gene family in <italic>Bemisia tabaci</italic> MEAM1 (Middle East-Asia Minor1 species) is poorly studied. In the study, we identified 15 myosin genes in <italic>B. tabaci</italic> MEAM1 based on a genome database. Myosin genes can be divided into ten classes, including subfamilies I, II, III, V, VI, VII, IX, XV, XVIII, XX in <italic>B. tabaci</italic> MEAM1. The amounts of myosin in Class I are the largest of the isoforms. Expression profiling of myosins by quantitative real-time PCR revealed that their expression differed among developmental stages and different tissues of <italic>B. tabaci</italic> MEAM1. The diversely may be related to the development characteristics of <italic>B. tabaci</italic> MEAM1. The <italic>BtaMyo-IIIb-like X1</italic> was highly expressed in nymphs 4 instar which may be related to the development process before metamorphosis. Our outcome contributes to the basis for further research on myosin gene function in <italic>B. tabaci</italic> MEAM1 and homologous myosins in other biology.</p> </abstract>

Ultrasonography validation for early alteration of diaphragm echodensity and function in the mdx mouse model of Duchenne muscular dystrophy.

The mdx mouse model of Duchenne muscular dystrophy is characterized by functional and structural alterations of the diaphragm since early stages of pathology, closely resembling patients’ condition. In recent years, ultrasonography has been proposed as a useful longitudinal non-invasive technique to assess mdx diaphragm dysfunction and evaluate drug efficacy over time. To date, only a few preclinical studies have been conducted. Therefore, an independent validation of this method by different laboratories is needed to increase results reliability and reduce biases. Here, we performed diaphragm ultrasonography in 3- and 6-month-old mdx mice, the preferred age-window for pharmacology studies. The alteration of diaphragm function over time was measured as diaphragm ultrasound movement amplitude. At the same time points, a first-time assessment of diaphragm echodensity was performed, as an experimental index of progressive loss of contractile tissue. A parallel evaluation of other in vivo and ex vivo dystrophy-relevant readouts was carried out. Both 3- and 6-month-old mdx mice showed a significant decrease in diaphragm amplitude compared to wild type (wt) mice. This index was well-correlated either with in vivo running performance or ex vivo isometric tetanic force of isolated diaphragm. In addition, diaphragms from 6-month-old dystrophic mice were also highly susceptible to eccentric contraction ex vivo. Importantly, we disclosed an age-dependent increase in echodensity in mdx mice not observed in wt animals, which was independent from abdominal wall thickness. This was accompanied by a notable increase of pro-fibrotic TGF-β1 levels in the mdx diaphragm and of non-muscle tissue amount in diaphragm sections stained by hematoxylin & eosin. Our findings corroborate the usefulness of diaphragm ultrasonography in preclinical drug studies as a powerful tool to monitor mdx pathology progression since early stages.

Identification of Let-7f-5p as a novel biomarker of recurrence in non-muscle invasive bladder cancer.

Among patients diagnosed with non-muscle invasive bladder cancer (NMIBC), 30% to 70% experience recurrences within 6 to 12 years of diagnosis. The need to screen for these events every 3 to 6 months and ultimately annually by cystoscopy makes bladder cancer one of the most expensive malignancies to manage. The purpose of this study was to identify reproducible prognostic microRNAs in resected non-muscle invasive bladder tumor tissue that are predictive of the recurrent tumor phenotype as potential biomarkers and molecular therapeutic targets. Two independent cohorts of NMIBC patients were analyzed using a biomarker discovery and validation approach, respectively. miRNA Let-7f-5p showed the strongest association with recurrence across both cohorts. Let-7f-5p levels in urine and plasma were both found to be significantly correlated with levels in tumor tissue. We assessed the therapeutic potential of targeting Lin28, a negative regulator of Let-7f-5p, with small-molecule inhibitor C1632. Lin28 inhibition significantly increased levels of Let-7f-5p expression and led to significant inhibition of viability and migration of HTB-2 cells. We have identified Let-7f-5p as a miRNA biomarker of recurrence in NMIBC tumors. We further demonstrate that targeting Lin28, a negative regulator of Let-7f-5p, represents a novel potential therapeutic opportunity in NMIBC.

MP17-03 MICRORNA LET-7F-5P IS A NOVEL BIOMARKER OF RECURRENCE AND A POTENTIAL THERAPEUTIC OPPORTUNITY IN NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (MP17)1 Apr 2020MP17-03 MICRORNA LET-7F-5P IS A NOVEL BIOMARKER OF RECURRENCE AND A POTENTIAL THERAPEUTIC OPPORTUNITY IN NON-MUSCLE INVASIVE BLADDER CANCER Kevin Shee*, Carmen J Marsit, Alan R Schned, Jason Pettus, David A Armstrong, Margaret R Karagas, Todd W Miller, John D Seigne, and Angeline S Andrew Kevin Shee*Kevin Shee* More articles by this author , Carmen J MarsitCarmen J Marsit More articles by this author , Alan R SchnedAlan R Schned More articles by this author , Jason PettusJason Pettus More articles by this author , David A ArmstrongDavid A Armstrong More articles by this author , Margaret R KaragasMargaret R Karagas More articles by this author , Todd W MillerTodd W Miller More articles by this author , John D SeigneJohn D Seigne More articles by this author , and Angeline S AndrewAngeline S Andrew More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000842.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: 50% to 75% of patients with non-muscle invasive bladder cancer (NMIBC) experience recurrences within 6 to 12 years of diagnosis, and the cost of screening makes bladder cancer one of the most expensive malignancies. The objective of this study was to identify reproducible prognostic miRNAs in resected non-muscle invasive bladder tumor tissue that are predictive of the recurrent tumor phenotype as potential biomarkers and molecular therapeutic targets. METHODS: NMIBC patient tumors from the New Hampshire Population Cohort (n=178) and Dartmouth Hitchcock Medical Center (n=38) were assayed for ∼800 miRNAs. Recurrence free survival (RFS) analyses using a multivariable model adjusted for sex, age, multiplicity, tumor size, stage, and grade were performed. Urine and plasma samples were obtained from available patients from the validation cohort and analyzed for miRNA expression. Lin28, a known negative regulator of Let-7f-5p, Lin28 primers and small molecule inhibitor Lin28 1632 were obtained and assayed using RT-PCR, cell viability, and scratch assays in NMIBC cell line HTB-2. RESULTS: miRNA Let-7f-5p showed the strongest association with recurrence across both the NH population cohort (p=0.039) and the DHMC TaHG/T1 cohort (p=0.065). Survival analyses stratified by median Let-7f-5p expression confirmed longer RFS in the patients with high Let-7f-5p for both discovery and validation cohorts (p=0.011 and 0.065; Figure 1A/B). Let-7f-5p was found to have potential clinical utility as a biomarker, and levels in urine and plasma were both found to be significantly correlated with levels in tumor tissue (P=0.0004 and 0.014, respectively). Finally, inhibition of Lin28 with Lin28 1632 significantly increased levels of Let-7f-5p expression (p=0.0003; Figure 1C) and led to significant inhibition of viability and migration of HTB-2 cells (both p<0.0001; Figures 1D/E). CONCLUSIONS: In this study, we have identified Let-7f-5p as a novel miRNA biomarker of recurrence in NMIBC tumors, and show that patients with high Let-7f-5p have longer RFS. We further show that targeting Lin28, a negative regulator of Let-7f-5p, represents a novel therapeutic opportunity in NMIBC. Source of Funding: Grant numbers R21CA182659, K07CA102327, CA182659, LM009012, GM103506 and GM103534, RR024475, RR028309, CA102327, CA121382, CA141017, CA099500, CA82354, CA078609, ES00002, 5P42ES05947, RR018787, and ES07373 from the National Cancer Institute, NIH, from the National Institute of Environmental Health Sciences, NIH, the National Center for Research Resources, NIH, and the National Institute of General Medical Sciences, NIH. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e226-e226 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kevin Shee* More articles by this author Carmen J Marsit More articles by this author Alan R Schned More articles by this author Jason Pettus More articles by this author David A Armstrong More articles by this author Margaret R Karagas More articles by this author Todd W Miller More articles by this author John D Seigne More articles by this author Angeline S Andrew More articles by this author Expand All Advertisement PDF downloadLoading ...

Functional Differences in MYH7b That Contribute to Distinct Biological Roles across Species and in Health and Disease

Myosin heavy chain 7b (MYH7b) is a recently identified sarcomeric myosin gene family member that has a unique expression pattern in mammals. MYH7b protein is absent from the heart and skeletal muscle of mammals and is only expressed in specialized muscles and, surprisingly, in the inner ear and brain. Additionally, compound heterozygous mutations in MYH7b (one in the motor domain and one in the rod domain) cause hearing loss in humans by an unknown mechanism (Haraksingh et al., BMC Genomics, 2014) indicating that MYH7b has an important yet undefined role in mammalian non-muscle tissue. Intriguingly, in snakes, MYH7b protein is abundant in cardiac and skeletal muscles. Despite these divergent roles in mammals and reptiles, MYH7b shares high sequence identity between these vertebrate classes, so it is unclear how mammalian MYH7b function may deviate in non-muscle tissues from its expected sarcomeric role. To address this question, we have generated recombinant protein for human MYH7b, human MYH7b with the motor domain hearing loss mutation (D515N), and python MYH7b, and have measured their biochemical and biophysical properties. We have observed differences in the steady-state actin-activated ATPase activities and actin translocation as assessed by in vitro motility assays between these three recombinant proteins. Comparing the motor activity of human MYH7b and python MYH7b provides both an evolutionary perspective and functional analysis of highly conserved myosins that operate in different cell types. Further, comparing the function of human wild type MYH7b and D515N MYH7b provides greater insight into the etiology of human hearing loss.

Tripeptide Arg-Gly-Asp (RGD) modifies the molecular mechanical properties of the non-muscle myosin IIA in human bone marrow-derived myofibroblasts seeded in a collagen scaffold.

Mesenchymal stem cells (MSCs) were obtained from human bone marrow and amplified in cultures supplemented with human platelet lysate in order to generate myofibroblasts. When MSCs were seeded in solid collagen scaffolds, they differentiated into myofibroblasts that were observed to strongly bind to the substrate, forming a 3D cell scaffold network that developed tension and shortening after KCl stimulation. Moreover, MSC-laden scaffolds recapitulated the Frank-Starling mechanism so that active tension increased in response to increases in the initial length of the contractile system. This constituted a bioengineering tissue that exhibited the contractile properties observed in both striated and smooth muscles. By using the A. F. Huxley formalism, we determined the myosin crossbridge (CB) kinetics of attachment (f1) and detachment (g1 and g2), maximum myosin ATPase activity, molar myosin concentration, unitary CB force and maximum CB efficiency. CB kinetics were dramatically slow, characterizing the non-muscle myosin type IIA (NMMIIA) present in myofibroblasts. When MSCs were seeded in solid collagen scaffolds functionalized with Arg-Gly-Asp (RGD), contractility increased and CB kinetics were modified, whereas the unitary NMMIIA-CB force and maximum CB efficiency did not change. In conclusion, we provided a non-muscle bioengineering tissue whose molecular mechanical characteristics of NMMIIA were very close to those of a non-muscle contractile tissue such as the human placenta.

Isolation and characterization of myogenic precursor cells from human cremaster muscle

Human myogenic precursor cells have been isolated and expanded from a number of skeletal muscles, but alternative donor biopsy sites must be sought after in diseases where muscle damage is widespread. Biopsy sites must be relatively accessible, and the biopsied muscle dispensable. Here, we aimed to histologically characterize the cremaster muscle with regard number of satellite cells and regenerative fibres, and to isolate and characterize human cremaster muscle-derived stem/precursor cells in adult male donors with the objective of characterizing this muscle as a novel source of myogenic precursor cells. Cremaster muscle biopsies (or adjacent non-muscle tissue for negative controls; N = 19) were taken from male patients undergoing routine surgery for urogenital pathology. Myosphere cultures were derived and tested for their in vitro and in vivo myogenic differentiation and muscle regeneration capacities. Cremaster-derived myogenic precursor cells were maintained by myosphere culture and efficiently differentiated to myotubes in adhesion culture. Upon transplantation to an immunocompromised mouse model of cardiotoxin-induced acute muscle damage, human cremaster-derived myogenic precursor cells survived to the transplants and contributed to muscle regeneration. These precursors are a good candidate for cell therapy approaches of skeletal muscle. Due to their location and developmental origin, we propose that they might be best suited for regeneration of the rhabdosphincter in patients undergoing stress urinary incontinence after radical prostatectomy.

Muscle wasting in the presence of disease, why is it so variable?

Skeletal muscle wasting is a common clinical feature of many chronic diseases and also occurs in response to single acute events. The accompanying loss of strength can lead to significant disability, increased care needs and have profound negative effects on quality of life. As muscle is the most abundant source of amino acids in the body, it appears to function as a buffer for fuel and substrates that can be used to repair damage elsewhere and to feed the immune system. In essence, the fundamentals of muscle wasting are simple: less muscle is made than is broken down. However, although well-described mechanisms modulate muscle protein turnover, significant individual differences in the amount of muscle lost in the presence of a given severity of disease complicate the understanding of underlying mechanisms and suggest that individuals have different sensitivities to signals for muscle loss. Furthermore, the rate at which muscle protein is turned over under normal conditions means that clinically significant muscle loss can occur with changes in the rate of protein synthesis and/or breakdown that are too small to be measurable. Consequently, the changes in expression of factors regulating muscle turnover required to cause a decline in muscle mass are small and, except in cases of rapid wasting, there is no consistent pattern of change in the expression of factors that regulate muscle mass. MicroRNAs are fine tuners of cell phenotype and are therefore ideally suited to cause the subtle changes in proteome required to tilt the balance between synthesis and degradation in a way that causes clinically significant wasting. Herein we present a model in which muscle loss as a consequence of disease in non-muscle tissue is modulated by a set of microRNAs, the muscle expression of which is associated with severity of disease in the non-muscle tissue. These microRNAs alter fundamental biological processes including the synthesis of ribosomes and mitochondria leading to reduced protein synthesis and increased protein breakdown, thereby freeing amino acids from the muscle. We argue that the variability in muscle loss observed in the human population arises from at least two sources. The first is from pre-existing or disease-induced variation in the expression of microRNAs controlling the sensitivity of muscle to the atrophic signal and the second is from the expression of microRNAs from imprinted loci (i.e. only expressed from the maternally or paternally inherited allele) and may control the rate of myonuclear recruitment. In the absence of disease, these factors do not correlate with muscle mass, since there is no challenge to the established balance. However, in the presence of such a challenge, these microRNAs determine the rate of decline for a given disease severity. Together these mechanisms provide novel insight into the loss of muscle mass and its variation in the human population. The involvement of imprinted loci also suggests that genes that regulate early development also contribute to the ability of individuals to resist muscle loss in response to disease.

Tropomyosin 3.5 protects the F-actin networks required for tissue biomechanical properties.

Tropomyosins (Tpms) stabilize F-actin and regulate interactions with other actin-binding proteins. The eye lens changes shape in order to focus light to transmit a clear image, and thus lens organ function is tied to its biomechanical properties, presenting an opportunity to study Tpm functions in tissue mechanics. Mouse lenses contain Tpm3.5 (also known as TM5NM5), a previously unstudied isoform encoded by Tpm3, which is associated with F-actin on lens fiber cell membranes. Decreased levels of Tpm3.5 lead to softer and less mechanically resilient lenses that are unable to resume their original shape after compression. While cell organization and morphology appear unaffected, Tmod1 dissociates from the membrane in Tpm3.5-deficient lens fiber cells resulting in reorganization of the spectrin-F-actin and α-actinin-F-actin networks at the membrane. These rearranged F-actin networks appear to be less able to support mechanical load and resilience, leading to an overall change in tissue mechanical properties. This is the first in vivo evidence that a Tpm protein is essential for cell biomechanical stability in a load-bearing non-muscle tissue, and indicates that Tpm3.5 protects mechanically stable, load-bearing F-actin in vivoThis article has an associated First Person interview with the first author of the paper.

The Myosin II Inhibitor, Blebbistatin, Ameliorates FeCl3-induced Arterial Thrombosis via the GSK3β-NF-κB Pathway.

Arterial thrombosis and its related diseases are major healthcare problems worldwide. Blebbistatin is an inhibitor of myosin II, which plays an important role in thrombosis. The aim of our study is to explore the effect and potential mechanism of blebbistatin on arterial thrombosis. A ferric chloride (FeCl3) solution at a concentration of 5% was used to induce carotid artery thrombosis in mice. Immunohistochemistry and immunofluorescence staining were used to detect the expression or activation of non-muscle myosin heavy chain IIA (NMMHC IIA), tissue factor (TF), GSK3β and NF-κB. Blebbistatin (1 mg/kg, i.p.) significantly reduced carotid artery thrombosis induced by FeCl3 solution in mice, inhibited NMMHC IIA expression and impeded TF expression via the GSK3β-NF-κB signalling pathway in mouse arterial vascular tissues. The present study demonstrates that blebbistatin may impede TF expression partly via the Akt/GSK3β-NF-κB signalling pathways in the endothelium in a FeCl3 model, shedding new insights into the pathogenesis of arterial thrombosis and providing new clues for the development of antithrombotic drugs.

403. Engineering Micro-Dystrophin AAV Vector for Clinical Translation

Duchenne muscular dystrophy (DMD) is the most severe muscular dystrophy affecting boys. It is caused by the loss of a cellular structural protein called dystrophin. Dystrophin preserves the integrity of the muscle cell membrane by providing a molecular link between the cytoskeleton and the extracellular matrix. The absence of dystrophin destabilizes the muscle membrane and promotes muscle death. AAV micro-dystrophin gene therapy has transformative potentials to significantly ameliorate DMD. Unfortunately, AAV therapy is challenged by destructive cellular immunity to newly expressed dystrophin. To overcome this obstacle, we engineered a double restrictive AAV microgene vector. Specifically, we limited off-target expression in non-muscle tissue with a muscle specific promoter and removed untoward expression in antigen-presenting cells using miR142-3p, a hematopoietic specific microRNA target sequence. Compared with the original AAV microgene vector, the newly engineered vector significantly reduced infiltration of CD4+ and CD8+ T cells in the canine DMD model. Our results suggest that the dual restriction strategy may greatly enhance clinical translation of AAV micro-dystrophin gene therapy.

Roles of Nebulin Family Members in the Heart.

The members of the nebulin protein family, including nebulin, nebulette, LASP-1, LASP-2, and N-RAP, contain various numbers of nebulin repeats and bind to actin, but are otherwise heterogeneous with regard to size, expression pattern, and function. This review focuses on the roles of nebulin family members in the heart. Nebulin is the largest member predominantly expressed in skeletal muscle, where it stretches along the thin filament. In heart, nebulin is detectable only at low levels and its absence has no apparent effects. Nebulette is similar in structure to the nebulin C-terminal Z-line region and specifically expressed in heart. Nebulette gene mutations have been identified in dilated cardiomyopathy patients and transgenic mice overexpressing nebulette mutants partially recapitulate the human pathology. In contrast, nebulette knockout mice show no functional phenotype, but exhibit Z-line widening. LASP-2 is an isoform of nebulette expressed in multiple tissues, including the heart. It is present in the Z-line and intercalated disc and able to bind and cross-link filamentous actin. LASP-1 is similar in structure to LASP-2, but expressed only in non-muscle tissue. N-RAP is present in myofibril precursors during myofibrillogenesis and thought to be involved in myofibril assembly, while it is localized at the intercalated disc in adult heart. Additional in vivo models are required to provide further insights into the functions of nebulin family members in the heart.

Oxygen Consumption of Brown Adipose Tissue (BAT) and Skeletal Muscle is Inversely Related

During exercise, the muscles' energy demand increases with increasing work load. In humans, the relationship between work load and oxygen uptake is linear until the maximal oxygen uptake (VO2max) is reached and higher exercise intensity requires additional anaerobic energy supply. VO2max is thought to reflect the maximum oxygen transport capacity of the cardiovascular system.We show that at room temperature normal mice could triple running speed at 25% inclination after reaching VO2max in spite of very modest increase of anaerobic muscle metabolism. In mice with cardiac dysfunction due to cardiac disruption of the Serca2 gene (S2KO), VO2max was reduced from week4 to week6 after gene disruption in parallel with progression of cardiac dysfunction. However, S2KO mice maintained maximal running speed at the same the level as the controls. Thus, paradoxically, running economy was better in S2KO than in controls. In S2KO, blood lactate was almost double of that of controls and respiratory exchange ratio was near 1, indicating greater reliance on anaerobic metabolism. However, heat production was lower in S2KO than in controls as reflected by tail temperature. Activity of BAT measured by fluoro-deoxyglucose using PET was reduced by 60±7% during running in controls and by 82±3% in running S2KO mice.In mice, the oxidative metabolism in non-muscle tissue, mainly in BAT, is reduced during exercise to provide more oxygen to the working muscles. This redistribution of oxygen delivery leaves the total VO2 unchanged over a wide range of exercise intensities. When cardiac output and VO2max are abnormally low, exercise intensity can be maintained since muscles can utilize the oxygen normally used by non-muscle tissue such as BAT. We conclude that oxygen consumption of skeletal muscle and BAT is regulated in a reciprocal way.

The Effect of Endoscopic Administration of Autologous Porcine Muscle-derived Cells Into the Urethral Sphincter

To verify the fate of autologous porcine myogenic cells after endoscopic administration into the urethral sphincter. This study was performed on pig animal models. The muscle-derived cells (MDCs) were isolated and identified. After the third passage, the 6 × 10(7) of PKH26 labeled cells were injected into the urethral sphincter using a urethrocystoscope. The urethras were collected after 28 days. To analyze the fate of injected cells, the PKH26 presence, the desmin expression, and the distribution of acetylcholine receptors were evaluated in the tissue sections. Moreover, the maximal urethral closure pressure (MUCP) was assessed in experimental and control groups at day 1 and day 28. The isolated porcine MDCs expressed desmin and were able to differentiate into myotubes in vitro. At day 28 after the transplantation, the depots of PKH26-positive cells were observed in the muscular layer, but also in the submucosa. The staining for desmin revealed that cells located in the muscle layer were integrated with muscle fibers that possessed acetylcholine receptors. However, cells administered into nonmuscle tissue did not express desmin. Urethral pressure profilometry demonstrated no significant differences between MUCP in the transplanted group in comparison to the control group at day 28. The present study demonstrates the successful endoscopic transplantation of myogenic cells into the urethral sphincter. The experiments indicated the key importance of precise cell administration in terms of their fate after the injection.

Cholesterol Depletion in Adipocytes Causes Caveolae Collapse Concomitant with Proteosomal Degradation of Cavin-2 in a Switch-Like Fashion

Caveolae, little caves of cell surfaces, are enriched in cholesterol, a certain level of which is required for their structural integrity. Here we show in adipocytes that cavin-2, a peripheral membrane protein and one of 3 cavin isoforms present in caveolae from non-muscle tissue, is degraded upon cholesterol depletion in a rapid fashion resulting in collapse of caveolae. We exposed 3T3-L1 adipocytes to the cholesterol depleting agent methyl-β-cyclodextrin, which results in a sudden and extensive degradation of cavin-2 by the proteasome and a concomitant movement of cavin-1 from the plasma membrane to the cytosol along with loss of caveolae. The recovery of cavin-2 at the plasma membrane is cholesterol-dependent and is required for the return of cavin-1 from the cytosol to the cell surface and caveolae restoration. Expression of shRNA directed against cavin-2 also results in a cytosolic distribution of cavin-1 and loss of caveolae. Taken together, these data demonstrate that cavin-2 functions as a cholesterol responsive component of caveolae that is required for cavin-1 localization to the plasma membrane, and caveolae structural integrity.

Protein Intake Does Not Increase Vastus Lateralis Muscle Protein Synthesis during Cycling

This study aimed to investigate the effect of protein ingestion on leg protein turnover and vastus lateralis muscle protein synthesis during bicycle exercise and recovery. Eight healthy males participated in two experiments in which they ingested either a carbohydrate solution (CHO) providing 0.49 g·kg(-1)·h(-1), or a carbohydrate and protein solution (CHO + P) providing 0.49 and 0.16 g·kg(-1)·h(-1), during 3 h of bicycle exercise and 3 h of recovery. Leg protein turnover was determined from stable isotope infusion (l-[ring-C6]phenylalanine), femoral-arterial venous blood sampling, and blood flow measurements. Muscle protein synthesis was calculated from the incorporation of l-[ring-C6]phenylalanine into protein. Consuming protein during exercise increased leg protein synthesis and decreased net leg protein breakdown; however, protein ingestion did not increase protein synthesis within the highly active vastus lateralis muscle (0.029%·h(-1), ± 0.004%·h(-1), and 0.030%·h(-1), ± 0.003%·h(-1), in CHO and CHO + P, respectively; P = 0.88). In contrast, consuming protein, during exercise and recovery, increased postexercise vastus lateralis muscle protein synthesis by 51% ± 22% (0.070%·h(-1), ± 0.003%·h(-1), and 0.105%·h(-1), ± 0.013%·h(-1), in CHO and CHO+P, respectively; P < 0.01). Furthermore, leg protein net balance was negative during recovery with CHO intake, whereas positive leg protein net balance was achieved with CHO+P intake. We conclude that consuming protein during prolonged bicycle exercise does not increase protein synthesis within highly active leg muscles. However, protein intake may have stimulated protein synthesis within less active leg muscles and/or other nonmuscle leg tissue. Finally, protein supplementation, during exercise and recovery, enhanced postexercise muscle protein synthesis and resulted in positive leg protein net balance.