Altered biomechanical properties of carotid arteries in two mouse models of muscular dystrophy

2007 ◽  
Vol 103 (2) ◽  
pp. 664-672 ◽  
Author(s):  
W. W. Dye ◽  
R. L. Gleason ◽  
E. Wilson ◽  
J. D. Humphrey
Keyword(s):  
Muscular Dystrophy ◽  
Axial Force ◽  
Carotid Arteries ◽  
Structural Integrity ◽  
Muscle Cells ◽  
Biomechanical Properties ◽  
Axial Stiffness ◽  
Wild Type ◽  
Functional Responses

Muscular dystrophy is characterized by skeletal muscle weakness and wasting, but little is known about possible alterations to the vasculature. Many muscular dystrophies are caused by a defective dystrophin-glycoprotein complex (DGC), which plays an important role in mechanotransduction and maintenance of structural integrity in muscle cells. The DGC is a group of membrane-associated proteins, including dystrophin and sarcoglycan-δ, that helps connect the cytoskeleton of muscle cells to the extracellular matrix. In this paper, mice lacking genes encoding dystrophin ( mdx) or sarcoglycan-δ ( sgcd−/−) were studied to detect possible alterations to vascular wall mechanics. Pressure-diameter and axial force-length tests were performed on common carotid arteries from mdx, sgcd−/−, and wild-type mice in active (basal) and passive smooth muscle states, and functional responses to three vasoactive compounds were determined at constant pressure and length. Apparent biomechanical differences included the following: mdx and sgcd−/− arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries exhibiting elevated circumferential stresses, and mdx and sgcd−/− arteries generated elevated axial loads and stresses in axial force-length tests. Interestingly, however, mdx and sgcd−/− arteries also had significantly lower in vivo axial stretches than did the wild type. Accounting for this possible adaptation largely eliminated the apparent differences in circumferential and axial stiffness, thus suggesting that loss of DGC proteins may induce adaptive biomechanical changes that can maintain overall wall mechanics in response to normal loads. Nevertheless, there remains a need to understand better possible vascular adaptations in response to sustained altered loads in patients with muscular dystrophy.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

In Vivo Imaging of Dynamic Shear Modulus of Rodent Mammary Tumors Using Ultrasound

2012 ◽  
Author(s):  
Yue Wang ◽  
Michael Insana
Keyword(s):  
In Vivo Imaging ◽  
Normal Tissue ◽  
Breast Tissue ◽  
Structural Integrity ◽  
Biomechanical Properties ◽  
Dynamic Shear Modulus ◽  
Living Tissue ◽  
Dynamic Shear

Biomechanical properties of living tissue are very important in maintaining normal tissue function, cellular and extracellular structural integrity. Therefore, the quantitative determination of biomechanical properties of breast tissue, especially in vivo, serves an important role in clinical diagnosis.

Thr164Ile polymorphism of the human β2-adrenoceptor exhibits blunted desensitization of cardiac functional responses in vivo

2003 ◽  
Vol 285 (5) ◽  
pp. H2034-H2038 ◽  
Author(s):  
Heike Bruck ◽  
Kirsten Leineweber ◽  
Anke Ulrich ◽  
Joachim Radke ◽  
Gerd Heusch ◽  
...  
Keyword(s):  
Heart Rate ◽  
Oral Treatment ◽  
Wild Type ◽  
Functional Responses ◽  
Maximum Increase ◽  
Cardiac Responses ◽  
Agonist Stimulation

In subjects heterozygous for Thr164Ile β2-adrenoceptor (β2AR) polymorphism, cardiac responses to β2AR agonist stimulation are blunted. In this study, we investigated agonist-induced desensitization of Thr164Ile β2ARs. For this purpose, we assessed in six subjects with heterozygous Thr164Ile β2ARs and in 10 subjects with homozygous wild-type (WT) β2ARs the effects of 2-wk oral treatment with 3 × 5 mg/day terbutaline on terbutaline infusion-induced increases in heart rate (HR) and contractility [measured as shortening of HR-corrected duration of electromechanical systole (QS2c)]. Compared with WT β2AR subjects, Thr164Ile subjects exhibited a blunted terbutaline-induced maximum increase in HR (WT 32 ± 4 beats/min, Thr164Ile 19 ± 3 beats/min, P < 0.05) and contractility (WT –54 ± 2 ms, Thr164Ile –37 ± 6 ms, P < 0.05). Two-week oral terbutaline treatment desensitized cardiac β2AR responses to terbutaline infusion (increase in HR: WT 10 ± 2 beats/min, Thr164Ile 8 ± 4 beats/min; increase in contractility: WT –22 ± 5 ms Thr164Ile: –17 ± 6 ms); however, the extent of desensitization was larger in WT than Thr164Ile β2AR subjects. Thus, after 2-wk oral terbutaline treatment cardiac, β2AR responses did not differ anymore between WT and Thr164Ile β2AR subjects. We conclude that agonist-induced desensitization of cardiac β2ARs is more pronounced in WT than Thr164Ile subjects. Thus cardiac Thr164Ile subjects appear to be somewhat protected against agonist-induced desensitization.

Biomechanical properties of carotid arteries from wild-type mice and dystrophin and sarcoglycan-δ knockout mice

2006 ◽  
Vol 39 ◽  
pp. S322
Author(s):  
S. Baek ◽  
R.L. Gleason ◽  
W. Dye ◽  
E. Wilson ◽  
J.D. Humphrey
Keyword(s):  
Knockout Mice ◽  
Carotid Arteries ◽  
Biomechanical Properties ◽  
Wild Type

scholarly journals The Hydrogenase Cytochrome b Heme Ligands of Azotobacter vinelandii Are Required for Full H2 Oxidation Capability

2000 ◽  
Vol 182 (12) ◽  
pp. 3429-3436 ◽  
Author(s):  
Laura Meek ◽  
Daniel J. Arp
Keyword(s):  
Methylene Blue ◽  
Structural Integrity ◽  
Azotobacter Vinelandii ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Oxidation Activity ◽  
Intact Cells ◽  
Methylene Blue Reduction ◽  
Partial Activity

ABSTRACT The hydrogenase in Azotobacter vinelandii, like other membrane-bound [NiFe] hydrogenases, consists of a catalytic heterodimer and an integral membrane cytochrome b. The histidines ligating the hemes in this cytochrome b were identified by H2 oxidation properties of altered proteins produced by site-directed mutagenesis. Four fully conserved and four partially conserved histidines in HoxZ were substituted with alanine or tyrosine. The roles of these histidines in HoxZ heme binding and hydrogenase were characterized by O2-dependent H2 oxidation and H2-dependent methylene blue reduction in vivo. Mutants H33A/Y (H33 replaced by A or Y), H74A/Y, H194A, H208A/Y, and H194,208A lost O2-dependent H2 oxidation activity, H194Y and H136A had partial activity, and H97Y,H98A and H191A had full activity. These results suggest that the fully conserved histidines 33, 74, 194, and 208 are ligands to the hemes, tyrosine can serve as an alternate ligand in position 194, and H136 plays a role in H2 oxidation. In mutant H194A/Y, imidazole (Imd) rescued H2 oxidation activity in intact cells, which suggests that Imd acts as an exogenous ligand. The heterodimer activity, quantitatively determined as H2-dependent methylene blue reduction, indicated that the heterodimers of all mutants were catalytically active. H33A/Y had wild-type levels of methylene blue reduction, but the other HoxZ ligand mutants had significantly less than wild-type levels. Imd reconstituted full methylene blue reduction activity in mutants H194A/Y and H208A/Y and partial activity in H194,208A. These results indicate that structural and functional integrity of HoxZ is required for physiologically relevant H2 oxidation, and structural integrity of HoxZ is necessary for full heterodimer-catalyzed H2 oxidation.

scholarly journals Peptide-Functionalized Dendrimer Nanocarriers for Targeted Microdystrophin Gene Delivery

Pharmaceutics ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2159
Author(s):  
Jessica Hersh ◽  
José Manuel Condor Capcha ◽  
Camila Iansen Irion ◽  
Guerline Lambert ◽  
Mauricio Noguera ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Gene Delivery ◽  
Nuclear Membrane ◽  
Targeted Delivery ◽  
Intracellular Transport ◽  
Muscle Cells ◽  
Skeletal Muscle Cells

Gene therapy is a good alternative for determined congenital disorders; however, there are numerous limitations for gene delivery in vivo including targeted cellular uptake, intracellular trafficking, and transport through the nuclear membrane. Here, a modified G5 polyamidoamine (G5 PAMAM) dendrimer–DNA complex was developed, which will allow cell-specific targeting to skeletal muscle cells and transport the DNA through the intracellular machinery and the nuclear membrane. The G5 PAMAM nanocarrier was modified with a skeletal muscle-targeting peptide (SMTP), a DLC8-binding peptide (DBP) for intracellular transport, and a nuclear localization signaling peptide (NLS) for nuclear uptake, and polyplexed with plasmid DNA containing the GFP-tagged microdystrophin (µDys) gene. The delivery of µDys has been considered as a therapeutic modality for patients suffering from a debilitating Duchenne muscular dystrophy (DMD) disorder. The nanocarrier–peptide–DNA polyplexes were prepared with different charge ratios and characterized for stability, size, surface charge, and cytotoxicity. Using the optimized nanocarrier polyplexes, the transfection efficiency in vitro was determined by demonstrating the expression of the GFP and the µDys protein using fluorescence and Western blotting studies, respectively. Protein expression in vivo was determined by injecting an optimal nanocarrier polyplex formulation to Duchenne model mice, mdx4Cv. Ultimately, these nanocarrier polyplexes will allow targeted delivery of the microdystrophin gene to skeletal muscle cells and result in improved muscle function in Duchenne muscular dystrophy patients.

Inhibition of TGF-beta receptor signaling in osteoblasts leads to decreased bone remodeling and increased trabecular bone mass

Development ◽  
1999 ◽  
Vol 126 (19) ◽  
pp. 4267-4279 ◽  
Author(s):  
E. Filvaroff ◽  
A. Erlebacher ◽  
J. Ye ◽  
S.E. Gitelman ◽  
J. Lotz ◽  
...  
Keyword(s):  
Bone Formation ◽  
Transgenic Mice ◽  
Bone Mass ◽  
Trabecular Bone ◽  
Bone Remodeling ◽  
Biomechanical Properties ◽  
Tgf Beta ◽  
Wild Type ◽  
Trabecular Bone Mass

Transforming growth factor-beta (TGF-beta) is abundant in bone matrix and has been shown to regulate the activity of osteoblasts and osteoclasts in vitro. To explore the role of endogenous TGF-(beta) in osteoblast function in vivo, we have inhibited osteoblastic responsiveness to TGF-beta in transgenic mice by expressing a cytoplasmically truncated type II TGF-beta receptor from the osteocalcin promoter. These transgenic mice develop an age-dependent increase in trabecular bone mass, which progresses up to the age of 6 months, due to an imbalance between bone formation and resorption during bone remodeling. Since the rate of osteoblastic bone formation was not altered, their increased trabecular bone mass is likely due to decreased bone resorption by osteoclasts. Accordingly, direct evidence of reduced osteoclast activity was found in transgenic mouse skulls, which had less cavitation and fewer mature osteoclasts relative to skulls of wild-type mice. These bone remodeling defects resulted in altered biomechanical properties. The femurs of transgenic mice were tougher, and their vertebral bodies were stiffer and stronger than those of wild-type mice. Lastly, osteocyte density was decreased in transgenic mice, suggesting that TGF-beta signaling in osteoblasts is required for normal osteoblast differentiation in vivo. Our results demonstrate that endogenous TGF-beta acts directly on osteoblasts to regulate bone remodeling, structure and biomechanical properties.

Annexin A2 Deficient Mice Are Resistant to Pathogenic Effects of Antiphospholipid Antibodies in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 421-421
Author(s):  
Zurina Romay-Penabad ◽  
Guadalupe Montiel-Manzano ◽  
Elizabeth Pappalardo ◽  
Katherine A. Hajjar ◽  
Tuya Shilagard ◽  
...  
Keyword(s):  
Antiphospholipid Antibodies ◽  
Cell Adhesion Molecule ◽  
Carotid Arteries ◽  
Thrombus Formation ◽  
Annexin A2 ◽  
Target Cells ◽  
Wild Type ◽  
Deficient Mice ◽  
Pathogenic Effects

Abstract Background: Thrombosis is an important cause of morbidity and mortality in Antiphospholipid Syndrome (APS) and in SLE patients with antiphospholipid antibodies (aPL). APL recognize β2 glycoprotein I (β2GPI)-bound to receptor (s) in endothelial cells (EC) and other target cells (i.e. platelets, monocytes) and trigger an intracellular signalling and a pro-coagulant and pro-inflammatory phenotype [i e.expression of tissue factor (TF), vascular cell adhesion molecule-1 (VCAM-1)] that lead to thrombosis. There is in vitro evidence that annexin A2 (A2), a receptor for tissue plasminogen activator (tPA) and plasminogen – and possibly other proteins such as toll-like receptors or the receptor for apolipoprotein E2′ - may be binding β2GPI on the membrane of target cells. Here, we examined the involvement of A2 in aPL-mediated pathogenic effects in vivo. We studied the effects of aPL Abs on thrombus formation, VCAM-1 expression in aortas of mice, and TF function in carotid artery homogenates in annexin A2 deficient (−/−) mice. Methods: A2 (−/−) mice and the corresponding wild-type (WT) mice, in groups of 10, were injected i.p. twice (0 and 48 hours later) with IgG from a patient with APS (IgG-APS) or with control IgG (IgG-NHS). Seventy-two hours after the first injection, several procedures were done in each mice: dynamics of thrombus formation (thrombus size), TF function in homogenates of carotid arteries, and c) VCAM-1 expression in the aortas using quantum dot nano crystals and two-photon excitation laser scanning microscopy. In addition, we examined the effect of an anti-A2 antibody on aPL-induced expression of intercellular cell-adhesion molecule (ICAM-1), E-selectin and TF acvitity on cultured endothelial cells (EC). Results: The titers of aCL and anti-β2GPI Abs in the sera of the mice at the time of surgery were medium-high positive in A2 (−/−) mice and in wild type mice injected with IgG-APS. Thrombus sizes were significantly larger in WT mice injected with IgG-APS when compared to similar type of mice treated with IgG-NHS (p=0.003). The size of thrombus in A2 (−/−) mice injected with IgG-APS was significantly smaller than mean thrombus size in WT mice injected with IgG-APS (p:0.0005). However, thrombus size in A2 (−/−) mice was larger in mice injected with IgG-APS when compared to same type of mice treated with control IgG-NHS (p=0.003), indicating a partial but significant abrogation of the thrombogenic effect. TF activity was significantly larger in WT mice treated with IgG-APS when compared to mice injected with IgG-NHS. Importantly, TF activity in carotid arteries homogenates of annexin A2 (−/−) mice injected with IgG-APS was significantly decreased (by 52%) when compared to wild type mice treated with IgG-APS. The expression of VCAM-1 in aorta of annexin A2 (−/−) ex vivo was also significantly reduced compared to LPS-treated mice (positive control) (p= 0.01). Interestingly, anti-A2 antibody significantly decreased aPL-induced expression of ICAM-1, E-sel and TF on cultured EC. Conclusions: Altogether these data indicate for the first time that A2 is involved in vivo pathogenic effects of aPL Abs. These findings may have important implications to devise new targeted and more specific therapeutic approaches to block the pathogenic effects of aPL Abs in patients with APS and SLE.

scholarly journals Development of a high-throughput screen to identify small molecule enhancers of sarcospan for the treatment of Duchenne muscular dystrophy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Cynthia Shu ◽  
Ariana N. Kaxon-Rupp ◽  
Judd R. Collado ◽  
Robert Damoiseaux ◽  
Rachelle H. Crosbie
Keyword(s):  
Gene Expression ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Calcium Channel ◽  
Muscle Cell ◽  
High Throughput ◽  
Promoter Activity ◽  
Muscle Cells ◽  
Wild Type ◽  
Protein Levels

Abstract Background Duchenne muscular dystrophy (DMD) is caused by loss of sarcolemma connection to the extracellular matrix. Transgenic overexpression of the transmembrane protein sarcospan (SSPN) in the DMD mdx mouse model significantly reduces disease pathology by restoring membrane adhesion. Identifying SSPN-based therapies has the potential to benefit patients with DMD and other forms of muscular dystrophies caused by deficits in muscle cell adhesion. Methods Standard cloning methods were used to generate C2C12 myoblasts stably transfected with a fluorescence reporter for human SSPN promoter activity. Assay development and screening were performed in a core facility using liquid handlers and imaging systems specialized for use with a 384-well microplate format. Drug-treated cells were analyzed for target gene expression using quantitative PCR and target protein expression using immunoblotting. Results We investigated the gene expression profiles of SSPN and its associated proteins during myoblast differentiation into myotubes, revealing an increase in expression after 3 days of differentiation. We created C2C12 muscle cells expressing an EGFP reporter for SSPN promoter activity and observed a comparable increase in reporter levels during differentiation. Assay conditions for high-throughput screening were optimized for a 384-well microplate format and a high-content imager for the visualization of reporter levels. We conducted a screen of 3200 compounds and identified seven hits, which include an overrepresentation of L-type calcium channel antagonists, suggesting that SSPN gene activity is sensitive to calcium. Further validation of a select hit revealed that the calcium channel inhibitor felodipine increased SSPN transcript and protein levels in both wild-type and dystrophin-deficient myotubes, without increasing differentiation. Conclusions We developed a stable muscle cell line containing the promoter region of the human SSPN protein fused to a fluorescent reporter. Using the reporter cells, we created and validated a scalable, cell-based assay that is able to identify compounds that increase SSPN promoter reporter, transcript, and protein levels in wild-type and dystrophin-deficient muscle cells.

Sign in / Sign up

Export Citation Format

Share Document