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Muscle & Nerve Oct 2016Duchenne muscular dystrophy (DMD) is caused by loss of the structural protein, dystrophin, resulting in muscle fragility. Muscle stem cell (MuSC) transplantation is a...
INTRODUCTION
Duchenne muscular dystrophy (DMD) is caused by loss of the structural protein, dystrophin, resulting in muscle fragility. Muscle stem cell (MuSC) transplantation is a potential therapy for DMD. It is unknown whether donor-derived muscle fibers are structurally innervated.
METHODS
Green fluorescent protein (GFP)-expressing MuSCs were transplanted into the tibials anterior of adult dystrophic mdx/mTR mice. Three weeks later the neuromuscular junction was labeled by immunohistochemistry.
RESULTS
The percent overlap between pre- and postsynaptic immunolabeling was greater in donor-derived GFP(+) myofibers, and fewer GFP(+) myofibers were identified as denervated compared with control GFP(-) fibers (P = 0.001 and 0.03). GFP(+) fibers also demonstrated acetylcholine receptor fragmentation and expanded endplate area, indicators of muscle reinnervation (P = 0.008 and 0.033).
CONCLUSION
It is unclear whether GFP(+) fibers are a result of de novo synthesis or fusion with damaged endogenous fibers. Either way, donor-derived fibers demonstrate clear histological innervation. Muscle Nerve 54: 763-768, 2016.
Topics: Animals; Mice; Mice, Inbred mdx; Mice, Transgenic; Muscle Cells; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Animal; Muscular Dystrophy, Duchenne; Stem Cell Transplantation
PubMed: 26998682
DOI: 10.1002/mus.25115 -
Cells Jan 2022Elevated blood free fatty acids (FFAs), as seen in obesity, impair insulin action leading to insulin resistance and Type 2 diabetes mellitus. Several serine/threonine...
Elevated blood free fatty acids (FFAs), as seen in obesity, impair insulin action leading to insulin resistance and Type 2 diabetes mellitus. Several serine/threonine kinases including JNK, mTOR, and p70 S6K cause serine phosphorylation of the insulin receptor substrate (IRS) and have been implicated in insulin resistance. Activation of AMP-activated protein kinase (AMPK) increases glucose uptake, and in recent years, AMPK has been viewed as an important target to counteract insulin resistance. We reported previously that carnosic acid (CA) found in rosemary extract (RE) and RE increased glucose uptake and activated AMPK in muscle cells. In the present study, we examined the effects of CA on palmitate-induced insulin-resistant L6 myotubes and 3T3L1 adipocytes. Exposure of cells to palmitate reduced the insulin-stimulated glucose uptake, GLUT4 transporter levels on the plasma membrane, and Akt activation. Importantly, CA attenuated the deleterious effect of palmitate and restored the insulin-stimulated glucose uptake, the activation of Akt, and GLUT4 levels. Additionally, CA markedly attenuated the palmitate-induced phosphorylation/activation of JNK, mTOR, and p70S6K and activated AMPK. Our data indicate that CA has the potential to counteract the palmitate-induced muscle and fat cell insulin resistance.
Topics: 3T3-L1 Cells; AMP-Activated Protein Kinases; Abietanes; Adipocytes; Animals; Cell Line; Fatty Acids, Nonesterified; Glucose; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; JNK Mitogen-Activated Protein Kinases; Mice; Models, Biological; Muscle Cells; Muscle Fibers, Skeletal; Palmitates; Phosphorylation; Phosphoserine; Proto-Oncogene Proteins c-akt; Rats; Ribosomal Protein S6 Kinases, 70-kDa; TOR Serine-Threonine Kinases
PubMed: 35011728
DOI: 10.3390/cells11010167 -
Communications Biology Nov 2023Optical stimulation and control of muscle cell contraction opens up a number of interesting applications in hybrid robotic and medicine. Here we show that recently...
Optical stimulation and control of muscle cell contraction opens up a number of interesting applications in hybrid robotic and medicine. Here we show that recently designed molecular phototransducer can be used to stimulate C2C12 skeletal muscle cells, properly grown to exhibit collective behaviour. C2C12 is a skeletal muscle cell line that does not require animal sacrifice Furthermore, it is an ideal cell model for evaluating the phototransducer pacing ability due to its negligible spontaneous activity. We study the stimulation process and analyse the distribution of responses in multinuclear cells, in particular looking at the consistency between stimulus and contraction. Contractions are detected by using an imaging software for object recognition. We find a deterministic response to light stimuli, yet with a certain distribution of erratic behaviour that is quantified and correlated to light intensity or stimulation frequency. Finally, we compare our optical stimulation with electrical stimulation showing advantages of the optical approach, like the reduced cell stress.
Topics: Animals; Muscle Fibers, Skeletal; Muscle Contraction; Electric Stimulation; Light; Robotics
PubMed: 37952040
DOI: 10.1038/s42003-023-05538-y -
Vascular Pharmacology Oct 2023Vascular endothelial and smooth muscle cell dysfunction proceed the development of numerous vascular diseases, such as atherosclerosis. Both estrogen and progesterone... (Review)
Review
Vascular endothelial and smooth muscle cell dysfunction proceed the development of numerous vascular diseases, such as atherosclerosis. Both estrogen and progesterone receptors are present on vascular endothelial and smooth muscle cells, and therefore it has been postulated that these compounds may affect vascular function. It has been well-established that estrogen is a vasoprotective compound, however, the effects of progesterone on vascular function are not well understood. This narrative review summarizes the current research investigating the impact of both endogenous progesterone, and exogenous synthetic progestin on vascular endothelial and smooth muscle cell function and identifies discrepancies on their effects in vitro and in vivo. We speculate that an inverted-U dose response curve may exist between nitric oxide bioavailability and progesterone concentration, and that the androgenic properties of a progestin may influence vascular function. Future research is needed to discern the effects of both endogenous progesterone and exogenous progestin on vascular endothelial and smooth muscle cell function with consideration for the impacts of progesterone/progestin dose, and progestin type.
Topics: Humans; Progestins; Progesterone; Progesterone Congeners; Estrogens; Endothelial Cells; Atherosclerosis; Myocytes, Smooth Muscle
PubMed: 37591444
DOI: 10.1016/j.vph.2023.107209 -
International Journal of Molecular... Mar 2023Obesity and elevated blood free fatty acid (FFA) levels lead to impaired insulin action causing insulin resistance in skeletal muscle, and contributing to the...
Obesity and elevated blood free fatty acid (FFA) levels lead to impaired insulin action causing insulin resistance in skeletal muscle, and contributing to the development of type 2 diabetes mellitus (T2DM). Mechanistically, insulin resistance is associated with increased serine phosphorylation of the insulin receptor substrate (IRS) mediated by serine/threonine kinases including mTOR and p70S6K. Evidence demonstrated that activation of the energy sensor AMP-activated protein kinase (AMPK) may be an attractive target to counteract insulin resistance. We reported previously that rosemary extract (RE) and the RE polyphenol carnosic acid (CA) activated AMPK and counteracted the FFA-induced insulin resistance in muscle cells. The effect of rosmarinic acid (RA), another polyphenolic constituent of RE, on FFA-induced muscle insulin resistance has never been examined and is the focus of the current study. Muscle cell (L6) exposure to FFA palmitate resulted in increased serine phosphorylation of IRS-1 and reduced insulin-mediated (i) Akt activation, (ii) GLUT4 glucose transporter translocation, and (iii) glucose uptake. Notably, RA treatment abolished these effects, and restored the insulin-stimulated glucose uptake. Palmitate treatment increased the phosphorylation/activation of mTOR and p70S6K, kinases known to be involved in insulin resistance and RA significantly reduced these effects. RA increased the phosphorylation of AMPK, even in the presence of palmitate. Our data indicate that RA has the potential to counteract the palmitate-induced insulin resistance in muscle cells, and further studies are required to explore its antidiabetic properties.
Topics: Humans; Insulin Resistance; Ribosomal Protein S6 Kinases, 70-kDa; AMP-Activated Protein Kinases; Diabetes Mellitus, Type 2; Glucose; Muscle, Skeletal; Insulin; TOR Serine-Threonine Kinases; Muscle Cells; Phosphorylation; Palmitates; Rosmarinic Acid
PubMed: 36982168
DOI: 10.3390/ijms24065094 -
International Journal of Molecular... Sep 2021Muscular dystrophies are a heterogeneous group of inherited diseases characterized by the progressive degeneration and weakness of skeletal muscles, leading to... (Review)
Review
Muscular dystrophies are a heterogeneous group of inherited diseases characterized by the progressive degeneration and weakness of skeletal muscles, leading to disability and, often, premature death. To date, no effective therapies are available to halt or reverse the pathogenic process, and meaningful treatments are urgently needed. From this perspective, it is particularly important to establish reliable in vitro models of human muscle that allow the recapitulation of disease features as well as the screening of genetic and pharmacological therapies. We herein review and discuss advances in the development of in vitro muscle models obtained from human induced pluripotent stem cells, which appear to be capable of reproducing the lack of myofiber proteins as well as other specific pathological hallmarks, such as inflammation, fibrosis, and reduced muscle regenerative potential. In addition, these platforms have been used to assess genetic correction strategies such as gene silencing, gene transfer and genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as well as to evaluate novel small molecules aimed at ameliorating muscle degeneration. Furthermore, we discuss the challenges related to in vitro drug testing and provide a critical view of potential therapeutic developments to foster the future clinical translation of preclinical muscular dystrophy studies.
Topics: Animals; Cell Differentiation; Drug Discovery; Dystrophin; Genetic Therapy; Humans; Induced Pluripotent Stem Cells; Muscle Cells; Muscular Dystrophies; Muscular Dystrophy, Animal
PubMed: 34502539
DOI: 10.3390/ijms22179630 -
International Journal of Molecular... Aug 2020Human mesenchymal stem cells (hMSCs) have shown their multipotential including differentiating towards endothelial and smooth muscle cell lineages, which triggers a new...
BACKGROUND
Human mesenchymal stem cells (hMSCs) have shown their multipotential including differentiating towards endothelial and smooth muscle cell lineages, which triggers a new interest for using hMSCs as a putative source for cardiovascular regenerative medicine. Our recent publication has shown for the first time that purinergic 2 receptors are key players during hMSC differentiation towards adipocytes and osteoblasts. Purinergic 2 receptors play an important role in cardiovascular function when they bind to extracellular nucleotides. In this study, the possible functional role of purinergic 2 receptors during MSC endothelial and smooth muscle differentiation was investigated.
METHODS AND RESULTS
Human MSCs were isolated from liposuction materials. Then, endothelial and smooth muscle-like cells were differentiated and characterized by specific markers via Reverse Transcriptase-PCR (RT-PCR), Western blot and immunochemical stainings. Interestingly, some purinergic 2 receptor subtypes were found to be differently regulated during these specific lineage commitments: P2Y4 and P2Y14 were involved in the early stage commitment while P2Y1 was the key player in controlling MSC differentiation towards either endothelial or smooth muscle cells. The administration of natural and artificial purinergic 2 receptor agonists and antagonists had a direct influence on these differentiations. Moreover, a feedback loop via exogenous extracellular nucleotides on these particular differentiations was shown by apyrase digest.
CONCLUSIONS
Purinergic 2 receptors play a crucial role during the differentiation towards endothelial and smooth muscle cell lineages. Some highly selective and potent artificial purinergic 2 ligands can control hMSC differentiation, which might improve the use of adult stem cells in cardiovascular tissue engineering in the future.
Topics: Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Endothelial Cells; Female; Gene Expression Regulation; Humans; Lipectomy; Mesenchymal Stem Cells; Myocytes, Smooth Muscle; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Young Adult
PubMed: 32867347
DOI: 10.3390/ijms21176210 -
Toxins May 2018Chronic kidney disease (CKD) is associated with profound vascular remodeling, which accelerates the progression of cardiovascular disease. This remodeling is... (Review)
Review
Chronic kidney disease (CKD) is associated with profound vascular remodeling, which accelerates the progression of cardiovascular disease. This remodeling is characterized by intimal hyperplasia, accelerated atherosclerosis, excessive vascular calcification, and vascular stiffness. Vascular smooth muscle cell (VSMC) dysfunction has a key role in the remodeling process. Under uremic conditions, VSMCs can switch from a contractile phenotype to a synthetic phenotype, and undergo abnormal proliferation, migration, senescence, apoptosis, and calcification. A growing body of data from experiments in vitro and animal models suggests that uremic toxins (such as inorganic phosphate, indoxyl sulfate and advanced-glycation end products) may directly impact the VSMCs' physiological functions. Chronic, low-grade inflammation and oxidative stress-hallmarks of CKD-are also strong inducers of VSMC dysfunction. Here, we review current knowledge about the impact of uremic toxins on VSMC function in CKD, and the consequences for pathological vascular remodeling.
Topics: Animals; Humans; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Toxins, Biological; Uremia
PubMed: 29844272
DOI: 10.3390/toxins10060218 -
International Journal of Molecular... Apr 2022Pulmonary artery hypertension (PAH) is a complex and progressive disease characterized by pulmonary vascular remodeling. Our previous study confirmed that...
Pulmonary artery hypertension (PAH) is a complex and progressive disease characterized by pulmonary vascular remodeling. Our previous study confirmed that NONRATT015587.2 could promote the proliferation of PASMCs and pulmonary vascular remodeling. However, the exact mechanism by which NONRATT015587.2 promotes PASMC proliferation is unclear. Bioinformatics analysis revealed that p21 is located at the downstream target of NONRATT015587.2. NONRATT015587.2 expression and localization were analyzed by PCR and fluorescence hybridization. Proliferation was detected by Cell Counting Kit‑8, flow cytometry and western blotting. In the current study, a monocrotaline (MCT)‑induced PAH rat model and cultured pulmonary artery smooth muscle cells (PASMCs) were used to elucidate the exact mechanism of NONRATT015587.2 in pulmonary vascular remodeling, alongside the effect following metformin (MET) treatment on vascular remodeling and smooth muscle cell proliferation. The results demonstrated that NONRATT015587.2 expression was upregulated in the MCT group and reduced in the MET + MCT group. In addition, NONRATT015587.2 could promote the proliferation of PASMCs. The expression levels of p21 were reduced in the MCT group, but increased in the MCT + MET group. Additionally, the expression of NONRATT015587.2 was upregulated in platelet‑derived growth factor‑BB (PDGF‑BB)‑induced PASMCs, whereas that of p21 was downregulated. Following MET treatment, the expression of NONRATT015587.2 was downregulated and that of p21 was upregulated, which inhibited the proliferation of PASMCs. After overexpression of NONRATT015587.2 in vitro, the proliferation effect of PASMCs was consistent with exogenous PDGF‑BB treatment, and MET reversed this effect. NONRATT015587.2 silencing inhibited the proliferation of PASMCs. In addition, p21 silencing reversed the inhibitory effect of NONRATT015587.2 silencing on the proliferation of PASMCs. However, the proliferation of PASMCs was inhibited following MET treatment when NONRATT015587.2 and p21 were silenced at the same time. Thus, NONRATT015587.2 promoted the proliferation of PASMCs by targeting p21, and MET inhibited the proliferation of PASMCs by upregulating p21 mediated via NONRATT015587.2.
Topics: Animals; Cell Proliferation; Cells, Cultured; In Situ Hybridization, Fluorescence; Metformin; Myocytes, Smooth Muscle; Pulmonary Artery; Rats; Rats, Sprague-Dawley
PubMed: 35147202
DOI: 10.3892/ijmm.2022.5104 -
Biomaterials Jun 2019The use of biochemical signaling to derive smooth muscle cells (SMCs) from mesenchymal stem cells (MSCs) has been explored, but the induction of a fully functional SMC...
The use of biochemical signaling to derive smooth muscle cells (SMCs) from mesenchymal stem cells (MSCs) has been explored, but the induction of a fully functional SMC phenotype remains to be a major challenge. Cell morphology has been shown to regulate MSC differentiation into various lineages, including SMCs. We engineered substrates with microgrooves to induce cell elongation to study the mechanism underlying the MSC shape modulation in SMC differentiation. In comparison to those on flat substrates, MSCs cultured on engineered substrates were elongated with increased aspect ratios for both cell body and nucleus, as well as augmented cytoskeletal tensions. Biochemical studies indicated that the microgroove-elongated cells expressed significantly higher levels of SMC markers. MicroRNA analyses showed that up-regulation of miR-145 and the consequent repression of KLF4 in these elongated cells promoted MSC-to-SMC differentiation. Rho/ROCK inhibitions, which impair cytoskeletal tension, attenuated cell and nuclear elongations and disrupted the miR-145/KLF4 regulation for SMC differentiation. Furthermore, cell traction force measurements showed that miR-145 is essential for the functional contractility in the microgroove-induced SMC differentiation. Collectively, our findings demonstrate that, through a Rho-ROCK/miR-145/KLF4 pathway, the elongated cell shape serves as a decisive geometric cue to direct MSC differentiation into functional SMCs.
Topics: Biomarkers; Cell Differentiation; Cell Shape; Dimethylpolysiloxanes; Humans; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Mesenchymal Stem Cells; MicroRNAs; Models, Biological; Myocytes, Smooth Muscle; Signal Transduction; Transforming Growth Factor beta1; Up-Regulation
PubMed: 30884320
DOI: 10.1016/j.biomaterials.2019.03.003