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2.
Int J Mol Sci ; 25(6)2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38542301

RESUMO

FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent inherited muscle disorders and is linked to the inappropriate expression of the DUX4 transcription factor in skeletal muscles. The deregulated molecular network causing FSHD muscle dysfunction and pathology is not well understood. It has been shown that the hypoxia response factor HIF1α is critically disturbed in FSHD and has a major role in DUX4-induced cell death. In this study, we further explored the relationship between DUX4 and HIF1α. We found that the DUX4 and HIF1α link differed according to the stage of myogenic differentiation and was conserved between human and mouse muscle. Furthermore, we found that HIF1α knockdown in a mouse model of DUX4 local expression exacerbated DUX4-mediated muscle fibrosis. Our data indicate that the suggested role of HIF1α in DUX4 toxicity is complex and that targeting HIF1α might be challenging in the context of FSHD therapeutic approaches.


Assuntos
Distrofia Muscular Facioescapuloumeral , Animais , Humanos , Camundongos , Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo
3.
FASEB J ; 38(3): e23461, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38317639

RESUMO

Amyotrophic lateral sclerosis is a fatal neurodegenerative disorder characterized by progressive skeletal muscle denervation and loss of motor neurons that results in muscle atrophy and eventual death due to respiratory failure. Previously, we identified a novel SOD1L84F variation in a familial ALS case. In this study, we examined the functional consequences of SOD1L84F overexpression in the mouse motor neuron cell line (NSC-34). The cells expressing SOD1L84F showed increased oxidative stress and increased cell death. Interestingly, SOD1L84F destabilized the native dimer and formed high molecular weight SDS-resistant protein aggregates. Furthermore, SOD1L84F also decreased the percentage of differentiated cells and significantly reduced neurite length. A plethora of evidence suggested active involvement of skeletal muscle in disease initiation and progression. We observed differential processing of the mutant SOD1 and perturbations of cellular machinery in NSC-34 and muscle cell line C2C12. Unlike neuronal cells, mutant protein failed to accumulate in muscle cells probably due to the activated autophagy, as evidenced by increased LC3-II and reduced p62. Further, SOD1L84F altered mitochondrial dynamics only in NSC-34. In addition, microarray analysis also revealed huge variations in differentially expressed genes between NSC-34 and C2C12. Interestingly, SOD1L84F hampered the endogenous FUS autoregulatory mechanism in NSC-34 by downregulating retention of introns 6 and 7 resulting in a two-fold upregulation of FUS. No such changes were observed in C2C12. Our findings strongly suggest the differential processing and response towards the mutant SOD1 in neuronal and muscle cell lines.


Assuntos
Esclerose Amiotrófica Lateral , Superóxido Dismutase-1 , Animais , Camundongos , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Modelos Animais de Doenças , Camundongos Transgênicos , Células Musculares/metabolismo , Mutação , Superóxido Dismutase-1/genética
4.
Int J Mol Sci ; 25(4)2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38396901

RESUMO

TMEM16A is a Ca2+-activated Cl- channel expressed in various species and tissues. In mammalian skeletal muscle precursors, the activity of these channels is still poorly investigated. Here, we characterized TMEM16A channels and investigated if the pharmacological activation of Piezo1 channels could modulate the TMEM16A currents in mouse myogenic precursors. Whole-cell patch-clamp recordings combined with the pharmacological agents Ani9, T16inh-A01 and Yoda1 were used to characterize TMEM16A-mediated currents and the possible modulatory effect of Piezo1 activity on TMEM16A channels. Western blot analysis was also carried out to confirm the expression of TMEM16A and Piezo1 channel proteins. We found that TMEM16A channels were functionally expressed in fusion-competent mouse myogenic precursors. The pharmacological blockage of TMEM16A inhibited myocyte fusion into myotubes. Moreover, the specific Piezo1 agonist Yoda1 positively regulated TMEM16A currents. The findings demonstrate, for the first time, a sarcolemmal TMEM16A channel activity and its involvement at the early stage of mammalian skeletal muscle differentiation. In addition, the results suggest a possible role of mechanosensitive Piezo1 channels in the modulation of TMEM16A currents.


Assuntos
Anoctamina-1 , Canais de Cloreto , Células Musculares , Animais , Camundongos , Anoctamina-1/metabolismo , Anoctamina-1/fisiologia , Transporte Biológico , Cálcio/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Canais Iônicos/metabolismo , Mamíferos/metabolismo , Células Musculares/metabolismo
5.
Eur J Pharmacol ; 967: 176351, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38290568

RESUMO

Doxorubicin is widely used for the treatment of human cancer, but its clinical use is limited by a cumulative dose-dependent cardiotoxicity. However, the mechanism of doxorubicin-induced cardiac atrophy and failure remains to be fully understood. In this study, we tested whether the specific NADPH oxidase 2 (Nox2) inhibitor GSK2795039 attenuates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy, leading to the amelioration of cardiac atrophy and dysfunction in chronic doxorubicin-induced cardiomyopathy. Mice were randomized to receive saline, doxorubicin (2.5 mg/kg, every other day, 6 times) or doxorubicin plus GSK2795039 (2.5 mg/kg, twice a day, 9 weeks). Left ventricular (LV) total wall thickness and LV ejection fraction were decreased in doxorubicin-treated mice compared with saline-treated mice and the decreases were prevented by the treatment of the specific Nox2 inhibitor GSK2795039. The ratio of total heart weight to tibia length and myocyte cross-sectional area were decreased in doxorubicin-treated mice, and the decreases were attenuated by the GSK2795039 treatment. In doxorubicin-treated mice, myocardial Nox2 and 4-hydroxynonenal levels were increased, myocardial expression of GAP43, tyrosine hydroxylase and norepinephrine transporter, markers of sympathetic nerve terminals, was decreased, and these changes were prevented by the GSK2795039 treatment. The ratio of LC3 II/I, a marker of autophagy, and Atg5, Atg12 and Atg12-Atg5 conjugate proteins were increased in doxorubicin-treated mice, and the increases were attenuated by the GSK2795039 treatment. These findings suggest that inhibition of Nox2 by GSK2795039 attenuates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy, thereby ameliorating cardiac atrophy and dysfunction after chronic doxorubicin treatment.


Assuntos
Aminopiridinas , Doxorrubicina , Células Musculares , Sulfonamidas , Animais , Camundongos , Atrofia/induzido quimicamente , Autofagia , Doxorrubicina/efeitos adversos , NADPH Oxidase 2
6.
Vaccine ; 42(6): 1259-1267, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38281898

RESUMO

Coronavirus Disease 2019 (COVID-19) vaccines protect the public and limit viral spread. However, inactivated viral vaccines use the whole virus particle, which contains many non-capsid proteins that may cause adverse immune responses. A report has found that the ADP-ribose-binding domains of SARS-CoV-2 non-structural protein 3 (NSP3) and human poly(ADP-ribose) polymerase family member 14 (PARP14) share a significant degree of homology. Here, we further show that antibodies against 2019 novel SARS-like coronavirus (SARS-CoV-2) NSP3 can bind human PARP14 protein. However, when G159R + G162R mutations were introduced into NSP3, the antibody titer against human PARP14 decreased 14-fold. Antibodies against SARS-CoV-2 NSP3 can cross-react with human skeletal muscle cells and astrocytes, but not human embryonic kidney 293T cells. However, when G159R + G162R mutations were introduced into NSP3, the cross-reaction was largely inhibited. The results imply that COVID-19 patients with high antibody titers against NSP3 may have high risks of muscular and/or neurological complications. And the possible strategies to improve the safety of inactivated viral vaccines are also discussed.


Assuntos
COVID-19 , Vacinas Virais , Humanos , SARS-CoV-2/genética , Proteínas não Estruturais Virais/química , Vacinas contra COVID-19 , Anticorpos , Células Musculares/metabolismo , Neuroglia/metabolismo
7.
Acupunct Med ; 42(1): 39-43, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37916461

RESUMO

OBJECTIVE: To examine for the in vitro existence of contractile nodules on the taut band of muscle fibers where myofascial trigger points (MTrPs) are located (using cell culture). METHODS: Sixteen male Sprague-Dawley rats (7 weeks old) were randomly divided into experimental and control groups. A blunt striking injury and eccentric exercise were applied to the gastrocnemius muscle of rats in the experimental group once a week for 8 weeks to establish an MTrP model. Subsequently, the rats were reared normally and rested for 4 weeks. After modeling, the skeletal muscles at the MTrPs (and non-MTrPs at the same anatomical position) were extracted from the two groups of rats for in vitro cell culture experiments of single muscle fibers. Potential contractile nodules in the MTrP group were exposed to different concentrations of acetylcholinesterase, whereas non-MTrP cells were exposed to acetylcholine. The morphological changes of muscle cells in each group were observed. RESULTS: By culturing MTrP cells in vitro, large contractile nodules remained in single MTrP muscle fibers, whereas some contractile nodules were twisted and deformed. After the addition of different acetylcholinesterase concentrations, no obvious morphological changes were observed in the contractile nodules in the MTrP group. After the non-MTrP cells were exposed to different acetylcholine concentrations, no significant morphological changes were observed in the single muscle fibers. CONCLUSION: MTrP cells can continue to maintain contractile morphology in vitro, but whether the recovery of such contractile nodules is related to acetylcholine remains uncertain.


Assuntos
Síndromes da Dor Miofascial , Pontos-Gatilho , Masculino , Ratos , Animais , Acetilcolinesterase , Síndromes da Dor Miofascial/terapia , Acetilcolina , Ratos Sprague-Dawley , Músculo Esquelético , Células Musculares
8.
Photochem Photobiol Sci ; 23(1): 107-118, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38057632

RESUMO

PURPOSE: Reactive oxygen species and mitochondrial dysfunction play a crucial role in the pathophysiology of Duchenne muscular dystrophy (DMD). The light-emitting diode therapy (LEDT) showed beneficial effects on the dystrophic muscles. However, the mechanisms of this therapy influence the molecular pathways in the dystrophic muscles, particularly related to antioxidant effects, which still needs to be elucidated. The current study provides muscle cell-specific insights into the effect of LEDT, 48 h post-irradiation, on oxidative stress and mitochondrial parameters in the dystrophic primary muscle cells in culture. METHODS: Dystrophic primary muscle cells were submitted to LEDT, at multiple wavelengths (420 nm, 470 nm, 660 nm and 850 nm), 0.5 J dose, and evaluated after 48 h based on oxidative stress markers, antioxidant enzymatic system and biogenesis, and functional mitochondrial parameters. RESULTS: The mdx muscle cells treated with LEDT showed a significant reduction of H2O2 production and 4-HNE, catalase, SOD-2, and GR levels. Upregulation of UCP3 was observed with all wavelengths while upregulation of PGC-1α and a slight upregulation of electron transport chain complexes III and V was only observed following 850 nm LEDT. In addition, the mitochondrial membrane potential and mitochondrial mass mostly tended to be increased following LEDT, while parameters like O2·- production tended to be decreased. CONCLUSION: The data shown here highlight the potential of LEDT as a therapeutic agent for DMD through its antioxidant action by modulating PGC-1α and UCP3 levels.


Assuntos
Antioxidantes , Músculo Esquelético , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Músculo Esquelético/efeitos da radiação , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Células Musculares/metabolismo
9.
Laryngoscope ; 134(1): 272-282, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37436167

RESUMO

OBJECTIVES: No curative injectable therapy exists for unilateral vocal fold paralysis. Herein, we explore the early implications of muscle-derived motor-endplate expressing cells (MEEs) for injectable vocal fold medialization after recurrent laryngeal nerve (RLN) injury. METHODS: Yucatan minipigs underwent right RLN transection (without repair) and muscle biopsies. Autologous muscle progenitor cells were isolated, cultured, differentiated, and induced to form MEEs. Three weeks after the injury, MEEs or saline were injected into the paralyzed right vocal fold. Outcomes including evoked laryngeal electromyography (LEMG), laryngeal adductor pressure, and acoustic vocalization data were analyzed up to 7 weeks post-injury. Harvested porcine larynges were examined for volume, gene expression, and histology. RESULTS: MEE injections were tolerated well, with all pigs demonstrating continued weight gain. Blinded analysis of videolaryngoscopy post-injection revealed infraglottic fullness, and no inflammatory changes. Four weeks after injection, LEMG revealed on average higher right distal RLN activity retention in MEE pigs. MEE-injected pigs on average had vocalization durations, frequencies, and intensities higher than saline pigs. Post-mortem, the MEE-injected larynges revealed statistically greater volume on quantitative 3D ultrasound, and statistically increased expression of neurotrophic factors (BDNF, NGF, NTF3, NTF4, NTN1) on quantitative PCR. CONCLUSIONS: Minimally invasive MEE injection appears to establish an early molecular and microenvironmental framework to encourage innate RLN regeneration. Longer follow-up is needed to determine if early findings will translate into functional contraction. LEVEL OF EVIDENCE: NA Laryngoscope, 134:272-282, 2024.


Assuntos
Laringe , Traumatismos do Nervo Laríngeo Recorrente , Paralisia das Pregas Vocais , Animais , Suínos , Prega Vocal , Porco Miniatura , Paralisia das Pregas Vocais/terapia , Eletromiografia , Nervo Laríngeo Recorrente/cirurgia , Células Musculares , Músculos Laríngeos/inervação
10.
Adv Healthc Mater ; 13(9): e2303485, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38150609

RESUMO

The integration of additive manufacturing technologies with the pyrolysis of polymeric precursors enables the design-controlled fabrication of architected 3D pyrolytic carbon (PyC) structures with complex architectural details. Despite great promise, their use in cellular interaction remains unexplored. This study pioneers the utilization of microarchitected 3D PyC structures as biocompatible scaffolds for the colonization of muscle cells in a 3D environment. PyC scaffolds are fabricated using micro-stereolithography, followed by pyrolysis. Furthermore, an innovative design strategy using revolute joints is employed to obtain novel, compliant structures of architected PyC. The pyrolysis process results in a pyrolysis temperature- and design-geometry-dependent shrinkage of up to 73%, enabling the geometrical features of microarchitected compatible with skeletal muscle cells. The stiffness of architected PyC varies with the pyrolysis temperature, with the highest value of 29.57 ± 0.78 GPa for 900 °C. The PyC scaffolds exhibit excellent biocompatibility and yield 3D cell colonization while culturing skeletal muscle C2C12 cells. They further induce good actin fiber alignment along the compliant PyC construction. However, no conclusive myogenic differentiation is observed here. Nevertheless, these results are highly promising for architected PyC scaffolds as multifunctional tissue implants and encourage more investigations in employing compliant architected PyC structures for high-performance tissue engineering applications.


Assuntos
Engenharia Tecidual , Tecidos Suporte , Tecidos Suporte/química , Engenharia Tecidual/métodos , Carbono , Células Musculares , Impressão Tridimensional
11.
Proc Natl Acad Sci U S A ; 121(1): e2314585121, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38147545

RESUMO

Fatty expansion is one of the features of muscle degeneration due to muscle injuries, and its presence interferes with muscle regeneration. Specifically, poor clinical outcomes have been linked to fatty expansion in rotator cuff tears and repairs. Our group recently found that fibroblast growth factor 8b (FGF-8b) inhibits adipogenic differentiation and promotes myofiber formation of mesenchymal stem cells in vitro. This led us to hypothesize that FGF-8b could similarly control the fate of muscle-specific cell populations derived from rotator cuff muscle involved in muscle repair following rotator cuff injury. In this study, we isolate fibro-adipogenic progenitor cells (FAPs) and satellite stem cells (SCs) from rat rotator cuff muscle tissue and analyzed the effects of FGF-8b supplementation. Utilizing a cell plating protocol, we successfully isolate FAPs-rich fibroblasts (FIBs) and SCs-rich muscle progenitor cells (MPCs). Subsequently, we demonstrate that FIB adipogenic differentiation can be inhibited by FGF-8b, while MPC myogenic differentiation can be enhanced by FGF-8b. We further demonstrate that phosphorylated ERK due to FGF-8b leads to the inhibition of adipogenesis in FIBs and SCs maintenance and myofiber formation in MPCs. Together, these findings demonstrate the powerful potential of FGF-8b for rotator cuff repair by altering the fate of muscle undergoing degeneration.


Assuntos
Lesões do Manguito Rotador , Manguito Rotador , Ratos , Animais , Manguito Rotador/cirurgia , Adipogenia , Fator 8 de Crescimento de Fibroblasto , Lesões do Manguito Rotador/cirurgia , Células Musculares , Desenvolvimento Muscular
12.
Cell Death Dis ; 14(12): 787, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-38040704

RESUMO

Lipotoxicity, the accumulation of lipids in non-adipose tissues, alters the metabolic transcriptome and mitochondrial metabolism in skeletal muscle. The mechanisms involved remain poorly understood. Here we show that lipotoxicity increased histone deacetylase 4 (HDAC4) and histone deacetylase 5 (HDAC5), which reduced the expression of metabolic genes and oxidative metabolism in skeletal muscle, resulting in increased non-oxidative glucose metabolism. This metabolic reprogramming was also associated with impaired apoptosis and ferroptosis responses, and preserved muscle cell viability in response to lipotoxicity. Mechanistically, increased HDAC4 and 5 decreased acetylation of p53 at K120, a modification required for transcriptional activation of apoptosis. Redox drivers of ferroptosis derived from oxidative metabolism were also reduced. The relevance of this pathway was demonstrated by overexpression of loss-of-function HDAC4 and HDAC5 mutants in skeletal muscle of obese db/db mice, which enhanced oxidative metabolic capacity, increased apoptosis and ferroptosis and reduced muscle mass. This study identifies HDAC4 and HDAC5 as repressors of skeletal muscle oxidative metabolism, which is linked to inhibition of cell death pathways and preservation of muscle integrity in response to lipotoxicity.


Assuntos
Histona Desacetilases , Células Musculares , Camundongos , Animais , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Processamento de Proteína Pós-Traducional , Morte Celular
14.
Cells ; 12(23)2023 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-38067180

RESUMO

Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease associated with progressive muscle atrophy, paralysis, and eventually death. Growing evidence demonstrates that the pathological process leading to ALS is the result of multiple altered mechanisms occurring not only in MNs but also in other cell types inside and outside the central nervous system. In this context, the involvement of skeletal muscle has been the subject of a few studies on patients and ALS animal models. In this work, by using primary myocytes derived from the ALS transgenic hSOD1(G93A) mouse model, we observed that the myogenic capability of such cells was defective compared to cells derived from control mice expressing the nonpathogenic hSOD1(WT) isoform. The correct in vitro myogenesis of hSOD1(G93A) primary skeletal muscle cells was rescued by the addition of a conditioned medium from healthy hSOD1(WT) myocytes, suggesting the existence of an in trans activity of secreted factors. To define a dataset of molecules participating in such safeguard action, we conducted comparative metabolomic profiling of a culture medium collected from hSOD1(G93A) and hSOD1(WT) primary myocytes and report here an altered secretion of amino acids and lipid-based signaling molecules. These findings support the urgency of better understanding the role of the skeletal muscle secretome in the regulation of the myogenic program and mechanisms of ALS pathogenesis and progression.


Assuntos
Esclerose Amiotrófica Lateral , Doença dos Neurônios Motores , Camundongos , Humanos , Animais , Esclerose Amiotrófica Lateral/metabolismo , Neurônios Motores/patologia , Camundongos Transgênicos , Superóxido Dismutase-1/metabolismo , Doença dos Neurônios Motores/metabolismo , Células Musculares/metabolismo , Metaboloma
15.
J Cell Sci ; 136(23)2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-38126809

RESUMO

Regulation of glucose transport, which is central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter (also known as SLC2A4) in the plasma membrane (PM) of fat and muscle cells. Physiologic signals [such as activated insulin receptor or AMP-activated protein kinase (AMPK)] increase PM GLUT4. Here, we show that the distribution of GLUT4 between the PM and interior of human muscle cells is dynamically maintained, and that AMPK promotes PM redistribution of GLUT4 by regulating exocytosis and endocytosis. Stimulation of exocytosis by AMPK is mediated by Rab10 and the Rab GTPase-activating protein TBC1D4. APEX2 proximity mapping reveals that GLUT4 traverses both PM-proximal and PM-distal compartments in unstimulated muscle cells, further supporting retention of GLUT4 by a constitutive retrieval mechanism. AMPK-stimulated translocation involves GLUT4 redistribution among the same compartments traversed in unstimulated cells, with a significant recruitment of GLUT4 from the Golgi and trans-Golgi network compartments. Our comprehensive proximal protein mapping provides an integrated, high-density, whole-cell accounting of the localization of GLUT4 at a resolution of ∼20 nm that serves as a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in a physiologically relevant cell type.


Assuntos
Transportador de Glucose Tipo 4 , Células Musculares , Proteoma , Humanos , Proteínas Quinases Ativadas por AMP , Membrana Celular , Músculos , Transportador de Glucose Tipo 4/metabolismo
16.
J Vis Exp ; (200)2023 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-37929978

RESUMO

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of α-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster, and then examining the microtubule networks in the neuromuscular junction and muscle cells, we accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/genética , Katanina/genética , Katanina/metabolismo , Larva/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Microtúbulos/metabolismo , Junção Neuromuscular/metabolismo , Células Musculares/metabolismo
17.
Commun Biol ; 6(1): 1165, 2023 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-37963972

RESUMO

In arterial myocytes, the canonical function of voltage-gated CaV1.2 and KV2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, KV2.1 also plays a sex-specific role by promoting the clustering and activity of CaV1.2 channels. However, the impact of KV2.1 protein organization on CaV1.2 function remains poorly understood. We discovered that KV2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of KV2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the KV2.1 clustering site (KV2.1S590A) eliminated KV2.1 macro-clustering and sex-specific differences in CaV1.2 cluster size and activity. We propose that the degree of KV2.1 clustering tunes CaV1.2 channel function in a sex-specific manner in arterial myocytes.


Assuntos
Células Musculares , Canais de Potássio Shab , Masculino , Feminino , Humanos , Canais de Potássio Shab/genética , Canais de Potássio Shab/metabolismo , Fosforilação , Miócitos de Músculo Liso/metabolismo
19.
Development ; 150(22)2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-37902086

RESUMO

Neuronal differentiation is regulated by neuronal activity. Here, we analyzed dendritic and axonal growth of Basket cells (BCs) and non-Basket cells (non-BCs) using sparse transfection of channelrhodopsin-YFP and repetitive optogenetic stimulation in slice cultures of rat visual cortex. Neocortical interneurons often display axon-carrying dendrites (AcDs). We found that the AcDs of BCs and non-BCs were, on average, the most complex dendrites. Further, the AcD configuration had an influence on BC axonal development. Axons originating from an AcD formed denser arborizations with more terminal endings within the dendritic field of the parent cell. Intriguingly, this occurred already in unstimulated BCs, and complexity was not increased further by optogenetic stimulation. However, optogenetic stimulation exerted a growth-promoting effect on axons emerging from BC somata. The axons of non-BCs neither responded to the AcD configuration nor to the optogenetic stimulation. The results suggest that the formation of locally dense BC plexuses is regulated by spontaneous activity. Moreover, in the AcD configuration, the AcD and the axon it carries mutually support each other's growth.


Assuntos
Axônios , Interneurônios , Animais , Ratos , Células Epiteliais , Células Musculares , Dendritos
20.
J Am Soc Mass Spectrom ; 34(11): 2567-2574, 2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37812744

RESUMO

Several analytical challenges make it difficult to accurately measure coenzyme A (CoA) metaboforms, including insufficient stability and a lack of available metabolite standards. Consequently, our understanding of CoA biology and the modulation of human diseases may be nascent. CoA's serve as lipid precursors, energy intermediates, and mediators of post-translational modifications of proteins. Here, we present a liquid chromatography-mass spectrometry (LC-MS) approach to measure malonyl-CoA, acetyl-CoA, and succinyl-CoA in complex biological samples. Additionally, we evaluated workflows to increase sample stability. We used reference standards to optimize CoA assay sensitivity and test CoA metabolite stability as a function of the reconstitution solvent. We show that using glass instead of plastic sample vials decreases CoA signal loss and improves the sample stability. We identify additives that improve CoA stability and facilitate accurate analysis of CoA species across large sample sets. We apply our optimized workflow to biological samples of skeletal muscle cells cultured under hypoxic and normoxia conditions. Together, our workflow improves the detection and identification of CoA species through targeted analysis in complex biological samples.


Assuntos
Acil Coenzima A , Malonil Coenzima A , Humanos , Malonil Coenzima A/metabolismo , Acetilcoenzima A/metabolismo , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Células Musculares/química , Células Musculares/metabolismo
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