Search Results (91)

Ventricular remodeling occurring in heart failure (HF) involves structural disarray of the sarcolemma T-tubule (TT)–sarcoplasmic reticulum (SR) dyad junctions, thereby disrupting the close apposition of L-type Ca²⁺ channels (Ca_V1.2) with ryanodine receptors (RyR2) that trigger SR Ca²⁺ release and myofilament contraction. In a rat ischemic heart failure model expressing low thyroid hormone (TH) function, we used 3D stochastic optical reconstruction microscopy (STORM) to image RyR2 clusters with Ca_V1.2 channels, and the associated protein junctophilin-2 (Jph2). We tested whether treatment with T3, the biologically active form of TH, throughout progression of the disease would preserve T-tubule structure and dyadic ion channel organization. Confocal microscopy of isolated cardiomyocytes (CMs) stained with ANEPPS membrane dye showed significantly decreased TT density in diseased CMs while T3 treatment attenuated TT disorganization. 3D STORM images of dyadic ion channels labeled with fluorescent-tagged antibodies to RyR-Dylight550, Jph-CF647 and Ca_V1.2/IgG-Dylight488 were captured. A density-based algorithm defined RyR2 clusters, and a 400 nm spherical 3D volume of interest around each RyR2 cluster’s centroid determined the number of Ca_V1.2 and Jph2 localizations associated with each RyR2 cluster. Analysis revealed significant reduction in RyR2 cluster size and number with reduced co-localized Jph2 in failing CMs. T3 treatment increased RyR2 cluster numbers and cluster volumes albeit non-significantly, with increased co-clustering of Jph2. The number of Ca_V1.2 co-localized with RyR2 clusters trended lower in the failing CMs. These results support maintaining TH homeostasis in optimizing the nanoscale organization of Ca²⁺ ion channels in triggering Ca²⁺ release and myofibrillar contraction in patients with heart disease. Full article

Repetitive temperature fluctuations during transportation and storage promote ice crystal formation in salmon flesh, leading to protein denaturation, lipid oxidation, and quality loss. Tuna skin, a major by-product of tuna processing, is a potential source of antifreeze peptides (AFPs) but remains underutilized. This study examined the cryoprotective effects of tuna skin-derived AFPs on salmon cubes subjected to repeated freeze–thaw cycles. Cubes treated with AFPs from three groups of protein hydrolysates prepared using trypsin, pepsin, or neutral protease were evaluated for texture, color, water holding capacity (WHC), volatile odor profiles, protein conformation, biochemical indices, and microstructure. AFP treatment improved textural properties, maintained color stability, and reduced thawing, cooking, and centrifugal losses. The neutral protease-treated group exhibited the optimal cryoprotective ability and it also limited aldehyde and sulfide accumulation, preserved the retention rate of α-helix structure at 49% which was higher than 39% in controls, and enhanced Ca²⁺-ATPase activity to 1.75 μmol Pi·mg⁻¹·h⁻¹ with a 45.8% increase compared to controls, and significantly inhibited protein and lipid oxidation. Microstructural analysis showed compact fibers and intact sarcolemma in the neutral protease-treated group samples, contrasting with severe disruption in controls. This study showed that tuna skin AFPs mitigate freeze–thaw damage in salmon cubes by stabilizing proteins and reducing oxidative deterioration, highlighting their potential as natural, healthy cryoprotectants for seafood preservation, meeting the growing demand of the food industry for clean-label, low-calorie preservation solutions, while advancing the circular economy of aquatic processing via the valorization of tuna skin by-products for high-value seafood applications. Full article

The depolarization of the sarcolemma is one of the first effects of unloading on skeletal muscle. We hypothesized that unloading-induced activation of the dihydropyridine receptor (DHPR), a voltage-sensitive L-type Ca²⁺ channel, and depolarization of the sarcolemma trigger intracellular Ca²⁺ release from the sarcoplasmic reticulum and activation of Ca²⁺-dependent signaling pathways, resulting in muscle atrophy. Nifedipine, a DHPR calcium channel blocker, was used to study the role of DHPR in the regulation of signaling pathways during three days of rat soleus muscle unloading/hindlimb suspension. Inhibition of the DHPR during unloading attenuates the decrease in soleus muscle contractile properties, prevents the accumulation of ATP, ROS, and Ca²⁺ content in the sarcoplasm and the mitochondria, and blocks the decrease in PGC1alpha mRNA expression and Junctophilin-1 (JP1) proteolysis. In nifedipine-treated rats, the improvement of the unloaded soleus muscle contractile properties could be mediated by blocking the calpain-mediated degradation of the cytoskeletal proteins. DHPR blocking could be one of the future directions for the preservation of contractile properties of inactive/unloaded muscle. Full article

Delayed reperfusion of an ischemic heart is known to impair the recovery of cardiac function, and the occurrence of intracellular Ca²⁺ overload in the myocardium is considered to play a critical role in the development of ischemia-reperfusion (I/R) injury. Since Ca²⁺/Mg²⁺ ecto-ATPase, which is activated by millimolar concentrations of Ca²⁺ or Mg²⁺, has been shown to serve as a Ca²⁺ gating mechanism for the entry of Ca²⁺ and subsequent development of intracellular Ca²⁺ overload, we investigated the role of depression in Ca²⁺/Mg²⁺ ecto-ATPase activity by polyclonal antibodies against Ca²⁺/Mg²⁺ ecto-ATPase in promoting the recovery of cardiac function in isolated perfused rat hearts upon subjection to I/R injury. Incubation of sarcolemma (SL) membranes with immune serum or purified IgG antibody fraction was found to depress both Ca²⁺-ATPase and Mg²⁺-ATPase activities. Pretreatment of hearts with immune serum or purified antibodies was observed to improve the recovery of cardiac function and depress the SL Ca²⁺/Mg²⁺ ecto-ATPase activities in hearts subjected to I/R injury. A marked increase in myocardial Ca²⁺ content in I/R hearts was also attenuated by immune serum treatment. Furthermore, treatment of cardiomyocytes from normal hearts with immune serum or purified antibodies reduced the ATP-induced increase in intracellular Ca²⁺ concentration. These results suggest that improvement in the recovery of cardiac function in hearts subjected to I/R injury by polyclonal Ca²⁺/Mg²⁺ ecto-ATPase antibodies may be due to the attenuation of intracellular Ca²⁺ overload. Full article

The Repulsive Guidance Molecule a (RGMa) is a multifunctional GPI-anchored protein localized in the sarcolemma and sarcoplasm of the adult skeletal muscle cell. Our research group showed that RGMa overexpression can promote myoblast fusion and induce hypertrophic muscle fibers during in vitro differentiation. Here, we report that RGMa is expressed in primary skeletal muscle cells cultured in vitro, showing a nuclear localization, revealed by immunostaining with an antibody targeting its C-terminal region (C-RGMa). While RGMa was detected in the nuclei, its canonical receptor, Neogenin, was predominantly found in the perinuclear region. Nuclear RGMa was absent in Neogenin-knockdown cells, suggesting that Neogenin mediates its nuclear transport. Functional assays suggested that RGMa promotes primary skeletal muscle cell viability and proliferation and supports their myogenic commitment. These findings reveal a previously unrecognized nuclear function of RGMa–Neogenin signaling and provide new insights into the regulation of skeletal muscle cell behavior in vitro. Full article

Dysferlin is a large transmembrane protein that is mutated or absent in Limb Girdle Muscular Dystrophy Type R2 (LGMD R2). Although it may have several functions in healthy skeletal muscle, most research on dysferlin has addressed its roles in repair of the sarcolemma and in maintaining proper control of Ca²⁺ homeostasis at the triad junction, where it concentrates. Here, we review the literature on the role of dysferlin in both membrane repair and in Ca²⁺ homeostasis, with a focus on the latter. We propose that pathophysiology in LGMD R2 is in part the result of increased leak of Ca²⁺ at the triad junction, which in turn reduces the amplitude of Ca²⁺ transients and, by activating Ca²⁺-induced Ca²⁺ release, or CICR, at the triad junction, induces Ca²⁺ waves. We discuss the mechanisms that regulate Ca²⁺ leak and Ca²⁺ levels at the triad junction under physiological and pathophysiological conditions. Our results suggest that suppression of abnormal leak and CICR may be therapeutic for LGMD R2 and other diseases of muscle linked to dysregulation of Ca²⁺ homeostasis. Full article

Dysferlinopathies are progressive muscular dystrophies caused by DYSF mutations, leading to impaired membrane repair, chronic inflammation, lipid accumulation, and muscle degeneration. No approved therapies currently halt the progression of this disease. Here, we evaluated the effects of daily oral administration of pulverized Boldo (Peumus boldus) leaves, commonly used as a nutraceutical, to blAJ mice, a model of dysferlinopathy. Symptomatic bIAJ mice were treated for four weeks with Boldo and presented significantly improved grip strength and restored endothelial-dependent vasodilation. Muscle perfusion and capillary density in the gastrocnemius were both enhanced by treatment. Histological analyses revealed that Boldo prevented myofiber atrophy, reduced centrally nucleated fibers, and improved muscle tissue architecture. Lipid accumulation observed in blAJ muscles was absent in Boldo-treated mice. At the cellular level, Boldo normalized sarcolemma membrane permeability (dye uptake) and reduced mRNA levels of inflammasome components (NLRP3, ASC, and IL-1β), suggesting anti-inflammatory activity. These findings indicate that Boldo improves vascular and muscle integrity, supporting its potential as a complementary therapeutic strategy for dysferlinopathy. Full article

Cardiomyocyte hypertrophy is regulated by several factors, including the ADP-ribosylation factor (Arf) family of small G proteins, among others. For instance, ArfGAP with dual pleckstrin homology domains 1 (Adap1) exerts an anti-hypertrophic effect in cultured cardiomyocytes. Its homologous protein, Adap2, is also expressed in the heart but its role remains elusive. To elucidate its function, we investigated the effects of adenoviral-mediated overexpression of Adap2 in cultured neonatal rat ventricular myocytes under both basal and pro-hypertrophic conditions, employing a range of microscopy and biochemical techniques. Despite minimal detection in neonatal rat hearts, Adap2 was found to be well expressed in adult rat hearts, being predominantly localized at the membrane fraction. In contrast to Adap1, overexpression of Adap2 provokes the robust accumulation of β1-integrin at the cellular surface of cultured cardiomyocytes. Interestingly, overexpressed Adap2 relocalizes at the sarcolemma and increases the size of cardiomyocytes upon phenylephrine stimulation, despite attenuating Erk1/2 phosphorylation and Nppa gene expression. Under these same conditions, cardiomyocytes overexpressing Adap2 also express higher level of detyrosinated tubulin, a marker of hypertrophic response. These findings provide new insights into the pro-hypertrophic function of Adap2 in cardiomyocytes. Full article

The skeletal muscle is a complex organ mainly composed of multinucleated fibres responsible for contractile activity, but it also contains postnatal myogenic stem cells (i.e., satellite cells), connective cells and nervous cells. The skeletal muscle is severely affected by aging, undergoing a progressive reduction in muscle mass, strength and endurance in a condition known as sarcopenia. The mechanisms underlying sarcopenia still need to be completely clarified, but they are undoubtedly multifactorial, involving all cell types constituting the skeletal muscle. Immunohistochemistry has widely been used to investigate skeletal muscle aging, identifying age-related molecular alterations in the various myofibre components, as well as in the satellite cells and peri-fibre environment. The wide range of immunohistochemical data reported in this review is proof of the primary role played by this long-established, yet modern, technique. Its high specificity for the molecules of interest, and the possibility of imaging and quantifying the signal in the real histological or cytological sites where these molecules are located and active, makes immunohistochemistry a unique and irreplaceable tool among the laboratory techniques in biomedicine. Full article

Chondrocytes maintain cartilage integrity through coordinated regulation of extracellular matrix (ECM) synthesis and remodeling. These processes depend on ECM dynamic interactions, mediated by integrin-based focal adhesions and associated cytoskeletal components. While the roles of core adhesion proteins are well described, the involvement of sarcoglycans (SGs) remains unclear in chondrocytes. Drawing parallels from striated muscle, where the SG subcomplex stabilizes the sarcolemma, we hypothesized that SGs similarly integrate into chondrocyte adhesion complexes. This study investigated the SGs (α, β, γ, δ) expression with cytoskeletal and adhesion proteins, including actin and vinculin, in human chondrocytes cultured by immunofluorescence, qPCR, and siRNA-mediated silencing. All four SG isoforms were expressed in the cytoplasmic and membrane domains, with enrichment at focal adhesion sites. Double labeling revealed SG colocalization with F-actin stress fibers and vinculin, indicating integration into the core adhesion complex. Silencing of each SG resulted in disrupted actin stress fibers, diffuse vinculin distribution, reduced focal plaque number, and a change in cell morphology. These findings support the hypothesis that SGs regulate actin cytoskeletal dynamics and focal contact stabilization. Loss of SG function compromises chondrocyte shape and adhesion, highlighting the importance of these glycoproteins also in non-muscle cells. Full article

The stability of the sarcolemma is severely impaired in a series of genetic neuromuscular diseases defined as muscular dystrophies. These are characterized by the centralization of skeletal muscle syncytial nuclei, the replacement of muscle fibers with fibrotic tissue, the release of inflammatory cytokines, and the disruption of muscle protein homeostasis, ultimately leading to necrosis and loss of muscle functionality. A specific subgroup of muscular dystrophies is associated with genetic defects in components of the dystrophin–glycoprotein complex (DGC), which plays a crucial role in linking the cytosol to the skeletal muscle basement membrane. In these cases, dystrophin-associated proteins fail to correctly localize to the sarcolemma, resulting in dystrophy characterized by an uncontrolled increase in protein degradation, which can ultimately lead to cell death. In this review, we explore the role of intracellular degradative pathways—primarily the ubiquitin–proteasome and autophagy–lysosome systems—in the progression of DGC-linked muscular dystrophies. The DGC acts as a hub for numerous signaling pathways that regulate various cellular functions, including protein homeostasis. We examine whether the loss of structural stability within the DGC affects key signaling pathways that modulate protein recycling, with a particular emphasis on autophagy. Full article

Limb–girdle muscular dystrophy R1 (LGMDR1) is characterized by progressive proximal muscle weakness due to mutations in the CAPN3 gene. Little is known about CAPN3’s function in muscle, but its loss results in aberrant sarcomere formation. Human muscle structure was analyzed in this study, with observations including integrin β1D isoform (ITGβ1D) mislocalization, a lack of Talin-1 (TLN1) in the sarcolemma and the irregular expression of focal adhesion kinase (FAK) in LGMDR1 muscles, suggesting a lack of integrin activation with an altered sarcolemma, extracellular matrix (ECM) assembly and signaling pathway deregulation, which may cause frailty in LGMDR1 muscle fibers. Additionally, altered nuclear morphology, centrosome distribution and microtubule organization have been found in muscle cells derived from LGMDR1 patients. Full article

Delayed reperfusion of the ischemic heart (I/R) is known to impair the recovery of cardiac function and produce a wide variety of myocardial defects, including ultrastructural damage, metabolic alterations, subcellular Ca²⁺-handling abnormalities, activation of proteases, and changes in cardiac gene expression. Although I/R injury has been reported to induce the formation of reactive oxygen species (ROS), inflammation, and intracellular Ca²⁺ overload, the generation of oxidative stress is considered to play a critical role in the development of cardiac dysfunction. Increases in the production of superoxide, hydroxyl radicals, and oxidants, such as hydrogen peroxide and hypochlorous acid, occur in hearts subjected to I/R injury. In fact, mitochondria are a major source of the excessive production of ROS in I/R hearts due to impairment in the electron transport system as well as activation of xanthine oxidase and NADPH oxidase. Nitric oxide synthase, mainly present in the endothelium, is also activated due to I/R injury, leading to the production of nitric oxide, which, upon combination with superoxide radicals, generates nitrosative stress. Alterations in cardiac function, sarcolemma, sarcoplasmic reticulum Ca²⁺-handling activities, mitochondrial oxidative phosphorylation, and protease activation due to I/R injury are simulated upon exposing the heart to the oxyradical-generating system (xanthine plus xanthine oxidase) or H₂O₂. On the other hand, the activation of endogenous antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, and the concentration of a transcription factor (Nrf2), which modulates the expression of various endogenous antioxidants, is depressed due to I/R injury in hearts. Furthermore, pretreatment of hearts with antioxidants such as catalase plus superoxide dismutase, N-acetylcysteine, and mercaptopropionylglycerine has been observed to attenuate I/R-induced subcellular Ca²⁺ handling and changes in Ca²⁺-regulatory activities; additionally, it has been found to depress protease activation and improve the recovery of cardiac function. These observations indicate that oxidative stress is intimately involved in the pathological effects of I/R injury and different antioxidants attenuate I/R-induced subcellular alterations and improve the recovery of cardiac function. Thus, we are faced with the task of developing safe and effective antioxidants as well as agents for upregulating the expression of endogenous antioxidants for the therapy of I/R injury. Full article

Mitochondrial diseases are complex disorders caused by nuclear or mitochondrial DNA mutations, leading to oxidative phosphorylation deficiency and excessive production of reactive oxygen species (ROS). While ROS have been well established in the pathogenesis of these diseases, the role of reactive nitrogen species (RNS) remains unclear. In this study, we performed a quantitative analysis of muscle fibers to investigate the relationship between protein nitration and mitochondrial abnormalities (mitochondrial proliferation and cytochrome-c oxidase (COX) deficiency) and factors like genotype, muscle damage, and age. A total of 1961 muscle fibers (303 from 4 controls and 1658 from 29 patients with mitochondrial diseases) were analyzed by immunostaining for nitro-tyrosine. Contrary to previous findings, which identified nitro-tyrosine only in small muscle vessels, we observed a broader distribution affecting the sarcolemma and sarcoplasm. Using multivariate techniques, we identified a significant correlation between protein nitration and mitochondrial proliferation but found no associations with COX deficiency, age, muscle damage, or genotype. These findings suggest that nitrative stress may contribute to mitochondrial dysfunction or play a role in signaling processes that induce mitochondrial biogenesis. Our results provide new insights into the molecular mechanisms of mitochondrial diseases and highlight the potential relevance of protein nitration. Full article

DUX4 is typically a repressed transcription factor, but its aberrant activation in Facioscapulohumeral Muscular Dystrophy (FSHD) leads to cell death by disrupting muscle homeostasis. This disruption affects crucial processes such as myogenesis, sarcolemma integrity, gene regulation, oxidative stress, immune response, and many other biological pathways. Notably, these disrupted processes have been associated, in other pathological contexts, with the presence of connexin (Cx) hemichannels—transmembrane structures that mediate communication between the intracellular and extracellular environments. Thus, hemichannels have been implicated in skeletal muscle atrophy, as observed in human biopsies and animal models of Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, and Dysferlinopathies, suggesting a potentially shared mechanism of muscle atrophy that has not yet been explored in FSHD. Despite various therapeutic strategies proposed to manage FSHD, no treatment or cure is currently available. This review summarizes the current understanding of the mechanisms underlying FSHD progression, with a focus on hormones, inflammation, reactive oxygen species (ROS), and mitochondrial function. Additionally, it explores the potential of targeting hemichannels as a therapeutic strategy to slow disease progression by preventing the spread of pathogenic factors between muscle cells. Full article