Intracellular amyloid formation in muscle cells of Aβ-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification

Background: The amyloid β-peptide is a ubiquitous peptide, which is prone to aggregate forming soluble toxic oligomers and insoluble less-toxic aggregates. The intrinsic and external/environmental factors that determine Aβ aggregation in vivo are poorly understood, as well as the cellular meaning of this process itself. Genetic data as well as cell biological and biochemical evidence strongly support the hypothesis that Aβ is a major player in the onset and development of Alzheimer's disease. In addition, it is also known that Aβ is involved in Inclusion Body Myositis, a common myopathy of the elderly in which the peptide accumulates intracellularly. Results: In the present work, we found that intracellular Aβ aggregation in muscle cells of Caenorhabditis elegans overexpressing Aβ peptide is affected by two single amino acid substitutions, E22G (Arctic) and V18A (NIC). Both variations show decrease intracellular amyloidogenesis compared to wild type Aβ. We show that intracellular amyloid aggregation of wild type Aβ is accelerated by Cu2+ and diminished by copper chelators. Moreover, we demonstrate through toxicity and behavioral assays that Aβ-transgenic worms display a higher tolerance to Cu2+ toxic effects and that this resistance may be linked to the formation of amyloid aggregates. Conclusion: Our data show that intracellular Aβ amyloid aggregates may trap excess of free Cu2+ buffering its cytotoxic effects and that accelerated intracellular Aβ aggregation may be part of a cell protective mechanism

Role of Matricellular CCN Proteins in Skeletal Muscle: Focus on CCN2/CTGF and Its Regulation by Vasoactive Peptides

The Cellular Communication Network (CCN) family of matricellular proteins comprises six proteins that share conserved structural features and play numerous biological roles. These proteins can interact with several receptors or soluble proteins, regulating cell signaling pathways in various tissues under physiological and pathological conditions. In the skeletal muscle of mammals, most of the six CCN family members are expressed during embryonic development or in adulthood. Their roles during the adult stage are related to the regulation of muscle mass and regeneration, maintaining vascularization, and the modulation of skeletal muscle fibrosis. This work reviews the CCNs proteins’ role in skeletal muscle physiology and disease, focusing on skeletal muscle fibrosis and its regulation by Connective Tissue Growth factor (CCN2/CTGF). Furthermore, we review evidence on the modulation of fibrosis and CCN2/CTGF by the renin-angiotensin system and the kallikrein-kinin system of vasoactive peptides

Role of hypoxia in skeletal muscle fibrosis: Synergism between hypoxia and TGF-beta signaling upregulates CCN2/CTGF expression specifically in muscle fibers

Several skeletal muscle diseases are characterized by fibrosis, the excessive accumulation of extracellular matrix. Transforming growth factor-β (TGF-β) and connective tissue growth factor (CCN2/CTGF) are two profibrotic factors augmented in fibrotic skeletal muscle, together with signs of reduced vasculature that implies a decrease in oxygen supply. We observed that fibrotic muscles are characterized by the presence of positive nuclei for hypoxia-inducible factor-1α (HIF-1α), a key mediator of the hypoxia response. However, it is not clear how a hypoxic environment could contribute to the fibrotic phenotype in skeletal muscle. We evaluated the role of hypoxia and TGF-β on CCN2 expression in vitro. Fibroblasts, myoblasts and differentiated myotubes were incubated with TGF-β1 under hypoxic conditions. Hypoxia and TGF-β1 induced CCN2 expression synergistically in myotubes but not in fibroblasts or undifferentiated muscle progenitors. This induction requires HIF-1α and the Smad-independent TGF-β signaling pathway. We performed in vivo experiments using pharmacological stabilization of HIF-1α or hypoxia-induced via hindlimb ischemia together with intramuscular injections of TGF-β1, and we found increased CCN2 expression. These observations suggest that hypoxic signaling together with TGF-β signaling, which are both characteristics of a fibrotic skeletal muscle environment, induce the expression of CCN2 in skeletal muscle fibers and myotubes.status: publishe

Episodic Binge-like Ethanol Reduces Skeletal Muscle Strength Associated with Atrophy, Fibrosis, and Inflammation in Young Rats

Binge Drinking (BD) corresponds to episodes of ingestion of large amounts of ethanol in a short time, typically ≤2 h. BD occurs across all populations, but young and sports-related people are especially vulnerable. However, the short- and long-term effects of episodic BD on skeletal muscle function have been poorly explored. Young rats were randomized into two groups: control and episodic Binge-Like ethanol protocol (BEP) (ethanol 3 g/kg IP, 4 episodes of 2-days ON-2-days OFF paradigm). Muscle function was evaluated two weeks after the last BEP episode. We found that rats exposed to BEP presented decreased muscle strength and increased fatigability, compared with control animals. Furthermore, we observed that skeletal muscle from rats exposed to BEP presented muscle atrophy, evidenced by reduced fiber size and increased expression of atrophic genes. We also observed that BEP induced fibrotic and inflammation markers, accompanied by mislocalization of nNOSµ and high levels of protein nitration. Our findings suggest that episodic binge-like ethanol exposure alters contractile capacity and increases fatigue by mechanisms involving atrophy, fibrosis, and inflammation, which remain for at least two weeks after ethanol clearance. These pathological features are common to several neuromuscular diseases and might affect muscle performance and health in the long term

ALS skeletal muscle shows enhanced TGF-β signaling, fibrosis and induction of fibro/adipogenic progenitor markers

<div><p>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which upper and lower motoneurons degenerate leading to muscle wasting, paralysis and eventually death from respiratory failure. Several studies indicate that skeletal muscle contributes to disease progression; however the molecular mechanisms remain elusive. Fibrosis is a common feature in skeletal muscle under chronic damage conditions such as those caused by muscular dystrophies or denervation. However, the exact mechanisms of fibrosis induction and the cellular bases of this pathological response are unknown. We show that extracellular matrix (ECM) components are augmented in skeletal muscles of symptomatic hSOD1^G93A mice, a widely used murine model of ALS. These mice also show increased TGF-β1 mRNA levels, total Smad3 protein levels and p-Smad3 positive nuclei. Furthermore, platelet-derived growth factor receptor-α (PDGFRα), Tcf4 and α-smooth muscle actin (α-SMA) levels are augmented in the skeletal muscle of symptomatic hSOD1^G93A mice. Additionally, the fibro/adipogenic progenitors (FAPs), which are the main producers of ECM constituents, are also increased in these pathogenic conditions. Therefore, FAPs and ECM components are more abundant in symptomatic stages of the disease than in pre-symptomatic stages. We present evidence that fibrosis observed in skeletal muscle of symptomatic hSOD1^G93A mice is accompanied with an induction of TGF-β signaling, and also that FAPs might be involved in triggering a fibrotic response. Co-localization of p-Smad3 positive cells together with PDGFRα was observed in the interstitial cells of skeletal muscles from symptomatic hSOD1^G93A mice. Finally, the targeting of pro-fibrotic factors such as TGF-β, CTGF/CCN2 and platelet-derived growth factor (PDGF) signaling pathway might be a suitable therapeutic approach to improve muscle function in several degenerative diseases.</p></div

Extracellular matrix (ECM) components are augmented in gastrocnemius muscle from symptomatic hSOD1^G93A mice.

<p><b>(A)</b> Fibronectin and collagen-III were detected by western-blot in protein extracts from wild-type mice (60 days old), pre-symptomatic (60 days old) hSOD1^G93A age-matched mice, wild-type (120 days old), and symptomatic (120 days old) hSOD1^G93A age matched mice. GAPDH protein levels are shown as loading control. <b>(B-C)</b> Protein levels of fibronectin and collagen-III were quantified using densitometric analysis. Values correspond to the mean ± SEM of four animals for each experimental condition. One-way ANOVA, ** p<0.05; *** p<0.001, n.s: not significant.</p

FAPs markers are increased in gastrocnemius muscle from symptomatic hSOD1^G93A mice.

<p><b>(A)</b> PDGFRα, Tcf4 and α-SMA were detected by western-blot in protein extracts from wild-type (60 days old), pre-symptomatic (60 days old) hSOD1^G93A age-matched mice, wild-type (120 days old), and symptomatic (120 days old) hSOD1^G93A age-matched mice. GAPDH protein levels are shown as loading control. <b>(B-I)</b> Tcf4 (white and green) and laminin (white and red) were detected by indirect immunofluorescence in cross-sections of gastrocnemius muscle from wild-type (120 days old) and hSOD1^G93A symptomatic (120 days old) age-matched mice, bar corresponds to 50 μm, nuclei were stained with Hoechst. Arrows show Tcf4-positive nuclei. <b>(J)</b> Tcf4-positive cells were quantified in gastrocnemius muscle from wild-type (120 days old) and symptomatic (120 days old) hSOD1^G93A age-matched mice, values correspond to the mean ± SEM of three animals for each experimental condition. One-way ANOVA, ** p<0.005. <b>(K-L)</b> PDGFRα (green) and WGA (red) <b>(M-N)</b> PDGFRα and p-Smad3 (red) were detected by indirect immunofluorescence in cross-sections of gastrocnemius muscle from wild-type (120 days old) and hSOD1^G93A symptomatic (120 days old) age-matched mice, bar corresponds to 50 μm, nuclei were stained with Hoechst. Arrows and asterisk show PDGFRα/p-Smad3 co-localization and PDGFRα^-/p-Smad3⁺ cells, respectively.</p

Muscle architecture is altered in gastrocnemius muscle from symptomatic hSOD1^G93A mice.

<p><b>(A-D)</b> Sirius red staining of gastrocnemius muscle from wild-type (120 days old) and symptomatic (120 days old) hSOD1^G93A age-matched mice seen under polarized light in cross-sections. Bar corresponds to 100 μm. <b>(E)</b> Fiber diameter in wild-type (120 days old; black circles) and symptomatic hSOD1^G93A (120 days old; black squares) mice, values correspond to the mean ± SEM of three animals for each experimental condition. Two-way ANOVA, *** p<0.001, n.s: not significant.</p

Extracellular matrix (ECM) components deposition in gastrocnemius muscle from symptomatic hSOD1^G93A mice.

<p><b>(A-D)</b> H&E staining. <b>(E-H)</b> Sirius red staining. <b>(I-L)</b> fibronectin (red) and <b>(M-P)</b> collagen-I (green) were detected by indirect immunofluorescence in cross-sections of gastrocnemius muscle from wild-type (60 days old), hSOD1^G93A pre-symptomatic (60 days old), wild-type (120 days old), and hSOD1^G93A symptomatic (120 days old) age-matched mice. Bar corresponds to 50 μm. Representative images of three mice per condition.</p