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Methylmercury myotoxicity targets formation of the myotendinous junction.

ABSTRACT: Methylmercury (MeHg) is a ubiquitous environmental contaminant and developmental toxicant known to cause a variety of persistent motor and cognitive deficits. While previous research has focused predominantly on neurotoxic MeHg effects, emerging evidence points to a myotoxic role whereby MeHg induces defects in muscle development and maintenance. A genome wide association study for developmental sensitivity to MeHg in Drosophila has revealed several conserved muscle morphogenesis candidate genes that function in an array of processes from myoblast migration and fusion to myotendinous junction (MTJ) formation and myofibrillogenesis. Here, we investigated candidates for a role in mediating MeHg disruption of muscle development by evaluating morphological and functional phenotypes of the indirect flight muscles (IFMs) in pupal and adult flies following 0, 5, 10, and 15 μM MeHg exposure via feeding at the larval stage. Developmental MeHg exposure induced a dose-dependent increase in muscle detachments (myospheres) within dorsal bundles of the IFMs, which paralleled reductions eclosion and adult flight behaviors. These effects were selectively phenocopied by altered expression of kon-tiki (kon), a chondroitin sulfate proteoglycan 4/NG2 homologue and a central component of MTJ formation. MeHg elevated kon transcript expression at a crucial window of IFM development and transgene overexpression of kon could also phenocopy myosphere phenotypes and eclosion and flight deficits. Finally, the myosphere phenotype resulting from 10 μM MeHg was partially rescued in a background of reduced kon expression using a targeted RNAi approach. Our findings implicate a component of the MTJ as a MeHg toxicity target which broaden the understanding of how motor deficits can emerge from early life MeHg exposure.

SUBMITTER: Peppriell AE

PROVIDER: S-EPMC7530093 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Methylmercury (MeHg) is a ubiquitous environmental contaminant and neurotoxicant that has long been known to cause a variety of motor deficits. These motor deficits have primarily been attributed to MeHg targeting of developing neurons and induction of oxidative stress and calcium dysregulation. Few studies have looked at how MeHg may be affecting fundamental signaling mechanisms in development, particularly in developing muscle. Studies in Drosophila recently revealed that MeHg perturbs embryonic muscle formation and upregulates Notch target genes, reflected predominantly by expression of the downstream transcriptional repressor Enhancer of Split mdelta [E(spl)m?]. An E(spl)m? reporter gene shows expression primarily in the myogenic domain, and both MeHg exposure and genetic upregulation of E(spl)m? can disrupt embryonic muscle development. Here, we tested the hypothesis that developing muscle is targeted by MeHg via upregulation of E(spl)m? using genetic modulation of E(spl)m? expression in combination with MeHg exposure in developing flies. Developmental MeHg exposure causes a decreased rate of eclosion that parallels gross disruption of indirect flight muscle (IFM) development. An increase in E(spl) expression across the pupal stages, with preferential E(spl)m? upregulation occurring at early (p5) stages, is also observed. E(spl)m? overexpression in myogenic lineages under the Mef2 promoter was seen to phenocopy eclosion and IFM effects of developmental MeHg exposure; whereas reduced expression of E(spl)m? shows rescue of eclosion and IFM morphology effects of MeHg exposure. No effects were seen on eclosion with E(spl)m? overexpression in neural and gut tissues. Our data indicate that muscle development is a target for MeHg and that E(spl)m? is a muscle-specific mediator of this myotoxicity. This research advances our knowledge of the target pathways that mediate susceptibility to MeHg toxicity, as well as a potential muscle development-specific role for E(spl)m?.

Project description:The myotendinous junction is a highly interdigitated interface designed to transfer muscle-generated force to tendon. Understanding how this interface is formed and organized, as well as identifying tendon- and muscle-specific extracellular matrix (ECM), is critical for designing effective regenerative therapies to restore functionality to damaged muscle-tendon units. However, a comparative analysis of the ECM proteome across this interface has not been conducted. The goal of this study was to resolve the distribution of ECM proteins that are uniformly expressed as well as those specific to each of the muscle, tendon, and junction tissues. The soleus muscles from 5-month-old wild-type C57BL/6 mice were harvested and dissected into the central muscle (M) away from tendon, the junction between muscle and tendon (J) and the tendon (T). Tissues were processed by either homogenizing in guanidine hydrochloride or fractionating to isolate the ECM from more soluble intracellular components and then analyzed using liquid chromatography-tandem mass spectrometry. Overall, we found that both tissue processing methods generated similar ECM profiles. Many ECM were found across the muscle-tendon unit, including type I collagen and associated fibril-regulating proteins. The ECM identified exclusively in M were primarily related to the basal lamina, whereas those specific to T and J tissue included thrombospondins and other matricellular ECM. Type XXII collagen (COL22A1) was restricted to J, and we identified COL5A3 as a potential marker of the muscle-tendon interface. Immunohistochemical analysis of key proteins confirmed the restriction of some basal lamina proteins to M, tenascin-C to T, and COL22A1 to J. COL5A3, PRELP, and POSTN were visualized in the tissue surrounding the junction, suggesting that these proteins play a role in stabilizing the interface. This comparative map provides a guide for tissue-specific ECM that can facilitate the spatial visualization of M, J, and T tissues and inform musculoskeletal regenerative therapies.

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Methylmercury myotoxicity targets formation of the myotendinous junction.

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