Alcoholic myopathies are characterized by neuromusculoskeletal symptoms such as compromised movement and weakness. Although these symptoms have been attributed to neurological damage, EtOH may also target skeletal muscle. EtOH exposure during zebrafish primary muscle development or adulthood results in smaller muscle fibers. However, the effects of EtOH exposure on skeletal muscle during the growth period that follows primary muscle development are not well understood. We determined the effects of EtOH exposure on muscle during this phase of development. Strikingly, muscle fibers at this stage are acutely sensitive to EtOH treatment: EtOH induces muscle degeneration. The severity of EtOH-induced muscle damage varies but muscle becomes more refractory to EtOH as muscle develops. NF-kB induction in muscle indicates that EtOH triggers a pro-inflammatory response. EtOH-induced muscle damage is p53-independent. Uptake of Evans blue dye shows that EtOH treatment causes sarcolemmal instability before muscle fiber detachment. Dystrophin-null sapje mutant zebrafish also exhibit sarcolemmal instability. We tested whether Trichostatin A (TSA), which reduces muscle degeneration in sapje mutants, would affect EtOH-treated zebrafish. We found that TSA and EtOH are a lethal combination. EtOH does, however, exacerbate muscle degeneration in sapje mutants. EtOH also disrupts adhesion of muscle fibers to their extracellular matrix at the myotendinous junction: some detached muscle fibers retain beta-Dystroglycan indicating failure of muscle end attachments. Overexpression of Paxillin, which reduces muscle degeneration in zebrafish deficient for beta-Dystroglycan, is not sufficient to rescue degeneration. Taken together, our results suggest that EtOH exposure has pleiotropic deleterious effects on skeletal muscle.

Muscle development and homeostasis are critical for normal muscle function. A key aspect of muscle physiology during development, growth, and homeostasis is modulation of protein turnover, the balance between synthesis and degradation of muscle proteins. Protein degradation depends upon lysosomal pH, generated and maintained by proton pumps. Sphingolipid transporter 1 (spns1), a highly conserved gene encoding a putative late endosome/lysosome carbohydrate/H+ symporter, plays a pivotal role in maintaining optimal lysosomal pH and spns1−/− mutants undergo premature senescence. However, the impact of dysregulated lysosomal pH on muscle development and homeostasis is not well understood. We found that muscle development proceeds normally in spns1−/− mutants prior to the onset of muscle degeneration. Dysregulation of the extracellular matrix (ECM) at the myotendinous junction (MTJ) coincided with the onset of muscle degeneration in spns1−/− mutants. Expression of the ECM proteins laminin 111 and MMP-9 was upregulated. Upregulation of laminin 111 mitigated the severity of muscle degeneration, as inhibition of adhesion to laminin 111 exacerbated muscle degeneration in spns1−/− mutants. MMP-9 upregulation was induced by tnfsf12 signaling, but abrogation of MMP-9 did not impact muscle degeneration in spns1−/− mutants. Taken together, these data indicate that dysregulated lysosomal pH impacts expression of ECM proteins at the myotendinous junction.