Synthesis and Evaluation of 4-Hydroxycoumarin Imines as Inhibitors of Class II Myosins.

Inhibitors of muscle myosin ATPases are needed to treat conditions that could be improved by promoting muscle relaxation. The lead compound for this study ((3-(N-butylethanimidoyl)ethyl)-4-hydroxy-2H-chromen-2-one; BHC) was previously discovered to inhibit skeletal myosin II. BHC and 34 analogues were synthesized to explore structure-activity relationships. The properties of analogues, including solubility, stability, and toxicity, suggest that the BHC scaffold may be useful for developing therapeutics. Inhibition of actin-activated ATPase activity of fast skeletal and cardiac muscle myosin II, inhibition of skeletal muscle contractility ex vivo, and slowing of in vitro actin-sliding velocity were measured. Several analogues with aromatic side arms showed improved potency (half-maximal inhibitory concentration (IC50) <1 µM) and selectivity (≥12-fold) for skeletal myosin versus cardiac myosin compared to BHC. Several analogues blocked neurotransmission, suggesting that they are selective for nonmuscle myosin II over skeletal myosin. Competition and molecular docking studies suggest that BHC and blebbistatin bind to the same site on myosin.

Postnatal Restriction of Activity-Induced Ca2+ Responses to Schwann Cells at the Neuromuscular Junction Are Caused by the Proximo-Distal Loss of Axonal Synaptic Vesicles during Development.

Terminal or perisynaptic Schwann cells (TPSCs) are nonmyelinating, perisynaptic glial cells at the neuromuscular junction (NMJ) that respond to neural activity by increasing intracellular calcium (Ca2+) and regulate synaptic function. The onset of activity-induced TPSC Ca2+ responses, as well as whether axonal Schwann cells (ASCs) along the nerve respond to nerve stimulation during development, is unknown. Here, we show that phrenic nerve stimulation in developing male and female mice elicited Ca2+ responses in both ASCs and TPSCs at embryonic day 14. ASC responses were lost in a proximo-distal gradient over time, but could continue to be elicited by bath application of neurotransmitter, suggesting that a loss of release rather than a change in ASC competence accounted for this response gradient. Similar to those of early postnatal TPSCs, developing ASC/TPSC responses were mediated by purinergic P2Y1 receptors. The loss of ASC Ca2+ responses was correlated to the proximo-distal disappearance of synaptophysin immunoreactivity and synaptic vesicles in phrenic axons. Accordingly, developing ASC Ca2+ responses were blocked by botulinum toxin. Interestingly, the loss of ASC Ca2+ responses was also correlated to the proximo-distal development of myelination. Finally, compared with postnatal TPSCs, neonatal TPSCs and ASCs displayed Ca2+ signals in response to lower frequencies and shorter durations of nerve stimulation. Together, these results with GCaMP3-expressing Schwann cells provide ex vivo evidence that both axons and presynaptic terminals initially exhibit activity-induced vesicular release of neurotransmitter, but that the subsequent loss of axonal synaptic vesicles accounts for the postnatal restriction of vesicular release to the NMJ.SIGNIFICANCE STATEMENT Neural activity regulates multiple aspects of development, including myelination. Whether the excitation of developing neurons in vivo results in the release of neurotransmitter from both axons and presynaptic terminals is unclear. Here, using mice expressing the genetically encoded calcium indicator GCaMP3 in Schwann cells, we show that both terminal/perisynaptic Schwann cells at the diaphragm neuromuscular junction and axonal Schwann cells along the phrenic nerve exhibit activity-induced calcium responses early in development, mediated by the vesicular release of ATP from the axons of motor neurons acting on P2Y1 receptors. These ex vivo findings corroborate classic in vitro studies demonstrating transmitter release by developing axons, and thus represent a tool to study the mechanisms and significance of this process during embryonic development.