Analysis of spontaneous and nerve-evoked calcium transients in intact extraocular muscles in vitro

Abstract

Extraocular muscles (EOMs) have unique calcium handling properties, yet little is known about the dynamics of calcium events underlying ultrafast and tonic contractions in myofibers of intact EOMs. Superior oblique EOMs of juvenile chickens were dissected with their nerve attached, maintained in oxygenated Krebs buffer, and loaded with fluo-4. Spontaneous and nerve stimulation-evoked calcium transients were recorded and, following calcium imaging, some EOMs were double-labeled with rhodamine-conjugated alpha-bungarotoxin (rhBTX) to identify EOM myofiber types. EOMs showed two main types of spontaneous calcium transients, one slow type (calcium waves with 1/2max duration of 2–12 s, velocity of 25–50 μm/s) and two fast “flash-like” types (Type 1, 30–90 ms; Type 2, 90–150 ms 1/2max duration). Single pulse nerve stimulation evoked fast calcium transients identical to the fast (Type 1) calcium transients. Calcium waves were accompanied by a local myofiber contraction that followed the calcium transient wavefront. The magnitude of calcium-wave induced myofiber contraction far exceeded those of movement induced by nerve stimulation and associated fast calcium transients. Tetrodotoxin eliminated nerve-evoked transients, but not spontaneous transients. Alpha-bungarotoxin eliminated both spontaneous and nerve-evoked fast calcium transients, but not calcium waves, and caffeine increased wave activity. Calcium waves were observed in myofibers lacking spontaneous or evoked fast transients, suggestive of multiply-innervated myofibers, and this was confirmed by double-labeling with rhBTX. We propose that the abundant spontaneous calcium transients and calcium waves with localized contractions that do not depend on innervation may contribute to intrinsic generation of tonic functions of EOMs.

Differential Effects of Phosphatase Inhibitors on the Calcium Homeostasis and Migration of HaCaT Keratinocytes

“…These observations suggest that the Ca 2+ release processes in cells next to the scratch are less frequent but last longer and result in a greater change in [Ca 2+ ] i as compared with untouched cells. Other cell types like Cajal [24] and extraocular muscle cells [25] also show spontaneous calcium elevations, which are similar to those observed here on HaCaT cells considering both their amplitude and their time course.…”

Adaptability of the Immature Ocular Motor Control System: Unilateral IGF-1 Medial Rectus Treatment

“…Complementarily, en plaque endplates are more centrally located on myofibers where fast myosins are expressed and exhibit fast-twitch characteristics. [33][34][35][36] Based on this common broad grouping of myofiber types, we additionally labeled myofibers with an antibody to slow MyHC to distinguish broadly between fast and slow myofiber types (Figs. 5, 6).…”

Adaptation of Slow Myofibers: The Effect of Sustained BDNF Treatment of Extraocular Muscles in Infant Nonhuman Primates

“…In addition to influencing the properties of neurons, neurotrophic factors also influence the properties of muscles. 16,40,77,78 Muscle-derived BDNF is expressed in the developing EOM and retrogradely transported to oculomotor neurons. 18,25 Brain-derived neurotrophic factor mRNA is also present in adult mouse EOM.…”

“…82 The distribution pattern of BDNF along the length of the EOM has interesting functional implications, as BDNF myofiber staining was strongest in areas of the muscle hypothesized to be more involved in the tonic or step phase of muscle contraction. 83,84 Large twitch movements are hypothesized to be executed predominantly by the large en plaque endplates on fast myofibers of the global layer, 38,40 which was the region with the lowest expression level of BDNF.…”

“…30,[35][36][37] Fast myofibers predominately have centrally located en plaque endings, and slow fibers have smaller en grappe endings along their length, although both myofibers types can be multiply innervated and have both types of neuromuscular junctions. [38][39][40] To simplify the complexity of EOM for analysis, we stained EOM fibers for slow myosin to help differentiate between neuromuscular junctions on these two broad myofiber types.…”