Alternating bipolar field stimulation identifies muscle fibers with defective excitability but maintained local Ca(2+) signals and contraction.

Abstract

BackgroundMost cultured enzymatically dissociated adult myofibers exhibit spatially uniform (UNI) contractile responses and Ca2+ transients over the entire myofiber in response to electric field stimuli of either polarity applied via bipolar electrodes. However, some myofibers only exhibit contraction and Ca2+ transients at alternating (ALT) ends in response to alternating polarity field stimulation. Here, we present for the first time the methodology for identification of ALT myofibers in primary cultures and isolated muscles, as well as a study of their electrophysiological properties.Results We used high-speed confocal microscopic Ca2+ imaging, electric field stimulation, microelectrode recordings, immunostaining, and confocal microscopy to characterize the properties of action potential-induced Ca2+ transients, contractility, resting membrane potential, and staining of T-tubule voltage-gated Na+ channel distribution applied to cultured adult myofibers. Here, we show for the first time, with high temporal and spatial resolution, that normal control myofibers with UNI responses can be converted to ALT response myofibers by TTX addition or by removal of Na+ from the bathing medium, with reappearance of the UNI response on return of Na+. Our results suggest disrupted excitability as the cause of ALT behavior and indicate that the ALT response is due to local depolarization-induced Ca2+ release, whereas the UNI response is triggered by action potential propagation over the entire myofiber. Consistent with this interpretation, local depolarizing monopolar stimuli give uniform (propagated) responses in UNI myofibers, but only local responses at the electrode in ALT myofibers. The ALT responses in electrically inexcitable myofibers are consistent with expectations of current spread between bipolar stimulating electrodes, entering (hyperpolarizing) one end of a myofiber and leaving (depolarizing) the other end of the myofiber. ALT responses were also detected in some myofibers within intact isolated whole muscles from wild-type and MDX mice, demonstrating that ALT responses can be present before enzymatic dissociation.ConclusionsWe suggest that checking for ALT myofiber responsiveness by looking at the end of a myofiber during alternating polarity stimuli provides a test for compromised excitability of myofibers, and could be used to identify inexcitable, damaged or diseased myofibers by ALT behavior in healthy and diseased muscle.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0076-8) contains supplementary material, which is available to authorized users.