Hernández-Ochoa Erick O, Vanegas Camilo, Iyer Shama R, Lovering Richard M, Schneider Martin F
Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, 108 N. Greene Street, Baltimore, MD 21201 USA.
Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, MD 21201 USA.
Skelet Muscle. 2016 Feb 5;6:6. doi: 10.1186/s13395-016-0076-8. eCollection 2016.
Most cultured enzymatically dissociated adult myofibers exhibit spatially uniform (UNI) contractile responses and Ca(2+) 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 Ca(2+) 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.
We used high-speed confocal microscopic Ca(2+) imaging, electric field stimulation, microelectrode recordings, immunostaining, and confocal microscopy to characterize the properties of action potential-induced Ca(2+) 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 Ca(2+) 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.
We 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.
大多数经酶解分离培养的成年肌纤维在通过双极电极施加的任意极性电场刺激下，在整个肌纤维上表现出空间均匀（UNI）的收缩反应和Ca(2+)瞬变。然而，一些肌纤维在交替极性场刺激下仅在交替（ALT）末端表现出收缩和Ca(2+)瞬变。在此，我们首次展示了在原代培养物和分离肌肉中鉴定ALT肌纤维的方法，以及对其电生理特性的研究。
我们使用高速共聚焦显微镜Ca(2+)成像、电场刺激、微电极记录、免疫染色和共聚焦显微镜来表征动作电位诱导的Ca(2+)瞬变、收缩性、静息膜电位以及应用于培养成年肌纤维的T管电压门控Na(+)通道分布的染色特性。在此，我们首次以高时间和空间分辨率表明，具有UNI反应的正常对照肌纤维可通过添加TTX或从浴液中去除Na(+)而转变为ALT反应肌纤维，在恢复Na(+)时UNI反应重新出现。我们的结果表明兴奋性破坏是ALT行为的原因，并表明ALT反应是由于局部去极化诱导的Ca(2+)释放，而UNI反应是由动作电位在整个肌纤维上的传播触发的。与此解释一致，局部去极化单极刺激在UNI肌纤维中产生均匀（传播）反应，但在ALT肌纤维中仅在电极处产生局部反应。电不可兴奋肌纤维中的ALT反应与双极刺激电极之间电流传播的预期一致，电流进入（超极化）肌纤维的一端并离开（去极化）肌纤维的另一端。在来自野生型和MDX小鼠的完整分离全肌肉中的一些肌纤维中也检测到了ALT反应，表明ALT反应可在酶解分离之前出现。
我们认为，在交替极性刺激期间观察肌纤维末端来检查ALT肌纤维反应性，可作为肌纤维兴奋性受损的一项测试，并可用于通过健康和患病肌肉中的ALT行为来识别不可兴奋、受损或患病的肌纤维。
