Voltage sensor movements of CaV1.1 during an action potential in skeletal muscle fibers.

The skeletal muscle L-type Ca2+ channel (CaV1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca2+ release. CaV1.1 contains four distinct voltage-sensing domains (VSDs), yet the contribution of each VSD to AP-evoked Ca2+ release remains unknown. To investigate the role of VSDs in excitation-contraction coupling (ECC), we encoded cysteine substitutions on each S4 voltage-sensing segment of CaV1.1, expressed each construct via in vivo gene transfer electroporation, and used in cellulo AP fluorometry to track the movement of each CaV1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of CaV1.1 voltage sensor charge movement in response to an AP waveform. Then we characterize the fluorescently labeled channels' VSD fluorescence signal responses to an AP and compare them with the waveforms of the electrically measured charge movement, the optically measured free myoplasmic Ca2+, and the calculated rate of Ca2+ release from the sarcoplasmic reticulum for an AP, the physiological signal for skeletal muscle fiber activation. A considerable fraction of the fluorescence signal for each VSD occurred after the time of peak Ca2+ release, and even more occurred after the earlier peak of electrically measured charge movement during an AP, and thus could not directly reflect activation of Ca2+ release or charge movement, respectively. However, a sizable fraction of the fluorometric signals for VSDs I, II, and IV, but not VSDIII, overlap the rising phase of charge moved, and even more for Ca2+ release, and thus could be involved in voltage sensor rearrangements or Ca2+ release activation.

Optical Recording of Action Potential Initiation and Propagation in Mouse Skeletal Muscle Fibers.

Skeletal muscle fibers have been used to examine a variety of cellular functions and pathologies. Among other parameters, skeletal muscle action potential (AP) propagation has been measured to assess the integrity and function of skeletal muscle. In this work, we utilize 1-(3-sulfonatopropyl)-4[ß[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine, a potentiometric dye, and mag-fluo-4, a low-affinity intracellular Ca2+indicator, to noninvasively and reliably measure AP conduction velocity in skeletal muscle. We used remote extracellular bipolar electrodes to generate an alternating polarity electric field that initiates an AP at either end of the fiber. Using enzymatically dissociated flexor digitorum brevis (FDB) fibers and high-speed line scans, we determine the conduction velocity to be ∼0.4 m/s. We applied these methodologies to FDB fibers under elevated extracellular potassium conditions and confirmed that the conduction velocity is significantly reduced in elevated [K+]o. Because our recorded velocities for FDB fibers were much slower than previously reported for other muscle groups, we compared the conduction velocity in FDB fibers to that of extensor digitorum longus (EDL) fibers and measured a significantly faster velocity in EDL fibers than FDB fibers. As a basis for this difference in conduction velocity, we found a similarly higher level of expression of Na+ channels in EDL than in FDB fibers. In addition to measuring the conduction velocity, we can also measure the passive electrotonic potentials elicited by pulses by applying tetrodotoxin and have constructed a circuit model of a skeletal muscle fiber to predict passive polarization of the fiber by the field stimuli. Our predictions from the model fiber closely resemble the recordings acquired from in vitro assays. With these techniques, we can examine how various pathologies and mutations affect skeletal muscle AP propagation. Our work demonstrates the utility of using 1-(3-sulfonatopropyl)-4[ß[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine or mag-fluo-4 to noninvasively measure AP initiation and conduction.

Acute Elevated Glucose Promotes Abnormal Action Potential-Induced Ca2+ Transients in Cultured Skeletal Muscle Fibers.

A common comorbidity of diabetes is skeletal muscle dysfunction, which leads to compromised physical function. Previous studies of diabetes in skeletal muscle have shown alterations in excitation-contraction coupling (ECC)-the sequential link between action potentials (AP), intracellular Ca2+ release, and the contractile machinery. Yet, little is known about the impact of acute elevated glucose on the temporal properties of AP-induced Ca2+ transients and ionic underlying mechanisms that lead to muscle dysfunction. Here, we used high-speed confocal Ca2+ imaging to investigate the temporal properties of AP-induced Ca2+ transients, an intermediate step of ECC, using an acute in cellulo model of uncontrolled hyperglycemia (25 mM, 48 h.). Control and elevated glucose-exposed muscle fibers cultured for five days displayed four distinct patterns of AP-induced Ca2+ transients (phasic, biphasic, phasic-delayed, and phasic-slow decay); most control muscle fibers show phasic AP-induced Ca2+ transients, while most fibers exposed to elevated D-glucose displayed biphasic Ca2+ transients upon single field stimulation. We hypothesize that these changes in the temporal profile of the AP-induced Ca2+ transients are due to changes in the intrinsic excitable properties of the muscle fibers. We propose that these changes accompany early stages of diabetic myopathy.