bioRxiv2022
Sodium (Na+) and potassium (K+) movements during repetitive stimulation of skeletal muscle fibers leads to lowered transmembrane Na+ and K+ gradients. Impaired calcium release resulting from the predicted reduction of the action potential (AP) overshoot (OS) has been suggested as a causative factor of muscle fatigue. To test this hypothesis, we used a double grease-gap method and simultaneously recorded membrane action potentials (MAPs) and Ca2+ release (as Ca2+ transients), elicited by single pulses or short trains of pulses (100 Hz, 100 ms), in rested fibers polarized to membrane potentials (Vm) between -100 to -55 mV, and exposed to various extracellular Na+ concentrations ([Na+]o; 115, 90, 60 and 40 mM). In single stimulation experiments, we found that at physiological Vm (-100 mV), Ca2+ release was mostly immune to [Na+]o reductions up to 60 mM (~1/2 the physiological value). In contrast, at 40 mM Na+o Ca2+ release was reduced by 80%, notwithstanding robust MAPs with large OS (~30 mV) were recruited in this conditions. At Vm between -100 and -60 mV, a 20% reduction of [Na+]o (115 to 90 mM) had no major detrimental effects on Ca2+ release. Instead, depolarization-dependent potentiation of Ca2+ transients, with a maximum at -65 mV, was observed at both 115 and 90 mM Na+o. Potentiation was smaller at 90 mM Na+o. At both [Na+]o, maximally potentiated Ca2+ transients (i.e. at -60 mV) were recruited by MAPS with reduced OSs. In contrast, Ca2+ release was significantly depressed and no potentiation was observed at Vm between -100 to -70 mV when [Na+]o was reduced 60 mM. At extreme Na+o (40 mM), Ca2+ release recorded at Vm between -100 and -70 mV was almost obliterated; nonetheless robust MAPs, with OSs of ~25 mV, were recruited. Extreme depolarizations significantly depressed Ca2+ release at all [Na+]o tested. The Vm leading to Ca2+ release depression was more negative the lower the [Na+]o (-55, -60 and -70 for 115, 90 and mM Na+o, respectively). Fiber exposed to 115-60 mM Na+o can sustain normal Ca2+ release at a frequency of 100 Hz when polarized between -100 and -80 mV. Depolarizations beyond -80 mV lead to impaired Ca2+ release along the trains. In most cases, there was no correlation between changes in Ca2+ release and changes in OS. At 40 mM Na+o, only the 1st-3rd stimuli of trains recruited Ca2+ transients, which were significantly depressed vis a vis close to normal MAPs. Neither the OS nor the duration of MAPs are figures of merit predicting the amplitude of Ca2+ transients. At critical combinations of depolarization, [Na+]o, and stimulation frequency, potentiated Ca2+ transients are recruited by MAPS with small OSs; and conversely, partial or total decoupling of Ca2+ release from close to normal MAPs was observed. Depolarization and Na+o deprivation depressed Ca2+ release in a synergistic way; lowered [Na+]o increased the detrimental effects of depolarization on Ca2+ release, and depolarization render the ECC process more sensitivity to Na+o deprivation. Impaired TTS AP generation and/or conduction may explain the detrimental effects of depolarization and Na+o deprivation on Ca2+ release. The effects of increased K+o and Na+o deprivation on the force generation of rested fibers can be explained on the basis of the effects of membrane depolarization and Na+o deprivation on Ca2+ release. Definitions[ion]i, [ion]o: intracellular and extracellular ion concentrations; ion= Na+, K+, Ca+2. (in molar units) EFM-Na, EMF-K: electromotive force of Na+ and K+ (in mV) ENa, EK: equilibrium potential for Na+ and Na+ (in mV) Vm: membrane or holding potential (in mV) TTS: transverse tubular system. Ca-FWHM, Ca+2 transient full-width at half-maximum (in ms) MAP-FWHM: MAP full-width at half-maximum (in ms) REF: releasing effective time, time a MAP waveform is above -40 mV (in ms)