Effects of membrane depolarization and changes in extracellular [K(+)] on the Ca (2+) transients of fast skeletal muscle fibers. Implications for muscle fatigue

Abstract

Repetitive activation of skeletal muscle fibers leads to a reduced transmembrane K(+) gradient. The resulting membrane depolarization has been proposed to play a major role in the onset of muscle fatigue. Nevertheless, raising the extracellular K(+) K(+)(O) concentration ([K(+)](O)) to 10 mM potentiates twitch force of rested amphibian and mammalian fibers. We used a double Vaseline gap method to simultaneously record action potentials (AP) and Ca(2+) transients from rested frog fibers activated by single and tetanic stimulation (10 pulses, 100 Hz) at various [K(+)](O) and membrane potentials. Depolarization resulting from current injection or raised [K(+](O) produced an increase in the resting [Ca(2+)]. Ca(2+) transients elicited by single stimulation were potentiated by depolarization from -80 to -60 mV but markedly depressed by further depolarization. Potentiation was inversely correlated with a reduction in the amplitude, overshoot and duration of APs. Similar effects were found for the Ca(2+) transients elicited by the first pulse of 100 Hz trains. Depression or block of Ca(2+) transient in response to the 2nd to 10th pulses of 100 Hz trains was observed at smaller depolarizations as compared to that seen when using single stimulation. Changes in Ca(2+) transients along the trains were associated with impaired or abortive APs. Raising [K(+)](O) to 10 mM potentiated Ca(2+) transients elicited by single and tetanic stimulation, while raising [K(+)](O) to 15 mM markedly depressed both responses. The effects of 10 mM K(+)(O) on Ca(2+) transients, but not those of 15 mM K(+)(O), could be fully reversed by hyperpolarization. The results suggests that the force potentiating effects of 10 mM K(+)(O) might be mediated by depolarization dependent changes in resting [Ca(2+)] and Ca(2+) release, and that additional mechanisms might be involved in the effects of 15 mM K(+)(O) on force generation.

Fig. 1

Measurement of F_max in live fibers. A Fluorescence transient (FT) elicited by AP stimulation and fluorescence changes (SAT) in response to exposure to the saturation solution (arrow). The inset shows the Ca²⁺ transient calculated from the fluorescence transient. B Action potential (AP) and transmembrane potential changes in response to exposure to the saturation solution (arrow)

Fig. 2

Ca²⁺ transients calculated from fibres loaded with different Ca²⁺ dyes and stretched to different sarcomere lengths. A Normalized fluorescence transients from fibers loaded with Rhod-2 (R), Fluo-3 (F) and OGB-5N(O). B Ca²⁺ transients calculated from the data in A. C Normalized Ca²⁺ transients from B. The inset in C shows the same transients in an expanded time scale. D Comparison of fluorescence (ΔF/F) and Ca²⁺ transients ([Ca²⁺]) from a fibre loaded with OGB-5N. E Ca²⁺ transients calculated from OGB-5N transient from a fiber stretched at 4.5 μm and stimulated with a single pulse and a train of pulses. F Ca²⁺ transients calculated from OGB-5N transient from a fiber stretched at 3.6 μm and stimulated with a single pulse and a train of pulses. The inset in panel E compares Ca²⁺ transients shown in E (trace a) and F (trace b). G–I. Ca²⁺ and fluorescence transients elicited by 100 Hz stimulation in fibers loaded with Rhod-2 (G), Fluo-3 (H) and OGB-5N (I). Sarcomere length: 4.5 μm (A–E, G–I) and 3.6 μm (F)

Fig. 3

Effect of holding potential on free resting [Ca²⁺] calculated from OGB-5N (A–C) and Fluo-3 data (D–F). Traces 1–5 in A, B and D, E are the resting [Ca²⁺] recorded at: −100 (control), −80, −70, −60 and −55 mV, respectively. Trace 6 is the resting [Ca²⁺] recorded 3 min after repolarization to −100 mV. C and F are the average resting [Ca²⁺] as a function of membrane potential from 8 to 6 fibers, respectively. [OGB-5N]: 200 μM. [Fluo-3]: 100 μM. Sarcomere length: 4.3 ± 0.2 μm and 4.1 ± 0.3 μm for C and F, respectively. Records were taken ~3 min after changing membrane potential

Fig. 4

Calcium transients elicited by single stimulation at different membrane potentials. A Traces 1–4 are Ca²⁺ transients calculated from OGB-5N fluorescence transients elicited at membrane potentials of −100, −80, −60 and −55 mV, respectively. B AP’s corresponding to Ca²⁺ transients in A. Note differences in time scales. C Ca²⁺ transients in A presented in a normalized scale. D Calcium transients calculated from OGB-5N fluorescence transients elicited at −100 before applying the depolarizing protocol (thin trace) and 3 min after repolarizing the fiber (thick trace). Sarcomere length: 4.5 μm

Fig. 5

Effects of membrane potential on AP and Ca²⁺ transients parameters. A Peak Ca²⁺ transient as a function of membrane potential. The dashed line represents the peak Ca²⁺ transient at −100 mV. B FDHM of Ca²⁺ transients recorded at various membrane potentials. C Depression of AP overshoot with membrane depolarization. D FDHM of AP’s elicited from various membrane potentials. Symbols and bars represent the mean ± ES (n = 5)

Fig. 6

Effects of membrane depolarization on Ca²⁺ transients elicited by repetitive stimulation. A–D Ca²⁺ transients elicited by 100 Hz stimulation in a fiber held at −100, −80, −60 and −55 mV, respectively. E–H First and last Ca²⁺ transients of A–D displayed in expanded time scales, respectively. The dashed lines in A–H indicate the resting [Ca²⁺]. I–L Electrical records corresponding to the Ca²⁺ transients shown in A–D, respectively. M–P Expanded time presentation of AP’s recorded simultaneously with the Ca²⁺ transients shown in E–H, respectively. Different voltage scales were used for M–P. The pulse amplitude was not changed. The dashed lines in I–P indicated the resting and zero potentials. The numbers in panels A–P indicate the position of responses along the train. Records were taken ~3 min after changing membrane potential

Fig. 7

Ca²⁺ transients elicited by single stimulation in a fiber exposed to 10 mM extracellular K⁺. A Superimposed Ca²⁺ transients calculated from fluorescence transients recorded in control conditions (−100 mV, 2.5 mM , trace 1), after depolarizing the fiber to −70 mV by current injection (2.5 mM , trace 2), and after exposing the fiber to 10 mM (trace 3). B Traces 1–3 are the APs corresponding to data in A. C Ca²⁺ transients recorded at −100 mV in a fiber exposed to 2.5 mM (trace 1) and 10 mM (trace 2). D Traces 1–2 are the APs corresponding to data in panel C. The dashed lines in B and D indicate the zero potential. Sarcomere length: 4.3 μm. Records were taken about 20 min after changing solutions

Fig. 8

Ca²⁺ transients in response to repetitive stimulation in a fiber exposed to 10 mM extracellular K⁺. A, B Ca²⁺ transients obtained in the presence of 2.5 mM at −100 and −70 mV, respectively. C Ca²⁺ transients obtained in the presence of 10 mM . D–F Traces 1, 2 and 10 represents the first, second and last Ca²⁺ transients in A–C, respectively. G–I AP trains eliciting the Ca²⁺ transients in panels A–C, respectively. J–L Traces 1, 2 and 10 are the APs triggering the Ca²⁺ transients in D–F, respectively. APs are shown in expanded time and voltage scales. Dashed lines in A–F indicated the resting [Ca²⁺]. Dashed lines in G–L represent the resting and zero potential. Records were taken ~20 min after changing solutions

Fig. 9

Depression of Ca²⁺ transients in a fiber exposed to 15 mM . A, B Trains of Ca²⁺ transients obtained in the presence of 2.5 mM extracellular K⁺ in a fiber polarized to −100 (A) and −70 mV (B). C Ca²⁺ transients obtained in the same fiber 20 min after exposure to 15 mM extracellular K⁺. D–F First (1) and last (10) Ca²⁺ transients of trains in A–C, respectively, shown in an expanded time scale. G–I Trains of APs eliciting the Ca²⁺ transients in A–C, respectively. J–L First (1) and last APs in G–I shown in an expanded time scale. Dashed lines in A–F indicate the resting [Ca²⁺]. Dashed lines in G–L indicate resting and zero potential

Fig. 10

Effects of 15 mM K⁺ on Ca²⁺ transients cannot be reversed by repolarization. A–C Ca²⁺ transients elicited by trains of AP’s in the same fiber maintained in control conditions (−100 mV, 2.5 mM K⁺, A); after exposure to 15 mM K+ and subsequent repolarization to −100 mV (B); and after returning to control conditions (C). D–F First (1) and last (2) Ca²⁺ transients in A–C, respectively, presented in an expanded time scale. G–I Trains of AP’s generating the Ca²⁺ transients in A–C, respectively. J–L First (1) and last (2) AP’s of the trains shown in G–I. Dashed lines in A–F indicate the resting [Ca²⁺]. Dashed lines in G–L indicate the resting and zero potential