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

Quiñonez M, González F, Morgado-Valle C, DiFranco M - J. Muscle Res. Cell. Motil. (2010)

Bottom Line: Similar effects were found for the Ca(2+) transients elicited by the first pulse of 100 Hz trains.Changes in Ca(2+) transients along the trains were associated with impaired or abortive APs.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Fisiología y Biofisíca del Músculo, IBE, UCV, Caracas, Venezuela. mquinonez@mednet.ucla.edu

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.

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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)
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Fig2: 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)

Mentions: We first verified the usefulness of high- (Rhod-2 and Fluo-3) and low affinity (OGB-5N) Ca2+ dyes to track fast Ca2+ transients elicited by single APs or short trains of APs, and determined the minimal fiber stretching assuring the effective prevention of possible mechanical artifacts in the presence of low EGTA concentrations ([EGTA]). Fluorescence transients from the 3 dyes, obtained in similar conditions in response to single stimulation, are shown superimposed in Fig. 2A. A normalized scale is used to highlight the kinetics differences among the fluorescence transients. As expected from their Kds, the slower and faster transients were recorded from fibers loaded with Rhod-2 and OGB-5N, respectively; whereas transients from fibers loaded with Fluo-3 displayed intermediate kinetics. See Table 2 for comparative kinetic parameters. Ca2+ transients calculated from fluorescence transients in Fig. 2A are shown in Fig. 2B and C. Ca2+ transients calculated from Rhod-2 and Fluo-3 transients display a remarkable “kinetic correction”, and approach the time course of Ca2+ transients calculated from OGB-5N transients, which reported the faster and larger free [Ca2+] changes. In addition to its kinetic limitation, and unexpectedly for a single binding site dye, Ca2+ transients from Rhod-2 display a two time constant decay. This behavior is in contrast to that found for Fluo-3 and OGN-5N, which display a monotonic decaying phase. The parameters characterizing Ca2+ transient calculated with Eq. 1 from fluorescence transients of the 3 dyes are shown in Table 2. The superiority of OGB-5N to track fast [Ca2+] changes is further stressed by superimposing fluorescence and Ca2+ data. As can be seen in Fig. 2D, Ca2+ transients are only slightly faster than OGB-5N fluorescence transients. This results from an acceleration of both the rising and falling phases, and a reduction of the time to peak of the Ca2+ transients as compared with the parameters of the corresponding fluorescence transient. This result suggests that at room temperature the reaction between OGB-5N and Ca2+ is close to equilibrium during the Ca2+ release.Fig. 2


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

Quiñonez M, González F, Morgado-Valle C, DiFranco M - J. Muscle Res. Cell. Motil. (2010)

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)
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Fig2: 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)
Mentions: We first verified the usefulness of high- (Rhod-2 and Fluo-3) and low affinity (OGB-5N) Ca2+ dyes to track fast Ca2+ transients elicited by single APs or short trains of APs, and determined the minimal fiber stretching assuring the effective prevention of possible mechanical artifacts in the presence of low EGTA concentrations ([EGTA]). Fluorescence transients from the 3 dyes, obtained in similar conditions in response to single stimulation, are shown superimposed in Fig. 2A. A normalized scale is used to highlight the kinetics differences among the fluorescence transients. As expected from their Kds, the slower and faster transients were recorded from fibers loaded with Rhod-2 and OGB-5N, respectively; whereas transients from fibers loaded with Fluo-3 displayed intermediate kinetics. See Table 2 for comparative kinetic parameters. Ca2+ transients calculated from fluorescence transients in Fig. 2A are shown in Fig. 2B and C. Ca2+ transients calculated from Rhod-2 and Fluo-3 transients display a remarkable “kinetic correction”, and approach the time course of Ca2+ transients calculated from OGB-5N transients, which reported the faster and larger free [Ca2+] changes. In addition to its kinetic limitation, and unexpectedly for a single binding site dye, Ca2+ transients from Rhod-2 display a two time constant decay. This behavior is in contrast to that found for Fluo-3 and OGN-5N, which display a monotonic decaying phase. The parameters characterizing Ca2+ transient calculated with Eq. 1 from fluorescence transients of the 3 dyes are shown in Table 2. The superiority of OGB-5N to track fast [Ca2+] changes is further stressed by superimposing fluorescence and Ca2+ data. As can be seen in Fig. 2D, Ca2+ transients are only slightly faster than OGB-5N fluorescence transients. This results from an acceleration of both the rising and falling phases, and a reduction of the time to peak of the Ca2+ transients as compared with the parameters of the corresponding fluorescence transient. This result suggests that at room temperature the reaction between OGB-5N and Ca2+ is close to equilibrium during the Ca2+ release.Fig. 2

Bottom Line: Similar effects were found for the Ca(2+) transients elicited by the first pulse of 100 Hz trains.Changes in Ca(2+) transients along the trains were associated with impaired or abortive APs.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Fisiología y Biofisíca del Músculo, IBE, UCV, Caracas, Venezuela. mquinonez@mednet.ucla.edu

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.

Show MeSH
Related in: MedlinePlus