IP(3)-dependent, post-tetanic calcium transients induced by electrostimulation of adult skeletal muscle fibers

Abstract

Tetanic electrical stimulation induces two separate calcium signals in rat skeletal myotubes, a fast one, dependent on Cav 1.1 or dihydropyridine receptors (DHPRs) and ryanodine receptors and related to contraction, and a slow signal, dependent on DHPR and inositol trisphosphate receptors (IP(3)Rs) and related to transcriptional events. We searched for slow calcium signals in adult muscle fibers using isolated adult flexor digitorum brevis fibers from 5-7-wk-old mice, loaded with fluo-3. When stimulated with trains of 0.3-ms pulses at various frequencies, cells responded with a fast calcium signal associated with muscle contraction, followed by a slower signal similar to one previously described in cultured myotubes. Nifedipine inhibited the slow signal more effectively than the fast one, suggesting a role for DHPR in its onset. The IP(3)R inhibitors Xestospongin B or C (5 µM) also inhibited it. The amplitude of post-tetanic calcium transients depends on both tetanus frequency and duration, having a maximum at 10-20 Hz. At this stimulation frequency, an increase of the slow isoform of troponin I mRNA was detected, while the fast isoform of this gene was inhibited. All three IP(3)R isoforms were present in adult muscle. IP(3)R-1 was differentially expressed in different types of muscle fibers, being higher in a subset of fast-type fibers. Interestingly, isolated fibers from the slow soleus muscle did not reveal the slow calcium signal induced by electrical stimulus. These results support the idea that IP(3)R-dependent slow calcium signals may be characteristic of distinct types of muscle fibers and may participate in the activation of specific transcriptional programs of slow and fast phenotype.

Figure 1.

Electrical stimuli induce a two-component Ca²⁺ signal in adult skeletal muscle fibers. Isolated muscle fibers from mouse FDB and loaded with Fluo3-AM were stimulated with a train of 270 pulses (0.3 ms each) at 45 Hz. Images were obtained by confocal microscopy. (A) We observed a first fast Ca²⁺ signal related to contraction when tetanic train was applied (gray bar in the graph) followed by a slower signal, observed as a delayed return to basal fluorescence levels after tetanic stimulus. Experiments were done in the presence of standard Krebs solution containing 1 mM calcium (filled squares, n = 11) or in absence of calcium with a medium supplemented with 0.5 mM EGTA (empty squares, n = 8). Experimental points could be fitted by a double exponential function as shown for a typical record in the lower graph (B). Images on the right (C) correspond to fiber fluorescence before, during, and after application of electrical stimulus (end of stimulation indicated by time = 0 s). (D) Single twitch can be fitted by a single exponential decay as shown for a representative record at left. On the right, an example of the signal obtained after stimulation with a short tetanus (45 Hz, 600 ms). Because of the small number of pulses, the slow signal is barely apparent, although the post-tetanic Ca²⁺ decay can still be fitted by a double exponential.

Figure 2.

The slow Ca²⁺ signal induced by electrical stimuli is dependent on DHPR and mediated by IP₃, and is not present in isolated fibers of the slow skeletal muscle soleus. Isolated muscle fibers from mouse FDB were obtained and treated as in Fig. 1, loaded with Fluo3-AM, and stimulated with a train of 270 pulses of 0.3 ms each at 45 Hz. (A) 25 µM Nifedipine completely inhibited the slow Ca²⁺ signal induced by a train of 270 pulses, 0.3 ms each at 45 Hz. Full symbols show the mean of control Ca²⁺ signals (n = 4). Empty symbols correspond to signals obtained after 15 min of fiber incubation with 25 µM Nifedipine (two representative signals shown in the graph from n = 4). (B) Incubation of fibers with 5 µM XeC (empty symbols, n = 4), which specifically blocks the Ca²⁺ release by IP₃R, inhibited the slow Ca²⁺ signal induced by electrical stimulation in adult muscle fibers (full symbols, n = 4). These experiments were done in the presence of 1 mM extracellular Ca²⁺. (C) Isolated muscle fibers from soleus were obtained and seeded by a method similar to that for FDB. Fibers were then stimulated with 270 pulses of 0.3 ms each at 45 and 10 Hz. Figure shows a representative signal (from n = 6). We observe no delayed return to basal levels, the signals resembling those of FDB fibers in the presence of inhibitors of the slow Ca²⁺ signal, indicating the absence of this slow Ca²⁺ signal in soleus fibers.

Figure 3.

The slow Ca²⁺ signal is dependent on frequency and duration of the electrical stimulus. (A) Individual isolated FDB fibers were stimulated with 270 pulses at 2, 5, and 10 Hz or at 10, 45, and 90 Hz (and in the inverted sequence 10, 5, and 2 Hz and 90, 45, 10 Hz). The magnitude of the slow Ca²⁺ signal was measured as the area under the curve in the post-tetanic region. The values are arbitrary units normalized against the area corresponding to 10 Hz. The graph shows the mean area of different fibers stimulated in both sequences of stimulation frequency. We observe a bell-shaped curve, depending on frequency, with a maximum at 10 Hz. Differences between each pair of frequencies, for each frequency series (i.e., 2 vs. 5, 2 vs. 10, and 5 vs. 10; 10 vs. 45, 10 vs. 90, and 45 vs. 90), are statistically significant (*, P < 0.05). (B) The magnitude of slow signal was dependent on the duration of electrical stimuli. The graph shows the mean values for different fibers stimulated at 45 Hz with 23, 68, and 270 pulses. The area values are arbitrary units normalized against signal corresponding to 270 pulses. We observe that the signal magnitude increases with the increase in the stimulus duration. (*, P < 0.05)

Figure 4.

Slow Ca²⁺ signal is observed in muscle fibers directly stimulated by the nerve. A nerve muscle preparation of an EDL muscle was loaded with Fluo3 -AM. The stimulation was performed by a pipette directly trough the nerve as illustrated in the schema (top left). In the right panel, we can see the fluorescence images of the muscle before, during, and after the stimulation through its nerve at 20 Hz for 600 ms. Application of the stimulus is indicated by time = 0 s. Each photograph had a scanning time of 1.57 s with no delay between them. We can see that after the end of tetanus, the fibers took several seconds to return to basal fluorescence levels as in the case for isolated fibers. In the graph at left, the average of four Ca²⁺ signals (filled squares) and the inhibition of the slow component obtained after incubation of nerve muscle preparation for 30 min with 20 µM of XeB (empty squares) are shown. The relative fluorescence levels are normalized to the maximum value obtained for each case.

Figure 5.

IP₃Rs subtypes are expressed in adult muscle at different intracellular locations. Immunofluorescence images for IP₃Rs and myosin heavy chain markers in different muscle slices are shown. IP₃R-3 was expressed in all muscle fiber types (A) and seems to be absent of most of the nuclear regions. It appears to be expressed in a striated pattern typical for sarcoplasmic reticulum proteins. IP₃R-2 also appears to be expressed in all muscle fibers and is expressed in some clusters at the core of the fiber and in the perinuclear regions (B, arrows). On the other hand, IP₃R-1 is not present in all muscle fibers, showing a mosaic expression pattern in all muscles studied: gastrocnemius (Gas), soleus, tibialis anterior (TA), and EDL (C–F). Immunofluorescence on serial cryosections of adult soleus mouse muscles using antibodies against type 1 IP₃R (G and J), slow MyHC-I (H), MyHC-II (I), MyHC-IIX (labeling all fibers except IIX, panel K), and MyHC-IIA (L). We observe that IP₃R -1 was absent in slow-type fibers, and its expression was restricted to a subgroup of fast fibers, corresponding to type IIX.

Figure 6.

Electrical stimulation induced an IP₃-dependent Troponin I-slow up-regulation and Troponin I-fast down-regulation in a frequency-dependent manner. Isolated muscle fibers from mouse FDB were obtained as in Fig. 1 and stimulated with a train of 270 pulses of 0.3 ms each at 45 Hz or 20 Hz. RNA was extracted at different times post-stimulation and cDNA obtained by RT-PCR. A set of fibers was incubated with 5 µM XeB for 30 min before stimulation (B and C). PCR was performed with primers specific for skeletal TnIs, TnIf, and β-actin, which serves to normalize TnI values. (A). We observe a significant increase of mRNA levels of TnIs two and 4 h after the stimulus (*, P < 0.05; **, P < 0.01); this increase was much larger for 20 Hz than for 45-Hz stimulation. The increase was not obtained (B) after 4 h when fibers were incubated with the specific inhibitor of IP₃R XeB. (C). TnIf significantly decreased (##, P < 0.01) 4 h after 20-Hz stimulus; 45-Hz stimulus shows no effect. The decrease was not obtained after 4 h when fibers were incubated with XeB.