No evidence for inositol 1,4,5-trisphosphate-dependent Ca2+ release in isolated fibers of adult mouse skeletal muscle

Abstract

The presence and role of functional inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) in adult skeletal muscle are controversial. The current consensus is that, in adult striated muscle, the relative amount of IP(3)Rs is too low and the kinetics of Ca(2+) release from IP(3)R is too slow compared with ryanodine receptors to contribute to the Ca(2+) transient during excitation-contraction coupling. However, it has been suggested that IP(3)-dependent Ca(2+) release may be involved in signaling cascades leading to regulation of muscle gene expression. We have reinvestigated IP(3)-dependent Ca(2+) release in isolated flexor digitorum brevis (FDB) muscle fibers from adult mice. Although Ca(2+) transients were readily induced in cultured C2C12 muscle cells by (a) UTP stimulation, (b) direct injection of IP(3), or (c) photolysis of membrane-permeant caged IP(3), no statistically significant change in calcium signal was detected in adult FDB fibers. We conclude that the IP(3)-IP(3)R system does not appear to affect global calcium levels in adult mouse skeletal muscle.

Figure 1.

UTP addition induces calcium release from cultured muscle cells but not from adult muscle fibers. Here, individual traces represent the [Ca²⁺]_i responses measured by Fura-2 video microscopy in single cells from the same field in a representative experiment. Similar results were obtained in at least three different experiments. Variations in [Ca²⁺]_i over time are represented by the ratio between the fluorescence intensities at 340- and 380-nm excitation wavelengths. (A) Changes in the 340/380 ratio of Fura-2 in C2C12 myotubes after UTP stimulation (100 µM). Top panels show the amount of calcium released at various time points (as indicated by a–d), with pseudocolors indicating the relative intensities. Each trace represents the calcium changes in an individual myotube (four different myotubes are shown, outlined in red, green, blue, and yellow). (B) Preincubation with 70 µM 2-APB inhibits [Ca²⁺]_i elicited by 100 µM UTP in C2C12 myotubes. The addition of ionomycin leads to a significant increase in intracellular calcium. (C) Response of adult fibers to the addition of 100 µM UTP. Traces of two individual fibers are shown. (D) Quantification of the response of C2C12 myotubes and adult single fibers (n = 25 for C2C12 and n = 10 for adult fibers). (E) Increase in [Ca²⁺]_i induced in two adult fibers by 10 mM caffeine. (F) The effect of caffeine is significantly reduced by the addition of 20 µM dantrolene, an inhibitor of RyR.

Figure 2.

Injection of IP₃ does not lead to an increase in intracellular calcium in adult skeletal muscle fibers. (A) Microinjection of 50 µM IP₃ mediated by patch clamp triggers a rapid Ca²⁺ response in a C2C12 myotube. Note that an increase in [Ca²⁺]_i is seen in the myotube injected with IP₃ (outlined in red) but not in noninjected neighboring myotube (outlined in green). (B) Microinjection of intracellular solution alone does not elicit any [Ca²⁺]_i variation in C2C12 myotubes. (C) Microinjection of IP₃ in adult single fibers does not induce any changes in [Ca²⁺]_i. As a positive control for injection, the membrane-impermeable dye Alexa red was injected simultaneously. Microinjection of ionomycin leads to a marked increase in the Fura-2 signal. Similar results were obtained in three different experiments; a total of 10 fibers were examined. (D) Microinjection of the fluorescent dye calcein was used to monitor the velocity of diffusion in adult single fibers. Four regions of interest are shown in a single fiber (yellow, red, green, and blue squares), with their respective increases in fluorescence in time shown in the graph (color of the line corresponds to the color of the square). The top panels are taken at two time points (indicated by a and b). Note that in all these experiments (A–D), the resting ratios differ from those in the experiments of Fig. 1, because the light source and the permissivity of the filters for both Fura-2 wavelengths were not the same in the two setups.

Figure 3.

Uncaging of IP₃ does not elicit appreciable calcium release in adult single fibers. (A) Pseudocolor images of FluoForte Ca²⁺-dependent fluorescence from C2C12 myoblasts at various time points in response to photorelease of caged IP₃. The shown images were obtained at 5.7 and 12.9 s after photo liberating IP₃ from its caged precursor. Traces show the time course of the relative percentage change in fluorescence (Δf/f₀) caused by intracellular Ca²⁺ increase for four different regions of interest (identified by the colored boxes). (B) Changes in [Ca²⁺]_i after photorelease of caged IP₃ in four FDB fibers. A minimal signal was detected in 11 out of 35 fibers. (C) Quantification of the peak relative increase of [Ca²⁺]_i after photorelease, as measured by the change in FluoForte intensity, in C2C12 muscle cells (n = 85 cells). (D) Same as C for adult muscle fibers isolated from 4-mo-old CD1 mice (left; n = 35) and 5-wk-old BALB/c mice (right; n = 24). The difference between fibers incubated with caged IP₃ and control fibers is not statistically significant.