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IP₃ receptor function and localization in myotubes: an unexplored Ca²⁺ signaling pathway in skeletal muscle

Jeanne A. Powell^1,*,, Maria Angelica Carrasco^2,*, Dany S. Adams¹, Beatrice Drouet³, Juan Rios², Marioly Müller², Manuel Estrada² and Enrique Jaimovich^2,*

¹ Department of Biological Sciences, Smith College, Northampton, MA 01063, USA
² Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Casilla 70005, Santiago 6530499, Chile
³ Inserm U-505, 15 rue de l’Ecole de Medecine, 75006, Paris, France
^* These authors have contributed equally to this work

View larger version (58K): [in a new window]   Fig. 1. IP3R isotypes. (A) Left panel: RT-PCR results of IP3R-1, 2 and 3 RNAs (type 2 and 3 appear as one band) in cultured muscle cells: rat myotubes (Mr) and the mouse muscle cell line, C2C12 (C2); rat CER, used as a positive control for IP3R-1. RT+ and RT-, with and without reverse transcriptase. Products were separated by electrophoresis in 0.8% agarose. Right panel: same experimental conditions as shown in the left panel, but 3% agarose was employed and three bands of expected sizes were obtained. Base pairs (bp) are noted in A and B. (B) First-strand cDNA was transcribed from total RNA of rat myotubes using random hexamers as primers. PCR was performed with primers specific for each IP3R isoform 1, 2 and 3. The products were separated by electrophoresis in 3% agarose. RT+ and RT- as in A. (C) Western blot analysis of IP3R-1 and 3 in skeletal muscle in culture. Nuclei isolated from the cell line C2C12, and homogenates from rat skeletal muscle (Mr) in primary culture were analyzed for the presence of types 1 and 3 IP3Rs. Thirty µg of protein from nuclei and 30 µg (Mr1) or 60 µg (Mr2) of rat myotube homogenate were incubated with: anti-IP3R-1 antibody (top panel), where 2 µg of rat (CER) were used as a positive control; or anti-IP3R-3 antibody (bottom panel), using 10 µg of HeLa homogenate as positive control.

View larger version (163K): [in a new window]   Fig. 2. Distribution of IP3Rs, Ca2+ATPase and -actinin in single optical sections (750±75 nm). Top panels: the SR labels for both IP3R (left panel) and Ca2+ATPase (middle panel) in a cross-striated pattern; the cross striations are superimposable (yellow in right panel). By contrast, only anti-IP3R stains the nuclear envelope region. Bottom panels: a different polyclonal anti-IP3R antibody (IP3R-M) (left panel) also reveals cross-striated staining. Middle panel, -actinin staining pattern; right panel, overlay of the two images.

View larger version (151K): [in a new window]   Fig. 3. Colocalization of muscle proteins and IP3R using confocal microscopy. (A,B,C) IP3R localized in the same region as -actinin of the Z line (single section, 710±71 nm). The composite, C, shows the yellow resulting from superimposition of green and red. (D,E,F) IP3R and myosin are found in alternating cross-striated bands. Panels are images of a single optical section (670±67 nm) showing IP3R (D), myosin fast form (E) and overlay (F). (G) Mature patterns of IP3R and 1 DHPR localization, in a single optical section (340±34 nm), showing overlay of green IP3Rs and red 1 DHPRs. Note that the dots (1) often overlap (arrows) or border (arrowheads) the edges of the IP3R striations.

View larger version (203K): [in a new window]   Fig. 4. IP3R in double cross striations and in the nuclear envelope region of cultured mouse myotubes; single optical sections, 530±53 nm. Top panel: IP3R staining is distributed in double cross striations (two striations per sarcomere) in a highly differentiated myotube. A connection between the cross striations of IP3Rs of the SR and the IP3Rs of the nuclear envelope region is suggested. Bottom panel shows another cell in which the continuity of SR and nuclear region IP3R staining is most evident (arrowheads). The orange tones represent the glow-over-under function of the microscope.

View larger version (63K): [in a new window]   Fig. 5. Schematic of immunocytochemical staining of cultured mouse skeletal muscle. Top panels: location of myosin filaments (A-band), actin filaments (I-band), Z-line, A-band and I-band SR, and T-tubules. The overlay cartoon shows T-tubules and SR in a striated, well-differentiated myotube; this organization of T-tubules leads to the striated appearance of staining for DHPRs (Fig. 3G). In the inset the distribution of IP3Rs is shown in green, prominent in the terminal cisternae of the I-band SR. Such a distribution would give a double-banded cross-striated pattern (Fig. 4). Bottom panel: known immunocytochemical staining patterns of selected proteins. The relationship between IP3R staining (green) and known proteins in red: Ca2+ATPase (Fig. 2), -actinin, myosin and 1 DHPR (Fig. 3). Colocalization of IP3R (green) and the red of Ca2+ATPase, -actinin and 1 DHPR results in yellow.

View larger version (131K): [in a new window]   Fig. 6. Calcium images of fluo-3 fluorescence in a mouse myotube. Confocal imaging allowed us to detect both a fast and a slow rise of intracellular Ca2+ concentration ([Ca2+]i). Basal fluorescence is shown at the top of the left panel. The next image was taken immediately after the bath solution was quickly changed to 47 mM K+; this solution remained in the bath throughout the time of the whole record. The subsequent images were taken every second or at times indicated. After the fast signal faded, a focus of fluorescence appeared in the right extreme of the myotube (8 seconds), and the slow propagation to the left of a Ca2+ wave became evident (8-15 seconds). Note that a low fluorescence increase (yellow-green) precedes high fluorescence (red) localized in confined regions, where nuclei are found. Some frames were omitted to make a more concise image. Total length of the myotube section, 173 µm.

View larger version (22K): [in a new window]   Fig. 7. Calcium signals in rat myotubes: effect of 2-APB and U-73122. Fluorescence images of a rat myotube loaded with fluo-3 were obtained as described in Materials and Methods. A region of the cell was selected and fluorescence intensity was quantified using previously described software (Estrada et al., 2000). High K+-containing solution (47 mM) was perfused. Top panel: myotube in control conditions; images were acquired every 232 milliseconds. At least two major components are evident in the fluorescence signal: a fast signal, associated with E-C coupling and a slow one linked to increases in cytoplasmic and nuclear Ca2+. Bottom panel: fluo-3 loaded myotubes pre-incubated for 30 minutes in the presence of 50 µM 2-APB (filled circles) or 20 minutes in the presence of 10 µM U-73122 (open squares) and depolarized in the presence of the drug. Images were acquired every second. Note that in both cases the slow component of the Ca2+ signal was almost completely inhibited.

View larger version (46K): [in a new window]   Fig. 8. KCl depolarization stimulates phosphorylation of ERKs 1/2 and CREB in rat myotubes in primary culture. (A) Top panel: representative western blots of ERKs of myotubes exposed to 84 mM KCl for the times indicated above the blots. Twenty µg of protein from whole cell lysates were analyzed by western blotting using an antibody that recognizes phosphorylated ERKs 1/2 (top blot). The blots were stripped and blotted with total ERKs antibody (bottom blot). Center panel: bars represent fold-induction of ERKs 1/2 phosphorylation (mean±s.e.m.) over control levels for three to six experiments. For A and B, *P<0.05, **P<0.001 (one-way analysis of variance followed by Dunnett’s multiple comparison post-test to compare the control to each of the conditions). Bottom panel: effect of 2-APB on ERKs phosphorylation. Myotubes were pretreated for 30 minutes with control vehicle or 50 µM 2-APB under resting conditions. Depolarization with 84 mM K+ was performed in the absence or presence of 2-APB and levels of P-ERKs were assayed at times shown. (B) Top panel: western blots of CREB phosphorylation following depolarization with high K+ solution for the times indicated. Fifty µg of protein from whole cell lysates were analyzed by western blotting with an antibody that recognizes CREB phosphorylated at serine 133. To correct for loading, a western blot with an antibody that recognizes the phosphorylated and nonphosphorylated forms of CREB was performed. Center panel: bars represent the fold induction (mean±s.e.m. for three to nine experiments) of CREB phosphorylation over control levels. Bottom panel: effect of 2-APB on CREB phosphorylation. Myotubes were pretreated for 30 minutes with control vehicle or 50 µM 2-APB under resting conditions. Depolarization with 84 mM K+ was performed in the absence or presence of 2-APB and levels of CREB and P-CREB were assayed at times shown.