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First published online 3 May 2005
doi: 10.1242/jcs.02341

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Regulation of skeletal muscle fiber type and slow myosin heavy chain 2 gene expression by inositol trisphosphate receptor 1

Department of Cell Biology and Anatomy, Chicago Medical School, 3333 Green Bay Road, North Chicago, Illinois 60064, USA

View larger version (49K): [in a new window]   Fig. 1. Immunodetection of IP3R1 in fast and slow muscle fibers. Embryonic day 13 (E13) pectoralis major and medial adductor myoblasts were isolated, cultured, and allowed to differentiate into muscle fibers for 7 days. On day 3 of incubation, E5 chick spinal cords were added to some of the cultures (+SC). Other cultures lacked innervation (–SC). IP3R1 was detected with a rabbit anti-IP3R1 antibody (+IP3R Ab) and a fluorescein-conjugated (green) secondary antibody. Control cultures lacked addition of the IP3R1 primary antibody (–IP3R Ab), but were incubated with the fluorescein-conjugated secondary antibody. Addition of spinal cords to muscle fiber cultures generates clusters of acetylcholine receptors in muscle fibers, which were detected by addition of rhodamine-conjugated (red) -bungarotoxin. Bar, 50 µm.

View larger version (18K): [in a new window]   Fig. 2. Western blot analysis of IP3R1 in PM and MA muscle fiber cultures. (A) Whole cell protein extracts (200 µg for IP3R1 and 10 µg for -actin) from innervated (+SC) and non-innervated (–SC) PM and medial adductor MA muscle fibers were electrophoresed and immunoblotted. IP3R1 and -actin were detected with a rabbit polyclonal antibody and mouse polyclonal antibody, respectively, followed by HRP-conjugated secondary antibodies and chemiluminescent substrates. (B) The abundance of IP3R1 in innervated (+SC) and non-innervated (–SC) PM and MA muscle fibers was quantified by using -actin as a loading control for normalization Innervation significantly (P<0.05) increased IP3R1 abundance in both PM and MA muscle fibers, and IP3R1 abundance was significantly (P<0.05) increased in innervated and non-innervated PM muscle fibers relative to that in MA muscle fibers. Bars represent mean±s.e.m., n=4.

View larger version (52K): [in a new window]   Fig. 3. Immunodetection of slow MyHC2 in muscle fibers in the presence of the IP3R1 inhibitors, 2-APB and xestospongin D. Innervated (+SC) and non-innervated (–SC) PM and MA muscle fibers were cultured in control medium (–2APB/Xesto) or in medium containing 100 µM 2-APB (+2APB) or 10 µM xestospongin D (+Xesto). Muscle fibers were immunostained for fast MyHCs with monoclonal antibody F59 and Texas Red-conjugated secondary antibody and for slow MyHC2 with monoclonal antibody S58 and a fluorescein-conjugated secondary antibody. Slow MyHC2 was detected in innervated MA muscle fibers in the absence and presence of 2-APB and xestospongin D. Slow MyHC2 was detected in PM muscle fibers only in the presence of both innervation and 2-APB or innervation and xestospongin D. Bar, 50 µm.

View larger version (11K): [in a new window]   Fig. 4. Effect of IP3R1 inhibition on protein kinase C (PKC) activity. PM muscle fibers were cultured with and without innervation by spinal cord (SC) explants. Medium in some of the cultures was supplemented with 100 µM 2-APB to inhibit IP3R1 activity. After 7 days in culture, PKC activities were determined as described in Materials and Methods. Both innervation and inhibition of IP3R1 activity significantly (P<0.05) reduced PKC activity. Bars represent mean (±s.e.m.) PKC activity (n=4).

View larger version (12K): [in a new window]   Fig. 5. Effect of innervation and IP3R1 inhibition on slow MyHC2 promoter activity. PM muscle fibers were cultured with and without spinal cord (SC) explants either in control medium or medium supplemented with 2-APB. Slow MyHC2 promoter activity was significantly (P<0.05) increased in innervated PM muscle fibers cultured in medium containing 2-APB. Innervation or IP3R1 inhibition alone did not significantly increase slow MyHC2 promoter activity. Bars represent mean (±s.e.m.) fold increase in activity compared to levels in the control (n=6).

View larger version (13K): [in a new window]   Fig. 6. Effect of innervation and IP3R1 inhibition on MEF2 and NFAT-dependent transcription. PM muscle fibers were transfected with MEF2 and NFAT promoter sensor constructs. Some of the cultures received spinal cord (SC) explants for innervation and half of the cultures were incubated in control medium or medium supplemented with 100 µM 2-APB to inhibit IP3R1 activity. Innervation significantly (P<0.05) increased both MEF2 and NFAT-dependent transcription in PM muscle fibers incubated in control medium. IP3R1 inhibition by 2-APB in non-innervated PM muscle fibers did not significantly affect MEF2-mediated transcription, but did significantly (P<0.05) reduce NFAT-dependent transcription. Inhibition of IP3R1 activity by 2-APB in innervated PM muscle fibers did not significantly affect MEF2-mediated transcription compared to innervated muscle fibers in control medium. NFAT-dependent transcription was significantly (P<0.05) reduced in innervated PM muscle fibers cultured in medium with 2-APB compared to control medium. Bars represent means±s.e.m., n=6.

View larger version (36K): [in a new window]   Fig. 7. Western blot analyses of NFATc1 and MEF2A in muscle fiber nuclear extracts. Innervated (SC) and non-innervated PM and MA muscle fibers were cultured in control medium or medium supplemented with 100 µM 2-APB. Muscle fiber nuclear extracts were prepared, electrophoresed and immunoblotted. NFATc1 and MEF2A were detected with respective primary antibodies and HRP-conjugated secondary antibodies. Inhibition of IP3R1 activity by 2-APB reduced NFAT abundance in nuclear extracts from all muscle fibers compared to the same cultures incubated in control medium. MEF2A nuclear abundance was not obviously affected by any of the other conditions tested.

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