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First published online November 24, 2004
doi: 10.1242/10.1242/jcs.01543

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Inhibition of ryanodine receptor 1 in fast skeletal muscle fibers induces a fast-to-slow muscle fiber type transition

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

View larger version (70K): [in a new window]   Fig. 1. Expression of slow MyHC2 in slow MA and fast PM muscle fibers. ED13 myoblasts from MA and PM muscles were incubated for 7 days. On the third day of incubation, some of the cultures received ED5 spinal cord explants to provide innervation. On day 4 of incubation, medium in some of the cultures was supplemented with 100 µM ryanodine. Muscle fibers were immunostained with F59 and S58 mAbs to detect fast MyHCs and slow MyHC2, respectively, followed by fluorochrome conjugated secondary antibodies. Slow MyHC2 was detected in innervated MA muscle fibers and in innervated PM muscle fibers cultured in medium containing 100 µM ryanodine.

View larger version (39K): [in a new window]   Fig. 2. Immunodetection of RyR1. (A) ED13 MA and PM muscle fibers with and without innervation were immunostained with an anti-RyR1 antibody and an FITC conjugated secondary antibody. Control for nonspecific immunostaining by the secondary antibody is included. (B) Protein extracts from ED13 PM and MA muscles in vivo as well as PM and MA muscle fibers cultured in the absence (-SC) and presence (+SC) of innervation were electrophoresed and blotted. RyR1 was detected using an anti-RyR1 antibody. -Actin was used as a loading control for normalization of RyR1 abundance and was detected using an anti-sarcomeric -actin antibody. (C) Western blots of RyR1 in extracts from ED13 PM and MA muscles in vivo as well as PM and MA muscle fibers cultured in the absence (-SC) and presence (+SC) of innervation were quantitated. PM muscle fibers contained significantly more RyR1 relative to MA muscle fibers (*P<0.03; **P<0.01). Bars represent means±s.e.m.; n=5.

View larger version (16K): [in a new window]   Fig. 3. PKC activity assays. Innervated and noninnervated PM and MA muscle fiber cultures incubated in control medium or medium containing 100 µM ryanodine were assayed for PKC activity. Results are expressed relative to PKC activities in noninnervated PM and MA muscle fibers incubated in control medium. PKC activities in PM muscle fibers in the presence of either innervation or 100 µM ryanodine were not significantly different. PM muscle fibers in the presence of both innervation and ryanodine had significantly less PKC activity (P<0.05). MA muscle fibers in the presence of innervation displayed significantly lower PKC activities (P<0.05). Bars represent means±s.e.m.; n=5.

View larger version (12K): [in a new window]   Fig. 4. Effects of ryanodine and innervation on slow MyHC2 promoter activity. PM and MA muscle fibers transfected with the slow MyHC2 promoter-luciferase construct, 2279SM2Luc, were innervated by spinal cord explants and cultured in medium containing 100 µM ryanodine. Slow MyHC2 promoter activities were normalized by cotransfection of pSVßGAL and expressed relative to promoter activities in noninnervated PM and MA muscle fibers incubated in control medium. Innervation significantly increased promoter activity in MA muscle fibers (P<0.05). Combinatorial effects of innervation and ryanodine significantly increased promoter activity in PM muscle fibers (P<0.05). Bars represent means±s.e.m.; n=6.

View larger version (12K): [in a new window]   Fig. 5. Effects of RyR1 inhibition on NFAT and MEF2-mediated transcription. PM muscle fibers transfected with either NFATLuc or MEF2Luc sensor constructs were co-cultured with spinal cord explants for innervation with and without 100 µM ryanodine in the culture medium. Data are expressed relative to sensor activities in noninnervated PM muscle fibers incubated in control medium. Innervation or ryanodine alone did not significantly increase NFAT or MEF2-mediated transcription, whereas both innervation and ryanodine significantly increased both NFAT and MEF2 transcriptional activity (P<0.05). SRE-mediated transcriptional activity did not significantly increase with ryanodine treatment or innervation, or both. Bars represent means±s.e.m.; n=6.

View larger version (51K): [in a new window]   Fig. 6. Electromobility shift and supershift assays of the slow MyHC2 NFAT binding site. (A) Nuclear extracts from innervated (SC) and noninnervated PM (lanes 2-5) and MA (lanes 6-9) muscle fibers incubated in control medium or medium containing 100 µM ryanodine (Ry) were incubated with the NFAT binding site oligonucleotide probe. Protein-DNA complexes were resolved in 5% nondenaturing polyacrylamide gel. A specific protein-DNA complex (arrowhead) formed from extracts of innervated PM muscle fibers incubated in medium containing ryanodine (lane 5) and from extracts of innervated MA muscle fibers (lanes 8 and 9). Lane 1 contained no nuclear extract. A faster migrating protein-DNA complex (asterisk) of unknown composition was present in each lane. (B) Inclusion of an anti-NFATc1 antibody in the binding reaction resulted in a supershifted protein-DNA complex (arrow). An anti-MEF2a antibody did not yield a supershifted complex.

View larger version (55K): [in a new window]   Fig. 7. Electromobility shift and supershift assays of the slow MyHC2 MEF2 binding site. (A) Nuclear extracts from innervated (SC) and noninnervated PM and MA muscle fibers were incubated with the MEF2 binding site oligonucleotide. Lane 1 contained no nuclear extract. Lanes 2-5 contained PM nuclear extract, and lanes 6-9 contained MA nuclear extracts. A protein-DNA complex (arrowhead) was identified in lanes 3-5 and 7-9 as formed by nuclear extracts from PM and MA muscle fibers either innervated or incubated in medium containing ryanodine. A faster migrating protein-DNA complex (asterisk) of unknown composition was present in each lane. (B) Inclusion of anti-MEF2A antibody in the binding reaction caused the formation of a supershifted complex (arrow). Inclusion of a NFATc1 antibody did not decrease the mobility of the MEF2A-containing complex.

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