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Overexpression of SMPX in adult skeletal muscle does not change skeletal muscle fiber type or size.

Eftestøl E, Alver TN, Gundersen K, Bruusgaard JC - PLoS ONE (2014)

Bottom Line: The fusion protein was localized predominantly in repetitive double stripes flanking the Z-disc, and was excluded from all nuclei.This localization would be consistent with SMPX being a mechanoreceptor, but not with SMPX playing a role as a transcription factor.In vivo overexpression of ectopic SMPX in skeletal muscle of adult mice gave no significant changes in fiber type distribution or cross sectional area, thus a role of SMPX in regulating muscle phenotype remains unclear.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, University of Oslo, Oslo, Norway.

ABSTRACT
Mechanical factors such as stretch are thought to be important in the regulation of muscle phenotype. Small muscle protein X-linked (SMPX) is upregulated by stretch in skeletal muscle and has been suggested to serve both as a transcription factor and a mechanosensor, possibly giving rise to changes in both fiber size and fiber type. We have used in vivo confocal imaging to study the subcellular localization of SMPX in skeletal muscle fibers of adult rats using a SMPX-EGFP fusion protein. The fusion protein was localized predominantly in repetitive double stripes flanking the Z-disc, and was excluded from all nuclei. This localization would be consistent with SMPX being a mechanoreceptor, but not with SMPX playing a role as a transcription factor. In vivo overexpression of ectopic SMPX in skeletal muscle of adult mice gave no significant changes in fiber type distribution or cross sectional area, thus a role of SMPX in regulating muscle phenotype remains unclear.

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Expression of SMPX plasmids, in vitro and in vivo.A) Western blot of SMPX-EGFP expression in HEK-293 cells stained with antibodies against EGFP. Lane 1: Negative control. Lane 2: EGFP only. Lane 3-6: SMPX-EGFP. B) In vivo fluorescence image of EDL muscle expressing pCMS-EGFP-Smpx. Scalebar is 500 microns. Inset: High magnification of single fibers. Scalebar is 50 microns. C) β-gal (left) and myosin heavy chain type 2A (right) stain on neighboring cross-sections from the same muscle as in B). β-gal expressing fibers marked with asterisks. Scale bar is 50 microns.
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pone-0099232-g001: Expression of SMPX plasmids, in vitro and in vivo.A) Western blot of SMPX-EGFP expression in HEK-293 cells stained with antibodies against EGFP. Lane 1: Negative control. Lane 2: EGFP only. Lane 3-6: SMPX-EGFP. B) In vivo fluorescence image of EDL muscle expressing pCMS-EGFP-Smpx. Scalebar is 500 microns. Inset: High magnification of single fibers. Scalebar is 50 microns. C) β-gal (left) and myosin heavy chain type 2A (right) stain on neighboring cross-sections from the same muscle as in B). β-gal expressing fibers marked with asterisks. Scale bar is 50 microns.

Mentions: In order to study the subcellular localization of SMPX, a fusion protein expressing SMPX in frame with EGFP (pEGFP-N1-Smpx) was made. Smpx was inserted at the N-terminal end of EGFP in the plasmid pEGFP-N1 (Clontech). We verified the expression of the SMPX-EGFP fusion protein by western blotting of transfected HEK-293 cells, with a band at the predicted size of approximately 40 kD, using a mouse antibody against GFP (Invitrogen). The negative control and EGFP only control showed no band at the correct size (Figure 1A). To induce in vivo overexpression of SMPX in adult muscle fibers, EGFP and Smpx driven by separate CMV promoters on the same plasmid (pCMS-EGFP-Smpx) were used. The contralateral leg was transfected with pCMS-EGFP (Clontech), serving as a sham control. For the fiber type and cross sectional area analysis a reporter plasmid (pAP-lacZ, gift from N. Gautam) expressing β-galactosidase (β-gal) was used to detect transfected fibers, since EGFP is difficult to detect on cryosections. β-gal was driven by the RSV promoter, while the SMPX-EGFP fusion protein, SMPX and EGFP were all driven by the CMV promoter.


Overexpression of SMPX in adult skeletal muscle does not change skeletal muscle fiber type or size.

Eftestøl E, Alver TN, Gundersen K, Bruusgaard JC - PLoS ONE (2014)

Expression of SMPX plasmids, in vitro and in vivo.A) Western blot of SMPX-EGFP expression in HEK-293 cells stained with antibodies against EGFP. Lane 1: Negative control. Lane 2: EGFP only. Lane 3-6: SMPX-EGFP. B) In vivo fluorescence image of EDL muscle expressing pCMS-EGFP-Smpx. Scalebar is 500 microns. Inset: High magnification of single fibers. Scalebar is 50 microns. C) β-gal (left) and myosin heavy chain type 2A (right) stain on neighboring cross-sections from the same muscle as in B). β-gal expressing fibers marked with asterisks. Scale bar is 50 microns.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4060999&req=5

pone-0099232-g001: Expression of SMPX plasmids, in vitro and in vivo.A) Western blot of SMPX-EGFP expression in HEK-293 cells stained with antibodies against EGFP. Lane 1: Negative control. Lane 2: EGFP only. Lane 3-6: SMPX-EGFP. B) In vivo fluorescence image of EDL muscle expressing pCMS-EGFP-Smpx. Scalebar is 500 microns. Inset: High magnification of single fibers. Scalebar is 50 microns. C) β-gal (left) and myosin heavy chain type 2A (right) stain on neighboring cross-sections from the same muscle as in B). β-gal expressing fibers marked with asterisks. Scale bar is 50 microns.
Mentions: In order to study the subcellular localization of SMPX, a fusion protein expressing SMPX in frame with EGFP (pEGFP-N1-Smpx) was made. Smpx was inserted at the N-terminal end of EGFP in the plasmid pEGFP-N1 (Clontech). We verified the expression of the SMPX-EGFP fusion protein by western blotting of transfected HEK-293 cells, with a band at the predicted size of approximately 40 kD, using a mouse antibody against GFP (Invitrogen). The negative control and EGFP only control showed no band at the correct size (Figure 1A). To induce in vivo overexpression of SMPX in adult muscle fibers, EGFP and Smpx driven by separate CMV promoters on the same plasmid (pCMS-EGFP-Smpx) were used. The contralateral leg was transfected with pCMS-EGFP (Clontech), serving as a sham control. For the fiber type and cross sectional area analysis a reporter plasmid (pAP-lacZ, gift from N. Gautam) expressing β-galactosidase (β-gal) was used to detect transfected fibers, since EGFP is difficult to detect on cryosections. β-gal was driven by the RSV promoter, while the SMPX-EGFP fusion protein, SMPX and EGFP were all driven by the CMV promoter.

Bottom Line: The fusion protein was localized predominantly in repetitive double stripes flanking the Z-disc, and was excluded from all nuclei.This localization would be consistent with SMPX being a mechanoreceptor, but not with SMPX playing a role as a transcription factor.In vivo overexpression of ectopic SMPX in skeletal muscle of adult mice gave no significant changes in fiber type distribution or cross sectional area, thus a role of SMPX in regulating muscle phenotype remains unclear.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, University of Oslo, Oslo, Norway.

ABSTRACT
Mechanical factors such as stretch are thought to be important in the regulation of muscle phenotype. Small muscle protein X-linked (SMPX) is upregulated by stretch in skeletal muscle and has been suggested to serve both as a transcription factor and a mechanosensor, possibly giving rise to changes in both fiber size and fiber type. We have used in vivo confocal imaging to study the subcellular localization of SMPX in skeletal muscle fibers of adult rats using a SMPX-EGFP fusion protein. The fusion protein was localized predominantly in repetitive double stripes flanking the Z-disc, and was excluded from all nuclei. This localization would be consistent with SMPX being a mechanoreceptor, but not with SMPX playing a role as a transcription factor. In vivo overexpression of ectopic SMPX in skeletal muscle of adult mice gave no significant changes in fiber type distribution or cross sectional area, thus a role of SMPX in regulating muscle phenotype remains unclear.

Show MeSH
Related in: MedlinePlus