Rescue of skeletal muscle alpha-actin-null mice by cardiac (fetal) alpha-actin

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Article

Reference

Rescue of skeletal muscle alpha-actin-null mice by cardiac (fetal) alpha-actin

NOWAK, Kristen J., et al.

Abstract

Skeletal muscle alpha-actin (ACTA1) is the major actin in postnatal skeletal muscle. Mutations of ACTA1 cause mostly fatal congenital myopathies. Cardiac alpha-actin (ACTC) is the major striated actin in adult heart and fetal skeletal muscle. It is unknown why ACTC and ACTA1 expression switch during development. We investigated whether ACTC can replace ACTA1 in postnatal skeletal muscle. Two ACTC transgenic mouse lines were crossed with Acta1 knockout mice (which all die by 9 d after birth). Offspring resulting from the cross with the high expressing line survive to old age, and their skeletal muscles show no gross pathological features. The mice are not impaired on grip strength, rotarod, or locomotor activity. These findings indicate that ACTC is sufficiently similar to ACTA1 to produce adequate function in postnatal skeletal muscle. This raises the prospect that ACTC reactivation might provide a therapy for ACTA1 diseases. In addition, the mouse model will allow analysis of the precise functional differences between ACTA1 and ACTC.

NOWAK, Kristen J., et al . Rescue of skeletal muscle alpha-actin-null mice by cardiac (fetal) alpha-actin. The Journal of Cell Biology , 2009, vol. 185, no. 5, p. 903-15

PMID : 19468071

DOI : 10.1083/jcb.200812132

Available at:

http://archive-ouverte.unige.ch/unige:5583

Disclaimer: layout of this document may differ from the published version.

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THE JOURNAL OF CELL BIOLOGY

JCB

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Supplemental Material

Nowak et al., http://www.jcb.org/cgi/content/full/jcb.200812132/DC1

Figure S1. Comparison of the amino acid sequences of the six human actins. PDB accession nos.: human ACTA1, AAH12597; human ACTC, CAG46594; human ACTA2, AAA51577; human ACTB, AAH16045; human ACTG, P63261; and human ACTG2, BAA00546. Amino acid residues differing between ACTA1 and ACTC are highlighted in blue, whereas amino acid residues differing between ACTA1 and ACTG1 are highlighted in green.

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Table S1. Antibody concentrations used for immunostaining and Western blotting

Antibody Source Clone Isotype Dilution

(immuno- staining)

Dilution (Western

blot)

Total actin Sigma-Aldrich AC-40 Mouse IgG1 NA 1:250,000

Sarcomeric actin Sigma-Aldrich 5C5 Mouse IgM 1:50 1:500,000

Skeletal muscle α-actin Clément et al., 1999 α-SKA1 Rabbit poly 1:100 1:20,000

Smooth muscle α-actin Sigma-Aldrich 1A4 Mouse IgG2a 1:200 NA

Sarcomeric tropomyosin Sigma-Aldrich CH1 Mouse IgG1 1:10 NA

Cardiac α-actin Research Diagnostics Ac1-20.4.2 Mouse IgG1 1:10 1:5,000

Sarcomeric α-actinin Sigma-Aldrich EA-53 Mouse IgG1 1:10 NA

Skeletal troponin-T Sigma-Aldrich JLT-12 Mouse IgG1 1:10 NA

Cardiac troponin-T Abcam 1F11 Mouse IgG2b 1:5 NA

Desmin Sigma-Aldrich DE-U-10 Mouse IgG1 1:10 NA

MHCI Novocastra NCL-MHCs Mouse IgG1 1:10 NA

MHCIIA DSMZ SC-71 Mouse IgG1 1:3 NA

MHCIIB DSMZ BF-F3 Mouse IgM 1:10 NA

MHCII Novocastra NCL-MHCf Mouse IgM 1:10 NA

MHCIIX Lucas et al., 2000 6H1 Mouse IgG1 1:2 NA

HPRT Santa Cruz Biotechnology, Inc. FL-218 Rabbit poly NA 1:1,000

Embryonic MHC, MYH3 Gambke and Rubinstein, 1984 2B6 Mouse IgG1 1:20 NA

Utrophin Blake et al., 1999 URD40 Goat poly 1:500 Na

DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures); NA, not applicable; poly, polyclonal.

References

Blake, D.J., R. Hawkes, M.A. Benson, and P.W. Beesley. 1999. Different dystrophin-like complexes are expressed in neurons and glia. J. Cell Biol. 147:645–658.

Clément, S., C. Chaponnier, and G. Gabbiani. 1999. A subpopulation of cardiomyocytes expressing alpha-skeletal actin is identified by a specific polyclonal antibody.

Circ. Res. 85:e51–e58.

Gambke, B., and N.A. Rubinstein. 1984. A monoclonal antibody to the embryonic myosin heavy chain of rat skeletal muscle. J. Biol. Chem. 259:12092–12100.

Lucas, C.A., L.H. Kang, and J.F. Hoh. 2000. Monospecific antibodies against the three mammalian fast limb myosin heavy chains. Biochem. Biophys. Res. Commun.

272:303–308.

Figure S2. Weights of wild-type and ACTC^Co/KO mice at different time points. Wild-type mice at 1 (n = 5), 4 (n = 20), 7 (n = 5), 12 (n = 9), and 18 mo (n = 18). ACTC^Co/KO mice at 1 (n = 8), 4 (n = 34), 7 (n = 6), 12 (n = 9), and 18 mo (n = 7). Error bars represent 6SEM. ***, P < 0.001.

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Table S2. MHC myofiber sizes in ACTC^Co/KO and wild-type mice

Muscle and age MHC fiber type Wild type ACTC^Co/KO P-value

µm µm

EDL 4.5 mo MHCIIB 37.57 ± 1.22 34.96 ± 0.67 0.123

EDL 4.5 mo MHCIIX 24.83 ± 1.63 21.61 ± 0.63 0.046^a

EDL 4.5 mo MHCIIA 19.66 ± 0.67 19.24 ± 0.58 0.805

EDL 10 mo MHCIIB 37.46 ± 1.54 37.12 ± 0.76 0.931

EDL 10 mo MHCIIX 27.96 ± 1.67 23.28 ± 0.81 0.017^a

EDL 10 mo MHCIIA 21.90 ± 1.47 19.87 ± 1.19 0.429

Soleus 4.5 mo MHCI 33.26 ± 1.42 34.98 ± 1.57 0.631

Soleus 4.5 mo MHCIIA 32.88 ± 1.75 25.49 ± 1.48 0.008^b

Soleus 10 mo MHCI 32.68 ± 1.92 35.33 ± 1.13 0.257

Soleus 10 mo MHCIIA 30.64 ± 1.14 29.82 ± 0.63 0.537

Muscles were collected from 4.5 (n = 8)- and 10-mo-old (n = 5) wild-type and ACTC^Co/KO mice and stained with anti-MHCI, anti-MHCIIA, anti-MHCIIB, and anti- MHCIIX antibodies.

aP < 0.05.

bP < 0.01.

Video 1. Video of a 1-yr-old ACTC^Co/KO and a wild-type mouse. Number 138 (written on the tail of one of the mice) is the wild-type mouse, and number 02 is the ACTC^Co/KO mouse. The ACTC^Co/KO mouse appears more active than the wild-type mouse and rears more often.