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

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.

Figure 1.

Immunostaining of quadriceps muscle of 7-mo-old ACTC transgenic mice. (A) ACTC staining shows that ACTC^Co mice express more ACTC than ACTC^Cr mice. (B) Immunostaining using ACTC and various MHC antibodies on ACTC^Co quadriceps tissue showing that the ACTC transgene is predominantly expressed in MHCIIB myofibers. Bars: (A) 50 µm; (B) 100 µm.

Figure 2.

Protein analyses using specific ACTC and ACTA1 antibodies. (A) Western blotting: Coomassie blue staining shows equal loading for myosin (∼220 kD) for all mouse quadriceps protein samples (lanes 1 and 2 = wild-type CBA/Ca;C57BL/6, lanes 3–5 = ACTC^Cr, lanes 6–9 = ACTC^Co, lanes 10–13 = ACTC^Co/KO, and lanes 14–16 = wild-type FVB/n;CBA/Ca;C57BL/6). Western blotting was performed using antibodies against total actin (all six actin isoforms; 42 kD), the striated actin isoforms only (both ACTC and ACTA1), ACTC only, or ACTA1 only. (B) Immunostaining of soleus muscles from 3-mo-old male ACTC^Co/KO and wild-type mice using anti-ACTC (red fluorescence) and anti-ACTA1 (green fluorescence) antibodies. The arrow indicates a muscle spindle positive for ACTC. Quadriceps, gastrocnemius, and EDL muscles were also examined and showed similar results (not depicted). (C) Representative flow cytometry of myofibers dissociated from the quadriceps muscles of 9-mo-old male wild-type and ACTC^Co/KO mice and stained using anti-ACTC and anti-ACTA1 antibodies (n = 12). Gastrocnemius, soleus, and EDL muscles were also examined and showed similar results (not depicted). Bar, 50 µm.

Figure 3.

Histology of skeletal muscle from male ACTC^Co/KO and wild-type mice. (A–D) Representative Gomori trichrome (A and B) and hematoxylin and eosin (C and D) staining of quadriceps muscle from 3.5-mo-old ACTC^Co/KO and wild-type mice. (E and F) Electron micrographs of quadriceps from 7-mo-old ACTC^Co/KO mice. E shows a longitudinal and F shows a cross-sectional electron micrograph. EDL and soleus muscles were also examined and produced similar results (not depicted). Bars: (A–D) 100 µm; (E and F) 0.5 µm.

Figure 4.

Enzyme histology of skeletal muscle from male ACTC^Co/KO and wild-type mice. (A–H) Representative NADH (A, B, E, and F) and cytochrome oxidase/succinate dehydrogenase enzyme histology (C, D, G, and H) of EDL (A–D) and soleus (E–H) muscles from 9-mo-old ACTC^Co/KO and wild-type mice. Bars, 100 µm.

Figure 5.

Mouse physiology: individual myofiber and whole-muscle analyses. (A and B) Skinned fiber analysis from EDL muscles from 3.5-mo-old male mice (n = 22 wild type; n = 23 ACTC^Co/KO) showing the force–Ca²⁺ relationship (A) and normalized maximum force (B). (C–E) Whole EDL muscle contractile experiments from 10-mo-old male mice (n = 8 for both wild-type and ACTC^Co/KO mice) demonstrating the force–frequency relationship (C), normalized maximal force (D), and force loss during fatiguing stimulation and postfatigue recovery (E). Error bars represent ±SEM. *, P < 0.05; **, P < 0.001.

Figure 6.

ACTC^Co/KO mice perform as well as or better than wild-type mice (and other genotypes tested) for grip strength, rotarod, and running wheel tests. (A–F) Mouse performance is given as measured by rotarod (A), grip strength (B), and running wheel analysis (mean speed [C], maximum speed [D], time spent running [E], and distance traveled [F]). n = 10, 11, or 12 for all groups of male 4–6-mo-old mice. Error bars represent ±SEM. *, P < 0.05; and **, P < 0.001 when ACTC^Co/KO mice were compared with all other genotypes.

Figure 7.

ACTC^Co/KO mice are more active than wild-type and other mice. (A–D) Locomotor activity over 30 min was measured, including distance traveled (A), time spent moving (B), number of rearings (C), and mean speed (D). n = 10, 11, or 12 for all groups of male 4–6-mo-old mice. Error bars represent ±SEM. *, P < 0.05; and **, P < 0.001 when ACTC^Co/KO mice were compared with wild-type mice.