Mice lacking skeletal muscle actin show reduced muscle strength and growth deficits and die during the neonatal period

Abstract

All four of the muscle actins (skeletal, cardiac, vascular, and enteric) in higher vertebrates show distinct expression patterns and display highly conserved amino acid sequences. While it is hypothesized that each of the muscle isoactins is specifically adapted to its respective tissue and that the minor variations among them have developmental and/or physiological relevance, the exact functional and developmental significance of these proteins remains largely unknown. In order to begin to assess these issues, we disrupted the skeletal actin gene by homologous recombination. All mice lacking skeletal actin die in the early neonatal period (day 1 to 9). These null animals appear normal at birth and can breathe, walk, and suckle, but within 4 days, they show a markedly lower body weight than normal littermates and many develop scoliosis. Null mice show a loss of glycogen and reduced brown fat that is consistent with malnutrition leading to death. Newborn skeletal muscles from null mice are similar to those of wild-type mice in size, fiber type, and ultrastructural organization. At birth, both hemizygous and homozygous null animals show an increase in cardiac and vascular actin mRNA in skeletal muscle, with no skeletal actin mRNA present in null mice. Adult hemizygous animals show an increased level of skeletal actin mRNA in hind limb muscle but no overt phenotype. Extensor digitorum longus (EDL) muscle isolated from skeletal-actin-deficient mice at day 2 to 3 showed a marked reduction in force production compared to that of control littermates, and EDL muscle from hemizygous animals displayed an intermediate force generation. Thus, while increases in cardiac and vascular smooth-muscle actin can partially compensate for the lack of skeletal actin in null mice, this is not sufficient to support adequate skeletal muscle growth and/or function.

FIG. 1.

Targeted disruption of the murine skeletal actin gene. (A) Targeting fragment. The exon-intron organization of the murine skeletal actin gene is shown at the top. Exons (E1 to E7) are represented by open boxes (noncoding exons) and dark boxes (coding exons). Details for the preparation of the targeting construct are presented in Materials and Methods. In brief, the BglI Site was converted to a BamHI site by using the appropriate linker. The targeting construct was obtained by the ligation of an HPRT minigene cassette into the new BamHI site within exon 2, and a TK cassette was linked to the 3′ end of the targeting construct. (B) Southern blot pattern of targeted and untargeted ES cells, with EcoRI used to digest the DNA and with the use of an EcoRI-NaeI probe. Note that the targeting event generates an 8.8-kb fragment due to the inclusion of the 3.1-kb HPRT cassette within exon 2. (C) Southern blot pattern used to genotype mice carrying the disrupted skeletal actin allele(s). DNA was digested with BglII and probed with an SmaI fragment that is contained within the 3′ end of the targeting fragment (0.7 kb) shown in panel A. Note that the targeted allele displays a 4.9-kb fragment that lacks the normal skeletal actin gene due to the presence of the 3.1-kb HPRT sequences.

FIG. 2.

Skeletal-actin-null mice do not survive the neonatal period and show marked growth retardation. (A) Homozygous null mice appear normal at birth but fail to thrive. Littermate pairs are shown at the ages indicated, with the wild-type mouse on the left and the skeletal-actin-null mouse on the right for each pair. While all pups appeared normal at birth, by day 3 and beyond, the skeletal-actin-deficient animals were clearly smaller than normal. Note the scoliosis in the day-5 skeletal-actin-null mouse. (B) Comparison of the body weights of normal (+/+), hemizygous (+/−), and homozygous (−/−) null mice 4 days after birth. The data are presented as the range (vertical line), mean (horizontal line), and standard deviation (black box). The genotypes of newborns from four litters of heterozygote mutings showed a Mendelian distribution: 11 +/+, 24 +/−, and 10 −/−. (C) Survival of skeletal-actin-null mice. Six litters consisting of 57 animals were monitored for 10 days. By day 6, only 1 of 11 homozygous null animals was alive, while 91% of the wild-type animals and 85% of the heterozygous null pups were alive.

FIG. 3.

Muscle actin mRNA content of limbs from wild-type (+/+), hemizygous (+/−), and homozygous (−/−) null mice 3 days after birth. GAPDH mRNA levels were also determined as an internal standard for loading.

FIG. 4.

Actin content of limbs from wild-type (+/+), hemizygous (+/−), and homozygous (−/−) null mice 3 days after birth. Three separate samples were assayed by immunoblotting. Total actin levels were assessed with MAb C4, vascular actin levels were determined with MAb 1A4, and striated actins (skeletal actin plus cardiac actin) were measured with MAb 5C5, as described in Materials and Methods.

FIG. 5.

Thigh muscles of 1-day-old wild-type (+/+) mice (A, C, and E) and homozygous null (−/−) mice (B, D, and F) show no consistent differences in myofiber diameter range, frequency of central nuclei (a feature of immaturity), or myofiber subtype differentiation. Central nuclei are common in panels B, C, D, and E. Mature fiber subtypes are present in panels E and F. The ranges of fiber diameter are similar in all panels; however, the intercellular space is greater in the −/− samples. Panels A and B are sections that were stained with hematoxylin and eosin, while panels C, D, E, and F are sections that were stained for myosin ATPase following incubation at pH 4.6, as described in Materials and Methods.

FIG. 6.

Newborn leg muscle was examined by electron microscopy as described in Materials and Methods. (A and B) A comparison of wild-type muscle (A) and skeletal-actin-null muscle (B) indicates that thin- and thick-filament organization of sarcomeres appears normal in homozygous null (−/−) mice at 1 day of age. (C) Cross-section of muscle of a skeletal-actin-null mouse showing the normal hexagonal organization of thick and thin filaments. Note that glycogen granules are depleted in the muscle sample from the null mouse.

FIG. 7.

By 4 days of age, skeletal-actin-null (−/−) mice show signs of starvation. (A and B) The midthorax cross sections of 4-day old wild-type (+/+) mice (A) exhibit normal amounts of brown fat (arrow), while those of the homozygous null (−/−) mice (B) exhibit a reduced area of brown fat (arrows). (D and E) Microscopic details demonstrate that homozygous null mice (D) had markedly less lipid (small arrows) in brown fat cells than wild-type animals (C) did. Similarly, there is a marked depletion of both liver and muscle glycogen in homozygous null mice (F) relative to wild-type mice (E); i.e., the magenta-colored grains corresponding to glycogen (large arrows) are abundant in panel E and absent in panel F. Note the smaller paraspinal muscle fibers with more frequent central nuclei found in null mice (D) at this age relative to those in wild-type mice (C). Panels C and D are sections that were stained with hematoxalin and eosin; panels E and F are sections that were stained for glycogen with periodic acid-Schiff stain.

FIG. 8.

EDL muscle from 1- to 2-day-old skeletal-actin-null (−/−) mice shows a reduced maximum tetanic tension. (A) Measurements of maximal force from electrically stimulated neonatal wild-type (+/+) (WT), heterozygous (+/−) (Het), or homozygous null (−/−) (Null) EDL muscles. Measurements were normalized to muscle length (F/L, in millinewtons per millimeter), body weight (F/BW, in millinewtons per milligram), or muscle length divided by body weight (F∗[L/BW], in millinewtons per square millimeter), where F represents force, L represents length, and BW represents body weight. (B) Raw data for measurements of force produced by maximally electrically stimulated EDL muscles (in millinewtons). In all cases, the skeletal-actin-null (−/−) muscle produced significantly less force than +/+ muscle, while (+/−) EDL muscles are intermediate. All values are means ± standard errors of the mean. Asterisks indicate results that are significantly different from values for the wild-type mice at P < 0.05 via t test.