Animal procedures and sample collection

Mice from an internal C57BL/6 J colony were allowed ad libitum access to pelleted rodent chow (Teklad F6 rodent diet, Harlan, Indianapolis, IN, USA) and tap water. Room temperature was maintained at 22°C with a 12 hour light/dark cycle. Male and female mice heterozygous for the myostatin null mutation were bred in monogamous pairs and litters were weaned at 21 days of age. Weaned mice were housed individually and genotyped with PCR based genotyping[29]. At 0 (birth), 21, and 42 and 70 days of age, 5 mice for each genotype by sex combination were euthanized by CO₂ asphyxiation and decapitation. Only 4 male wild type mice were collected at d 70. Pregnant dams were also euthanized by CO₂ asphyxiation and decapitation 14 days after breeding (d -7) and 5 fetuses collected to represent each genotype by sex combination. At d -7, the entire mouse body was collected. At d 0, muscles from both hindlimbs were combined. At all other time points, biceps femoris muscles were weighed and collected. Tissues were stored at -80°C until analysis.

RNA Extraction and cDNA synthesis

Total RNA was extracted using TRI-Reagent (Sigma-Aldrich, St. Louis, MO, USA) and BCP phase separation reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturers’ protocols. RNA purity and concentration were measured using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA). For cDNA synthesis, RNA was diluted to 500 ng/μl using RNase-free water and 1 μg of RNA was reversed transcribed using the qScript cDNA Super Mix (Quanta Biosciences, Inc, Gaithersburg, MD).

Quantitative real-time PCR

Statistical analysis

Data were analyzed using the Proc Mixed procedure in SAS version 9.2 (SAS Institute Inc., Cary, NC, USA). The model included the fixed effects of age (when appropriate), sex, and all 2- and 3-way interactions. Data are presented as least squares means ± 95% confidence intervals.

Body and muscle weights

Body and muscle weights during postnatal growth were compared between sexes and genotypes to illustrate differences in growth patterns. At day 0 and 21, body weights were similar among all genotype and sex combinations. Mice became heavier with age, and at d 70, myostatin null mice were heavier than wild type mice within sex while males were heavier than females within genotype (Figure 1A). Bicep femoris weights were similar among all treatment groups at d 21 and 42, but myostatin null muscles were heavier than wild type at d 70 (Figure 1B). These results are similar to previous reports of body and muscle weights of myostatin null mice[29, 32].

Expression of growth regulating genes in muscle during growth

To determine the expression of these genes during growth, the expression of all genes in wild type male mice was analyzed in comparison to d 70 expression (Figure 2). All genes were affected (P < 0.01) by age with an overall decline of expression with increasing age. For all genes except Mycn, expression was not different between d -7 and d 0; Mycn expression was reduced at d 0 compared with d -7. In all genes, expression was reduced (P < 0.05) at d 21, d 42 and d 70 compared with d -7 and d 0. Expression of Ezh2, Gpc3, Mdk, and Plagl1 did not decline further after d 21. However, the expression of Mest, and Peg3 was reduced (P < 0.05) at d 42 and 70 compared with d 21, with expression at the later time points being similar. For Mycn, expression was reduced (P < 0.05) at d 21 compared with d 42 with expression at d 70 being intermediate and similar to either time point. Therefore, the results of previous experiments detailing the expression of these genes in various organs[1, 2] are confirmed in skeletal muscle. Expression of these genes declined with age in wild type male mice.

Expression differences between sexes and genotypes

Expression data from d 21, 42 and 70 were compared with the average wild type male expression at d 21 to calculate fold changes. These data represent a consistent muscle group as opposed to the previous data that combine biceps femoris with whole hind limb or fetus (Figure 3). Expression of Mdk and Plagl1 did not decline with age from d 21 to 70. Expression of Ezh2, Gpc3, and Peg3 were all increased (P < 0.05) at d 21 compared with d 42 and 70, which were similar to each other. Expression of Mest was increased (P < 0.05) at d 21 compared with d 42 and at d 42 compared with d 70. Expression of Mycn actually increased with age as expression was increased (P < 0.05) at d 42 and 70 compared with d 21.

Expression of all genes was unaffected by sex. Furthermore, with the exception of Mdk, expression was also unaffected by genotype and interactions between genotype with age or sex with age. Expression of Mdk was increased (P ≤ 0.01) at d 21 in myostatin null mice compared with wild type, but was similar between genotypes at d 42 or 70 (Figure 3). Expression of Mdk was also increased (P ≤ 0.01) at d 70 in female mice compared with males, but was similar between sexes at d 21 and 42 (data not shown). Expression of Ezh2 at d 70 was also increased (P ≤ 0.01) in wild type mice compared with myostatin null. Therefore, despite differences in muscle weights at d 70 between wild type and myostatin null mice, only the expression of two genes differed at any time point investigated and in both cases, gene expression was increased in wild type, not the more heavily muscled myostatin null mice. Therefore, these data suggest that increases in satellite cell activity and hypertrophy noted in myostatin null mice is not likely due to increased activity in these growth promoting genes.

Nevertheless, we have confirmed that this suite of genes is downregulated with age in skeletal muscle similar to other organs. Ezh2, Mdk, Mest, Peg3, and Plagl1 are known to be involved in muscle regeneration and all of the genes are overly expressed in rhabdomyosarcoma, a skeletal muscle cancer[4–8, 23, 24, 26, 33]. It is possible that these genes aid in postnatal muscle growth by activation, proliferation, or differentiation of satellite cells. Ezh2 has an established role in satellite cell activation[24]; it can be speculated that Mdk, Mest, Peg3, and Plagl1 might have similar functions because they are also involved in muscle regeneration[23, 26]. Mycn has not been well characterized in muscle, but it promotes the proliferation of neural progenitor cells and inhibits neural differentiation and, therefore, might exhibit similar functions in muscle with regard to satellite cells[27]. Given these functions, it is expected that expression of these genes would decrease with age as there is less activation, proliferation, and differentiation of satellite cells and, thus, less need for the growth regulatory functions of these genes as growth slows. In contrast, Gpc3 is thought be a negative regulator of growth such that increased expression results in reduced proliferation[28]. Gpc3 functions in muscle have not been identified; however, Gpc3 is involved in liver regeneration such that expression peaks during the early and middle stages of the process[28]. This leads to the conclusion that Gpc3 expression might increase early in postnatal development to negatively regulate growth and proliferation, so overgrowth of organs and muscle does not occur, and decrease as growth slows because proliferation has decreased leading to less need for its regulatory functions.