Nur77 deletion impairs muscle growth during developmental myogenesis and muscle regeneration in mice.

Muscle atrophy is a prevalent condition in illness and aging. Identifying novel pathways that control muscle mass may lead to therapeutic advancement. We previously identified Nur77 as a transcriptional regulator of glycolysis in skeletal muscle. More recently, we showed that Nur77 expression also controls myofiber size in mice. It was unknown, however, whether Nur77's regulation of muscle size begins during developmental myogenesis or only in adulthood. To determine the importance of Nur77 throughout muscle growth, we examined myofiber size at E18.5, 3 weeks postnatal age, and in young adult mice. Using the global Nur77-/- mice, we showed that Nur77 deficiency reduced myofiber size as early as E18.5. The reduction in myofiber size became more pronounced by 3 weeks of age. We observed comparable reduction in myofiber size in young myofiber-specific Nur77-knockout mice. These findings suggest that Nur77's effect on muscle growth is intrinsic to its expression in differentiating myofibers, and not dependent on its expression in myogenic stem cells. To determine the importance of Nur77 expression in muscle accretion in mature mice, we generated an inducible-, muscle-specific, Nur77-deficient mouse model. We demonstrated that tamoxifen-induced deletion of Nur77 in 3-month-old mice reduced myofiber size. This change was accompanied by increased activity of Smad2 and FoxO3, two negative regulators of muscle mass. The role of Nur77 in muscle growth was further elaborated in the cardiotoxin-induced muscle regeneration model. Compared to wildtype mice, regenerated myofibers were smaller in Nur77-/- mice. However, when normalized to saline-injected muscle, the recovery of sarcoplasmic area was comparable between Nur77-/- and wildtype mice. These findings suggest that Nur77 deficiency compromises myofiber growth, but not the regenerative capacity of myogenic progenitor cells. Collectively, the findings presented here demonstrate Nur77 as an important regulator of muscle growth both during prenatal and postnatal myogenesis

Characterization of the inducible, muscle-specific, Nur77-null (imKO) mouse.

<p>(A) Schematic of the floxed-Nur77 allele. Open triangle—LoxP site. F₁, F₂, and R denote forward and reverse genotyping primers. (B) Confirmation of tamoxifen- and muscle-specific deletion of Nur77 by PCR genotyping. Upper panel—mice injected with tamoxifen (Tmx); lower panel—mice injected with vehicle (Veh). L—ladder, SKM—skeletal muscle, HRT—heart, LIV—liver, WAT—white adipose tissue. (C) Expression of <i>Nur77</i> and its target genes in quadriceps muscle of control (Fl) and imKO mice one week after tamoxifen injection. N = 4. (D-F) Mean myofiber CSA of EDL, gastrocnemius, and plantaris muscles. N = 6–8. (G) Gene expression from white quadriceps of 6-month-old Fl and imKO mice. (H) Immunoblot analysis of EDL lysate. (I) Quantitation of immunoblot. N = 7. White bars denote floxed controls, black bars denote imKO mice. **P<0.01, **P<0.01, ***P<0.001.</p

Cell cycle analysis of <i>Nur77</i>^<i>-/-</i> myogenic stem cells.

<p>(A) Measurement of wildtype and Nur77 knockout cells incorporating BrdU as a function of total DNA content. (B) Relative proportion of cells in different phases of cell cycle. N = 5. *P<0.05, **P<0.01.</p

Muscle-specific Nur77 deletion is sufficient to reduce myofiber size in 3-week-old mice.

<p>Mean myofiber CSA, fiber count, mid-muscle CSA, and fiber composition of plantaris (A-D) and soleus muscles (E-H). N = 5–6 for plantaris, 4–6 for soleus. *P<0.05, **P<0.01, ***P<0.001. Fl—floxed-Nur77 control, mKO—muscle-specific Nur77 knockout.</p

Nur77 deficiency reduces myofiber size by 3 weeks of age.

<p>(A-E) Body and muscle mass in 3-week-old global <i>Nur77</i>^<i>-/-</i> mice. N = 10–14. (F-H) Mean cross-sectional area (CSA), mean fiber count, and fiber composition of EDL muscle in wildtype and <i>Nur77</i>^<i>-/-</i> mice. N = 6. (I-J) Mean fiber count and fiber composition of plantaris muscle (N = 6–7). *P<0.05, **P<0.01, ***P<0.001. WT—wildtype, KO—knockout, TA—tibialis anterior, Gastr—gastrocnemius, BM—body mass, EDL—extensor digitorum longus.</p

Characteristics of imKO mice.

<p>Characteristics of imKO mice.</p

Cardiotoxin-induced muscle regeneration in Nur77-deficient mice.

<p>(A) H&E stains of cryosections from TA muscle isolated 15 days after normal saline (NS) or cardiotoxin (CTX) injection. Scale bar = 500 μm. Mean myofiber CSA, recovery of myofiber CSA, and percentage of myofibers with central nuclei from global Nur77 KO (B-E) and mKO (F-G) mice. N = 6–7 for B-E, 5–6 for F-G. *P<0.05, **P<0.01, ***P<0.001.</p

Nur77 deficiency reduces secondary myofiber size in E18.5 mouse hindlimb.

<p>(A) Representative immunostaining of E18.5 mouse hindlimb. Red—all myofibers (MY32-reactive), green—primary myofibers (A4.84-reactive). Secondary myofibers are identified as red fibers that were not green. F- fibula, Tib—tibialis, P—peroneus, E—EDL, TA—tibialis anterior. (B) Relative abundance of primary and secondary myofibers in E18.5 EDL, TA, and peroneus muscles. (C) CSA of secondary myofibers in the EDL. N = 7–8. ***P<0.001.</p

Establishing a stable, repeatable platform for measuring changes in sperm DNA methylation

Abstract Background Several independent research groups have shown that alterations in human sperm methylation profiles correlate with decreased fecundity and an increased risk of poor embryo development. Moving these initial findings from the lab into a clinical setting where they can be used to measure male infertility though requires a platform that is stable and robust against batch effects that can occur between sample runs. Operating parameters must be established, performance characteristics determined, and guidelines set to ensure repeatability and accuracy. The standard for technical validation of a lab developed test (LDT) in the USA comes from the Clinical Laboratory Improvement Amendments (CLIA). However, CLIA was introduced in 1988, before the advent of genome-wide profiling and associated computational analysis. This, coupled with its intentionally general nature, makes its interpretation for epigenetic assays non-trivial. Results Here, we present an interpretation of the CLIA technical validation requirements for profiling DNA methylation and calling aberrant methylation using the Illumina Infinium platform (e.g., the 450HM and MethylationEPIC). We describe an experimental design to meet these requirements, the experimental results obtained, and the operating parameters established. Conclusions The CLIA guidelines, although not intended for high-throughput assays, can be interpreted in a way that is consistent with modern epigenetic assays. Based on such an interoperation, Illumina’s Infinium platform is quite amenable to usage in a clinical setting for diagnostic work

Nur77 deletion impairs muscle growth during developmental myogenesis and muscle regeneration in mice

Muscle atrophy is a prevalent condition in illness and aging. Identifying novel pathways that control muscle mass may lead to therapeutic advancement. We previously identified Nur77 as a transcriptional regulator of glycolysis in skeletal muscle. More recently, we showed that Nur77 expression also controls myofiber size in mice. It was unknown, however, whether Nur77's regulation of muscle size begins during developmental myogenesis or only in adulthood. To determine the importance of Nur77 throughout muscle growth, we examined myofiber size at E18.5, 3 weeks postnatal age, and in young adult mice. Using the global Nur77-/- mice, we showed that Nur77 deficiency reduced myofiber size as early as E18.5. The reduction in myofiber size became more pronounced by 3 weeks of age. We observed comparable reduction in myofiber size in young myofiber-specific Nur77-knockout mice. These findings suggest that Nur77's effect on muscle growth is intrinsic to its expression in differentiating myofibers, and not dependent on its expression in myogenic stem cells. To determine the importance of Nur77 expression in muscle accretion in mature mice, we generated an inducible-, muscle-specific, Nur77-deficient mouse model. We demonstrated that tamoxifen-induced deletion of Nur77 in 3-month-old mice reduced myofiber size. This change was accompanied by increased activity of Smad2 and FoxO3, two negative regulators of muscle mass. The role of Nur77 in muscle growth was further elaborated in the cardiotoxin-induced muscle regeneration model. Compared to wildtype mice, regenerated myofibers were smaller in Nur77-/- mice. However, when normalized to saline-injected muscle, the recovery of sarcoplasmic area was comparable between Nur77-/- and wildtype mice. These findings suggest that Nur77 deficiency compromises myofiber growth, but not the regenerative capacity of myogenic progenitor cells. Collectively, the findings presented here demonstrate Nur77 as an important regulator of muscle growth both during prenatal and postnatal myogenesis