Identification of FHL1 as a regulator of skeletal muscle mass: implications for human myopathy

Regulators of skeletal muscle mass are of interest, given the morbidity and mortality of muscle atrophy and myopathy. Four-and-a-half LIM protein 1 (FHL1) is mutated in several human myopathies, including reducing-body myopathy (RBM). The normal function of FHL1 in muscle and how it causes myopathy remains unknown. We find that FHL1 transgenic expression in mouse skeletal muscle promotes hypertrophy and an oxidative fiber-type switch, leading to increased whole-body strength and fatigue resistance. Additionally, FHL1 overexpression enhances myoblast fusion, resulting in hypertrophic myotubes in C2C12 cells, (a phenotype rescued by calcineurin inhibition). In FHL1-RBM C2C12 cells, there are no hypertrophic myotubes. FHL1 binds with the calcineurin-regulated transcription factor NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1), enhancing NFATc1 transcriptional activity. Mutant RBM-FHL1 forms aggregate bodies in C2C12 cells, sequestering NFATc1 and resulting in reduced NFAT nuclear translocation and transcriptional activity. NFATc1 also colocalizes with mutant FHL1 to reducing bodies in RBM-afflicted skeletal muscle. Therefore, via NFATc1 signaling regulation, FHL1 appears to modulate muscle mass and strength enhancement

Skeletal muscle NOX4 is required for adaptive responses that prevent insulin resistance

Reactive oxygen species (ROS) generated during exercise are considered integral for the health-promoting effects of exercise. However, the precise mechanisms by which exercise and ROS promote metabolic health remain unclear. Here, we demonstrate that skeletal muscle NADPH oxidase 4 (NOX4), which is induced after exercise, facilitates ROS-mediated adaptive responses that promote muscle function, maintain redox balance, and prevent the development of insulin resistance. Conversely, reductions in skeletal muscle NOX4 in aging and obesity contribute to the development of insulin resistance. NOX4 deletion in skeletal muscle compromised exercise capacity and antioxidant defense and promoted oxidative stress and insulin resistance in aging and obesity. The abrogated adaptive mechanisms, oxidative stress, and insulin resistance could be corrected by deleting the H2O2-detoxifying enzyme GPX-1 or by treating mice with an agonist of NFE2L2, the master regulator of antioxidant defense. These findings causally link NOX4-derived ROS in skeletal muscle with adaptive responses that promote muscle function and insulin sensitivity

AKT signaling promotes DNA damage accumulation and proliferation in polycystic kidney disease

Polycystic kidney disease (PKD) results in the formation of renal cysts that can impair function leading to renal failure. DNA damage accumulates in renal epithelial cells in PKD but the molecular mechanisms are unclear and are investigated here. Phosphoinositide 3- kinase (PI3K)/AKT signaling activates mammalian target of rapamycin complex 1 (mTORC1) and hyperactivation of mTORC1 is a common event in PKD, however, mTORC1 inhibitors have yielded disappointing results in clinical trials. Here we demonstrate AKT and mTORC1 hyperactivation in two representative murine PKD models (renal epithelial-specific Inpp5e knockout and collecting duct-specific Pkd1 deletion) and identify a downstream signaling network that contributes to DNA damage accumulation. Inpp5e- and Pkd1-null renal epithelial cells showed DNA damage including double-stranded DNA breaks associated with increased replication fork numbers, multinucleation and centrosome amplification. mTORC1 activated CAD, which promotes de novo pyrimidine synthesis, to sustain cell proliferation. AKT, but not mTORC1, inhibited the DNA repair/replication fork origin firing regulator TOPBP1, which impacts on DNA damage and cell proliferation. Notably, Inpp5e- and Pkd1-null renal epithelial cell spheroid formation defects were rescued by AKT inhibition. These data reveal that AKT hyperactivation contributes to DNA damage accumulation in multiple forms of PKD and cooperates with mTORC1 to promote cell proliferation. Hyperactivation of AKT may play a causal role in PKD by regulating DNA damage and cell proliferation, independent of mTORC1, and AKT inhibition may be a novel therapeutic approach for PKD

FHL1 reduces fibrosis and fat deposition in dystrophic <i>FRG1</i> mice.

<p>Representative images of transverse sections of (A) quadriceps and (B) trapezius muscle from 12-week-old wild type, <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice stained with Masson’s trichrome to detect fibrosis within muscle. The percentage area of fibrosis staining in muscle was quantified from wild type (n = 4–6), <i>FRG1</i> (n = 4–6) and <i>FRG1</i>/<i>FHL1</i> (n = 5–6) mice. Representative images of transverse muscle sections from the (C) quadriceps and (D) trapezius muscle stained with Oil Red O to detect fat deposits within muscle. The percentage area of fat deposition in muscle was quantified in wild type (n = 3–4), <i>FRG1</i> (n = 4) and <i>FRG1</i>/<i>FHL1</i> (n = 4) mice. Data represent the mean ± SEM; *p&lt;0.05; **p&lt;0.005; ***p&lt;0.0005 determined by two-tailed Student’s T-test. Scale bars = 100μm.</p

Generation of <i>FRG1</i> and <i>FRG1/FHL1</i> mice.

<p>(A) Immunoblot analysis of protein expression in the tibialis anterior, quadriceps, triceps and trapezius muscles from wild type, <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice. HA-tagged FHL1 was detected using a HA-specific antibody; β-tubulin immunoblotting and ponceau red staining of membranes were used as loading controls. (B) Relative FRG1 and FHL1 expression levels in the tibialis anterior, quadriceps, triceps and trapezius muscles from wild type, <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice. Protein expression was quantified using densitometry. Quantitative RT-PCR analysis of FRG1 (C) and FHL1 (D) mRNA in muscles from wild type, <i>FRG1</i> and <i>FRG1/FHL1</i> mice. Data represent the mean from n≥4 mice/genotype; *p&lt;0.05, **p&lt;0.005, ***p&lt;0.001 determined by two-tailed student’s T-test.</p

FHL1 enhances myoblast fusion in <i>FRG1</i> mice.

<p>(A) Representative images of longitudinal sections of triceps muscle from 12-week old wild type, <i>FRG1</i> and <i>FRG1/FHL1</i> mice co-stained for dystrophin to outline the muscle fiber membrane and DAPI to detect nuclei. Boxed region indicates area shown in high magnification image inset. Scale bars = 100μm. (B) The number of nuclei per mm of muscle fiber was counted as a measure of myoblast fusion at 12 weeks (wild type n = 3, <i>FRG1</i> n = 4, <i>FRG1/FHL1</i> n = 4). Data represent the mean ± SEM; ns not significant, *p&lt;0.05determined by two-tailed Student’s T-test.</p