UBR2 targets myosin heavy chain IIb and IIx for degradation: Molecular mechanism essential for cancer-induced muscle wasting.

Cancer cachexia is a lethal metabolic syndrome featuring muscle wasting with preferential loss of fast-twitching muscle mass through an undefined mechanism. Here, we show that cancer induces muscle wasting by selectively degrading myosin heavy chain (MHC) subtypes IIb and IIx through E3 ligase UBR2-mediated ubiquitylation. Induction of MHC loss and atrophy in C2C12 myotubes and mouse tibialis anterior (TA) by murine cancer cells required UBR2 up-regulation by cancer. Genetic gain or loss of UBR2 function inversely altered MHC level and muscle mass in TA of tumor-free mice. UBR2 selectively interacted with and ubiquitylated MHC-IIb and MHC-IIx through its substrate recognition and catalytic domain, respectively, in C2C12 myotubes. Elevation of UBR2 in muscle of tumor-bearing or free mice caused loss of MHC-IIb and MHC-IIx but not MHC-I and MHC-IIa or other myofibrillar proteins, including α-actin, troponin, tropomyosin, and tropomodulin. Muscle-specific knockout of UBR2 spared KPC tumor-bearing mice from losing MHC-IIb and MHC-IIx, fast-twitching muscle mass, cross-sectional area, and contractile force. The rectus abdominis (RA) muscle of patients with cachexia-prone cancers displayed a selective reduction of MHC-IIx in correlation with higher UBR2 levels. These data suggest that UBR2 is a regulator of MHC-IIb/IIx essential for cancer-induced muscle wasting, and that therapeutic interventions can be designed by blocking UBR2 up-regulation by cancer.

Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle.

Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or ßcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions.

Argininosuccinate Lyase Deficiency Causes an Endothelial-Dependent Form of Hypertension.

Primary hypertension is a major risk factor for ischemic heart disease, stroke, and chronic kidney disease. Insights obtained from the study of rare Mendelian forms of hypertension have been invaluable in elucidating the mechanisms causing primary hypertension and development of antihypertensive therapies. Endothelial cells play a key role in the regulation of blood pressure; however, a Mendelian form of hypertension that is primarily due to endothelial dysfunction has not yet been described. Here, we show that the urea cycle disorder, argininosuccinate lyase deficiency (ASLD), can manifest as a Mendelian form of endothelial-dependent hypertension. Using data from a human clinical study, a mouse model with endothelial-specific deletion of argininosuccinate lyase (Asl), and in vitro studies in human aortic endothelial cells and induced pluripotent stem cell-derived endothelial cells from individuals with ASLD, we show that loss of ASL in endothelial cells leads to endothelial-dependent vascular dysfunction with reduced nitric oxide (NO) production, increased oxidative stress, and impaired angiogenesis. Our findings show that ASLD is a unique model for studying NO-dependent endothelial dysfunction in human hypertension.

Redox regulation of autophagy in skeletal muscle.

Autophagy is a cellular degradative pathway that involves the delivery of cytoplasmic components, including proteins and organelles, to the lysosome for degradation. Autophagy is implicated in the maintenance of skeletal muscle; increased autophagy leads to muscle atrophy while decreased autophagy leads to degeneration and weakness. A growing body of work suggests that reactive oxygen species (ROS) are important cellular signal transducers controlling autophagy. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and mitochondria are major sources of ROS generation in skeletal muscle that are likely regulating autophagy through different signaling cascades based on localization of the ROS signals. This review aims to provide insight into the redox control of autophagy in skeletal muscle. Understanding the mechanisms by which ROS regulate autophagy will provide novel therapeutic targets for skeletal muscle diseases.

FBN1 contributing to familial congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a relatively common, life--threatening birth defect. We present a family with recurrent CDH--paraesophageal and central--for whom exome sequencing (ES) revealed a frameshift mutation (c.4969_4970insA, p.Ile1657Asnfs*30) in the fibrillin 1 gene (FBN1) that causes Marfan syndrome. A diagnosis of Marfan syndrome had not been considered previously in this family. However, a review of the literature demonstrated that FBN1 mutations have an unusual pattern of CDH in which paraesophageal hernias are particularly common. Subsequent clinical evaluations revealed evidence for ectopia lentis in affected family members supporting a clinical diagnosis of Marfan syndrome. Since only two other cases of familial CDH have been described in association with FBN1 mutations, we investigated an oligogenic hypothesis by examining ES data for deleterious sequence changes in other CDH-related genes. This search revealed putatively deleterious sequence changes in four other genes that have been shown to cause diaphragm defects in humans and/or mice--FREM1, DES, PAX3 and MET. It is unclear whether these changes, alone or in aggregate, are contributing to the development of CDH in this family. However, their individual contribution is likely to be small compared to that of the frameshift mutation in FBN1. We conclude that ES can be used to identify both major and minor genetic factors that may contribute to CDH. These results also suggest that ES should be considered in the diagnostic evaluation of individuals and families with CDH, particularly when other diagnostic modalities have failed to reveal a molecular etiology.

Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal degenerative muscle disease resulting from mutations in the dystrophin gene. Increased oxidative stress and altered Ca(2+) homeostasis are hallmarks of dystrophic muscle. While impaired autophagy has recently been implicated in the disease process, the mechanisms underlying the impairment have not been elucidated. Here we show that nicotinamide adenine dinucleotide phosphatase (Nox2)-induced oxidative stress impairs both autophagy and lysosome formation in mdx mice. Persistent activation of Src kinase leads to activation of the autophagy repressor mammalian target of rapamycin (mTOR) via PI3K/Akt phosphorylation. Inhibition of Nox2 or Src kinase reduces oxidative stress and partially rescues the defective autophagy and lysosome biogenesis. Genetic downregulation of Nox2 activity in the mdx mouse decreases reactive oxygen species (ROS) production, abrogates defective autophagy and rescues histological abnormalities and contractile impairment. Our data highlight mechanisms underlying the pathogenesis of DMD and identify NADPH oxidase and Src kinase as potential therapeutic targets.

Sphingomyelinase promotes oxidant production and skeletal muscle contractile dysfunction through activation of NADPH oxidase.

Elevated concentrations of sphingomyelinase (SMase) have been detected in a variety of diseases. SMase has been shown to increase muscle derived oxidants and decrease skeletal muscle force; however, the sub-cellular site of oxidant production has not been elucidated. Using redox sensitive biosensors targeted to the mitochondria and NADPH oxidase (Nox2), we demonstrate that SMase increased Nox2-dependent ROS and had no effect on mitochondrial ROS in isolated FDB fibers. Pharmacological inhibition and genetic knockdown of Nox2 activity prevented SMase induced ROS production and provided protection against decreased force production in the diaphragm. In contrast, genetic overexpression of superoxide dismutase within the mitochondria did not prevent increased ROS production and offered no protection against decreased diaphragm function in response to SMase. Our study shows that SMase induced ROS production occurs in specific sub-cellular regions of skeletal muscle; however, the increased ROS does not completely account for the decrease in muscle function.