Jessica L. Simmers

Losartan restores skeletal muscle remodeling and protects against disuse atrophy in sarcopenia.

Losartan improves muscle remodeling and protects against immobilization atrophy by mediating pathways critical for muscle homeostasis. Losartan Comes of Age The Little Old Lady from Pasadena describes a diminutive woman of advanced years who aggressively drives her Dodge around a southern California city. In popular culture, people link long life spans with being “little”; yet, shortened stature is only one physical change associated with aging. Another, less jocular, transformation is loss of muscle mass and strength—called sarcopenia—which can cause disability and predicts impending death in older adults. Burks et al. now identify losartan, an angiotensin II receptor antagonist commonly used to treat high blood pressure, as a new drug candidate for treating sarcopenia. Although the causes of sarcopenia are poorly understood, transforming growth factor–β (TGF-β) may contribute to faulty repair in aged muscle. Burks et al. used losartan to antagonize TGF-β signaling in an aged mouse model of sarcopenia. Losartan treatment improved muscle remodeling after injury and protected sarcopenic muscle from further loss of muscle mass caused by immobilization; these effects were mediated via two signaling circuits critical for skeletal muscle homeostasis: the TGF-β and insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathways. These observations suggest that treatment with losartan, a Food and Drug Administration (FDA)–approved drug, may benefit sarcopenia patients and allow little old ladies everywhere to continue their street racing for many years to come. Go granny go. Sarcopenia, a critical loss of muscle mass and function because of the physiological process of aging, contributes to disability and mortality in older adults. It increases the incidence of pathologic fractures, causing prolonged periods of hospitalization and rehabilitation. The molecular mechanisms underlying sarcopenia are poorly understood, but recent evidence suggests that increased transforming growth factor–β (TGF-β) signaling contributes to impaired satellite cell function and muscle repair in aged skeletal muscle. We therefore evaluated whether antagonism of TGF-β signaling via losartan, an angiotensin II receptor antagonist commonly used to treat high blood pressure, had a beneficial impact on the muscle remodeling process of sarcopenic mice. We demonstrated that mice treated with losartan developed significantly less fibrosis and exhibited improved in vivo muscle function after cardiotoxin-induced injury. We found that losartan not only blunted the canonical TGF-β signaling cascade but also modulated the noncanonical TGF-β mitogen-activated protein kinase pathway. We next assessed whether losartan was able to combat disuse atrophy in aged mice that were subjected to hindlimb immobilization. We showed that immobilized mice treated with losartan were protected against loss of muscle mass. Unexpectedly, this protective mechanism was not mediated by TGF-β signaling but was due to an increased activation of the insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. Thus, blockade of the AT1 (angiotensin II type I) receptor improved muscle remodeling and protected against disuse atrophy by differentially regulating the TGF-β and IGF-1/Akt/mTOR signaling cascades, two pathways critical for skeletal muscle homeostasis. Thus, losartan, a Food and Drug Administration–approved drug, may prove to have clinical benefits to combat injury-related muscle remodeling and provide protection against disuse atrophy in humans with sarcopenia.

A quantitative proteomic approach for identification of potential biomarkers in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. In this study, our objective was to identify differentially regulated proteins in HCC through a quantitative proteomic approach using iTRAQ. More than 600 proteins were quantitated of which 59 proteins were overexpressed and 92 proteins were underexpressed in HCC as compared to adjacent normal tissue. Several differentially expressed proteins were not implicated previously in HCC. A subset of these proteins (six each from upregulated and downregulated groups) was further validated using immunoblotting and immunohistochemical labeling. Some of the overexpressed proteins with no previous description in the context of HCC include fibroleukin, interferon induced 56 kDa protein, milk fat globule-EGF factor 8, and myeloid-associated differentiation marker. Interestingly, all the enzymes of urea metabolic pathway were dramatically downregulated. Immunohistochemical labeling confirmed differential expression of fibroleukin, myeloid associated differentiation marker and ornithine carbamoyl transferase in majority of HCC samples analyzed. Our results demonstrate quantitative proteomics as a robust discovery tool for the identification of differentially regulated proteins in cancers.

Activation of serum/glucocorticoid-induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy

Maintaining skeletal muscle mass is essential for general health and prevention of disease progression in various neuromuscular conditions. Currently, no treatments are available to prevent progressive loss of muscle mass in any of these conditions. Hibernating mammals are protected from muscle atrophy despite prolonged periods of immobilization and starvation. Here, we describe a mechanism underlying muscle preservation and translate it to non‐hibernating mammals. Although Akt has an established role in skeletal muscle homeostasis, we find that serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) regulates muscle mass maintenance via downregulation of proteolysis and autophagy as well as increased protein synthesis during hibernation. We demonstrate that SGK1 is critical for the maintenance of skeletal muscle homeostasis and function in non‐hibernating mammals in normal and atrophic conditions such as starvation and immobilization. Our results identify a novel therapeutic target to combat loss of skeletal muscle mass associated with muscle degeneration and atrophy.

A critical role for muscle ring finger-1 in acute lung injury-associated skeletal muscle wasting

RATIONALE Acute lung injury (ALI) is a debilitating condition associated with severe skeletal muscle weakness that persists in humans long after lung injury has resolved. The molecular mechanisms underlying this condition are unknown. OBJECTIVES To identify the muscle-specific molecular mechanisms responsible for muscle wasting in a mouse model of ALI. METHODS Changes in skeletal muscle weight, fiber size, in vivo contractile performance, and expression of mRNAs and proteins encoding muscle atrophy-associated genes for muscle ring finger-1 (MuRF1) and atrogin1 were measured. Genetic inactivation of MuRF1 or electroporation-mediated transduction of miRNA-based short hairpin RNAs targeting either MuRF1 or atrogin1 were used to identify their role in ALI-associated skeletal muscle wasting. MEASUREMENTS AND MAIN RESULTS Mice with ALI developed profound muscle atrophy and preferential loss of muscle contractile proteins associated with reduced muscle function in vivo. Although mRNA expression of the muscle-specific ubiquitin ligases, MuRF1 and atrogin1, was increased in ALI mice, only MuRF1 protein levels were up-regulated. Consistent with these changes, suppression of MuRF1 by genetic or biochemical approaches prevented muscle fiber atrophy, whereas suppression of atrogin1 expression was without effect. Despite resolution of lung injury and down-regulation of MuRF1 and atrogin1, force generation in ALI mice remained suppressed. CONCLUSIONS These data show that MuRF1 is responsible for mediating muscle atrophy that occurs during the period of active lung injury in ALI mice and that, as in humans, skeletal muscle dysfunction persists despite resolution of lung injury.

Loss of sarcolemmal nNOS is common in acquired and inherited neuromuscular disorders

Objective: Neuronal nitric oxide synthase (nNOS), normally expressed at the sarcolemmal membrane, is known to be mislocalized to the sarcoplasm in several forms of muscular dystrophy. Our objectives were to characterize further the range of patients manifesting aberrant nNOS sarcolemmal immunolocalization and to study nNOS localization in animal models of nondystrophic myopathy. Methods: We carried out a retrospective cross-sectional study. We performed immunofluorescent staining for nNOS on biopsy specimens from 161 patients with acquired and nondystrophin inherited neuromuscular conditions. The localization of sarcolemmal nNOS correlated with mobility and functional status. Muscle specimens from mouse models of steroid-induced and starvation-related atrophy were studied for qualitative and quantitative nNOS expression. Results: Sarcolemmal nNOS staining was abnormal in 42% of patients with inherited myopathic conditions, 25% with acquired myopathic conditions, 57% with neurogenic conditions, and 93% with hypotonia. Interestingly, we found significant associations between mobility status or muscle function and sarcolemmal nNOS expression. Furthermore, mouse models of catabolic stress also demonstrated mislocalization of sarcolemmal nNOS. Conclusion: Our analyses indicate that nNOS mislocalization is observed in a broad range of nondystrophic neuromuscular conditions associated with impaired mobility status and catabolic stress. Our findings suggest that the assessment of sarcolemmal localization of nNOS represents an important tool for the evaluation of muscle biopsies of patients with a variety of inherited and acquired forms of neuromuscular disorders.

Denervation atrophy is independent from Akt and mTOR activation and is not rescued by myostatin inhibition

The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to ActRIIB, a receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization) and denervation (sciatic nerve resection) atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo-treated mice. Our results suggest that this protection from disuse atrophy is regulated by serum and glucocorticoid-induced kinase (SGK) rather than by Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that, despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy.

18O Labeling for a Quantitative Proteomic Analysis of Glycoproteins in Hepatocellular Carcinoma

IntroductionQuantitative proteomics using tandem mass spectrometry is an attractive approach for identification of potential cancer biomarkers. Fractionation of complex tissue samples into subproteomes prior to mass spectrometric analyses increases the likelihood of identifying cancer-specific proteins that might be present in low abundance. In this regard, glycosylated proteins are an interesting class of proteins that are already established as biomarkers for several cancers.Materials and MethodsIn this study, we carried out proteomic profiling of tumor and adjacent non-cancer liver tissues from hepatocellular carcinoma (HCC) patients. Glycoprotein enrichment from liver samples using lectin affinity chromatography and subsequent 18O/16O labeling of peptides allowed us to obtain relative abundance levels of lectin-bound proteins. As a complementary approach, we also examined the relative expression of proteins in HCC without glycoprotein enrichment. Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. We identified and quantitated over 200 proteins from the lectin-based approach. Interesting among these were fetuin, cysteine-rich protein 1, serpin peptidase inhibitor, leucine-rich alpha-2-glycoprotein 1, melanoma cell adhesion molecule, and heparan sulfate proteoglycan-2. Using lectin affinity followed by PNGase F digestion coupled to 18O labeling, we identified 34 glycosylation sites with consensus sequence N-X-T/S. Western blotting and immunohistochemical staining were carried out for several proteins to confirm mass spectrometry results.ConclusionThis study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC.

Generation of a cre recombinase-conditional Nos1ap over-expression transgenic mouse

Polymorphic non-coding variants at the NOS1AP locus have been associated with the common cardiac, metabolic and neurological traits and diseases. Although, in vitro gene targeting-based cellular and biochemical studies have shed some light on NOS1AP function in cardiac and neuronal tissue, to enhance our understanding of NOS1AP function in mammalian physiology and disease, we report the generation of cre recombinase-conditional Nos1ap over-expression transgenic mice (Nos1apTg). Conditional transgenic mice were generated by the pronuclear injection method and three independent, single-site, multiple copies integration event-based founder lines were selected. For heart-restricted over-expression, Nos1apTg mice were crossed with Mlc2v-cre and Nos1ap transcript over-expression was observed in left ventricles from Nos1apTg; Mlc2v-cre F1 mice. We believe that with the potential of conditional over-expression, Nos1apTg mice will be a useful resource in studying NOS1AP function in various tissues under physiological and disease states.

G.P.84 Mislocalization of nNOS alters the interactions between nNOS and proteins involved with muscle contraction in steroid-induced myopathy

Abstract Neuronal nitric oxide synthase (nNOS) synthesizes nitric oxide (NO) thereby modulating contractile force, blood flow, and glucose metabolism among other processes in skeletal muscle. In healthy muscle, nNOS localizes to the sarcolemma interacting with members of the dystrophin–glycoprotein complex (DGC). Disruption of the DGC results in loss of nNOS from the sarcolemma; however, mislocalization of nNOS also occurs without obvious changes to the DGC. We have shown that nNOS is lost from the sarcolemma in patients with a variety of inherited and acquired neuropathies and with an intact DGC. Reduction of muscle function correlates with loss of sarcolemmal nNOS in these patients. Similarly, we found mislocalization of nNOS in a mouse model of steroid-induced myopathy (SIM) in the context of normal nNOS expression, a maintained DGC, reduced muscle strength, and delayed recovery from muscle fatigue. Using a proteomic approach, we assessed nNOS binding partners and changes in interaction following steroid treatment. We identify 151 proteins interacting with nNOS; 62% of them show no changes in binding with nNOS in SIM mice. Annotation of these interactors reveals nNOS association with mitochondrial proteins and proteins related to glucose metabolism. However, 38% of identified proteins show altered interaction with nNOS following steroid treatment. Cytoskeletal proteins comprise a large subset of these interactors including proteins that form the sarcomere such as α-actinin-3 (ACTN3) and myozenin-1 (MYOZ1). Additionally, a small subset of proteins, associated with the sarcoplasmic reticulum, has altered nNOS interaction in SIM mice. Our data demonstrates that, in conjunction with loss of sarcolemmal nNOS, the interaction between nNOS and proteins involved in muscle contraction is altered in steroid-induced myopathy. These findings are of clinical interest because muscle weakness and muscle fatigue is a common feature of many neuromuscular disorders.

T.O.6 Myostatin inhibitor ActIIb rescues atrophy and protects muscle growth signaling pathways in immobilization but not denervation

Abstract Myostatin, a member of the TGFβ signaling family, acts as a natural inhibitor of skeletal muscle growth via binding to the ActIIb receptor on the cell surface of myofibers. Because myostatin inhibition in a normal mouse induces profound muscle hypertrophy, we investigated the use of a soluble ActIIb receptor as a treatment for immobilization and denervation atrophy in mice. We found that immobilized mice were protected from atrophy after three weeks of administration of ActIIb as compared to the placebo treated group. In contrast, ActIIb treatment did not rescue muscle atrophy in denervated mice. Molecular analysis of the skeletal muscle from immobilized mice demonstrated that ActIIb treatment leads to the protection of several pro-growth pathways. We found that mice treated with ActIIb maintained mTOR signaling while the placebo immobilized group showed a significant decrease in many components of this pathway. The ActIIb treated group was also protected from the loss of anti-apoptotic and pro-growth Jak/Stat signaling markers as compared to placebo immobilized mice. In contrast, and in agreement with the muscle phenotype, molecular analysis of the denervated mice showed a loss of anti-apoptotic and an increase in pro-autophagic markers suggesting that these pathways mediate the loss of muscle mass. In addition, denervated mice demonstrated a pronounced disregulation of pro-growth markers in the Jak/Stat signaling cascade. Surprisingly, denervated mice revealed upregulation of many components of mTOR signaling cascade. As this pathway is associated with hypertrophy, we suggest that this is a compensatory mechanism to prevent further atrophy. These results highlight that myostatin inhibition is a valid therapeutic option for conditions associated with disuse atrophy, but not for denervation induced atrophy. Furthermore, our results indicate that different molecular mechanisms drive loss of muscle mass in immobilization and denervation atrophy.