Sarcopenia in the Cirrhotic Patient: Current Knowledge and Future Directions.

Cirrhosis predisposes to abnormalities in energy, hormonal, and immunological homeostasis. Disturbances in these metabolic processes create susceptibility to sarcopenia or pathological muscle wasting. Sarcopenia is prevalent in cirrhosis and its presence portends significant adverse outcomes including the length of hospital stay, infectious complications, and mortality. This highlights the importance of identification of at-risk individuals with early nutritional, therapeutic and physical therapy intervention. This manuscript summarizes literature relevant to sarcopenia in cirrhosis, describes current knowledge, and elucidates possible future directions.

The accumulation of muscle RING finger-1 in regenerating myofibers: Implications for muscle repair in immune-mediated necrotizing myopathy.

Background: Muscle RING finger-1 (MuRF-1) plays a key role in the degradation of skeletal muscle proteins. We hypothesize the involvement of MuRF-1 in immune-mediated necrotizing myopathy (IMNM). Methods: Muscle biopsies from patients with IMNM (n = 37) were analyzed and compared to biopsies from patients with dermatomyositis (DM, n = 13), dysferlinopathy (n = 9) and controls (n = 7) using immunostaining. Results: MuRF-1 staining could be observed in IMNM, DM and dysferlinopathy biopsies, whereas the percentage of MuRF-1 positive myofibers was significantly higher in IMNM than in dysferlinopathy (p = 0.0448), and positively correlated with muscle weakness and disease activity in IMNM and DM. Surprisingly, MuRF-1 staining predominantly presented in regenerating fibers but not in atrophic fibers. Moreover, MuRF-1-positive fibers tended to be distributed around necrotic myofibers and myofibers with sarcolemma membrane attack complex deposition. Abundant MuRF-1 expression in IMNM and DM was associated with rapid activation of myogenesis after muscle injury, whereas relatively low expression of MuRF-1 in dysferlinopathy may be attributed to damaged muscle regeneration. Conclusions: MuRF-1 accumulated in regenerating myofibers, which may contribute to muscle injury repair in IMNM and DM. MuRF-1 staining may help clinicians differentiate IMNM and dysferlinopathy.

L-Arginine/nitric oxide regulates skeletal muscle development via muscle fibre-specific nitric oxide/mTOR pathway in chickens.

L-Arginine (L-Arg), the precursor of nitric oxide (NO), plays an important role in muscle function. Fast-twitch glycolytic fibres are more susceptible to age-related atrophy than slow-twitch oxidative fibres. The effect of L-Arg/NO on protein metabolism of fast- and slow-twitch muscle fibres was evaluated in chickens. In Exp. 1, 48 chicks at 1 day old were divided into 4 groups of 12 birds and subjected to 4 treatments: basal diet without supplementation or supplemented with 1% L-Arg, and water supplemented with or without L-nitro-arginine methyl ester (L-NAME, 18.5 mM). In Exp. 2, 48 chicks were divided into 4 groups of 12 birds fed with the basal diet and subjected to the following treatments: tap water (control), tap water supplemented with L-NAME (18.5 mM), or molsidomine (MS, 0.1 mM), or 18.5 mM L-NAME + 0.1 mM MS (NAMS). The regulatory effect of L-Arg/NO was further investigated in vitro with myoblasts obtained from chicken embryo pectoralis major (PM) and biceps femoris (BF). In vivo, dietary L-Arg supplementation increased breast (+14.94%, P < 0.05) and thigh muscle mass (+23.40%, P < 0.05); whereas, MS treatment had no detectable influence. However, L-NAME treatment blocked the beneficial influence of L-Arg on muscle development. L-Arg decreased (P < 0.05) protein synthesis rate, phosphorylated mTOR and ribosomal protein S6 kinase beta-1 (p70S6K) levels in breast muscle, which was recovered by L-NAME treatment. In vitro, L-Arg or sodium nitroprusside (SNP) reduced protein synthesis rate, suppressed phosphorylated mTOR/p70S6K and decreased atrogin-1 and muscle RING finger 1 (MuRF1) in myoblasts from PM muscle (P < 0.05). L-NAME abolished the inhibitory effect of L-Arg on protein synthesis and the mTOR/p70S6K pathway. However, myoblasts from BF muscle showed the weak influence. Moreover, blocking the mTOR/p70S6K pathway with rapamycin suppressed protein synthesis of the 2 types of myoblasts; whereas, the protein expression of atrogin-1 and MuRF1 levels were restricted only in myoblasts from PM muscle. In conclusion, L-Arg/NO/mTOR/p70S6K pathway enhances protein accumulation and muscle development in fast-twitch glycolytic muscle in chickens. L-Arg/NO regulates protein turnover in a muscle fibre specific way, which highlights the potential clinical application in fast-twitch glycolytic muscle fibres.

Alterations in Autophagy and Mammalian Target of Rapamycin (mTOR) Pathways Mediate Sarcopenia in Patients with Cirrhosis.

Background and aims: The pathophysiology of sarcopenia in cirrhosis is poorly understood. We aimed to evaluate the histological alterations in the muscle tissue of patients with cirrhosis and sarcopenia, and identify the regulators of muscle homeostasis. Methods: Computed tomography images at third lumbar vertebral level were used to assess skeletal muscle index (SMI) in 180 patients. Sarcopenia was diagnosed based on the SMI cut-offs from a population of similar ethnicity. Muscle biopsy was obtained from the vastus lateralis in 10 sarcopenic patients with cirrhosis, and the external oblique in five controls (voluntary kidney donors during nephrectomy). Histological changes were assessed by hematoxylin and eosin staining and immunohistochemistry for phospho-FOXO3, phospho-AKT, phospho-mTOR, and apoptosis markers (annexin V and caspase 3). The messenger ribonucleic acid (mRNA) expressions for MSTN, FoxO3, markers of ubiquitin-proteasome pathway (FBXO32, TRIM63), and markers of autophagy (Beclin-1 and LC3) were also quantified. Results: The prevalence of sarcopenia was 14.4%. Muscle histology in sarcopenics showed atrophic angulated fibers (P = 0.002) compared to controls. Immunohistochemistry showed a significant loss of expression of phospho-mTOR (P = 0.026) and an unaltered phospho-AKT (P = 0.089) in sarcopenic patients. There were no differences in the immunostaining for annexin-V, caspase-3, and phospho-FoxO3 between the two groups. The mRNA expressions of MSTN and Beclin-1 were higher in sarcopenics (P = 0.04 and P = 0.04, respectively). The two groups did not differ in the mRNA levels for TRIM63, FBXO32, and LC3. Conclusions: Significant muscle atrophy, increase in autophagy, MSTN gene expression, and an impaired mTOR signaling were seen in patients with sarcopenia and cirrhosis.

The Role of Muscle Ring Finger-1 (MuRF1), MuRF2, MuRF3, and Atrogin-1 on Bone Microarchitecture In Vivo.

Ubiquitin proteasome system was found to contribute to bone loss by regulating bone turnover and metabolism, by modulating osteoblast differentiation and bone formation as well as formation of osteoclasts that contribute to bone resorption. Muscle Ring Finger (MuRF) are novel ubiquitin ligases, which are muscle specific and have not been much implicated in the bone but have been implicated in several human diseases including heart failure and skeletal muscle atrophy. This study is aimed at understanding the role of MuRF1, MuRF2, MuRF3 and Atrogin which are distinct MuRF family proteins in bone homeostasis. Wildtype, heterozygous and homozygous mice of each of the isoforms were used and the bone microarchitecture and mechanical properties were assessed using microCT and biomechanics. MuRF1 depletion was found to alter cortical properties in both males and females, but only trabecular spacing in the females. MuRF2 depletion let to no changes in the cortical and trabecular properties but change in the strain to yield in the females. Depletion of MuRF3 led to decrease in the cortical properties in the females and increase in the trabecular properties in the males. Atrogin depletion was found to reduce cortical properties in both males and females, whereas some trabecular properties were found to be reduced in the females. Each muscle-specific ligase was found to alter the bone structure and mechanical properties in a distinct a sex-dependent manner.

MuRF1 deficiency prevents age-related fat weight gain, possibly through accumulation of PDK4 in skeletal muscle mitochondria in older mice.

Recent studies show that muscle mass and metabolic function are interlinked. Muscle RING finger 1 (MuRF1) is a critical muscle-specific ubiquitin ligase associated with muscle atrophy. Yet, the molecular target of MuRF1 in atrophy and aging remains unclear. We examined the role of MuRF1 in aging, using MuRF1-deficient (MuRF1-/- ) mice in vivo, and MuRF1-overexpressing cell in vitro. MuRF1 deficiency partially prevents age-induced skeletal muscle loss in mice. Interestingly, body weight and fat mass of more than 7-month-old MuRF1-/- mice were lower than in MuRF1+/+ mice. Serum and muscle metabolic parameters and results of indirect calorimetry suggest significantly higher energy expenditure and enhanced lipid metabolism in 3-month-old MuRF1-/- mice than in MuRF1+/+ mice, resulting in suppressed adipose tissue gain during aging. Pyruvate dehydrogenase kinase 4 (PDK4) is crucial for a switch from glucose to lipid metabolism, and the interaction between MuRF1 and PDK4 was examined. PDK4 protein levels were elevated in mitochondria from the skeletal muscle in MuRF1-/- mice. In vitro, MuRF1 interacted with PDK4 but did not induce degradation through ubiquitination. Instead, SUMO posttranscriptional modification (SUMOylation) of PDK4 was detected in MuRF1-overexpressing cells, in contrast to cells without the RING domain of MuRF1. MuRF1 deficiency enhances lipid metabolism possibly by upregulating PDK4 localization into mitochondrial through prevention of SUMOylation. Inhibition of MuRF1-mediated PDK4 SUMOylation is a potential therapeutic target for age-related dysfunction of lipid metabolism and muscle atrophy.

Hydrogen sulphide ameliorating skeletal muscle atrophy in db/db mice via Muscle RING finger 1 S-sulfhydration.

Muscle atrophy occurs in many pathological states, including cancer, diabetes and sepsis, whose results primarily from accelerated protein degradation and activation of the ubiquitin-proteasome pathway. Expression of Muscle RING finger 1 (MuRF1), an E3 ubiquitin ligase, was increased to induce the loss of muscle mass in diabetic condition. However, hydrogen sulphide (H2 S) plays a crucial role in the variety of physiological functions, including antihypertension, antiproliferation and antioxidant. In this study, db/db mice and C2C12 myoblasts treated by high glucose and palmitate and oleate were chose as animal and cellular models. We explored how exogenous H2 S attenuated the degradation of skeletal muscle via the modification of MuRF1 S-sulfhydration in db/db mice. Our results show cystathionine-r-lyase expression, and H2 S level in skeletal muscle of db/db mice was reduced. Simultaneously, exogenous H2 S could alleviate ROS production and reverse expression of ER stress protein markers. Exogenous H2 S could decrease the ubiquitination level of MYOM1 and MYH4 in db/db mice. In addition, exogenous H2 S reduced the interaction between MuRF1 with MYOM1 and MYH4 via MuRF1 S-sulfhydration. Based on these results, we establish that H2 S prevented the degradation of skeletal muscle via MuRF1 S-sulfhydration at the site of Cys44 in db/db mice.

Role of Akirin1 in the regulation of skeletal muscle fiber-type switch.

Akirin1 is a highly conserved ubiquitously expressed nuclear protein. Owing to its strong nuclear localization signal and protein-protein interaction properties, Akirin1 has been speculated to regulate transcription of target genes as a cofactor. Previous studies have reported Akirin1 as a downstream target of myostatin, a potent negative regulator of myogenesis. Mice lacking myostatin displayed enhanced Akirin1 gene expression. Further, in vitro evidence has shown Akirin1 overexpression leads to hypertrophy in C2 C 12 myotubes. In this study, we used Akirin1 knockout mice as a model system to further elucidate the function of Akirin1 in fully differentiated skeletal muscle. Akirin1 knockout mice did not show any obvious phenotypic difference when compared with wild type. However, promoter-reporter assay suggested that Akirin1 regulated the transcription of muscle-specific RING finger 1 (MuRF-1), an important E3 ubiquitin ligase in skeletal muscle. Furthermore, ablation of Akirin1 resulted in increased type IIa and decreased type I muscle fibers, which was further supported by an increase in Myh2 and decrease in Myh7 gene expression. Also, histochemical studies for succinate dehydrogenase activity revealed a less oxidative muscle in the absence of Akirin1. Together, our study suggests a novel role of Akirin1 in maintaining the muscle fiber type and regulation of the metabolic activity of the skeletal muscle.

Tumor necrosis factor-like weak inducer of apoptosis regulates quadriceps muscle atrophy and fiber-type alteration in a rat model of chronic obstructive pulmonary disease.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), weakness and muscle mass loss of the quadriceps muscle has been demonstrated to predict survival and mortality rates of patients. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), as a member of the TNF superfamily, has recently been identified as a key regulator of skeletal muscle wasting and metabolic dysfunction. So our aim was to study the role of TWEAK during quadriceps muscle atrophy and fiber-type transformation in COPD model rats and its possible pathway. METHODS: Forty-four healthy male adult Wistar rats were randomly divided into two groups: A normal control group (n = 16) and a COPD model group (n = 28). The COPD group was exposed to cigarette smoke for 90 d and injected with porcine pancreatic elastase on day 15, whereas the control group was injected with saline alone. Following treatment, weights of the quadriceps muscles were measured and hematoxylin and eosin staining was performed to identify structural changes in lung and quadriceps muscle tissue. Immunohistochemical staining was also conducted to determine the localization of TWEAK, nuclear factor (NF)-κB, muscle ring finger (MuRF)-1 and proliferator-activated coactivator (PGC)-1a proteins in the quadriceps muscle, and western blotting was used to assess the level of protein expression. RESULTS: Compared with controls, COPD model rats exhibited significantly lower quadriceps muscle weight (P < 0.05) accompanied by fiber atrophy and disordered fiber arrangement, a wide gap between adjacent muscle fibers, a significant reduction in nuclear number (P < 0.05) and an uneven size distribution. The proportion of fiber types was also significantly altered (P < 0.05). In addition, TWEAK expression in the quadriceps muscle of COPD model rats was significantly higher than that in control rats (P < 0.05), and was significantly associated with quadriceps atrophy and fiber-type alteration (P < 0.05). Levels of NF-κB, MuRF1 and PGC-1α expression also significantly differed between the two groups (P < 0.05). CONCLUSIONS: Collectively these data suggest that increased levels of TWEAK may lead to skeletal muscle atrophy and fiber-type alteration, which in turn may be associated with activation of the ubiquitin-proteasome pathway, involving NF-κB, MuRF1 and PGC-1α as potential regulatory factors. These preliminary results in rats suggest that TWEAK may be a therapeutic target for the treatment of muscle atrophy in COPD.

Post-translationally modified muscle-specific ubiquitin ligases as circulating biomarkers in experimental cancer cachexia.

Cancer cachexia is a severe wasting syndrome characterized by the progressive loss of lean body mass and systemic inflammation. Up to 80% of cancer patients experience cachexia, with 20-30% of cancer-related deaths directly linked to cachexia. Despite efforts to identify early cachexia and cancer relapse, clinically useful markers are lacking. Recently, we identified the role of muscle-specific ubiquitin ligases Atrogin-1 (MAFbx, FBXO32) and Muscle Ring Finger-1 in the pathogenesis of cardiac atrophy and hypertrophy. We hypothesized that during cachexia, the Atrogin-1 and MuRF1 ubiquitin ligases are released from muscle and migrate to the circulation where they could be detected and serve as a cachexia biomarker. To test this, we induced cachexia in mice using the C26 adenocarcinoma cells or vehicle (control). Body weight, tumor volume, and food consumption were measured from inoculation until ~day 14 to document cachexia. Western blot analysis of serum identified the presence of Atrogin-1 and MuRF1 with unique post-translational modifications consistent with mono- and poly- ubiquitination of Atrogin-1 and MuRF1 found only in cachectic serum. These findings suggest that both increased Atrogin-1 and the presence of unique post-translational modifications may serve as a surrogate marker specific for cachexia.

n-3 Fatty acids preserve muscle mass and insulin sensitivity in a rat model of energy restriction.

In obese subjects, the loss of fat mass during energy restriction is often accompanied by a loss of muscle mass. The hypothesis that n-3 PUFA, which modulate protein homoeostasis via effects on insulin sensitivity, could contribute to maintain muscle mass during energy restriction was tested in rats fed a high-fat diet (4 weeks) rich in 18 : 1 n-9 (oleic acid, OLE-R), 18 : 3 n-3 (α-linolenic acid, ALA-R) or n-3 long-chain (LC-R) fatty acid and then energy restricted (8 weeks). A control group (OLE-ad libitum (AL)) was maintained with AL diet throughout the study. Rats were killed 10 min after an i.v. insulin injection. All energy-restricted rats lost weight and fat mass, but only the OLE-R group showed a significant muscle loss. The Gastrocnemius muscle was enriched with ALA in the ALA-R group and with LC-PUFA in the ALA-R and LC-R groups. The proteolytic ubiquitin-proteasome system was differentially affected by energy restriction, with MAFbx and muscle ring finger-1 mRNA levels being decreased in the LC-R group (-30 and -20 %, respectively). RAC-α serine/threonine-protein kinase and insulin receptor substrate 1 phosphorylation levels increased in the LC-R group (+70 %), together with insulin receptor mRNA (+50 %). The ALA-R group showed the same overall activation pattern as the LC-R group, although to a lesser extent. In conclusion, dietary n-3 PUFA prevent the loss of muscle mass associated with energy restriction, probably by an improvement in the insulin-signalling pathway activation, in relation to enrichment of plasma membranes in n-3 LC-PUFA.

Asparagine reduces the mRNA expression of muscle atrophy markers via regulating protein kinase B (Akt), AMP-activated protein kinase α, toll-like receptor 4 and nucleotide-binding oligomerisation domain protein signalling in weaning piglets after lipopolysaccharide challenge.

Pro-inflammatory cytokines are critical in mechanisms of muscle atrophy. In addition, asparagine (Asn) is necessary for protein synthesis in mammalian cells. We hypothesised that Asn could attenuate lipopolysaccharide (LPS)-induced muscle atrophy in a piglet model. Piglets were allotted to four treatments (non-challenged control, LPS-challenged control, LPS+0·5 % Asn and LPS+1·0 % Asn). On day 21, the piglets were injected with LPS or saline. At 4 h post injection, piglet blood and muscle samples were collected. Asn increased protein and RNA content in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). However, Asn had no effect on the protein abundance of MAFbx and MuRF1. In addition, Asn decreased muscle AMP-activated protein kinase (AMPK) α phosphorylation, but increased muscle protein kinase B (Akt) and Forkhead Box O (FOXO) 1 phosphorylation. Moreover, Asn decreased the concentrations of TNF-α, cortisol and glucagon in plasma, and TNF-α mRNA expression in muscles. Finally, Asn decreased mRNA abundance of muscle toll-like receptor (TLR) 4 and nucleotide-binding oligomerisation domain protein (NOD) signalling-related genes, and regulated their negative regulators. The beneficial effects of Asn on muscle atrophy may be associated with the following: (1) inhibiting muscle protein degradation via activating Akt and inactivating AMPKα and FOXO1; and (2) decreasing the expression of muscle pro-inflammatory cytokines via inhibiting TLR4 and NOD signalling pathways by modulation of their negative regulators.

Control of skeletal muscle atrophy in response to disuse: clinical/preclinical contentions and fallacies of evidence.

Muscle wasting resulting wholly or in part from disuse represents a serious medical complication that, when prolonged, can increase morbidity and mortality. Although much knowledge has been gained over the past half century, the underlying etiology by which disuse alters muscle proteostasis remains enigmatic. Multidisciplinary and novel methodologies are needed to fill gaps and overcome barriers to improved patient care. The present review highlights seminal concepts from a symposium at Experimental Biology 2016. These proceedings focus on 1) the role of insulin resistance in mediating disuse-induced changes in muscle protein synthesis (MPS) and breakdown (MPB), as well as cross-talk between carbohydrate and protein metabolism; 2) the relative importance of MPS/MPB in mediating involuntary muscle loss in humans and animals; 3) interpretative limitations associated with MPS/MPB "markers," e.g., MuRF1/MAFbx mRNA; and finally, 4) how OMIC technologies can be leveraged to identify molecular pathways (e.g., ATF4, p53, p21) mediating disuse atrophy. This perspective deals primarily with "simple atrophy" due to unloading. Nonetheless, it is likely that disuse is a pervasive contributor to muscle wasting associated with catabolic disease-related atrophy (i.e., due to associated sedentary behaviour of disease burden). Key knowledge gaps and challenges are identified to stimulate discussion and identify opportunities for translational research. Data from animal and human studies highlight both similarities and differences. Integrated preclinical and clinical research is encouraged to better understand the metabolic and molecular underpinnings and translational relevance,for disuse atrophy. These approaches are crucial to clinically prevent or reverse muscle atrophy, thereby reestablishing homeostasis and recovery.

Insulin down-regulates the expression of ubiquitin E3 ligases partially by inhibiting the activity and expression of AMP-activated protein kinase in L6 myotubes.

While insulin is an anabolic hormone, AMP-activated protein kinase (AMPK) is not only a key energy regulator, but it can also control substrate metabolism directly by inducing skeletal muscle protein degradation. The hypothesis of the present study was that insulin inhibits AMPK and thus down-regulates the expression of the ubiquitin E3 ligases, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) in skeletal muscle cells. Differentiated L6 myotubes were treated with 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside (AICAR) and/or compound C to stimulate and/or block AMPK respectively. These treatments were also conducted in the presence or absence of insulin and the cells were analysed by western blot and quantitative real-time PCR. In addition, nucleotide levels were determined using HPLC. The activation of AMPK with AICAR enhanced the mRNA levels of MAFbx and MuRF1. Insulin reduced the phosphorylation and activity AMPK, which was accompanied by reduced MAFbx and MuRF1 mRNA levels. Using a protein kinase B (PKB/Akt) inhibitor, we found that insulin regulates AMPK through the activation of Akt. Furthermore, insulin down-regulated AMPK α2 mRNA. We conclude that insulin inhibits AMPK through Akt phosphorylation in L6 myotubes, which may serve as a possible signalling pathway for the down-regulation of protein degradation. In addition, decreased expression of AMPK α2 may partially participate in inhibiting the activity of AMPK.

Angiotensin II Induces Skeletal Muscle Atrophy by Activating TFEB-Mediated MuRF1 Expression.

RATIONALE: Skeletal muscle wasting with accompanying cachexia is a life threatening complication in congestive heart failure. The molecular mechanisms are imperfectly understood, although an activated renin-angiotensin aldosterone system has been implicated. Angiotensin (Ang) II induces skeletal muscle atrophy in part by increased muscle-enriched E3 ubiquitin ligase muscle RING-finger-1 (MuRF1) expression, which may involve protein kinase D1 (PKD1). OBJECTIVE: To elucidate the molecular mechanism of Ang II-induced skeletal muscle wasting. METHODS AND RESULTS: A cDNA expression screen identified the lysosomal hydrolase-coordinating transcription factor EB (TFEB) as novel regulator of the human MuRF1 promoter. TFEB played a key role in regulating Ang II-induced skeletal muscle atrophy by transcriptional control of MuRF1 via conserved E-box elements. Inhibiting TFEB with small interfering RNA prevented Ang II-induced MuRF1 expression and atrophy. The histone deacetylase-5 (HDAC5), which was directly bound to and colocalized with TFEB, inhibited TFEB-induced MuRF1 expression. The inhibition of TFEB by HDAC5 was reversed by PKD1, which was associated with HDAC5 and mediated its nuclear export. Mice lacking PKD1 in skeletal myocytes were resistant to Ang II-induced muscle wasting. CONCLUSION: We propose that elevated Ang II serum concentrations, as occur in patients with congestive heart failure, could activate the PKD1/HDAC5/TFEB/MuRF1 pathway to induce skeletal muscle wasting.

The ubiquitin ligase MuRF1 regulates PPARα activity in the heart by enhancing nuclear export via monoubiquitination.

The transcriptional regulation of peroxisome proliferator-activated receptor (PPAR) α by post-translational modification, such as ubiquitin, has not been described. We report here for the first time an ubiquitin ligase (muscle ring finger-1/MuRF1) that inhibits fatty acid oxidation by inhibiting PPARα, but not PPARß/δ or PPARγ in cardiomyocytes in vitro. Similarly, MuRF1 Tg+ hearts showed significant decreases in nuclear PPARα activity and acyl-carnitine intermediates, while MuRF1-/- hearts exhibited increased PPARα activity and acyl-carnitine intermediates. MuRF1 directly interacts with PPARα, mono-ubiquitinates it, and targets it for nuclear export to inhibit fatty acid oxidation in a proteasome independent manner. We then identified a previously undescribed nuclear export sequence in PPARα, along with three specific lysines (292, 310, 388) required for MuRF1's targeting of nuclear export. These studies identify the role of ubiquitination in regulating cardiac PPARα, including the ubiquitin ligase that may be responsible for this critical regulation of cardiac metabolism in heart failure.

Non-targeted metabolomics analysis of cardiac Muscle Ring Finger-1 (MuRF1), MuRF2, and MuRF3 in vivo reveals novel and redundant metabolic changes.

The muscle-specific ubiquitin ligases MuRF1, MuRF2, MuRF3 have been reported to have overlapping substrate specificities, interacting with each other as well as proteins involved in metabolism and cardiac function. In the heart, all three MuRF family proteins have proven critical to cardiac responses to ischemia and heart failure. The non-targeted metabolomics analysis of MuRF1-/-, MuRF2-/-, and MuRF3-/- hearts was initiated to investigate the hypothesis that MuRF1, MuRF2, and MuRF3 have a similarly altered metabolome, representing alterations in overlapping metabolic processes. Ventricular tissue was flash frozen and quantitatively analyzed by GC/MS using a library built upon the Fiehn GC/MS Metabolomics RTL Library. Non-targeted metabolomic analysis identified significant differences (via VIP statistical analysis) in taurine, myoinositol, and stearic acid for the three MuRF-/- phenotypes relative to their matched controls. Moreover, pathway enrichment analysis demonstrated that MuRF1-/- had significant changes in metabolite(s) involved in taurine metabolism and primary acid biosynthesis while MuRF2-/- had changes associated with ascorbic acid/aldarate metabolism (via VIP and t-test analysis vs. sibling-matched wildtype controls). By identifying the functional metabolic consequences of MuRF1, MuRF2, and MuRF3 in the intact heart, non-targeted metabolomics analysis discovered common pathways functionally affected by cardiac MuRF family proteins in vivo. These novel metabolomics findings will aid in guiding the molecular studies delineating the mechanisms that MuRF family proteins regulate metabolic pathways. Understanding these mechanism is an important key to understanding MuRF family proteins' protective effects on the heart during cardiac disease.

Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1.

Muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1 were identified more than 10 years ago as two muscle-specific E3 ubiquitin ligases that are increased transcriptionally in skeletal muscle under atrophy-inducing conditions, making them excellent markers of muscle atrophy. In the past 10 years much has been published about MuRF1 and MAFbx with respect to their mRNA expression patterns under atrophy-inducing conditions, their transcriptional regulation, and their putative substrates. However, much remains to be learned about the physiological role of both genes in the regulation of mass and other cellular functions in striated muscle. Although both MuRF1 and MAFbx are enriched in skeletal, cardiac, and smooth muscle, this review will focus on the current understanding of MuRF1 and MAFbx in skeletal muscle, highlighting the critical questions that remain to be answered.

Mechanisms of cisplatin-induced muscle atrophy.

Fatigue is the most common side effect of chemotherapy. However, the mechanisms of "muscle fatigue" induced by anti-cancer drugs are not fully understood. We therefore investigated the muscle-atrophic effect of cisplatin, a platinum-based anti-cancer drug, in mice. C57BL/6J mice were treated with cisplatin (3mg/kg, i.p.) or saline for 4 consecutive days. On Day 5, hindlimb and quadriceps muscles were isolated from mice. The loss of body weight and food intake under the administration of cisplatin was the same as those in a dietary restriction (DR) group. Under the present conditions, the administration of cisplatin significantly decreased not only the muscle mass of the hindlimb and quadriceps but also the myofiber diameter, compared to those in the DR group. The mRNA expression levels of muscle atrophy F-box (MAFbx), muscle RING finger-1 (MuRF1) and forkhead box O3 (FOXO3) were significantly and further increased by cisplatin treated group, compared to DR. Furthermore, the mRNA levels of myostatin and p21 were significantly upregulated by the administration of cisplatin, compared to DR. On the other hand, the phosphorylation of Akt and FOXO3a, which leads to the blockade of the upregulation of MuRF1 and MAFbx, was significantly and dramatically decreased by cisplatin. These findings suggest that the administration of cisplatin increases atrophic gene expression, and may lead to an imbalance between protein synthesis and protein degradation pathways, which would lead to muscle atrophy. This phenomenon could, at least in part, explain the mechanism of cisplatin-induced muscle fatigue.

Hyperhomocysteinemia attenuates angiogenesis through reduction of HIF-1α and PGC-1α levels in muscle fibers during hindlimb ischemia.

Hyperhomocysteinemia (HHcy) is associated with elderly frailty, skeletal muscle injury and malfunction, reduced vascular integrity and function, and mortality. Although HHcy has been implicated in the impairment of angiogenesis after hindlimb ischemia in murine models, the underlying mechanisms are still unclear. We hypothesized that HHcy compromises skeletal muscle perfusion, collateral formation, and arteriogenesis by diminishing postischemic vasculogenic responses in muscle fibers. To test this hypothesis, we created femoral artery ligation in wild-type and heterozygous cystathionine ß-synthase (CBS(+/-)) mice (a model for HHcy) and assessed tissue perfusion, collateral vessel formation, and skeletal muscle function using laser-Doppler perfusion imaging, barium angiography, and fatigue tests. In addition, we assessed postischemic levels of VEGF and levels of its muscle-specific regulators: hypoxia-inducible factor (HIF)-1α and peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α. The observations indicated dysregulation of VEGF, HIF-1α, and PGC-1α levels in ischemic skeletal muscles of CBS(+/-) mice. Concomitant with the reduced ischemic angiogenic responses, we also observed diminished leptin expression and attenuated Akt signaling in ischemic muscle fibers of CBS(+/-) mice. Moreover, there was enhanced atrogene, ubiquitin ligases that conjugate proteins for degradation during muscle atrophy, transcription, and reduced muscle function after ischemia in CBS(+/-) mice. These results suggest that HHcy adversely affects muscle-specific ischemic responses and contributes to muscle frailty.