Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2

The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4-18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild-type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing.     

MuRF1-dependent regulation of systemic carbohydrate metabolism as revealed from transgenic mouse studies

Under various pathophysiological muscle-wasting conditions, such as diabetes and starvation, a family of ubiquitin ligases, including muscle-specific RING-finger protein 1 (MuRF1), are induced to target muscle proteins for degradation via ubiquitination. We have generated transgenic mouse lines over-expressing MuRF1 in a skeletal muscle-specific fashion (MuRF1-TG mice) in an attempt to identify the in vivo targets of MuRF1. MuRF1-TG lines were viable, had normal fertility and normal muscle weights at eight weeks of age. Comparison of quadriceps from MuRF1-TG and wild type mice did not reveal elevated multi-ubiquitination of myosin as observed in human patients with muscle wasting. Instead, MuRF1-TG mice expressed lower levels of pyruvate dehydrogenase (PDH), a mitochondrial key enzyme in charge of glycolysis, and of its regulator PDK2. Furthermore, yeast two-hybrid interaction studies demonstrated the interaction of MuRF1 with PDH, PDK2, PDK4, PKM2 (all participating in glycolysis) and with phosphorylase β (PYGM) and glycogenin (both regulating glycogen metabolism). Consistent with the idea that MuRF1 may regulate carbohydrate metabolism, MuRF1-TG mice had twofold elevated insulin blood levels and lower hepatic glycogen contents. To further examine MuRF1's role for systemic carbohydrate regulation, we performed glucose tolerance tests (GTT) in wild type and MuRF1-TG mice. During GTT, MuRF1-TG mice developed striking hyperinsulinaemia and hepatic glycogen stores, that were depleted at basal levels, became rapidly replenished. Taken together, our data demonstrate that MuRF1 expression in skeletal muscle re-directs glycogen synthesis to the liver and stimulates pancreatic insulin secretion, thereby providing a regulatory feedback loop that connects skeletal muscle metabolism with the liver and the pancreas during metabolic stress.     

Maintenance of muscle mass and load‐induced growth in Muscle RING Finger 1 null mice with age

Age‐related loss of muscle mass occurs to varying degrees in all individuals and has a detrimental effect on morbidity and mortality. Muscle RING Finger 1 (MuRF1), a muscle‐specific E3 ubiquitin ligase, is believed to mediate muscle atrophy through the ubiquitin proteasome system (UPS). Deletion of MuRF1 (KO) in mice attenuates the loss of muscle mass following denervation, disuse, and glucocorticoid treatment; however, its role in age‐related muscle loss is unknown. In this study, skeletal muscle from male wild‐type (WT) and MuRF1 KO mice was studied up to the age of 24 months. Muscle mass and fiber cross‐sectional area decreased significantly with age in WT, but not in KO mice. In aged WT muscle, significant decreases in proteasome activities, especially 20S and 26S β5 (20–40% decrease), were measured and were associated with significant increases in the maladaptive endoplasmic reticulum (ER) stress marker, CHOP. Conversely, in aged MuRF1 KO mice, 20S or 26S β5 proteasome activity was maintained or decreased to a lesser extent than in WT mice, and no increase in CHOP expression was measured. Examination of the growth response of older (18 months) mice to functional overload revealed that old WT mice had significantly less growth relative to young mice (1.37‐ vs. 1.83‐fold), whereas old MuRF1 KO mice had a normal growth response (1.74‐ vs. 1.90‐fold). These data collectively suggest that with age, MuRF1 plays an important role in the control of skeletal muscle mass and growth capacity through the regulation of cellular stress.     

MURF1 deficiency suppresses unloading-induced effects on osteoblasts and osteoclasts to lead to bone loss

Loss of mechanical stress or unloading causes disuse osteoporosis that leads to fractures and deteriorates body function and affects mortality rate in aged population. This bone loss is due to reduction in osteoblastic bone formation and increase in osteoclastic bone resorption. MuRF1 is a muscle RING finger protein which is involved in muscle wasting and its expression is enhanced in the muscle of mice subjected to disuse condition such as hind limb unloading (HU). However, whether MuRF1 is involved in bone loss due to unloading is not known. We therefore examined the effects of MuRF1 deficiency on unloading-induced bone loss. We conducted hind limb unloading of MuRF1 KO mice and wild-type control mice. Unloading induced about 60% reduction in cancellous bone volume (BV/TV) in WT mice. In contrast, MuRF1 deficiency suppressed unloading-induced cancellous bone loss. The cortical bone mass was also reduced by unloading in WT mice. In contrast, MuRF1 deficiency suppressed this reduction in cortical bone mass. To understand whether the effects of MuRF1 deficiency suppress bone loss is on the side of bone formation or bone resorption, histomorphometry was conducted. Unloading reduced bone osteoblastic formation rate (BFR) in WT. In contrast, MuRF1 deficiency suppressed this reduction. Regarding bone resorption, unloading increased osteoclast number in WT. In contrast, MURF1 deficiency suppressed this osteoclast increase. These data indicated that the ring finger protein, MURF1 is involved in disuse-induced bone loss in both of the two major bone remodeling activities, osteoblastic bone formation and osteoclastic bone resorption.     

Deficiency of inducible nitric oxide synthase attenuates immobilization-induced skeletal muscle atrophy in mice

The present study examined the effects of inducible nitric oxide synthase (iNOS) deficiency on skeletal muscle atrophy in single leg-immobilized iNOS knockout (KO) and wild-type (WT) mice. The left leg was immobilized for 1 wk, and the right leg was used as the control. Muscle weight and contraction-stimulated glucose uptake were reduced by immobilization in WT mice, which was accompanied with increased iNOS expression in skeletal muscle. Deficiency of iNOS attenuated muscle weight loss and the reduction in contraction-stimulated glucose uptake by immobilization. Phosphorylation of Akt, mTOR, and p70S6K was reduced to a similar extent by immobilization in both WT and iNOS KO mice. Immobilization decreased FoxO1 phosphorylation and increased mRNA and protein levels of MuRF1 and atrogin-1 in WT mice, which were attenuated in iNOS KO mice. Aconitase and superoxide dismutase activities were reduced by immobilization in WT mice, and deficiency of iNOS normalized these enzyme activities. Increased nitrotyrosine and carbonylated protein levels by immobilization in WT mice were reversed in iNOS KO mice. Phosphorylation of ERK and p38 was increased by immobilization in WT mice, which was reduced in iNOS KO mice. Immobilization-induced muscle atrophy was also attenuated by an iNOS-specific inhibitor N(6)-(1-iminoethyl)-l-lysine, and this finding was accompanied by increased FoxO1 phosphorylation and reduced MuRF1 and atrogin-1 levels. These results suggest that deficiency of iNOS attenuates immobilization-induced skeletal muscle atrophy through reduced oxidative stress, and iNOS-induced oxidative stress may be required for immobilization-induced skeletal muscle atrophy.     

Genome-wide Mechanosensitive MicroRNA (MechanomiR) Screen Uncovers Dysregulation of Their Regulatory Networks in the mdm Mouse Model of Muscular Dystrophy

Muscular dystrophies (MDs) are a heterogeneous group of genetic and neuromuscular disorders, which result in severe loss of motor ability and skeletal muscle mass and function. Aberrant mechanotransduction and dysregulated-microRNA pathways are often associated with the progression of MD. Here, we hypothesized that dysregulation of mechanosensitive microRNAs (mechanomiRs) in dystrophic skeletal muscle plays a major role in the progression of MD. To test our hypothesis, we performed a genome-wide expression profile of anisotropically regulated mechanomiRs and bioinformatically analyzed their target gene networks. We assessed their functional roles in the advancement of MD using diaphragm muscles from mdm (MD with myositis) mice, an animal model of human tibial MD (titinopathy), and their wild-type littermates. We were able to show that ex vivo anisotropic mechanical stretch significantly alters the miRNA expression profile in diaphragm muscles from WT and mdm mice; as a result, some of the genes associated with MDs are dysregulated in mdm mice due to differential regulation of a distinct set of mechanomiRs. Interestingly, we found a contrasting expression pattern of the highly expressed let-7 family mechanomiRs, let-7e-5p and miR-98–5p, and their target genes associated with the extracellular matrix and TGF-β pathways, respectively, between WT and mdm mice. Gain- and loss-of-function analysis of let-7e-5p in myocytes isolated from the diaphragms of WT and mdm mice confirmed Col1a1, Col1a2, Col3a1, Col24a1, Col27a1, Itga1, Itga4, Scd1, and Thbs1 as target genes of let-7e-5p. Furthermore, we found that miR-98 negatively regulates myoblast differentiation. Our study therefore introduces additional biological players in the regulation of skeletal muscle structure and myogenesis that may contribute to unexplained disorders of MD.     

Reduction in mdx mouse muscle degeneration by low-intensity endurance exercise: a proteomic analysis in quadriceps muscle of exercised compared with sedentary mdx mice

In our recent study was shown a significant recovery of damaged skeletal muscle of mice with X-linked muscular dystrophy (mdx) following low-intensity endurance exercise, probably by reducing the degeneration of dystrophic muscle. Consequently, in the present work, we aimed to identify proteins involved in the observed reduction in degenerating fibres. To this end, we used proteomic analysis to evaluate changes in the protein profile of quadriceps dystrophic muscles of exercised compared with sedentary mdx mice. Four protein spots were found to be significantly changed and were identified as three isoforms of carbonic anhydrase 3 (CA3) and superoxide dismutase [Cu-Zn] (SODC). Protein levels of CA3 isoforms were significantly up-regulated in quadriceps of sedentary mdx mice and were completely restored to wild–type (WT) mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Protein levels of SODC were down-regulated in quadriceps of sedentary mdx mice and were significantly restored to WT mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Western blot data were in agreement with those obtained using proteomic analysis and revealed the presence of one more CA3 isoform that was significantly changed. Based on data found in the present study, it seems that low-intensity endurance exercise may in part contribute to reduce cell degeneration process in mdx muscles, by counteracting oxidative stress.     

Inverse relationship between PGC-1alpha protein expression and triacylglycerol accumulation in rodent skeletal muscle.

PGC-1alpha is a key regulator of tissue metabolism, including skeletal muscle. Because it has been shown that PGC-1alpha alters the capacity for lipid metabolism, it is possible that PGC-1alpha expression is regulated by the intramuscular lipid milieu. Therefore, we have examined the relationship between PGC-1alpha protein expression and the intramuscular fatty acid accumulation in hindlimb muscles of animals in which the capacity for fatty acid accumulation in muscle is increased (Zucker obese rat) or reduced [FAT/CD36 null (KO) mice]. Rates of palmitate incorporation into triacylglycerols were determined in perfused red (RG) and white gastrocnemius (WG) muscles of lean and obese Zucker rats and in perfused RG and WG muscles of FAT/CD36 KO and wild-type (WT) mice. In obese Zucker rats, the rate of palmitate incorporation into triacylglycerol depots in RG and WG muscles were 28 and 24% greater than in lean rats (P < 0.05). In FAT/CD36 KO mice, the rates of palmitate incorporation into triacylglycerol depots were lower in RG (-50%) and WG muscle (-24%) compared with the respective muscles in WT mice (P < 0.05). In the obese animals, PGC-1alpha protein content was reduced in both RG (-13%) and WG muscles (-15%) (P < 0.05). In FAT/CD36 KO mice, PGC-1alpha protein content was upregulated in both RG (+32%, P < 0.05) and WG muscles (+50%, P < 0.05). In conclusion, from studies in these two animal models, it appears that PGC-1alpha protein expression is inversely related to components of intramuscular lipid metabolism, because 1) PGC-1alpha protein expression is downregulated when triacylglycerol synthesis rates, an index of intramuscular lipid metabolism, are increased, and 2) PGC-1alpha protein expression is upregulated when triacylglycerol synthesis rates are reduced. Therefore, we speculate that the intramuscular lipid sensing may be involved in regulating the protein expression of PGC-1alpha in skeletal muscle.     

Lack of REDD1 reduces whole body glucose and insulin tolerance,and impairs skeletal muscle insulin signaling

A lack of the REDD1 promotes dysregulated growth signaling, though little has been established with respect to the metabolic role of REDD1. Therefore, the goal of this study was to determine the role of REDD1 on glucose and insulin tolerance, as well as insulin stimulated growth signaling pathway activation in skeletal muscle. First, intraperitoneal (IP) injection of glucose or insulin were administered to REDD1 wildtype (WT) versus knockout (KO) mice to examine changes in blood glucose over time. Next, alterations in skeletal muscle insulin (IRS-1, Akt, ERK 1/2) and growth (4E-BP1, S6K1, REDD1) signaling intermediates were determined before and after IP insulin treatment (10 min). REDD1 KO mice were both glucose and insulin intolerant when compared to WT mice, evident by higher circulating blood glucose concentrations and a greater area under the curve following IP injections of glucose or insulin. While the REDD1 KO exhibited significant though blunted insulin-stimulated increases (p < 0.05) in Akt S473 and T308 phosphorylation versus the WT mice, acute insulin treatment has no effect (p < 0.05) on REDD1 KO skeletal muscle 4E-BP1 T37/46, S6K1 T389, IRS-1 Y1222, and ERK 1/2 T202/Y204 phosphorylation versus the WT mice. Collectively, these novel data suggest that REDD1 has a more distinct role in whole body and skeletal muscle metabolism and insulin action than previously thought.     

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 (H₂S) 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 H₂S 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 H₂S level in skeletal muscle of db/db mice was reduced. Simultaneously, exogenous H₂S could alleviate ROS production and reverse expression of ER stress protein markers. Exogenous H₂S could decrease the ubiquitination level of MYOM1 and MYH4 in db/db mice. In addition, exogenous H₂S reduced the interaction between MuRF1 with MYOM1 and MYH4 via MuRF1 S‐sulfhydration. Based on these results, we establish that H₂S prevented the degradation of skeletal muscle via MuRF1 S‐sulfhydration at the site of Cys44 in db/db mice.     

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-β-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, nuleotide 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.     

Cardiac proteasome activity in muscle ring finger-1 null mice at rest and following synthetic glucocorticoid treatment

Muscle ring finger-1 (MuRF1) is a muscle-specific E3 ubiquitin ligase that has been implicated in the regulation of cardiac mass through its control of the ubiquitin proteasome system. While it has been suggested that MuRF1 is required for cardiac atrophy, a resting cardiac phenotype has not been reported in mice with a null deletion [knockout (KO)] of MuRF1. Here, we report that MuRF1 KO mice have significantly larger hearts than age-matched wild-type (WT) littermates at ≥ 6 mo of age and that loss of cardiac mass can occur in the absence of MuRF1. The objective of this study was to determine whether changes in proteasome activity were responsible for the cardiac phenotypes observed in MuRF1 KO mice. Cardiac function, architecture, and proteasome activity were analyzed at rest and following 28 days of dexamethasone (Dex) treatment in 6-mo-old WT and MuRF1 KO mice. Echocardiography demonstrated normal cardiac function in the enlarged hearts in MURF1 KO mice. At rest, heart mass and cardiomyocyte diameter were significantly greater in MuRF1 KO than in WT mice. The increase in cardiac size in MuRF1 KO mice was related to a decrease in proteasome activity and an increase in Akt signaling relative to WT mice. Dex treatment induced a significant loss of cardiac mass in MuRF1 KO, but not WT, mice. Furthermore, Dex treatment resulted in an increase in proteasome activity in KO, but a decrease in WT, mice. In contrast, Akt/mammalian target of rapamycin signaling decreased in MuRF1 KO mice and increased in WT mice in response to Dex treatment. These findings demonstrate that MuRF1 plays an important role in regulating cardiac size through alterations in protein turnover and that MuRF1 is not required to induce cardiac atrophy.     

Approaching the multifaceted nature of energy metabolism: inactivation of the cytosolic creatine kinases via homologous recombination in mouse embryonic stem cells

To study the physiological role of the creatine kinase/phosphocreatine (CK/PCr) system in cells and tissues with a high and fluctuating energy demand we have concentrated on the site-directed inactivation of the B- and M-CK genes encoding the cytosolic CK protein subunits. In our approach we used homologous recombination in mouse embryonic stem (ES) cells from strain 129/Sv. Using targeting constructs based on strain 129/Sv isogenic DNA we managed to ablate the essential exons of the B-CK and M-CK genes at reasonably high frequencies. ES clones with fully disrupted B-CK and two types of M-CK gene mutations, a null (M-CK^–) and leaky (M-CK¹) mutation, were used to generate chimaeric mutant mice via injection in strain C57BL/6 derived blastocysts. Chimaeras with the B-CK null mutation have no overt abnormalities but failed to transmit the mutation to their offspring. For the M-CK^– and M-CK¹ mutations successful transmission was achieved and heterozygous and homozygous mutant mice were bred. Animals deficient in MM-CK are phenotypically normal but lack muscular burst activity. Fluxes through the CK reaction in skeletal muscle are highly impaired and fast fibres show adaptation in cellular architecture and storage of glycogen. Mice homozygous for the leaky M-CK allele, which have 3-fold reduced MM-CK activity, show normal fast fibres but CK fluxes and burst activity are still not restored to wildtype levels.     

Lack of skeletal muscle liver kinase B1 alters gene expression,mitochondrial content,inflammation and oxidative stress without affecting high-fat diet-induced obesity or insulin resistance

Ad libitum high-fat diet (HFD) induces obesity and skeletal muscle metabolic dysfunction. Liver kinase B1 (LKB1) regulates skeletal muscle metabolism by controlling the AMP-activated protein kinase family, but its importance in regulating muscle gene expression and glucose tolerance in obese mice has not been established. The purpose of this study was to determine how the lack of LKB1 in skeletal muscle (KO) affects gene expression and glucose tolerance in HFD-fed, obese mice.KO and littermate control wild-type (WT) mice were fed a standard diet or HFD for 14 weeks. RNA sequencing, and subsequent analysis were performed to assess mitochondrial content and respiration, inflammatory status, glucose and insulin tolerance, and muscle anabolic signaling.KO did not affect body weight gain on HFD, but heavily impacted mitochondria-, oxidative stress-, and inflammation-related gene expression. Accordingly, mitochondrial protein content and respiration were suppressed while inflammatory signaling and markers of oxidative stress were elevated in obese KO muscles. KO did not affect glucose or insulin tolerance. However, fasting serum insulin and skeletal muscle insulin signaling were higher in the KO mice. Furthermore, decreased muscle fiber size in skmLKB1-KO mice was associated with increased general protein ubiquitination and increased expression of several ubiquitin ligases, but not muscle ring finger 1 or atrogin-1. Taken together, these data suggest that the lack of LKB1 in skeletal muscle does not exacerbate obesity or insulin resistance in mice on a HFD, despite impaired mitochondrial content and function and elevated inflammatory signaling and oxidative stress.     

Loss of P2X7 nucleotide receptor function leads to abnormal fat distribution in mice

The P2X7 receptor is an ATP-gated cation channel expressed by a number of cell types. We have shown previously that disruption of P2X7 receptor function results in downregulation of osteogenic markers and upregulation of adipogenic markers in calvarial cell cultures. In the present study, we assessed whether loss of P2X7 receptor function results in changes to adipocyte distribution and lipid accumulation in vivo. Male P2X7 loss-of-function (KO) mice exhibited significantly greater body weight and epididymal fat pad mass than wild-type (WT) mice at 9 months of age. Fat pad adipocytes did not differ in size, consistent with adipocyte hyperplasia rather than hypertrophy. Histological examination revealed ectopic lipid accumulation in the form of adipocytes and/or lipid droplets in several non-adipose tissues of older male KO mice (9–12 months of age). Ectopic lipid was observed in kidney, extraorbital lacrimal gland and pancreas, but not in liver, heart or skeletal muscle. Specifically, lacrimal gland and pancreas from 12-month-old male KO mice had greater numbers of adipocytes in perivascular, periductal and acinar regions. As well, lipid droplets accumulated in the renal tubular epithelium and lacrimal acinar cells. Blood plasma analyses revealed diminished total cholesterol levels in 9- and 12-month-old male KO mice compared with WT controls. Interestingly, no differences were observed in female mice. Moreover, there were no significant differences in food consumption between male KO and WT mice. Taken together, these data establish novel in vivo roles for the P2X7 receptor in regulating adipogenesis and lipid metabolism in an age- and sex-dependent manner.     

Targeting the Ubiquitin E3 Ligase MuRF1 to Inhibit Muscle Atrophy

Progressive muscle wasting, also known as myopathy or muscle atrophy is a debilitating and life-threatening disorder. Myopathy is a pathological condition of many diseases including cancer, diabetes, COPD, and AIDS and is a natural consequence of inactivity and aging (sarcopenia). Muscle atrophy occurs when there is a net loss of muscle mass resulting in a change in the balance between protein synthesis and protein degradation. The ubiquitin pathway and specific ubiquitin pathway enzymes have been directly implicated in the progression of atrophy. The ubiquitin E3 ligase Muscle-specific RING Finger E3 ligase (MuRF1) is upregulated and increases protein degradation and muscle wasting in numerous muscle atrophy models. The inhibition of MuRF1 could be a novel mechanism to prevent or reverse muscle wasting associated with various pathologies. We screened a small molecule library for inhibitors to MuRF1 activity and identified P013222, an inhibitor of MuRF1 autoubiquitylation. Further, P013222 was shown to inhibit MuRF1-dependent substrate ubiquitylation, and was active in inhibiting MuRF1 in a cellular atrophy model. Thus MuRF1 can be targeted in a specific manner and produce positive results in cellular atrophy models.     

Cytoarchitectural and metabolic adaptations in muscles with mitochondrial and cytosolic creatine kinase deficiencies

We have blocked creatine kinase (CK) mediated phosphocreatine (PCr) ATP transphosphorylation in mitochondria and cytosol of skeletal muscle by knocking out the genes for the mitochondrial (ScCKmit) and the cytosolic (M-CK) CK isoforms in mice. Animals which carry single or double mutations, if kept and tested under standard laboratory conditions, have surprisingly mild changes in muscle physiology. Strenuous ex vivo conditions were necessary to reveal that MM-CK absence in single and double mutants leads to a partial loss of tetanic force output. Single ScCKmit deficiency has no noticeable effects but in combination the mutations cause slowing of the relaxation rate. Importantly, our studies revealed that there is metabolic and cytoarchitectural adaptation to CK defects in energy metabolism. The effects involve mutation type-dependent alterations in the levels of AMP, IMP, glycogen and phosphomonoesters, changes in activity of metabolic enzymes like AMP-deaminase, alterations in mitochondrial volume and contractile protein (MHC isoform) profiles, and a hyperproliferation of the terminal cysternae of the SR (in tubular aggregates). This suggests that there is a compensatory resiliency of loss-of-function and redirection of flux distributions in the metabolic network for cellular energy in our mutants.     

LKB1 and the regulation of malonyl-CoA and fatty acid oxidation in muscle

5'-AMP-activated protein kinase (AMPK), by way of its inhibition of acetyl-CoA carboxylase (ACC), plays an important role in regulating malonyl-CoA levels and the rate of fatty acid oxidation in skeletal and cardiac muscle. In these tissues, LKB1 is the major AMPK kinase and is therefore critical for AMPK activation. The purpose of this study was to determine how the lack of muscle LKB1 would affect malonyl-CoA levels and/or fatty-acid oxidation. Comparing wild-type (WT) and skeletal/cardiac muscle-specific LKB1 knockout (KO) mice, we found that the 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR)-stimulated decrease in malonyl-CoA levels in WT heart and quadriceps muscles was entirely dependent on the presence of LKB1, as was the AICAR-induced increase in fatty-acid oxidation in EDL muscles in vitro, since these responses were not observed in KO mice. Likewise, the decrease in malonyl-CoA levels after muscle contraction was attenuated in KO gastrocnemius muscles, suggesting that LKB1 plays an important role in promoting the inhibition of ACC, likely by activation of AMPK. However, since ACC phosphorylation still increased and malonyl-CoA levels decreased in KO muscles (albeit not to the levels observed in WT mice), whereas AMPK phosphorylation was entirely unresponsive, LKB1/AMPK signaling cannot be considered the sole mechanism for inhibiting ACC during and after muscle activity. Regardless, our results suggest that LKB1 is an important regulator of malonyl-CoA levels and fatty acid oxidation in skeletal muscle.