Organ Crosstalk Contributes to Muscle Wasting in Chronic Kidney Disease.

Muscle wasting (ie, atrophy) is a serious consequence of chronic kidney disease (CKD) that reduces muscle strength and function. It reduces the quality of life for CKD patients and increases the risks of comorbidities and mortality. Current treatment strategies to prevent or reverse skeletal muscle loss are limited owing to the broad and systemic nature of the initiating signals and the multifaceted catabolic mechanisms that accelerate muscle protein degradation and impair protein synthesis and repair pathways. Recent evidence has shown how organs such as muscle, adipose, and kidney communicate with each other through interorgan exchange of proteins and RNAs during CKD. This crosstalk changes cell functions in the recipient organs and represents an added dimension in the complex processes that are responsible for muscle atrophy in CKD. This complexity creates challenges for the development of effective therapies to ameliorate muscle wasting and weakness in patients with CKD.

Muscle Atrophy in CKD: A Historical Perspective of Advancements in Its Understanding.

OBJECTIVE: This perspective reviews the seminal clinical and experimental observations that led to today's current mechanistic model of muscle protein loss (wasting) in patients with chronic kidney disease (CKD). RESULTS AND CONCLUSION: Early International Society of Renal Nutrition and Metabolism (ISRNM) meetings facilitated discussions and hypotheses about the causes of muscle wasting in CKD. It became widely recognized that wasting is common and correlated with increased risks of mortality and morbidity. Although anorexia and dietary restrictions contribute to muscle loss, several features of CKD-associated wasting cannot be explained by malnutrition alone. The protein catabolism-inducing actions of metabolic acidosis, inflammation, insulin resistance, endocrine disorders and uremic toxins were progressively identified. Continued research to understand the interactions of inflammation, anabolic resistance, mitochondrial dysfunction, exercise, and nutrition on muscle protein turnover in patients with CKD will hopefully accelerate discoveries and treatments to ameliorate muscle wasting as well as the progression of CKD.

The impact of senescence on muscle wasting in chronic kidney disease.

BACKGROUND: Muscle wasting is a common complication of chronic kidney disease (CKD) that is associated with higher mortality. Although the mechanisms of myofibre loss in CKD has been widely studied, the contribution of muscle precursor cell (MPC) senescence remains poorly understood. Senescent MPCs no longer proliferate and can produce proinflammatory factors or cytokines. In this study, we tested the hypothesis that the senescence associated secretory phenotype (SASP) of MPCs contributes to CKD-induced muscle atrophy and weakness. METHODS: CKD was induced in mice by 5/6th nephrectomy. Kidney function, muscle size, and function were measured, and markers of atrophy, inflammation, and senescence were evaluated using immunohistochemistry, immunoblots, or qPCR. To study the impact of senescence, a senolytics cocktail of dasatinib + quercetin (D&Q) was given orally to mice for 8 weeks. To investigate CKD-induced senescence at the cellular level, primary MPCs were incubated with serum from CKD or control subjects. The roles of specific proteins in MPC senescence were studied using adenoviral transduction, siRNA, and plasmid transfection. RESULTS: In the hindlimb muscles of CKD mice, (i) the senescence biomarker SA-ß-gal was sharply increased (~30-fold); (ii) the DNA damage response marker γ-H2AX was increased 1.9-fold; and (iii) the senescence pathway markers p21 and p16INK4a were increased 1.99-fold and 2.82-fold, respectively (all values, P < 0.05), whereas p53 was unchanged. γ-H2AX, p21, and p16INK4A were negatively correlated at P < 0.05 with gastrocnemius weight, suggesting a causal relationship with muscle atrophy. Administration of the senolytics cocktail to CKD mice for 8 weeks eliminated the disease-related elevation of p21, p16INK4a , and γ-H2AX, abolished positive SA-ß-gal, and depressed the high levels of the SASP cytokines, TNF-α, IL-6, IL-1ß, and IFN (all values, P < 0.05). Skeletal muscle weight, myofibre cross-sectional area, and grip function were improved in CKD mice receiving D&Q. Markers of protein degradation, inflammation, and MPCs dysfunction were also attenuated by D&Q treatment compared with the vehicle treatment in 5/6th nephrectomy mice (all values, P < 0.05). Uraemic serum induced senescence in cultured MPCs. Overexpression of FoxO1a in MPCs increased the number of p21+ senescent cells, and p21 siRNA prevented uraemic serum-induced senescence (P < 0.05). CONCLUSIONS: Senescent MPCs are likely to contribute to the development of muscle wasting during CKD by producing inflammatory cytokines. Limiting senescence with senolytics ameliorated muscle wasting and improved muscle strength in vivo and restored cultured MPC functions. These results suggest potential new therapeutic targets to improve muscle health and function in CKD.

Assessing Global Kidney Nutrition Care.

BACKGROUND AND OBJECTIVES: Nutrition intervention is an essential component of kidney disease management. This study aimed to understand current global availability and capacity of kidney nutrition care services, interdisciplinary communication, and availability of oral nutrition supplements. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The International Society of Renal Nutrition and Metabolism (ISRNM), working in partnership with the International Society of Nephrology (ISN) Global Kidney Health Atlas Committee, developed this Global Kidney Nutrition Care Atlas. An electronic survey was administered among key kidney care stakeholders through 182 ISN-affiliated countries between July and September 2018. RESULTS: Overall, 160 of 182 countries (88%) responded, of which 155 countries (97%) answered the survey items related to kidney nutrition care. Only 48% of the 155 countries have dietitians/renal dietitians to provide this specialized service. Dietary counseling, provided by a person trained in nutrition, was generally not available in 65% of low-/lower middle-income countries and "never" available in 23% of low-income countries. Forty-one percent of the countries did not provide formal assessment of nutrition status for kidney nutrition care. The availability of oral nutrition supplements varied globally and, mostly, were not freely available in low-/lower middle-income countries for both inpatient and outpatient settings. Dietitians and nephrologists only communicated "sometimes" on kidney nutrition care in ≥60% of countries globally. CONCLUSIONS: This survey reveals significant gaps in global kidney nutrition care service capacity, availability, cost coverage, and deficiencies in interdisciplinary communication on kidney nutrition care delivery, especially in lower-income countries.

Pathophysiological mechanisms leading to muscle loss in chronic kidney disease.

Loss of muscle proteins is a deleterious consequence of chronic kidney disease (CKD) that causes a decrease in muscle strength and function, and can lead to a reduction in quality of life and increased risk of morbidity and mortality. The effectiveness of current treatment strategies in preventing or reversing muscle protein losses is limited. The limitations largely stem from the systemic nature of diseases such as CKD, which stimulate skeletal muscle protein degradation pathways while simultaneously activating mechanisms that impair muscle protein synthesis and repair. Stimuli that initiate muscle protein loss include metabolic acidosis, insulin and IGF1 resistance, changes in hormones, cytokines, inflammatory processes and decreased appetite. A growing body of evidence suggests that signalling molecules secreted from muscle can enter the circulation and subsequently interact with recipient organs, including the kidneys, while conversely, pathological events in the kidney can adversely influence protein metabolism in skeletal muscle, demonstrating the existence of crosstalk between kidney and muscle. Together, these signals, whether direct or indirect, induce changes in the levels of regulatory and effector proteins via alterations in mRNAs, microRNAs and chromatin epigenetic responses. Advances in our understanding of the signals and processes that mediate muscle loss in CKD and other muscle wasting conditions will support the future development of therapeutic strategies to reduce muscle loss.

The International Society of Renal Nutrition and Metabolism Commentary on the National Kidney Foundation and Academy of Nutrition and Dietetics KDOQI Clinical Practice Guideline for Nutrition in Chronic Kidney Disease.

The Academy of Nutrition and Dietetics and the National Kidney Foundation collaborated to provide an update to the Clinical Practice Guidelines (CPG) for nutrition in chronic kidney disease (CKD). These guidelines provide a valuable update to many aspects of the nutrition care process. They include changes in the recommendations for nutrition screening and assessment, macronutrients, and targets for electrolytes and minerals. The International Society of Renal Nutrition and Metabolism assembled a special review panel of experts and evaluated these recommendations prior to public review. As one of the highlights of the CPG, the recommended dietary protein intake range for patients with diabetic kidney disease is 0.6-0.8 g/kg/day, whereas for CKD patients without diabetes it is 0.55-0.6 g/kg/day. The International Society of Renal Nutrition and Metabolism endorses the CPG with the suggestion that clinicians may consider a more streamlined target of 0.6-0.8 g/kg/day, regardless of CKD etiology, while striving to achieve intakes closer to 0.6 g/kg/day. For implementation of these guidelines, it will be important that all stakeholders work to detect kidney disease early to ensure effective primary and secondary prevention. Once identified, patients should be referred to registered dietitians or the region-specific equivalent, for individualized medical nutrition therapy to slow the progression of CKD. As we turn our attention to the new CPG, we as the renal nutrition community should come together to strengthen the evidence base by standardizing outcomes, increasing collaboration, and funding well-designed observational studies and randomized controlled trials with nutritional and dietary interventions in patients with CKD.

Exosome-Mediated miR-29 Transfer Reduces Muscle Atrophy and Kidney Fibrosis in Mice.

Our previous study showed that miR-29 attenuates muscle wasting in chronic kidney disease. Other studies found that miR-29 has anti-fibrosis activity. We hypothesized that intramuscular injection of exosome-encapsulated miR-29 would counteract unilateral ureteral obstruction (UUO)-induced muscle wasting and renal fibrosis. We used an engineered exosome vector, which contains an exosomal membrane protein gene Lamp2b that was fused with the targeting peptide RVG (rabies viral glycoprotein peptide). RVG directs exosomes to organs that express the acetylcholine receptor, such as kidney. The intervention of Exo/miR29 increased muscle cross-sectional area and decreased UUO-induced upregulation of TRIM63/MuRF1 and FBXO32/atrogin-1. Interestingly, renal fibrosis was partially depressed in the UUO mice with intramuscular injection of Exo/miR29. This was confirmed by decreased TGF-ß, alpha-smooth muscle actin, fibronectin, and collagen 1A1 in the kidney of UUO mice. When we used fluorescently labeled Exo/miR29 to trace the Exo/miR route in vivo and found that fluorescence was visible in un-injected muscle and in kidneys. We found that miR-29 directly inhibits YY1 and TGF-ß3, which provided a possible mechanism for inhibition of muscle atrophy and renal fibrosis by Exo/miR29. We conclude that Exo/miR29 ameliorates skeletal muscle atrophy and attenuates kidney fibrosis by downregulating YY1 and TGF-ß pathway proteins.

Precision Medicine for Nutritional Management in End-Stage Kidney Disease and Transition to Dialysis.

Chronic kidney disease (CKD) is a global public health burden. Dialysis is not only costly but may not be readily available in developing countries. Even in highly developed nations, many patients may prefer to defer or avoid dialysis. Thus, alternative options to dialysis therapy or to complement dialysis are needed urgently and are important objectives in CKD management that could have huge clinical and economic implications globally. The role of nutritional therapy as a strategy to slow CKD progression and uremia was discussed as early as the late 19th and early 20th century, but was only seriously explored in the 1970s. There is a revival of interest recently owing to encouraging data as well as the increase of precision medicine with an emphasis on a personalized approach to CKD management. Although part of the explanation for the inconclusive data may relate to variations in study design and dietary prescription, diversity in genetic make-up, variations in the non-nutritional management of CKD, intra-individual variations in responses to dietary and nondietary treatment, psychosocial factors, and dietary compliance issues, these all may contribute to the heterogeneous data and responses. This brings in the evolving concept of precision medicine, in which disease management should be tailored and individualized according not only to clinical manifestations but also to the genetic make-up and biologic responses to therapy, which may vary depending on genetic composition. Precision nutrition management also should take into account patient demographics, social, psychological, education, and compliance factors, which all may influence the therapeutic needs and responses to the nutritional therapy prescribed. In this review, we provide a novel concept of precision medicine in nutritional management in end-stage kidney disease with a transition to dialysis and propose how this may be the way forward for nutritional therapy in the CKD population.

Glucocorticoid-induced CREB activation and myostatin expression in C2C12 myotubes involves phosphodiesterase-3/4 signaling.

Muscle atrophy in metabolic conditions like chronic kidney disease (CKD) and diabetes are associated with glucocorticoid production, dysfunctional insulin/Akt/FoxO3 signaling and increased myostatin expression. We recently found that CREB, a transcription factor proposed to regulate myostatin expression, is highly phosphorylated in some wasting conditions. Based on a novel Akt-PDE3/4 signaling paradigm, we hypothesized that reduced Akt signaling contributes to CREB activation and myostatin expression. C2C12 myotubes were incubated with dexamethasone (Dex), an atrophy-inducing synthetic glucocorticoid. Akt/CREB signaling and myostatin expression were evaluated by immunoblot and qPCR analyses. Inhibitors of Akt, phosphodiesterase (PDE)-3/4, and protein kinase A (PKA) signaling were used to test our hypothesis. Incubating myotubes with Dex for 3-24 h inhibited Akt phosphorylation and enhanced CREB phosphorylation as well as myostatin mRNA and protein. Inhibition of PI3K/Akt signaling with LY294002 similarly increased CREB phosphorylation. Isobutyl-methylxanthine (IBMX, a pan PDE inhibitor), milrinone (PDE3 inhibitor) and rolipram (PDE4 inhibitor) augmented CREB phosphorylation and myostatin expression. Inhibition of protein kinase A by PKI reverted Dex- or IBMX-induced CREB phosphorylation and myostatin expression. Our study provides evidence supporting a newly identified mechanism by which a glucocorticoid-related reduction in Akt signaling contributes to myostatin expression via CREB activation.

miRNA-23a/27a attenuates muscle atrophy and renal fibrosis through muscle-kidney crosstalk.

BACKGROUND: The treatment of muscle wasting is accompanied by benefits in other organs, possibly resulting from muscle-organ crosstalk. However, how the muscle communicates with these organs is less understood. Two microRNAs (miRs), miR-23a and miR-27a, are located together in a gene cluster and regulate proteins that are involved in the atrophy process. MiR-23a/27a has been shown to reduce muscle wasting and act as an anti-fibrotic agent. We hypothesized that intramuscular injection of miR-23a/27a would counteract both muscle wasting and renal fibrosis lesions in a streptozotocin-induced diabetic model. METHODS: We generated an adeno-associated virus (AAV) that overexpresses the miR-23a∼27a∼24-2 precursor RNA and injected it into the tibialis anterior muscle of streptozotocin-induced diabetic mice. Muscle cross-section area (immunohistology plus software measurement) and muscle function (grip strength) were used to evaluate muscle atrophy. Fibrosis-related proteins were measured by western blot to monitor renal damage. In some cases, AAV-GFP was used to mimic the miR movement in vivo, allowing us to track organ redistribution by using the Xtreme Imaging System. RESULTS: The injection of AAV-miR-23a/27a increased the levels of miR-23a and miR-27a as well as increased phosphorylated Akt, attenuated the levels of FoxO1 and PTEN proteins, and reduced the abundance of TRIM63/MuRF1 and FBXO32/atrogin-1 in skeletal muscles. It also decreased myostatin mRNA and protein levels as well as the levels of phosphorylated pSMAD2/3. Provision of miR-23a/27a attenuates the diabetes-induced reduction of muscle cross-sectional area and muscle function. Curiously, the serum BUN of diabetic animals was reduced in mice undergoing the miR-23a/27a intervention. Renal fibrosis, evaluated by Masson trichromatic staining, was also decreased as were kidney levels of phosphorylated SMAD2/3, alpha smooth muscle actin, fibronectin, and collagen. In diabetic mice injected intramuscularly with AAV-GFP, GFP fluorescence levels in the kidneys showed linear correlation with the levels in injected muscle when examined by linear regression. Following intramuscular injection of AAV-miR-23a∼27a∼24-2, the levels of miR-23a and miR-27a in serum exosomes and kidney were significantly increased compared with samples from control virus-injected mice; however, no viral DNA was detected in the kidney. CONCLUSIONS: We conclude that overexpression of miR-23a/27a in muscle prevents diabetes-induced muscle cachexia and attenuates renal fibrosis lesions via muscle-kidney crosstalk. Further, this crosstalk involves movement of miR potentially through muscle originated exosomes and serum distribution without movement of AAV. These results could provide new approaches for developing therapeutic strategies for diabetic nephropathy with muscle wasting.

Palmitate-induced ER stress and inhibition of protein synthesis in cultured myotubes does not require Toll-like receptor 4.

Saturated fatty acids, such as palmitate, are elevated in metabolically dysfunctional conditions like type 2 diabetes mellitus. Palmitate has been shown to impair insulin sensitivity and suppress protein synthesis while upregulating proteolytic systems in skeletal muscle. Increased sarco/endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response may contribute to the palmitate-induced impairment of muscle protein synthesis. In some cell types, ER stress occurs through activation of the Toll-like receptor 4 (TLR4). Given the link between ER stress and suppression of protein synthesis, we investigated whether palmitate induces markers of ER stress and protein synthesis by activating TLR4 in cultured mouse C2C12 myotubes. Myotubes were treated with vehicle, a TLR4-specific ligand (lipopolysaccharides), palmitate, or a combination of palmitate plus a TLR4-specific inhibitor (TAK-242). Inflammatory indicators of TLR4 activation (IL-6 and TNFα) and markers of ER stress were measured, and protein synthesis was assessed using puromycin incorporation. Palmitate substantially increased the levels of IL-6, TNF-α, CHOP, XBP1s, and ATF 4 mRNAs and augmented the levels of CHOP, XBP1s, phospho-PERK and phospho-eIF2α proteins. The TLR4 antagonist attenuated both acute palmitate and LPS-induced increases in IL-6 and TNFα, but did not reduce ER stress signaling with either 6 h or 24 h palmitate treatment. Similarly, treating myotubes with palmitate for 6 h caused a 43% decline in protein synthesis consistent with an increase in phospho-eIF2α, and the TLR4 antagonist did not alter these responses. These results suggest that palmitate does not induce ER stress through TLR4 in muscle, and that palmitate impairs protein synthesis in skeletal muscle in part by induction of ER stress.

MicroRNA-23a and MicroRNA-27a Mimic Exercise by Ameliorating CKD-Induced Muscle Atrophy.

Muscle atrophy is a frequent complication of CKD, and exercise can attenuate the process. This study investigated the role of microRNA-23a (miR-23a) and miR-27a in the regulation of muscle mass in mice with CKD. These miRs are located in a gene cluster that is regulated by the transcription factor NFAT. CKD mice expressed less miR-23a in muscle than controls, and resistance exercise (muscle overload) increased the levels of miR-23a and miR-27a in CKD mice. Injection of an adeno-associated virus encoding the miR-23a/27a/24-2 precursor RNA into the tibialis anterior muscles of normal and CKD mice led to increases in mature miR-23a and miR-27a but not miR-24-2 in the muscles of both cohorts. Overexpression of miR-23a/miR-27a in CKD mice attenuated muscle loss, improved grip strength, increased the phosphorylation of Akt and FoxO1, and decreased the activation of phosphatase and tensin homolog (PTEN) and FoxO1 and the expression of TRIM63/MuRF1 and FBXO32/atrogin-1 proteins. Provision of miR-23a/miR-27a also reduced myostatin expression and downstream SMAD-2/3 signaling, decreased activation of caspase-3 and -7, and increased the expression of markers of muscle regeneration. Lastly, in silico miR target analysis and luciferase reporter assays in primary satellite cells identified PTEN and caspase-7 as targets of miR-23a and FoxO1 as a target of miR-27a in muscle. These findings provide new insights about the roles of the miR-23a/27a-24-2 cluster in CKD-induced muscle atrophy in mice and suggest a mechanism by which exercise helps to maintain muscle mass.

Glucocorticoids Alter CRTC-CREB Signaling in Muscle Cells: Impact on PGC-1α Expression and Atrophy Markers.

Muscle wasting associated with chronic diseases has been linked to decreased expression of PGC-1α and overexpression of PGC-1α counters muscle loss. CREB, in conjunction with the CREB-regulated transcription coactivator (CRTC2), is a positive modulator of PGC-1α transcription. We previously reported that PGC-1α expression is decreased in skeletal muscle of diabetic rats despite a high level of CREB phosphorylation (i.e., activation), suggesting that CRTC2-CREB signaling may be dysregulated. In this study, the relationship between CREB/CRTC signaling and PGC-1α expression was examined in L6 myotubes treated with dexamethasone (Dex, 48h) to induce atrophy. Dex decreased PGC-1α mRNA and protein as well as the levels of CRTC1 and CRTC2 in the nucleus. Dex also altered the nuclear levels of two known regulators of CRTC2 localization; the amount of calcinuerin catalytic A subunit (CnA) was decreased whereas SIK was increased. To assess PGC-1α transcription, muscle cells were transfected with a PGC-1α luciferase reporter plasmid (PGC-1α-Luc). Dex suppressed PGC-1α luciferase activity while both isobutylmethylxanthine (IBMX) and over-expression of CRTC1 or CRTC2 increased PGC-1α-Luc activity. Mutation of the CRE binding site from PGC-1α-Luc reporter attenuated the responses to both IBMX and the CRTC proteins. Consistent with the reporter gene results, overexpression of CRTC2 produced an increase in CRTC2 in the nucleus and in PGC-1α mRNA and PGC-1α protein. Overexpression of CRTC2 was not sufficient to prevent the decrease in PGC-1α mRNA or protein by Dex. In summary, these data suggest that attenuated CREB/CRTC signaling contributes to the decrease in PGC-1α expression during atrophy.

Muscle atrophy in patients with Type 2 Diabetes Mellitus: roles of inflammatory pathways, physical activity and exercise.

Muscle atrophy is caused by an imbalance in contractile protein synthesis and degradation which can be triggered by various conditions including Type 2 Diabetes Mellitus (T2DM). Reduced muscle quality in patients with T2DM adversely affects muscle function, the capacity to perform activities of daily living, quality of life and ultimately may increase the risk of premature mortality. Systemic inflammation initiated by obesity and prolonged overnutrition not only contributes to insulin resistance typical of T2DM, but also promotes muscle atrophy via decreased muscle protein synthesis and increased ubiquitin-proteasome, lysosomal-proteasome and caspase 3- mediated protein degradation. Emerging evidence suggests that the inflammation-sensitive Nuclear Factor κ B (NF-κB) and Signal Transducer and Activator of Transcription 3 (STAT3) pathways may contribute to muscle atrophy in T2DM. In contrast, exercise appears to be an effective tool in promoting muscle hypertrophy, in part due to its effect on systemic and local (skeletal muscle) inflammation. The current review discusses the role inflammation plays in muscle atrophy in T2DM and the role of exercise training in minimising the effect of inflammatory markers on skeletal muscle. We also report original data from a cohort of obese patients with T2DM compared to age-matched controls and demonstrate that patients with T2DM have 60% higher skeletal muscle expression of the atrophy transcription factor FoxO1. This review concludes that inflammatory pathways in muscle, in particular, NF-κB, potentially contribute to T2DM-mediated muscle atrophy. Further in-vivo and longitudinal human research is required to better understand the role of inflammation in T2DM-mediated atrophy and the anti-inflammatory effect of exercise training under these conditions.

Docosahexaenoic acid prevents palmitate-induced activation of proteolytic systems in C2C12 myotubes.

Saturated fatty acids like palmitate contribute to muscle atrophy in a number of conditions (e.g., type II diabetes) by altering insulin signaling. Akt is a key modulator of protein balance that inhibits the FoxO transcription factors (e.g., FoxO3) which selectively induce the expression of atrophy-inducing genes (atrogenes) in the ubiquitin-proteasome and autophagy-lysosome systems. Conversely, omega-3 polyunsaturated fatty acids have beneficial effects on insulin signaling and may preserve muscle mass. In an earlier report, the omega-3 fatty acid docosahexaenoic acid (DHA) protected myotubes from palmitate-induced atrophy; the mechanisms underlying the alterations in protein metabolism were not identified. This study investigated whether DHA prevents a palmitate-induced increase in proteolysis by restoring Akt/FoxO signaling. Palmitate increased the rate of protein degradation, while cotreatment with DHA prevented the response. Palmitate reduced the activation state of Akt and increased nuclear FoxO3 protein while decreasing its cytosolic level. Palmitate also increased the messenger RNAs (mRNAs) of two FoxO3 atrogene targets, the E3 ubiquitin ligase atrogin-1/MAFbx and the autophagy mediator Bnip3. DHA attenuated the effects of palmitate on Akt activation, FoxO3 localization and atrogene mRNAs. DHA, alone or in combination with palmitate and decreased the ratio of LC3B-II:LC3B-I protein as well as the rate of autophagosome formation, as indicated by reduced LC3B-II protein in the presence of 10 mmol/L methylamine, suggesting an independent effect of DHA on the macroautophagy pathway. These data indicate that palmitate induces myotube atrophy, at least in part, by activating multiple proteolytic systems and that DHA counters the catabolic effects of palmitate by restoring Akt/FoxO signaling.

miR-182 attenuates atrophy-related gene expression by targeting FoxO3 in skeletal muscle.

Skeletal muscle atrophy occurs in response to a variety of conditions including chronic kidney disease, diabetes, cancer, and elevated glucocorticoids. MicroRNAs (miR) may play a role in the wasting process. Activation of the forkhead box O3 (FoxO3) transcription factor causes skeletal muscle atrophy in patients, animals, and cultured cells by increasing the expression of components of the ubiquitin-proteasome and autophagy-lysosome proteolytic systems. To identify microRNAs that potentially modulate the atrophy process, an in silico target analysis was performed and miR-182 was predicted to target FoxO3 mRNA. Using a combination of immunoblot analysis, quantitative real-time RT-PCR, and FoxO3 3'-UTR luciferase reporter genes, miR-182 was confirmed to regulate FoxO3 expression in C2C12 myotubes. Transfection of miR-182 into muscle cells decreased FoxO3 mRNA 30% and FoxO3 protein 67% (P < 0.05) and also prevented a glucocorticoid-induced upregulation of multiple FoxO3 gene targets including MAFbx/atrogin-1, autophagy-related protein 12 (ATG12), cathepsin L, and microtubule-associated protein light chain 3 (LC3). Treatment of C2C12 myotubes with dexamethasone (Dex) (1 µM, 6 h) to induce muscle atrophy decreased miR-182 expression by 63% (P < 0.05). Similarly, miR-182 was decreased 44% (P < 0.05) in the gastrocnemius muscle of rats injected with streptozotocin to induce diabetes compared with controls. Finally, miR-182 was present in exosomes isolated from the media of C2C12 myotubes and Dex increased its abundance. These data identify miR-182 as an important regulator of FoxO3 expression that participates in the control of atrophy-inducing genes during catabolic diseases.

miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export.

Skeletal muscle atrophy is prevalent in chronic diseases, and microRNAs (miRs) may play a key role in the wasting process. miR-23a was previously shown to inhibit the expression of atrogin-1 and muscle RING-finger protein-1 (MuRF1) in muscle. It also was reported to be regulated by cytoplasmic nuclear factor of activated T cells 3 (NFATc3) in cardiomyocytes. The objective of this study was to determine if miR-23a is regulated during muscle atrophy and to evaluate the relationship between calcineurin (Cn)/NFAT signaling and miR-23a expression in skeletal muscle cells during atrophy. miR-23a was decreased in the gastrocnemius of rats with acute streptozotocin-induced diabetes, a condition known to increase atrogin-1 and MuRF1 expression and cause atrophy. Treatment of C2C12 myotubes with dexamethasone (Dex) for 48 h also reduced miR-23a as well as RCAN1.4 mRNA, which is transcriptionally regulated by NFAT. NFATc3 nuclear localization and the amount of miR-23a decreased rapidly within 1 h of Dex administration, suggesting a link between Cn signaling and miR-23a. The level of miR-23a was lower in primary myotubes from mice lacking the α- or ß-isoform of the CnA catalytic subunit than wild-type mice. Dex did not further suppress miR-23a in myotubes from Cn-deficient mice. Overexpression of CnAß in C2C12 myotubes prevented Dex-induced suppression of miR-23a. Finally, miR-23a was present in exosomes isolated from the media of C2C12 myotubes, and Dex increased its exosomal abundance. Dex did not alter the number of exosomes released into the media. We conclude that atrophy-inducing conditions downregulate miR-23a in muscle by mechanisms involving attenuated Cn/NFAT signaling and selective packaging into exosomes.