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 crosssectional 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-b, 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-b3, 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-b pathway proteins.

Chronic kidney disease (CKD) causes loss of lean body mass by multiple mechanisms. This study examines whether autophagy-mediated proteolysis contributes to CKD-induced muscle wasting. We tested autophagy in the muscle of CKD mice with plantaris muscle overloading to mimic resistance exercise or with acupuncture plus low-frequency electrical stimulation (Acu/LFES) treatment. In CKD muscle, Bnip3, Beclin-1, and LC3II mRNAs and proteins were increased compared with those in control muscle, indicating autophagosome-lysosome formation induction. Acu/LFES suppressed the CKD-induced upregulation of autophagy. However, overloading increased autophagy-related proteins in normal and CKD muscle. Serum from uremic mice induces autophagy formation but did not increase the myosin degradation or actin break down in cultured muscle satellite cells. We examined mitochondrial biogenesis, copy number, and ATP production in cultured myotubes, and found all three aspects to be decreased by uremic serum. Inhibition of autophagy partially reversed this decline in cultured myotubes. In CKD mice, the mitochondrial copy number, biogenesis marker peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), mitochondrial transcription factor A (TFAM), and mitochondrial fusion marker Mitofusin-2 (Mfn2) are decreased. Both muscle overloading and Acu/LFES increased mitochondrial copy number, and reversed the CKD-induced decreases in PGC-1α, TFAM, and Mfn2. We conclude that the autophagy is activated in the muscle of CKD mice. However, myofibrillar protein is not directly broken down through autophagy. Instead, CKD-induced upregulation of autophagy leads to dysfunction of mitochondria and decrease of ATP production.

Effective therapeutic strategies to treat CKD-induced muscle atrophy are urgently needed. Lowfrequency electrical stimulation (LFES) may be effective in preventing muscle atrophy, because LFES is an acupuncture technique that mimics resistance exercise by inducing muscle contraction. To test this hypothesis, we treated 5/6-nephrectomized mice (CKD mice) and control mice with LFES for 15 days. LFES prevented soleus and extensor digitorum longus muscle weight loss and loss of hind-limb muscle grip in CKD mice. LFES countered the CKD-induced decline in the IGF-1 signaling pathway and led to increases in markers of protein synthesis and myogenesis and improvement in muscle protein metabolism. In control mice, we observed an acute response phase immediately after LFES, during which the expression of inflammatory cytokines (IFN-g and IL-6) increased. Expression of the M1 macrophage marker IL-1b also increased acutely, but expression of the M2 marker arginase-1 increased 2 days after initiation of LFES, paralleling the change in IGF-1. In muscle cross-sections of LFES-treated mice, arginase-1 colocalized with IGF-1. Additionally, expression of microRNA-1 and -206, which inhibits IGF-1 translation, decreased in the acute response phase after LFES and increased at a later phase. We conclude that LFES ameliorates CKDinduced skeletal muscle atrophy by upregulation of the IGF-1 signaling pathway, which improves protein metabolism and promotes myogenesis. The upregulation of IGF-1 may be mediated by decreased expression of microRNA-1 and -206 and/or activation of M2 macrophages.

Using microarray analysis, we found that aging sarcopenia is associated with a sharp increase in the mRNA of the matricellular protein CCN1 (Cyr61/CTGF/Nov). CCN1 mRNA was upregulated 113-fold in muscle of aged vs. young rats. CCN1 protein was increased in aging muscle in both rats (2.8-fold) and mice (3.8-fold). When muscle progenitor cells (MPCs) were treated with recombinant CCN1, cell proliferation was decreased but there was no change in the myogenic marker myoD. However, the CCN1-treated MPCs did express a senescence marker (SA-βgal). Interestingly, we found CCN1 increased p53, p16(Ink4A), and pRP (hypophosphorylated retinoblastoma protein) protein levels, all of which can arrest cell growth in MPCs. When MPCs were treated with aged rodent serum CCN1 mRNA increased by sevenfold and protein increased by threefold suggesting the presence of a circulating regulator. Therefore, we looked for a circulating regulator. Wnt-3a, a stimulator of CCN1 expression, was increased in serum from elderly humans (2.6-fold) and aged rodents (2.0-fold) compared with young controls. We transduced C2C12 myoblasts with wnt-3a and found that CCN1 protein was increased in a time- and dose-dependent manner. We conclude that in aging muscle, the circulating factor wnt-3a acts to increase CCN1 expression, prompting muscle senescence by activating cell arrest proteins.

Mortality and morbidity are increased in patients with muscle atrophy resulting from catabolic diseases such as diabetes. At present there is no pharmacological treatment that successfully reverses muscle wasting from catabolic conditions. We hypothesized that acupuncture plus low frequency electric stimulation (Acu-LFES) would mimic the impact of exercise and prevent diabetes-induced muscle loss. Streptozotocin (STZ) was used to induce diabetes in mice. The mice were then treated with Acu-LFES for 15 minutes daily for 14 days. Acupuncture points were selected according to the WHO Standard Acupuncture Nomenclature guide. The needles were connected to an SDZ-II electronic acupuncture device delivering pulses at 20Hz and 1mA. Acu-LFES prevented soleus and EDL muscle weight loss and increased hind-limb muscle grip function in diabetic mice. Muscle regeneration capacity was significantly increased by Acu-LFES. The expression of Pax7, MyoD, myogenin and embryo myosin heavy chain (eMyHC) was significantly decreased in diabetic muscle vs. control muscle. The suppressed levels in diabetic muscle were reversed by Acu-LFES. The IGF-1 signaling pathway was also upregulated by Acu-LFES. Phosphorylation of Akt, mTOR and p70S6K were downregulated by diabetes leading to a decline in muscle mass, however, Acu-LFES countered the diabetes-induced decline. In addition, microRNA-1 and -206 were increased by Acu-LFES after 24 days of treatment. We conclude that Acu-LFES is effective in counteracting diabetes-induced skeletal muscle atrophy by increasing IGF-1 and its stimulation of muscle regeneration.