Abstract Muscle atrophy is commonly caused by various diseases but still lacks effective treatment in clinical practice. Here, an artificial circular RNA (circRNA) named circmiR‐29b, which is designed to be a molecular sponge for miR‐29b containing 12 imperfect bulged miR‐29b binding sites is constructed. CircmiR‐29b shows a favorable functional effect with respect to miR‐29b repression in a specific and drastic manner. CircmiR‐29b can protect against in vitro muscle atrophy induced by dexamethasone (Dex), angiotensin II (Ang II), and tumor necrosis factor alpha (TNF‐α). Besides, circmiR‐29b attenuates in vivo muscle atrophy induced by denervation, immobilization, and Ang II. More importantly, skeletal muscle specific gene therapy using circmiR‐29b is able to attenuate established muscle atrophy induced by spiral wire immobilization. This work has developed an engineered circmiR‐29b acting as an miR‐29b sponge, and offers a promising therapeutic tool to prevent muscle atrophy.
Skeletal muscle atrophy is a common clinical feature of many acute and chronic conditions. Circular RNAs (circRNAs) are covalently closed RNA transcripts that are involved in various physiological and pathological processes, but their role in muscle atrophy remains unknown. Global circRNA expression profiling indicated that circRNAs are involved in the pathophysiological processes of muscle atrophy. circTmeff1 is identified as a potential circRNA candidate that influences muscle atrophy. It is further identified that circTmeff1 is highly expressed in multiple types of muscle atrophy in vivo and in vitro. Moreover, the overexpression of circTmeff1 triggers muscle atrophy in vitro and in vivo, while the knockdown of circTmeff1 expression rescues muscle atrophy in vitro and in vivo. In particular, the knockdown of circTmeff1 expression partially rescues muscle mass in mice during established atrophic settings. Mechanistically, circTmeff1 directly interacts with TAR DNA-binding protein 43 (TDP-43) and promotes aggregation of TDP-43 in mitochondria, which triggers the release of mitochondrial DNA (mtDNA) into cytosol and activation of the cyclic GMP-AMP synthase (cGAS)/ stimulator of interferon genes (STING) pathway. Unexpectedly, TMEFF1-339aa is identified as a novel protein encoded by circTmeff1 that mediates its pro-atrophic effects. Collectively, the inhibition of circTmeff1 represents a novel therapeutic approach for multiple types of skeletal muscle atrophy.
Pathological cardiac hypertrophy occurs in response to numerous stimuli and precedes heart failure (HF). Therapies that ameliorate pathological cardiac hypertrophy are highly needed.The expression level of miR-30d was analyzed in hypertrophy models and serum of patients with chronic heart failure by qRT-PCR. Gain and loss-of-function experiments of miR-30d were performed in vitro. miR-30d gain of function were performed in vivo. Bioinformatics, western blot, luciferase assay, qRT-PCR, and immunofluorescence were performed to examine the molecular mechanisms of miR-30d.miR-30d was decreased in both murine and neonatal rat cardiomyocytes (NRCMs) models of hypertrophy. miR-30d overexpression ameliorated phenylephrine (PE) and angiotensin II (Ang II) induced hypertrophy in NRCMs, whereas the opposite phenotype was observed when miR-30d was downregulated. Consistently, the miR-30d transgenic rat was found to protect against isoproterenol (ISO)-induced pathological hypertrophy. Mechanistically, methyltransferase EZH2 could promote H3K27me3 methylation in the promotor region of miR-30d and suppress its expression during the pathological cardiac hypertrophy. miR-30d prevented pathological cardiac hypertrophy via negatively regulating its target genes MAP4K4 and GRP78 and inhibiting pro-hypertrophic nuclear factor of activated T cells (NFAT). Adeno-associated virus (AAV) serotype 9 mediated-miR-30d overexpression exhibited beneficial effects in murine hypertrophic model. Notably, miR-30d was reduced in serum of patients with chronic heart failure and miR-30d overexpression could significantly ameliorate pathological hypertrophy in human embryonic stem cell-derived cardiomyocytes.Overexpression of miR-30d may be a potential approach to treat pathological cardiac hypertrophy.This work was supported by the grants from National Key Research and Development Project (2018YFE0113500 to J Xiao), National Natural Science Foundation of China (82020108002 to J Xiao, 81900359 to J Li), the grant from Science and Technology Commission of Shanghai Municipality (20DZ2255400 and 21XD1421300 to J Xiao, 22010500200 to J Li), Shanghai Sailing Program (19YF1416400 to J Li), the "Dawn" Program of Shanghai Education Commission (19SG34 to J Xiao), the "Chen Guang" project supported by the Shanghai Municipal Education Commission and Shanghai Education Development Foundation (19CG45 to J Li).
Muscle atrophy is debilitating and can be induced by several stressors. Unfortunately, there are no effective pharmacological treatment until now. MicroRNA (miR)-29b is an important target that we identified to be commonly involved in multiple types of muscle atrophy. Although sequence-specific inhibition of miR-29b has been developed, in this study, we report a novel small-molecule miR-29b inhibitor that targets miR-29b hairpin precursor (pre-miR-29b) (Targapremir-29b-066 [TGP-29b-066]) considering both its three-dimensional structure and the thermodynamics of interaction between pre-miR-29b and the small molecule. This novel small-molecule inhibitor has been demonstrated to attenuate muscle atrophy induced by angiotensin II (Ang II), dexamethasone (Dex), and tumor necrosis factor α (TNF-α) in C2C12 myotubes, as evidenced by increase in the diameter of myotube and decrease in the expression of Atrogin-1 and MuRF-1. Moreover, it can also attenuate Ang II-induced muscle atrophy in mice, as evidenced by a similar increase in the diameter of myotube, reduced Atrogin-1 and MuRF-1 expression, AKT-FOXO3A-mTOR signaling activation, and decreased apoptosis and autophagy. In summary, we experimentally identified and demonstrated a novel small-molecule inhibitor of miR-29b that could act as a potential therapeutic agent for muscle atrophy.
Abstract Muscle atrophy is a frequently observed complication, characterized by the loss of muscle mass and strength, which diminishes the quality of life and survival. No effective therapy except exercise is currently available. In our previous study, repressing miR-29b has been shown to reduce muscle atrophy. In our current study, we have constructed artificially engineered extracellular vesicles for the delivery of CRISPR/Cas9 to target miR-29b (EVs-Cas9-29b). EVs-Cas9-29b has shown a favorable functional effect with respect to miR-29b repression in a specific and rapid manner by gene editing. In in vitro conditions, EVs-Cas9-29b could protect against muscle atrophy induced by dexamethasone (Dex), angiotensin II (AngII), and tumor necrosis factor-alpha (TNF-α). And EVs-Cas9-29b introduced in vivo preserved muscle function in the well-established immobilization and denervation-induced muscle atrophy mice model. Our work demonstrates an engineered extracellular vesicles delivery of the miR-29b editing system, which could be potentially used for muscle atrophy therapy.
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