Histone deacetylase (HDAC) inhibition in muscle cells enhances bone regeneration through the release of extracellular vesicles (EVs) containing specific microRNAs, according to a new study published in Nature Cell Death & Differentiation. The research demonstrates that inhibiting HDACs with Trichostatin A (TSA) in human skeletal muscle myoblasts (HSMMs) promotes the formation of myotubes and increases the expression of myogenic genes. These TSA-treated HSMMs release EVs (TSA-EVs) that exhibit enhanced properties and can prevent bone loss in ovariectomized mice, suggesting a novel therapeutic approach for osteoporosis and related bone disorders. The study highlights the role of muscle-derived EVs in mediating crosstalk between muscle and bone, offering new insights into the treatment of bone-related diseases.
HDAC Inhibition Enhances Myoblast Function and EV Secretion
To investigate the impact of HDAC inhibition on skeletal muscle cells, HSMMs were treated with TSA, resulting in a dose-dependent reduction in HDAC activity and an increase in histone H3K9 acetylation. Specifically, TSA at 50 nM significantly enhanced myotube fusion compared to untreated cells, as indicated by hypernucleated myotubes with increased cell size. qPCR testing revealed that TSA at 50 nM significantly enhanced the expression of Myod, Myf5, Myog, Myh2 and Mrf4 genes. These results indicate that HDAC inhibition with TSA promotes myotube formation and enhances myogenic gene expression.
EVs isolated from TSA-treated HSMM cultures exhibited a higher zeta potential and increased RNA quantity compared to EVs from untreated cells (UN-EVs). Immunoblotting analysis confirmed the presence of CD9, CD81, and Alix proteins in both groups of EVs. Furthermore, TSA-EVs were internalized more efficiently by human bone marrow mesenchymal stem cells (hBMSCs), suggesting that HDAC inhibition enhances EV properties and their uptake by target cells.
TSA-EVs Prevent Ovariectomized-Induced Bone Loss
To determine whether muscle-derived EVs could target bone tissue, DIR-labeled EVs were injected intravenously into mice. Time-lapse imaging showed a gradual increase in fluorescent signaling in the hind legs of mice treated with EVs, with stronger signal intensity observed in bone tissues such as the limbs and spine. These findings indicate the biodistribution and accumulation of HSMM-EVs in bone tissues.
In ovariectomized (OVX) mice, systemic injection of TSA-EVs every two days for eight weeks significantly increased bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), while reducing trabecular separation (Tb.Sp). Mechanical stress testing revealed that TSA-EV injection increased the maximum load, bone stiffness, breaking energy, and energy to ultimate load. Bone histomorphometry showed significantly higher mineral apposition rate (MAR) and bone formation rate (BFR/BS) in mice receiving TSA-EV injections. Immunofluorescence staining revealed increased expression of OSX and OPN in mice treated with TSA-EVs, demonstrating that TSA-EVs effectively promote osteogenesis in vivo and counteract bone loss.
TSA-EVs Promote Osteogenic Differentiation and Inhibit Osteoclast Formation
In vitro experiments showed that TSA-EV treatment significantly enhanced hBMSCs proliferation in a dose-dependent manner. The TSA-EV group exhibited a higher intensity of alkaline phosphatase (ALP) staining and increased alizarin red S (ARS) staining intensity, with prominent calcium nodule formation. qPCR analysis showed that the expression levels of Alp, Bglap, Col1a1, Spp1, and Bmp2 were markedly upregulated in the TSA-EV group. Furthermore, TSA-EVs significantly inhibited osteoclast formation compared to UN-EVs, as evidenced by reduced TRAP-positive cell area and decreased expression of osteoclast differentiation-related genes, including Cathepsin K (Ctsk), DC-STAMP, and Acid Phosphatase 5 (Acp5). These findings highlight the dual role of TSA-EVs in regulating bone remodeling.
MicroRNA Profile Alteration in HSMM-Derived EVs
MicroRNA expression profiling revealed distinct microRNA expression profiles in TSA-EVs compared to UN-EVs. The top 7 upregulated miRNAs identified in TSA-EVs were hsa-miR-10401-3p, hsa-miR-6514-5p, hsa-miR-96-5p, hsa-miR-183-5p, hsa-miR-589-3p, hsa-miR-873-3p, and hsa-miR-656-5p. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of their target genes revealed enrichment in terms related to osteogenesis and several signaling pathways associated with osteoblast differentiation, including the PI3K-Akt, MAPK, mTOR, and Wnt signaling pathways. Further validation using qPCR confirmed that hsa-miR-589-3p, hsa-miR-873-3p, and hsa-miR-6514-5p were significantly upregulated in TSA-EVs, with hsa-miR-873-3p demonstrating the highest expression fold change. Overexpression and knockdown experiments confirmed that miR-873-3p plays a crucial role in TSA-EV-induced osteogenesis.
TSA-EVs Mimic Exercise Effects on Bone Formation
To investigate whether TSA treatment of HSMMs mimics the effects of exercise, EVs were isolated from HSMMs, TSA-treated HSMMs, non-exercised OVX mice muscle, and exercised OVX mice muscle. qPCR results showed significantly higher levels of miR-873-3p in both TSA-HSMM-EV and Exercise-EV groups. In vitro experiments showed that both TSA-HSMM-EV and Exercise-EV groups significantly increased ALP activity compared to HSMM-EV and Non-Exercise-EV groups. In vivo experiments in OVX mice showed that both the Exercise and TSA-HSMM-EV groups had significant improvements in bone parameters, with higher MAR and BFR/BS. These results demonstrate that TSA-HSMM-EVs can effectively mimic the beneficial effects of exercise on bone formation.
Extracellular Vesicle Transfers HSMM-miR-873-3p to hBMSCs
Co-culture experiments using a Transwell system confirmed that HSMM-derived miR-873-3p can be transferred to hBMSCs, enhancing osteogenic activity in vitro. hBMSCs incubated with miR-873 mimics-transfected HSMMs exhibited an increase in proliferation rate, while those incubated with miR-873 inhibitor-transfected HSMMs showed inhibition. ALP and ARS staining showed elevated intensity in the miR-873 mimics group and decreased intensity in the miR-873 inhibitor group. These results suggest that elevated miR-873-3p in HSMMs contributes to increased miR-873-3p levels in hBMSCs, thereby enhancing osteogenic activity. Further experiments demonstrated that HSMM-derived miR-873-3p is transferred through EVs to hBMSCs, promoting the osteogenesis process.
Hsa-miR-873-3p Promotes Osteogenesis via Targeting CNN2
Bioinformatic analysis identified H2 calponin (CNN2) as a potential target gene of miR-873-3p. Luciferase reporter assays confirmed that CNN2 3’ UTR is a direct target of miR-873. qPCR analysis demonstrated decreased CNN2 expression in hBMSCs after incubation with miR-873 mimics-transfected HSMMs. Rescue experiments showed that inhibiting CNN2 in hBMSCs could abolish the negative role of miR-873-inhibitor on cell proliferation and osteogenesis. These results demonstrate that histone acetylation mediates HSMMs to produce EVs containing excessive miR-873-3p, which can be directly transferred to hBMSCs and promote osteogenesis by silencing CNN2.
