Canagliflozin inhibits PASMCs proliferation via regulating SGLT1/AMPK signaling and attenuates artery remodeling in MCT-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) was a devastating disease characterized by artery remodeling, ultimately resulting in right heart failure. The aim of this study was to investigate the effects of canagliflozin (CANA), a sodium-glucose cotransporter 2 inhibitor (SGLT2i) with mild SGLT1 inhibitory effects, on rats with PAH, as well as its direct impact on pulmonary arterial smooth muscle cells (PASMCs). PAH rats were induced by injection of monocrotaline (MCT) (40 mg/kg), followed by four weeks of treatment with CANA (30 mg/kg/day) or saline alone. Pulmonary artery and right ventricular (RV) remodeling and dysfunction in PAH were alleviated with CANA, as assessed by echocardiography. Hemodynamic parameters and structural of pulmonary arteriole, including vascular wall thickness and wall area, were reduced by CANA. RV hypertrophy index, cardiomyocyte hypertrophy, and fibrosis were decreased with CANA treatment. PASMCs proliferation was inhibited by CANA under stimulation by platelet-derived growth factor (PDGF)-BB or hypoxia. Activation of AMP kinase (AMPK) was induced by CANA treatment in cultured PASMCs in a time- and concentration-dependent manner. These effects of CANA were attenuated when treatment with compound C, an AMPK inhibitor. Abundant expression of SGLT1 was observed in PASMCs and pulmonary arteries, while SGLT2 expression was undetectable. SGLT1 increased in response to PDGF-BB or hypoxia stimulation, while PASMCs proliferation was inhibited and beneficial effects of CANA were counteracted by knockdown of SGLT1. Our research demonstrated for the first time that CANA inhibited the proliferation of PASMCs by regulating SGLT1/AMPK signaling and thus exerted an anti-proliferative effect on MCT-induced PAH.

[Application of extended trochanter osteotomy wire fixation combined with autologous bone graft in revision of total hip arthroplasty].

OBJECTIVE: To investigate the clinical effect of using lengthened trochanteric osteotomy wire fixation combined with autologous bone graft in patients undergoing revision total hip arthroplasty. METHODS: From December 2010 to December 2018, 18 patients underwent revision of total hip arthroplasty with extended trochanteric osteotomy wire fixation and autogenous bone graft, including 8 males and 10 females with an average age of (78.89±3.32) years old ranging from 68 to 82 years. The time from the initial replacement to the revision was 9 to 22 (16.33±2.93) years. The patients were followed up regularly after operation. The healing time of osteotomy, the time of full weight-bearing activity, Harris score of hip joint and complications were recorded. RESULTS: All 18 patients were followed up for 16 to 38 months with an average of (25.78±6.65) months. The incision length was 16 to 21 cm with an average of (18.89±1.32) cm; the operation time was 105 to 128 min with an average of (115.44±6.59) min, the bleeding volume was 240 to 285 ml with an average of (267.44±13.77) ml. The healing time of osteotomy was 12 to 18 weeks with an average of (15.61±1.75) weeks. Harris score of hip joint was (47.11±5.04) before operation, (76.39±3.85) during full weight-bearing activities, and (82.22±2.76) at the final follow-up(P<0.05). During the follow-up period, there were no complications such as limb shortening, infection, poor incision healing, prosthesis loosening and sinking, and periprosthetic fracture. CONCLUSION: In revision total hip arthroplasty, the use of extended trochanteric osteotomy wire fixation combined with autologous bone graft can achieve satisfactory clinical results, but the surgeon needs to make a systematic plan for the pre-revision, intraoperative and postoperative recovery.

[A follow up study on the treatment of tibial fractures with intramedullary interlocking nail through the suprapatellar approach].

OBJECTIVE: To investigate the short-term effect of suprapatellar interlocking intramedullary nail in the treatment of tibial fractures. METHODS: Eighty patients with tibial fractures treated from January 2016 to June 2018 were treated with interlocking intramedullary nail, who were divided into observation group (suprapatellar approach) and control group (patellar ligament approach) according to different surgical approaches. There were 40 cases in the observation group, including 28 males and 12 females, aged 28 to 67 years with a mean of (46.70±10.34) years. There were 40 cases in the control group, including 30 males and 10 females, aged 31 to 69 years with a mean of(49.38±10.74) years. The operation time, incision length, intraoperative C-arm X-ray fluoroscopy times, intraoperative blood loss, fracture healing time, postoperative active straight leg raise (SLR) time, hospital stay, visual analogue scale (VAS), knee pain rate and postoperative Hospital for Special Surgery (HSS) score were recorded and compared between two groups. RESULTS: All the patients were followed up, and the duration ranged from 19 to 38 months, with an average of(24.60±4.52) months. In the observation group, the operation time was(53.83± 7.01) min;the incision length was (3.98±0.83) cm;the number of intraoperative C-arm X-ray fluoroscopy was (18.90±1.75) times;the fracture healing time was (10.03±0.89) weeks;the postoperative active SLR time was (1.19±0.25) days;and the hospital stay was(6.73±1.06) days. The above indexes were better than those in the control group (P<0.05). In the latest follow-up, 34 cases got an excellent result, 5 good, 1 fair and 0 poorin the observation group. In the control group, 25 cases got an excellent result, 9 good, 6 fair and 0 poor. The curative effect of the observation group was better than that of the control group(P< 0.05). CONCLUSION: The treatment of tibial fractures with suprapatellar interlocking intramedullary nail has the advantages of less trauma and better recovery of knee function. It can obtain more satisfactory clinical results and can be further widely used.

Rationale and design of a mechanistic clinical trial of JAK inhibition to prevent ventilator-induced diaphragm dysfunction.

INTRODUCTION: Ventilator-induced diaphragm dysfunction (VIDD) is an important phenomenon that has been repeatedly demonstrated in experimental and clinical models of mechanical ventilation. Even a few hours of MV initiates signaling cascades that result in, first, reduced specific force, and later, atrophy of diaphragm muscle fibers. This severe, progressive weakness of the critical ventilatory muscle results in increased duration of MV and thus increased MV-associated complications/deaths. A drug that could prevent VIDD would likely have a major positive impact on intensive care unit outcomes. We identified the JAK/STAT pathway as important in VIDD and then demonstrated that JAK inhibition prevents VIDD in rats. We subsequently developed a clinical model of VIDD demonstrating reduced contractile force of isolated diaphragm fibers harvested after ∼7 vs ∼1 h of MV during a thoracic surgical procedure. MATERIALS AND METHODS: The NIH-funded clinical trial that has been initiated is a prospective, placebo controlled trial: subjects undergoing esophagectomy are randomized to receive 6 preoperative doses of the FDA-approved JAK inhibitor Tofacitinib (commonly used for rheumatoid arthritis) vs. placebo. The primary outcome variable will be the difference in the reduction that occurs in force generation of diaphragm single muscle fibers (normalized to their cross-sectional area), in the Tofacitinib vs. placebo subjects, over 6 h of MV. DISCUSSION: This trial represents a first-in-human, mechanistic clinical trial of a drug to prevent VIDD. It will provide proof-of-concept in human subjects whether JAK inhibition prevents clinical VIDD, and if successful, will support an ICU-based clinical trial that would determine whether JAK inhibition impacts clinical outcome variables such as duration of MV and mortality.

Long non-coding RNA DANCR modulates osteogenic differentiation by regulating the miR-1301-3p/PROX1 axis.

The balance of osteoblasts and marrow adipocytes from bone marrow mesenchymal stem cells (BM-MSCs) maintains bone health. Under aging or other pathological stimuli, BM-MSCs will preferentially differentiate into marrow adipocytes and reduce osteoblasts, leading to osteoporosis. Long non-coding RNA differentiation antagonizing non-protein coding RNA (DANCR) participates in the osteogenic differentiation of human BM-MSCs, but the mechanism by which DANCR regulates the osteogenic differentiation of human BM-MSCs has not been fully explained. We observed that DANCR and prospero homeobox 1 (PROX1) were downregulated during osteogenic differentiation of human BM-MSCs, while miR-1301-3p had an opposite trend. DANCR overexpression decreased the levels of alkaline phosphatase, RUNX2, osteocalcin, Osterix in BM-MSCs after osteogenic induction, but DANCR silencing had the opposite result. Moreover, DANCR sponged miR-1301-3p to regulate PROX1 expression. miR-1301-3p overexpression reversed the suppressive role of DANCR elevation on the osteogenic differentiation of human BM-MSCs. Also, PROX1 elevation abolished the promoting role of miR-1301-3p overexpression on the osteogenic differentiation of human BM-MSCs. In conclusion, DANCR suppressed the osteogenic differentiation of human BM-MSCs through the miR-1301-3p/PROX1 axis, offering a novel mechanism by which DANCR is responsible for the osteogenic differentiation of human BM-MSCs.

mTORC1 underlies age-related muscle fiber damage and loss by inducing oxidative stress and catabolism.

Aging leads to skeletal muscle atrophy (i.e., sarcopenia), and muscle fiber loss is a critical component of this process. The mechanisms underlying these age-related changes, however, remain unclear. We show here that mTORC1 signaling is activated in a subset of skeletal muscle fibers in aging mouse and human, colocalized with fiber damage. Activation of mTORC1 in TSC1 knockout mouse muscle fibers increases the content of morphologically abnormal mitochondria and causes progressive oxidative stress, fiber damage, and fiber loss over the lifespan. Transcriptomic profiling reveals that mTORC1's activation increases the expression of growth differentiation factors (GDF3, 5, and 15), and of genes involved in mitochondrial oxidative stress and catabolism. We show that increased GDF15 is sufficient to induce oxidative stress and catabolic changes, and that mTORC1 increases the expression of GDF15 via phosphorylation of STAT3. Inhibition of mTORC1 in aging mouse decreases the expression of GDFs and STAT3's phosphorylation in skeletal muscle, reducing oxidative stress and muscle fiber damage and loss. Thus, chronically increased mTORC1 activity contributes to age-related muscle atrophy, and GDF signaling is a proposed mechanism.

Logistic regression analysis of drug compliance and influencing factors in elderly osteoporosis patients.

A cross-sectional study was conducted to understand the medication compliance of elderly osteoporosis patients and to further analyze the influencing factors of drug compliance. The elderly osteoporosis patients who were admitted to the First People's Hospital of Huzhou, Zhejiang from March 2015 to January 2017 were selected as the research subjects. Subsequently, the three months, six months and 12 months of follow-up were performed from the group of subjects who prescribe anti-osteoporosis drugs to determine the patient's medication status. Hereon, a cross-sectional survey was conducted to investigate the status of drug compliance in elderly patients with osteoporosis. Moreover, multivariate logistic regression analysis was used to analyze the influencing factors of medication compliance. The discontinuation rates and causes were not the same in different time periods and the cumulative withdrawal rate was high (37%) within one year. A total of 492 cases of elderly osteoporosis patients were included in this study, whereas only 45.73% of patients had good drug compliance. Elderly patients with osteoporosis have poor medication compliance and education, marital status, and medication types all affect medicine-taking compliance of patients. Therefore, it is suggested that health education should be carried out, and psychological care of patients should also be strengthened. Meanwhile, the follow-up system for drug compliance should be established to improve medication compliance as well world wide.

The Signaling Network Resulting in Ventilator-induced Diaphragm Dysfunction.

Mechanical ventilation (MV) is a life-saving measure for those incapable of adequately ventilating or oxygenating without assistance. Unfortunately, even brief periods of MV result in diaphragm weakness (i.e., ventilator-induced diaphragm dysfunction [VIDD]) that may render it difficult to wean the ventilator. Prolonged MV is associated with cascading complications and is a strong risk factor for death. Thus, prevention of VIDD may have a dramatic impact on mortality rates. Here, we summarize the current understanding of the pathogenic events underlying VIDD. Numerous alterations have been proven important in both human and animal MV diaphragm. These include protein degradation via the ubiquitin proteasome system, autophagy, apoptosis, and calpain activity-all causing diaphragm muscle fiber atrophy, altered energy supply via compromised oxidative phosphorylation and upregulation of glycolysis, and also mitochondrial dysfunction and oxidative stress. Mitochondrial oxidative stress in fact appears to be a central factor in each of these events. Recent studies by our group and others indicate that mitochondrial function is modulated by several signaling molecules, including Smad3, signal transducer and activator of transcription 3, and FoxO. MV rapidly activates Smad3 and signal transducer and activator of transcription 3, which upregulate mitochondrial oxidative stress. Additional roles may be played by angiotensin II and leaky ryanodine receptors causing elevated calcium levels. We present, here, a hypothetical scaffold for understanding the molecular pathogenesis of VIDD, which links together these elements. These pathways harbor several drug targets that could soon move toward testing in clinical trials. We hope that this review will shape a short list of the most promising candidates.

Smad3 initiates oxidative stress and proteolysis that underlies diaphragm dysfunction during mechanical ventilation.

Prolonged use of mechanical ventilation (MV) leads to atrophy and dysfunction of the major inspiratory muscle, the diaphragm, contributing to ventilator dependence. Numerous studies have shown that proteolysis and oxidative stress are among the major effectors of ventilator-induced diaphragm muscle dysfunction (VIDD), but the upstream initiator(s) of this process remain to be elucidated. We report here that periodic diaphragm contraction via phrenic nerve stimulation (PNS) substantially reduces MV-induced proteolytic activity and oxidative stress in the diaphragm. We show that MV rapidly induces phosphorylation of Smad3, and PNS nearly completely prevents this effect. In cultured cells, overexpressed Smad3 is sufficient to induce oxidative stress and protein degradation, whereas inhibition of Smad3 activity suppresses these events. In rats subjected to MV, inhibition of Smad3 activity by SIS3 suppresses oxidative stress and protein degradation in the diaphragm and prevents the reduction in contractility that is induced by MV. Smad3's effect appears to link to STAT3 activity, which we previously identified as a regulator of VIDD. Inhibition of Smad3 suppresses STAT3 signaling both in vitro and in vivo. Thus, MV-induced diaphragm inactivity initiates catabolic changes via rapid activation of Smad3 signaling. An early intervention with PNS and/or pharmaceutical inhibition of Smad3 may prevent clinical VIDD.

The JAK-STAT pathway is critical in ventilator-induced diaphragm dysfunction.

Mechanical ventilation (MV) is one of the lynchpins of modern intensive-care medicine and is life saving in many critically ill patients. Continuous ventilator support, however, results in ventilation-induced diaphragm dysfunction (VIDD) that likely prolongs patients' need for MV and thereby leads to major associated complications and avoidable intensive care unit (ICU) deaths. Oxidative stress is a key pathogenic event in the development of VIDD, but its regulation remains largely undefined. We report here that the JAK-STAT pathway is activated in MV in the human diaphragm, as evidenced by significantly increased phosphorylation of JAK and STAT. Blockage of the JAK-STAT pathway by a JAK inhibitor in a rat MV model prevents diaphragm muscle contractile dysfunction (by ~85%, p < 0.01). We further demonstrate that activated STAT3 compromises mitochondrial function and induces oxidative stress in vivo, and, interestingly, that oxidative stress also activates JAK-STAT. Inhibition of JAK-STAT prevents oxidative stress-induced protein oxidation and polyubiquitination and recovers mitochondrial function in cultured muscle cells. Therefore, in ventilated diaphragm muscle, activation of JAK-STAT is critical in regulating oxidative stress and is thereby central to the downstream pathogenesis of clinical VIDD. These findings establish the molecular basis for the therapeutic promise of JAK-STAT inhibitors in ventilated ICU patients.

Coronin 6 regulates acetylcholine receptor clustering through modulating receptor anchorage to actin cytoskeleton.

The maintenance of a high density of neurotransmitter receptors at the postsynaptic apparatus is critical for efficient neurotransmission. Acetylcholine receptors (AChRs) are neurotransmitter receptors densely packed on the postsynaptic muscle membrane at the neuromuscular junction (NMJ) via anchoring onto the actin cytoskeletal network. However, how the receptor-associated actin is coordinately regulated is not fully understood. We report here that Coronin 6, a newly identified member of the coronin family, is highly enriched at adult NMJs and regulates AChR clustering through modulating the interaction between receptors and the actin cytoskeletal network. Experiments with cultured myotubes reveal that Coronin 6 is important for both agrin- and laminin-induced AChR clustering. Furthermore, Coronin 6 forms a complex with AChRs and actin in a manner dependent on its C-terminal region and a conserved Arg(29) residue at the N terminus, both of which are critical for the cytoskeletal anchorage of AChRs. Importantly, in vivo knockdown of Coronin 6 in mouse skeletal muscle fibers leads to destabilization of AChR clusters. Together, these findings demonstrate that Coronin 6 is a critical regulator of AChR clustering at the postsynaptic region of the NMJs through modulating the receptor-anchored actin cytoskeleton.

mTORC1 promotes denervation-induced muscle atrophy through a mechanism involving the activation of FoxO and E3 ubiquitin ligases.

Skeletal muscle mass and function are regulated by motor innervation, and denervation results in muscle atrophy. The activity of mammalian target of rapamycin complex 1 (mTORC1) is substantially increased in denervated muscle, but its regulatory role in denervation-induced atrophy remains unclear. At early stages after denervation of skeletal muscle, a pathway involving class II histone deacetylases and the transcription factor myogenin mediates denervation-induced muscle atrophy. We found that at later stages after denervation of fast-twitch muscle, activation of mTORC1 contributed to atrophy and that denervation-induced atrophy was mitigated by inhibition of mTORC1 with rapamycin. Activation of mTORC1 through genetic deletion of its inhibitor TSC1 (tuberous sclerosis complex 1) sensitized mice to denervation-induced muscle atrophy and suppressed the kinase activity of Akt, leading to activation of FoxO transcription factors and increasing the expression of genes encoding E3 ubiquitin ligases atrogin [also known as MAFbx (muscle atrophy F-box protein)] and MuRF1 (muscle-specific ring finger 1). Rapamycin treatment of mice restored Akt activity, suggesting that the denervation-induced increase in mTORC1 activity was producing feedback inhibition of Akt. Genetic deletion of the three FoxO isoforms in skeletal muscle induced muscle hypertrophy and abolished the late-stage induction of E3 ubiquitin ligases after denervation, thereby preventing denervation-induced atrophy. These data revealed that mTORC1, which is generally considered to be an important component of anabolism, is central to muscle catabolism and atrophy after denervation. This mTORC1-FoxO axis represents a potential therapeutic target in neurogenic muscle atrophy.

Diaphragm muscle atrophy in the mouse after long-term mechanical ventilation.

INTRODUCTION: Mechanical ventilation (MV) is a life-saving measure, but full ventilator support causes ventilator-induced diaphragm atrophy (VIDA). Previous studies of VIDA have relied on human biopsies or a rat model. If MV can induce diaphragm atrophy in mice, then mechanistic study of VIDA could be explored via genetic manipulation. RESULTS: We show that 18 hours of MV in mice results in a 15% loss of diaphragm weight and a 17% reduction in fiber cross-sectional area. Important catabolic cascades are activated in this mouse model: transcription of the ubiquitin ligases, atrogin and MuRF1, and the apoptotic marker, Bim, are increased; the marker of autophagy, LC3, is induced at the protein level and shows a punctate distribution in diaphragm muscle fibers. CONCLUSIONS: This mouse model recapitulates the key pathophysiological findings of other models of VIDA, and it will enable the genetic manipulation required to fully explore the mechanisms underlying this important process.

[Comparison between pavlik harness and bryant traction for femoral shaft fractures in infants].

OBJECTIVE: To compare clinical effects between Pavlik harness and Bryant traction in treating femoral shaft fractures in infants,including the time of hospitalization, expense of treatment, complications,time of bone union. METHODS: From May 2005 to August 2010,the clinical data of 42 infants with femoral shaft fractures were retrospectively analyzed. Among the patients, 23 cases were treated with Pavlik harness(Pavlik harness group),there were 14 males and 9 females,ranging in age from 1 to 12 months with an average of (5.5+/-2.4) months,including upper 1/3 segment of 16 cases and middle segment of 7 cases; transverse fracture of 18 cases and oblique fracture of 5 cases. The other 19 patients were treated with Bryant traction (Bryant traction group),there were 15 males and 4 females,ranging in age from 2 to 12 months with an average of (6.7+/-2.8) months,including upper 1/3 segment of 13 cases and middle segment of 6 cases;transverse fracture of 12 cases and oblique fracture of 7 cases. The time of hospitalization,expense of treatment,complications,time of bone union were analyzed in the patients. RESULTS: All patients were followed up with an average of 25.3 months (ranging from 19 to 30) in Pavlik harness group and 23.7 months (ranging from 17 to 28) in Bryant traction group. Time of hospitalization, expense of treatment in Pavlik harness group were respectively (0.4+/-0.7) d, (2147.7+/-64.9) yuan; and in Bryant traction group were respectively(27.1+/-2.2) d, (2741.3+/-227.6) yuan;there was significant difference between two groups(P<0.05). No complication was found in Pavlik harness group and 8 cases complicated with skin hydroa in Bryant traction group, there was significant difference between two groups (P<0.05). Time of bone union,difference of both lower extremities in Pavlik harness group were respectively (4.1+/-0.3)weeks, (6.3+/-4.1) mm;and in Bryant traction group were respectively (3.9+/-0.3) weeks, (7.6 +/-4.3) mm; 20 cases got bone healing in Pavlik harness group and 18 cases got bone healing in Bryant traction group;there was no significant difference between two groups (P>0.05). CONCLUSION: Compared with Bryant traction method,Pavlik harness method has obvious advantages in time of hospitalization, expense of treatment, complications in treating femoral shaft fractures in infants.

Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems.

Glycolysis is the initial step of glucose catabolism and is up-regulated in cancer cells (the Warburg Effect). Such shifts toward a glycolytic phenotype have not been explored widely in other biological systems, and the molecular mechanisms underlying the shifts remain unknown. With proteomics, we observed increased glycolysis in disused human diaphragm muscle. In disused muscle, lung cancer, and H(2)O(2)-treated myotubes, we show up-regulation of the rate-limiting glycolytic enzyme muscle-type phosphofructokinase (PFKm, >2 fold, P<0.05) and accumulation of lactate (>150%, P<0.05). Using microRNA profiling, we identify miR-320a as a regulator of PFKm expression. Reduced miR-320a levels (to ∼50% of control, P<0.05) are associated with the increased PFKm in each of these diverse systems. Manipulation of miR-320a levels both in vitro and in vivo alters PFKm and lactate levels in the expected directions. Further, miR-320a appears to regulate oxidative stress-induced PFKm expression, and reduced miR-320a allows greater induction of glycolysis in response to H(2)O(2) treatment. We show that this microRNA-mediated regulation occurs through PFKm's 3' untranslated region and that Ets proteins are involved in the regulation of PFKm via miR-320a. These findings suggest that oxidative stress-responsive microRNA-320a may regulate glycolysis broadly within nature.

Intrinsic apoptosis in mechanically ventilated human diaphragm: linkage to a novel Fos/FoxO1/Stat3-Bim axis.

Mechanical ventilation (MV) is a life-saving measure in many critically ill patients. However, prolonged MV results in diaphragm dysfunction that contributes to the frequent difficulty in weaning patients from the ventilator. The molecular mechanisms underlying ventilator-induced diaphragm dysfunction (VIDD) remain poorly understood. We report here that MV induces myonuclear DNA fragmentation (3-fold increase; P<0.01) and selective activation of caspase 9 (P<0.05) and Bcl2-interacting mediator of cell death (Bim; 2- to 7-fold increase; P<0.05) in human diaphragm. MV also statistically significantly down-regulates mitochondrial gene expression and induces oxidative stress. In cultured muscle cells, we show that oxidative stress activates each of the catabolic pathways thought to underlie VIDD: apoptotic (P<0.05), proteasomal (P<0.05), and autophagic (P<0.01). Further, silencing Bim expression blocks (P<0.05) oxidative stress-induced apoptosis. Overlapping the gene expression profiles of MV human diaphragm and H2O2-treated muscle cells, we identify Fos, FoxO1, and Stat3 as regulators of Bim expression as well as of expression of the catabolic markers atrogin and LC3. We thus identify a novel Fos/FoxO1/Stat3-Bim intrinsic apoptotic pathway and establish the centrality of oxidative stress in the development of VIDD. This information may help in the design of specific drugs to prevent this condition.

A histone deacetylase 4/myogenin positive feedback loop coordinates denervation-dependent gene induction and suppression.

Muscle activity contributes to formation of the neuromuscular junction and affects muscle metabolism and contractile properties through regulated gene expression. However, the mechanisms coordinating these diverse activity-regulated processes remain poorly characterized. Recently, it was reported that histone deacetylase 4 (HDAC4) can mediate denervation-induced myogenin and nicotinic acetylcholine receptor gene expression. Here, we report that HDAC4 is not only necessary for denervation-dependent induction of genes involved in synaptogenesis (nicotinic acetylcholine receptor and muscle-specific receptor tyrosine kinase) but also for denervation-dependent suppression of genes involved in glycolysis (muscle-specific enolase and phosphofructokinase). In addition, HDAC4 differentially regulates genes involved in muscle fiber type specification by inducing myosin heavy chain IIA and suppressing myosin heavy chain IIB. Consistent with these regulated gene profiles, HDAC4 is enriched in fast oxidative fibers of innervated tibialis anterior muscle and HDAC4 knockdown enhances glycolysis in cultured myotubes. HDAC4 mediates gene induction indirectly by suppressing the expression of Dach2 and MITR that function as myogenin gene corepressors. In contrast, HDAC4 is directly recruited to myocyte enhancer factor 2 sites within target promoters to mediate gene suppression. Finally, we discovered an HDAC4/myogenin positive feedback loop that coordinates gene induction and repression underlying muscle phenotypic changes after muscle denervation.

Activity-dependent gene regulation in skeletal muscle is mediated by a histone deacetylase (HDAC)-Dach2-myogenin signal transduction cascade.

Muscle activity contributes to muscle development and function largely by means of regulated gene expression. Many genes crucial to neuromuscular synapse formation, such as MuSK and nAChRs, are induced before muscle innervation or after muscle denervation, and this induction requires expression of the E-box binding, basic helix-loop-helix muscle-specific transcription factor, myogenin (Mgn). The mechanism by which muscle activity is coupled to gene expression is poorly defined. Here we report that inhibition of histone deacetylase (HDAC) activity attenuates the induction of activity-regulated genes in aneural myotubes and adult denervated muscle. The effect of HDAC inhibitors requires new protein synthesis, suggesting HDACs may regulate the expression of a Mgn transcriptional repressor. We identified Dach2 as a Mgn transcriptional repressor whose expression is dramatically reduced in an HDAC-dependent manner in developing aneural myotubes or adult denervated muscle. Dach2 overexpression in denervated muscle suppressed Mgn, nAChR, and MuSK gene induction, whereas Dach2 knockdown induced Mgn gene expression in innervated muscle and relieved Mgn promoter inhibition by HDAC inhibitors. Thus, a HDAC-Dach2-myogenin signaling pathway has been identified to decode nerve activity and control muscle gene expression in developing and adult skeletal muscle.