INAUGURAL ARTICLE by a Recently Elected Academy Member:Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A

Myostatin (MSTN) is a transforming growth factor-β (TGF-β) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-β family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis.

Myostatin (MSTN) is a secreted signaling molecule that normally acts to limit skeletal muscle growth (for review, see ref. 1). Mice lacking MSTN exhibit dramatic increases in muscle mass throughout the body, with individual muscles growing to about twice the normal size (2). MSTN appears to play two distinct roles in regulating muscle size, one to regulate the number of muscle fibers that are formed during development and a second to regulate the growth of those fibers postnatally. The sequence of MSTN has been highly conserved through evolution, with the mature MSTN peptide being identical in species as divergent as humans and turkeys (3). The function of MSTN has also been conserved, and targeted or naturally occurring mutations in MSTN have been shown to cause increased muscling in numerous species, including cattle (3–5), sheep (6), dogs (7), rabbits (8), rats (9), swine (10), goats (11), and humans (12). Numerous pharmaceutical and biotechnology companies have developed biologic agents capable of blocking MSTN activity, and these have been tested in clinical trials for a wide range of indications, including Duchenne and facioscapulohumeral muscular dystrophy, inclusion body myositis, muscle atrophy following falls and hip fracture surgery, age-related sarcopenia, Charcot–Marie–Tooth disease, and cachexia due to chronic obstructive pulmonary disease, end-stage kidney disease, and cancer.The finding that certain inhibitors of MSTN signaling can increase muscle mass even in Mstn^−/− mice revealed that the function of MSTN as a negative regulator of muscle mass is partially redundant with at least one other TGF-β family member (13, 14), and subsequent studies have identified activin A as one of these cooperating ligands (15, 16). MSTN and activin A share many key regulatory and signaling components. For example, the activities of both MSTN and activin A can be modulated extracellularly by naturally occurring inhibitory binding proteins, including follistatin (17, 18) and the follistatin-related protein, FSTL-3 or FLRG (19, 20). Moreover, MSTN and activin A also appear to share receptor components. Based on in vitro studies, MSTN is capable of binding initially to the activin type II receptors, ACVR2 and ACVR2B (also called ActRIIA and ActRIIB) (18) followed by engagement of the type I receptors, ALK4 and ALK5 (21). In previous studies, we presented genetic evidence supporting a role for both ACVR2 and ACVR2B in mediating MSTN signaling and regulating muscle mass in vivo. Specifically, we showed that mice expressing a truncated, dominant-negative form of ACVR2B in skeletal muscle (18) or carrying deletion mutations in Acvr2 and/or Acvr2b (13) have significantly increased muscle mass. One limitation of the latter study, however, was that we could not examine the consequence of complete loss of both receptors using the deletion alleles, as double homozygous mutants die early during embryogenesis (22). Moreover, the roles that the two type I receptors, ALK4 and ALK5, play in regulating MSTN and activin A signaling in muscle in vivo have not yet been documented using genetic approaches. Here, we present the results of studies in which we used floxed alleles for each of the type II and type I receptor genes in order to target these receptors alone and in combination in muscle fibers. We show that these receptors are functionally redundant and that signaling through each of these receptors contributes to the overall control of muscle mass.     

299名健康男童经鼻吸气压力调查

目的调查中国健康男性儿童经鼻吸气压力(SNIP)的范围。方法对299名5~12岁健康男童进行SNIP测量。记录每名参与者的身高、体重、出生日期及测试时间。结果 299名参与者SNIP的平均值为(76.9±22.6)cm H2O。5~6岁、7~8岁、9~10岁、11~12岁SNIP的平均值分别为(59.1±14.2)cm H2O、(76.9±19.5)cm H2O、(85.3±23.5)cm H2O、(83.0±22.6)cm H2O。5~6岁、7~8岁及9~10岁间SNIP有显著性差异(P0.05),9~10岁与11~12岁间SNIP无显著性差异(P=0.55)。SNIP与年龄、身高、体重及体质量指数间存在相关性(P0.01)。结论中国健康男童SNIP参考值与国外存在一定差异,有必要建立中国儿童SNIP正常参考值范围。     

The Spinal Control of Backward Locomotion

Animal locomotion requires changing direction, from forward to backward. Here, we tested the hypothesis that sensorimotor circuits within the spinal cord generate backward locomotion and adjust it to task demands. We collected kinematic and electromyography (EMG) data during forward and backward locomotion at different treadmill speeds before and after complete spinal transection in six adult cats (three males and three females). After spinal transection, five/six cats performed backward locomotion, which required tonic somatosensory input in the form of perineal stimulation. One spinal cat performed forward locomotion but not backward locomotion while two others stepped backward but not forward. Spatiotemporal adjustments to increasing speed were similar in intact and spinal cats during backward locomotion and strategies were similar to forward locomotion, with shorter cycle and stance durations and longer stride lengths. Patterns of muscle activations, including muscle synergies, were similar for forward and backward locomotion in spinal cats. Indeed, we identified five muscle synergies that were similar during forward and backward locomotion. Lastly, spinal cats also stepped backward on a split-belt treadmill, with the left and right hindlimbs stepping at different speeds. Therefore, our results show that spinal sensorimotor circuits generate backward locomotion but require additional excitability compared with forward locomotion. Similar strategies for speed modulation and similar patterns of muscle activations and muscle synergies during forward and backward locomotion are consistent with a shared spinal locomotor network, with sensory feedback from the limbs controlling the direction.SIGNIFICANCE STATEMENT Animal locomotion requires changing direction, including forward, sideways and backward. This paper shows that the center controlling locomotion within the spinal cord can produce a backward pattern when instructed by sensory signals from the limbs. However, the spinal locomotor network requires greater excitability to produce backward locomotion compared with forward locomotion. The paper also shows that the spinal network controlling locomotion in the forward direction also controls locomotion in the backward direction.     

超声引导下腹横肌平面阻滞在腹腔镜下胆囊切除术术后镇痛的疗效与评价

目的 探讨超声引导下腹横肌平面阻滞在腹腔镜下胆囊切除术术后镇痛的疗效与评价。方法 将2018年1月～2019年3月在我院肝胆外科行腹腔镜下胆囊切除术的60例患者随机分为两组,对照组使用气管插管全身麻醉,观察组使用气管插管全身麻醉联合超声引导下腹横肌平面阻滞,比较两组患者术后静息及咳嗽时的疼痛情况、术后镇静程度、不同时间点血压及心率变化、不良反应情况。结果 在静息及咳嗽状态下,观察组出PACU时、术后4 h、术后24 h的VAS疼痛评分均明显低于对照组(P0.05);观察组入PACU后5 min、10 min、15 min的镇静评分低于对照组(P0.05),而入PACU后30 min的镇静评分无明显差异(P0.05);两组在术中及入PACU后各时间点的MAP无明显差异(P0.05),观察组在入PACU 10 min的心率慢于对照组(P0.05),其他时间点无明显差异(P0.05);观察组术后头痛、头晕、嗜睡、恶心呕吐、皮肤瘙痒、颤抖等不良反应发生率与对照组相比无明显差异(P0.05)。结论 超声引导下腹横肌平面阻滞在腹腔镜下胆囊切除术术后镇痛的疗效显著,对血流动力学影响小,安全性高,具有积极的临床意义。     

Live-imaging of revertant and therapeutically restored dystrophin in the DmdEGFP-mdx mouse model for Duchenne muscular dystrophy

Background

Dmd^mdx, harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies.

Methods

We generated a Dmd^EGFP-mdx reporter allele carrying in cis the mdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 23 excision.

Results

Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx-23 mutation.

Conclusion

Intravital imaging of Dmd^EGFP-mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.