Muscle-specific Drp1 overexpression impairs skeletal muscle growth via translational attenuation

Mitochondrial fission and fusion are essential processes in the maintenance of the skeletal muscle function. The contribution of these processes to muscle development has not been properly investigated in vivo because of the early lethality of the models generated so far. To define the role of mitochondrial fission in muscle development and repair, we have generated a transgenic mouse line that overexpresses the fission-inducing protein Drp1 specifically in skeletal muscle. These mice displayed a drastic impairment in postnatal muscle growth, with reorganisation of the mitochondrial network and reduction of mtDNA quantity, without the deficiency of mitochondrial bioenergetics. Importantly we found that Drp1 overexpression activates the stress-induced PKR/eIF2α/Fgf21 pathway thus leading to an attenuated protein synthesis and downregulation of the growth hormone pathway. These results reveal for the first time how mitochondrial network dynamics influence muscle growth and shed light on aspects of muscle physiology relevant in human muscle pathologies.Skeletal muscle growth and mitochondrial metabolism are intimately linked. In myogenic precursor cells, mitochondrial mass, mtDNA copy number and mitochondrial respiration increase after the onset of myogenic differentiation;^{1, 2} furthermore, postnatal development of fast-twitch muscle is accompanied by an increase in mtDNA copy number³ and muscle regeneration is impaired when mitochondrial protein synthesis is inhibited with chloramphenicol.^{2, 4} These observations suggest that a change in the mitochondrial metabolism is necessary for proper muscle development. During myogenesis and postnatal development, the shape of mitochondria is also remodelled:^{3, 5, 6} in an elegant mouse model with fluorescent mitochondria it was shown that in young mice mitochondria of the extensor digitorum longus (EDL) muscle are shaped as elongated tubules oriented along the long axis of the muscle fibre, whereas in adult mice mitochondria are punctuated and organised into doublets.¹Mitochondrial network morphology is controlled by the balance between fusion and fission. In mammals, three large GTPases are involved in mitochondrial fusion: mitofusins 1 and 2 (Mfn1 and Mfn2) participate in the early steps of mitochondrial outer-membrane fusion, whereas the optic atrophy 1 protein (Opa1) is essential for inner-membrane fusion.⁷ Mitochondrial fission is mediated by the evolutionarily conserved dynamin-related protein 1 (Drp1).⁸ In humans, mutations in Mfn2 and Opa1 cause two neurodegenerative diseases – Charcot–Marie–Tooth type 2 A and dominant optic atrophy, respectively – and a mutation in Drp1 has been linked to neonatal lethality with multisystem failure.^{9, 10, 11} Moreover, Drp1 expression was reported to increase in a model of cachexia¹² and to contribute to muscle insulin resistance in obese and type 2 diabetic mice.^{13, 14}The importance of mitochondrial dynamics in muscle physiology has become increasingly clear. In skeletal muscle, mitochondria undergo fusion to share matrix content in order to support excitation–contraction coupling.¹⁵ The mitochondrial network is remodelled in atrophic conditions, and denervation and expression of fission machinery in adult myofibres is sufficient to cause muscle wasting.¹⁶ Moreover, mice lacking Mfn1 and 2 in fast-twitch muscles exhibit drastic growth defects and muscle atrophy before dying at 6–8 weeks of age.³ Animal models in which mitochondrial fission proteins are manipulated during skeletal muscle development are not yet available, but in vitro data demonstrate that regulation of Drp1 is critical for myogenesis: myoblasts differentiation requires nitric oxide-dependent inhibition of Drp1⁶ and pharmacological inhibition of Drp1 activity impairs myogenic differentiation.¹⁷To explore in vivo the role of Drp1 and mitochondrial shape in the developing muscle, we generated a transgenic mouse line specifically overexpressing Drp1 in skeletal muscle during myogenesis. These mice display strong impairments in mitochondrial network shape and in muscle growth. We show that the mechanism responsible for the growth defect involves inhibition of protein synthesis and activation of the Atf4 pathway.     

Myocyte-derived Tnfsf14 is a survival factor necessary for myoblast differentiation and skeletal muscle regeneration

Adult skeletal muscle tissue has a uniquely robust capacity for regeneration, which gradually declines with aging or is compromised in muscle diseases. The cellular mechanisms regulating adult myogenesis remain incompletely understood. Here we identify the cytokine tumor necrosis factor superfamily member 14 (Tnfsf14) as a positive regulator of myoblast differentiation in culture and muscle regeneration in vivo. We find that Tnfsf14, as well as its cognate receptors herpes virus entry mediator (HVEM) and lymphotoxin β receptor (LTβR), are expressed in both differentiating myocytes and regenerating myofibers. Depletion of Tnfsf14 or either receptor inhibits myoblast differentiation and promotes apoptosis. Our results also suggest that Tnfsf14 regulates myogenesis by supporting cell survival and maintaining a sufficient pool of cells for fusion. In addition, we show that Akt mediates the survival and myogenic function of Tnfsf14. Importantly, local knockdown of Tnfsf14 is found to impair injury-induced muscle regeneration in a mouse model, affirming an important physiological role for Tnfsf14 in myogenesis in vivo. Furthermore, we demonstrate that localized overexpression of Tnfsf14 potently enhances muscle regeneration, and that this regenerative capacity of Tnfsf14 is dependent on Akt signaling. Taken together, our findings reveal a novel regulator of skeletal myogenesis and implicate Tnfsf14 in future therapeutic development.Mature skeletal muscle tissue contains a resident population of stem cells that imparts a great capacity for regeneration. Upon injury, these quiescent satellite cells are reactivated and begin to proliferate.^{1, 2} Effective myogenesis depends on the daughter myoblasts successfully differentiating and fusing with each other to regenerate the characteristic multinucleated skeletal myofibers. This involves a number of highly regulated steps, including activation of myogenic genes, migration, cell–cell adhesion and alignment, and finally membrane fusion.^{3, 4, 5} The fundamental principles underlying each step are well-conserved across species.⁶ Pathologies may result from dysregulation of these processes, including the suite of muscular dystrophies, cachexia and sarcopenia. However, the complex signaling mechanisms underlying skeletal myogenesis are still not fully understood.It has long been accepted that the secreted factors influencing muscle cell regeneration in vivo are largely of immune cell origin; indeed, immune cells have been reported to reach concentrations over 100 000 cells/mm³ in regenerating muscle tissue.⁷ Recently, however, muscle cells are being revealed as prolific secretors of a wide variety of cytokines and growth factors,^{8, 9, 10, 11} including several that attract immune cells to regenerating muscle.⁷ Secretome studies show that myoblasts secrete different factors during proliferation than during differentiation, and even at different time points throughout differentiation.^{8, 10, 11} Another study identified numerous chemokine mRNAs expressed by differentiating myoblasts, which may be involved in regulating cell migration during myogenesis.⁹ However, functions of the newly identified muscle-secreted cytokines are mostly unexplored. Using RNAi, we conducted the first functional screen of cytokines for their impact on myogenic differentiation in C2C12 myoblasts, which allowed us to identify potential regulators of myogenesis in distinct functional groups.¹² These results suggest the intriguing possibility that muscle cell-secreted proteins have a previously under-appreciated role in modulating muscle development and regeneration.The function of cytokines in myogenesis is relevant to our understanding of not only basic muscle physiology, but also the diseases that negatively affect the health of muscle tissue, such as cachexia. Cachexia is characterized by extreme wasting of lean body mass and often occurs with an underlying chronic illness, such as cancer or congestive cardiac failure.¹³ Muscle atrophy during cachexic states ultimately stems from ubiquitin-mediated breakdown of myofibrils.¹⁴ Significantly, a well-documented association exists between cachexia and the dysregulation of cytokines, most notably the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin-1 (IL-1) and interleukin-6 (IL-6).^{14, 15}Tumor necrosis factor superfamily member 14 (Tnfsf14), also known as LIGHT (homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes), exists in two main forms: a type II transmembrane glycoprotein that projects extracellularly, and a soluble cytokine formed by cleavage of the extracellular portion of the protein off of the cell membrane.¹⁶ Through its receptors in the TNF receptor (TNFR) superfamily, HVEM (TNFRSF14) and lymphotoxin β receptor (LTβR), Tnfsf14 signaling is involved in lymphoid organ development and organization, as well as innate and adaptive immune responses.^{17, 18, 19} In recent years, Tnfsf14 has also emerged as a promising candidate for cancer immunotherapy.²⁰Tnfsf14 regulates cell survival and apoptosis in lymphocytes and tumor cells, and the cellular context determines whether Tnfsf14 is pro-survival or pro-apoptosis.^{20, 21, 22} Neither the expression nor the function of Tnfsf14 or its receptors has been reported in skeletal muscles. Our current study uncovers Tnfsf14 as a critical regulator of myoblast differentiation and muscle regeneration by governing myoblast survival, and implicates Tnfsf14 in potential therapeutic development for maintenance of muscle health.