Transcriptional and cytopathological hallmarks of FSHD in chronic DUX4-expressing mice.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by loss of repression of the DUX4 gene; however, the DUX4 protein is rare and difficult to detect in human muscle biopsies, and pathological mechanisms are obscure. FSHD is also a chronic disease that progresses slowly over decades. We used the sporadic, low-level, muscle-specific expression of DUX4 enabled by the iDUX4pA-HSA mouse to develop a chronic long-term muscle disease model. After 6 months of extremely low sporadic DUX4 expression, dystrophic muscle presented hallmarks of FSHD histopathology, including muscle degeneration, capillary loss, fibrosis, and atrophy. We investigated the transcriptional profile of whole muscle as well as endothelial cells and fibroadiopogenic progenitors (FAPs). Strikingly, differential gene expression profiles of both whole muscle and, to a lesser extent, FAPs, showed significant overlap with transcriptional profiles of MRI-guided human FSHD muscle biopsies. These results demonstrate a pathophysiological similarity between disease in muscles of iDUX4pA-HSA mice and humans with FSHD, solidifying the value of chronic rare DUX4 expression in mice for modeling pathological mechanisms in FSHD and highlighting the importance FAPs in this disease.

A novel P300 inhibitor reverses DUX4-mediated global histone H3 hyperacetylation, target gene expression, and cell death.

Facioscapulohumeral muscular dystrophy (FSHD) results from mutations causing overexpression of the transcription factor, DUX4, which interacts with the histone acetyltransferases, EP300 and CBP. We describe the activity of a new spirocyclic EP300/CBP inhibitor, iP300w, which inhibits the cytotoxicity of the DUX4 protein and reverses the overexpression of most DUX4 target genes, in engineered cell lines and FSHD myoblasts, as well as in an FSHD animal model. In evaluating the effect of iP300w on global histone H3 acetylation, we discovered that DUX4 overexpression leads to a dramatic global increase in the total amount of acetylated histone H3. This unexpected effect requires the C-terminus of DUX4, is conserved with mouse Dux, and may facilitate zygotic genome activation. This global increase in histone H3 acetylation is reversed by iP300w, highlighting the central role of EP300 and CBP in the transcriptional mechanism underlying DUX4 cytotoxicity and the translational potential of blocking this interaction.

BGP-15 improves contractile function of regenerating soleus muscle.

This study investigated the effect of the heat shock protein inducer O-[3-piperidino-2-hydroxy-1-propyl]-nicotinic amidoxime (BGP-15) on the morphology and contractile function of regenerating soleus muscles from mice. Cryolesioned soleus muscles from young mice treated daily with BGP-15 (15 mg/Kg) were evaluated on post-cryolesion day 10. At this time point, there was a significant decrease in the cross-sectional area of regenerating myofibers, maximal force, specific tetanic force, and fatigue resistance of regenerating soleus muscles. BGP-15 did not reverse the decrease in myofiber cross-sectional area but effectively prevented the reduction in tetanic force and fatigue resistance of regenerating muscles. In addition, BGP-15 treatment increased the expression of embryonic myosin heavy chain (e-MyHC), MyHC-II and MyHC-I in regenerating muscles. Although BGP-15 did not alter voltage dependent anion-selective channel 2 (VDAC2) expression in cryolesioned muscles, it was able to increase inducible 70-kDa heat shock protein (HSP70) expression. Our results suggest that BGP-15 improves strength recovery in regenerating soleus muscles by accelerating the re-expression of adult MyHC-II and MyHC-I isoforms and HSP70 induction. The beneficial effects of BGP-15 on the contractile function of regenerating muscles reinforce the potential of this molecule to be used as a therapeutic agent.

ß2-Adrenoceptor is involved in connective tissue remodeling in regenerating muscles by decreasing the activity of MMP-9.

We investigated the role of ß2-adrenoceptors in the connective tissue remodeling of regenerating muscles from ß2-adrenoceptor knockout (ß2KO) mice. Tibialis anterior muscles from ß2KO mice were cryolesioned and analyzed after 3, 10, and 21 days. Regenerating muscles from ß2KO mice showed a significant increase in the area density of the connective tissue and in the amount of collagen at 10 days compared with wild-type (WT) mice. A greater increase occurred in the expression levels of collagen I, III, and IV in regenerating muscles from ß2KO mice evaluated at 10 days compared with WT mice; this increase continued at 21 days, except for collagen III. Matrix metalloproteinase (MMP-2) activity increased to a similar extent in regenerating muscles from both ß2KO and WT mice at 3 and 10 days. This was also the case for MMP-9 activity in regenerating muscles from both ß2KO and WT mice at 3 days; however, at 10 days post-cryolesion, this activity returned to baseline levels only in WT mice. MMP-3 activity was unaltered in regenerating muscles at 10 days. mRNA levels of tumor necrosis factor-α increased in regenerating muscles from WT and ß2KO mice at 3 days and, at 10 days post-cryolesion, returned to baseline only in WT mice. mRNA levels of interleukin-6 increased in muscles from WT mice at 3 days post-cryolesion and returned to baseline at 10 days post-cryolesion but were unchanged in ß2KO mice. Our results suggest that the ß2-adrenoceptor contributes to collagen remodeling during muscle regeneration by decreasing MMP-9 activity.

Leucine supplementation improves regeneration of skeletal muscles from old rats.

The decreased regenerative capacity of old skeletal muscles involves disrupted turnover of proteins. This study investigated whether leucine supplementation in old rats could improve muscle regenerative capacity. Young and old male Wistar rats were supplemented with leucine; then, the muscles were cryolesioned and examined after 3 and 10 days. Leucine supplementation attenuated the decrease in the expression of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4E (eIF4E) in young and old muscles on day 3 post-injury and promoted an increase in the cross-sectional area of regenerating myofibers from both young and old soleus muscles on day 10 post-injury. This supplementation decreased the levels of ubiquitinated proteins and increased the proteasome activity in young regenerating muscles, but the opposite effect was observed in old regenerating muscles. Moreover, leucine decreased the inflammation area and induced an increase in the number of proliferating satellite cells in both young and old muscles. Our results suggest that leucine supplementation improves the regeneration of skeletal muscles from old rats, through the preservation of certain biological responses upon leucine supplementation. Such responses comprise the decrease in the inflammation area, increase in the number of proliferating satellite cells and size of regenerating myofibers, combined with the modulation of components of the phosphoinositide 3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway and ubiquitin-proteasome system.

Leucine supplementation accelerates connective tissue repair of injured tibialis anterior muscle.

This study investigated the effect of leucine supplementation on the skeletal muscle regenerative process, focusing on the remodeling of connective tissue of the fast twitch muscle tibialis anterior (TA). Young male Wistar rats were supplemented with leucine (1.35 g/kg per day); then, TA muscles from the left hind limb were cryolesioned and examined after 10 days. Although leucine supplementation induced increased protein synthesis, it was not sufficient to promote an increase in the cross-sectional area (CSA) of regenerating myofibers (p > 0.05) from TA muscles. However, leucine supplementation reduced the amount of collagen and the activation of phosphorylated transforming growth factor-ß receptor type I (TßR-I) and Smad2/3 in regenerating muscles (p < 0.05). Leucine also reduced neonatal myosin heavy chain (MyHC-n) (p < 0.05), increased adult MyHC-II expression (p < 0.05) and prevented the decrease in maximum tetanic strength in regenerating TA muscles (p < 0.05). Our results suggest that leucine supplementation accelerates connective tissue repair and consequent function of regenerating TA through the attenuation of TßR-I and Smad2/3 activation. Therefore, future studies are warranted to investigate leucine supplementation as a nutritional strategy to prevent or attenuate muscle fibrosis in patients with several muscle diseases.

The ß2-adrenoceptor agonist formoterol improves structural and functional regenerative capacity of skeletal muscles from aged rat at the early stages of postinjury.

Skeletal muscles from old rats fail to completely regenerate following injury. This study investigated whether pharmacological stimulation of ß2-adrenoceptors in aged muscles following injury could improve their regenerative capacity, focusing on myofiber size recovery. Young and aged rats were treated with a subcutaneous injection of ß2-adrenergic agonist formoterol (2 µg/kg/d) up to 10 and 21 days after soleus muscle injury. Formoterol-treated muscles from old rats evaluated at 10 and 21 days postinjury showed reduced inflammation and connective tissue but a similar number of regenerating myofibers of greater caliber when compared with their injured controls. Formoterol minimized the decrease in tetanic force and increased protein synthesis and mammalian target of rapamycin phosphorylation in old muscles at 10 days postinjury. Our results suggest that formoterol improves structural and functional regenerative capacity of regenerating skeletal muscles from aged rats by increasing protein synthesis via mammalian target of rapamycin activation. Furthermore, formoterol may have therapeutic benefits in recovery following muscle damage in senescent individuals.

GaAs 904-nm laser irradiation improves myofiber mass recovery during regeneration of skeletal muscle previously damaged by crotoxin.

This work investigated the effect of gallium arsenide (GaAs) irradiation (power: 5 mW; intensity: 77.14 mW/cm(2), spot: 0.07 cm(2)) on regenerating skeletal muscles damaged by crotoxin (CTX). Male C57Bl6 mice were divided into six groups (n = 5 each): control, treated only with laser at doses of 1.5 J or 3 J, CTX-injured and, CTX-injured and treated with laser at doses of 1.5 J or 3 J. The injured groups received a CTX injection into the tibialis anterior (TA) muscle. After 3 days, TA muscles were submitted to GaAs irradiation at doses of 1.5 or 3 J (once a day, during 5 days) and were killed on the eighth day. Muscle histological sections were stained with hematoxylin and eosin (H&E) in order to determine the myofiber cross-sectional area (CSA), the previously injured muscle area (PIMA) and the area density of connective tissue. The gene expression of MyoD and myogenin was detected by real-time PCR. GaAs laser at a dose of 3 J, but not 1.5 J, significantly increased the CSA of regenerating myofibers and reduced the PIMA and the area density of intramuscular connective tissue of CTX-injured muscles. MyoD gene expression increased in the injured group treated with GaAs laser at a dose of 1.5 J. The CTX-injured, 3-J GaAs laser-treated, and the CTX-injured and treated with 3-J laser groups showed an increase in myogenin gene expression when compared to the control group. Our results suggest that GaAs laser treatment at a dose of 3 J improves skeletal muscle regeneration by accelerating the recovery of myofiber mass.

High-voltage electrical stimulation improves nerve regeneration after sciatic crush injury.

BACKGROUND: Peripheral nerve injury causes prolonged functional limitation being a clinical challenge to identify resources that accelerates its recovery. OBJECTIVES: To investigate the effect of high-voltage electrical stimulation (HVES) on the morphometric and functional characteristics of the regenerated nerve after crush injury in rats. METHODS: Twenty Wistar rats were randomly allocated into 4 groups: Control (CON) - without injury and without HVES; Denervated (D) - sciatic nerve crush only; Denervated + HVES - sciatic nerve crush and HVES; SHAM - without injury but HVES. The HVES and SHAM groups were stimulated (100 Hz; minimum voltage of 100 V, 20 µs, 100 µs interpulse interval) for 30 min/day, 5 days/week. The sciatic functional index (SFI) was evaluated before the injury and at the 7th, 14th and 21st postoperatory (PO) days. Neural components and the area density of connective tissue, blood vessels and macrophages were analyzed. RESULTS: Axonal diameter was higher on the HVES than on D group, reaching almost 80% above the control values after 21 days (p<0.05). Fiber diameter and myelin sheath thickness were higher on the HVES than on D group (p<0.05) reaching 96.5% and 100% of the control values, respectively. Functional recovery at the 14th PO day was better on group HVES. The macrophages and connective tissue area density was lower on the HVES group, while blood vessels number did not differ among groups. CONCLUSIONS: The HVES accelerated the functional recovery, potentiated the nerve fibers maturation and decreased macrophages and connective tissue area density, suggesting acceleration of neural repair.

High-voltage electrical stimulation improves nerve regeneration after sciatic crush injury / Estimulação elétrica de alta voltagem favorece a regeneração nervosa após compressão do nervo isquiático

BACKGROUND: Peripheral nerve injury causes prolonged functional limitation being a clinical challenge to identify resources that accelerates its recovery. OBJECTIVES: To investigate the effect of high-voltage electrical stimulation (HVES) on the morphometric and functional characteristics of the regenerated nerve after crush injury in rats. METHODS: Twenty Wistar rats were randomly allocated into 4 groups: Control (CON) - without injury and without HVES; Denervated (D) - sciatic nerve crush only; Denervated + HVES - sciatic nerve crush and HVES; SHAM - without injury but HVES. The HVES and SHAM groups were stimulated (100 Hz; minimum voltage of 100 V, 20 μs, 100 μs interpulse interval) for 30 min/day, 5 days/week. The sciatic functional index (SFI) was evaluated before the injury and at the 7th, 14th and 21st postoperatory (PO) days. Neural components and the area density of connective tissue, blood vessels and macrophages were analyzed. RESULTS: Axonal diameter was higher on the HVES than on D group, reaching almost 80 percent above the control values after 21 days (p<0.05). Fiber diameter and myelin sheath thickness were higher on the HVES than on D group (p<0.05) reaching 96.5 percent and 100 percent of the control values, respectively. Functional recovery at the 14th PO day was better on group HVES. The macrophages and connective tissue area density was lower on the HVES group, while blood vessels number did not differ among groups. CONCLUSIONS: The HVES accelerated the functional recovery, potentiated the nerve fibers maturation and decreased macrophages and connective tissue area density, suggesting acceleration of neural repair.

CONTEXTUALIZAÇÃO: Lesões nervosas periféricas provocam limitação funcional prolongada, sendo um desafio para a clínica identificar recursos que acelerem sua recuperação. OBJETIVOS: Investigar a influência da estimulação elétrica de alta voltagem (EEAV) sobre a morfologia e a função do nervo regenerado após esmagamento em ratos. MÉTODOS: Vinte ratos Wistar foram divididos nos grupos: controle (CON) - sem lesão e sem EEAV; desnervado (D) - esmagamento do nervo isquiático; desnervado + EEAV (EEAV) - esmagamento do nervo e EEAV; SHAM - sem lesão, porém submetido à EEAV. Os grupos EEAV e SHAM foram estimulados (100 Hz, tensão mínima de 100 V; 20 μs e 100 μs interpulso) 30 min/dia, 5 dias/semana. O índice funcional do ciático (IFC) foi avaliado antes da lesão, nos 7º, 14º e 21º dias pós-operatório (PO). Componentes neurais, densidade de área de tecido conjuntivo, de vasos sanguíneos e macrófagos foram analisados. RESULTADOS: O diâmetro axonal foi maior no grupo EEAV que no grupo D, atingindo quase 80 por cento dos valores-controle após 21 dias (p<0,05). O diâmetro das fibras e espessura das bainhas de mielina foram maiores no grupo EEAV que no D (p<0,05), alcançando 96,5 por cento e 100 por cento dos valores-controle, respectivamente. A recuperação funcional no 14º dia PO foi melhor no grupo EEAV. A densidade de área de macrófagos e tecido conjuntivo foi menor no grupo EEAV, enquanto o número de vasos sanguíneos não diferiu entre os grupos. CONCLUSÕES: A EEAV acelerou a recuperação funcional, potencializou a maturação das fibras nervosas regeneradas e promoveu diminuição da densidade de área de macrófagos e tecido conjuntivo no nervo, sugerindo aceleração do reparo neural.

Mammalian target of rapamycin complex 1 is involved in differentiation of regenerating myofibers in vivo.

This work was undertaken to provide further insight into the role of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle regeneration, focusing on myofiber size recovery. Rats were treated or not with rapamycin, an mTORC1 inhibitor. Soleus muscles were then subjected to cryolesion and analyzed 1, 10, and 21 days later. A decrease in soleus myofiber cross-section area on post-cryolesion days 10 and 21 was accentuated by rapamycin, which was also effective in reducing protein synthesis in these freeze-injured muscles. The incidence of proliferating satellite cells during regeneration was unaltered by rapamycin, although immunolabeling for neonatal myosin heavy chain (MHC) was weaker in cryolesion+rapamycin muscles than in cryolesion-only muscles. In addition, the decline in tetanic contraction of freeze-injured muscles was accentuated by rapamycin. This study indicates that mTORC1 plays a key role in the recovery of muscle mass and the differentiation of regenerating myofibers, independently of necrosis and satellite cell proliferation mechanisms.