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, 10 (1), 4659

Sustained Elevation of MG53 in the Bloodstream Increases Tissue Regenerative Capacity Without Compromising Metabolic Function

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Sustained Elevation of MG53 in the Bloodstream Increases Tissue Regenerative Capacity Without Compromising Metabolic Function

Zehua Bian et al. Nat Commun.

Abstract

MG53 is a muscle-specific TRIM-family protein that presides over the cell membrane repair response. Here, we show that MG53 present in blood circulation acts as a myokine to facilitate tissue injury-repair and regeneration. Transgenic mice with sustained elevation of MG53 in the bloodstream (tPA-MG53) have a healthier and longer life-span when compared with littermate wild type mice. The tPA-MG53 mice show normal glucose handling and insulin signaling in skeletal muscle, and sustained elevation of MG53 in the bloodstream does not have a deleterious impact on db/db mice. More importantly, the tPA-MG53 mice display remarkable dermal wound healing capacity, enhanced muscle performance, and improved injury-repair and regeneration. Recombinant human MG53 protein protects against eccentric contraction-induced acute and chronic muscle injury in mice. Our findings highlight the myokine function of MG53 in tissue protection and present MG53 as an attractive biological reagent for regenerative medicine without interference with glucose handling in the body.

Conflict of interest statement

J.M. and T.T. have an equity interest in TRIM-edicine, Inc., which develops rhMG53 for treatment of human diseases. Patents on the use of MG53 are held by Rutgers University—Robert Wood Johnson Medical School. All other authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Mouse model with sustained elevation of MG53 in the bloodstream. a 1 µl sera derived from 3-month wild type (WT) and tPA-MG53 mice at 2 months (young), 12 months (middle) and 24 months (aged) were probed with anti-MG53 antibody. b Quantification of serum levels of MG53 in wild type and tPA-MG53 mice by western blot (n = 10, wild type; n = 9 for tPA-MG53, P < 0.0001). c Skeletal muscle (2 µg per lane) and heart (2 µg per lane) derived from tPA-MG53 and WT littermate mice were blotted with anti-MG53 antibody. Red arrows indicate doublet of MG53 and tPA-MG53. d H&E staining of vital organs from 32-month-old tPA-MG53 mice show normal tissue morphology. The pictures are representative of two other tPA-MG53 mice at 32 months age. Error bar represents the standard deviation and P value was generated by t test
Fig. 2
Fig. 2
Assessment of insulin signaling and glucose handling in tPA-MG53 and WT mice. a tPA-MG53 and WT littermate mice at 6 weeks were treated with HFD and the changes in body weight were followed for 10 weeks (n = 5 per group). b Glucose tolerance tests were conducted with tPA-MG53 and WT littermates at the age of 6 weeks (left) and 30 weeks (right). c Insulin-tolerance tests were conducted with tPA-MG53 and WT littermates at the age of 8 weeks (left) and 32 weeks (right). n = 6 for WT, n = 5 for tPA-MG53. d TA muscle (60 µg total protein per lane) derived tPA-MG53 and WT littermates were probed with antibodies against IRS-1, IR-β, Glut-4. GAPDH serves as loading control. e Quantification of protein expressions based on western blot. f tPA-MG53 and WT mice were treated with insulin (0.75 U/kg) for 15 min, fresh TA muscles were probed with antibodies against IRS-1, p-Akt, Akt, and GAPDH. Error bar represents the standard deviation
Fig. 3
Fig. 3
tPA-MG53 mice show increased healing capacity following ear-punch injury. a tPA-MG53 mice in a mixed genetic background of 129/Sv and C57BL/6J expressed elevated levels of circulating MG53 compared with WT littermates. 1.3 µl sera were loaded per lane and probed with anti-MG53 antibody. 0.1 ng of rhMG53 dissolved in serum derived from mg53−/− mice was used as reference standard. b Representative pictures of ear punch injury in WT (left panels) and tPA-MG53 mice (right panels) at different days post-injury. c IHC revealed the concentration of MG53 at the leading edge of ear-punch at 2 h after wounding in the tPA-MG53 mice. d Masson’s trichrome staining showed remarkable differences in the ear skin architecture between WT and tPA-MG53 mice, with wounds created acutely at 2 h (red arrow) and 14 days (yellow rectangle) following ear punch. e WT mice do not heal over the 10-day observation, the tPA-MG53 mice all healed completely at day 10. n = 10 per group. f Areas positive for trichrome staining were used as index for tissue fibrosis. Quantification revealed significant difference between WT and tPA-MG53 mice at 14 days post ear-punch injury. *p < 0.05; **p < 0.01. Error bar represents the standard deviation and p value was generated by t test
Fig. 4
Fig. 4
Increased muscle performance of the tPA-MG53 mice under stress conditions. a Gross anatomy of soleus, EDL and gastrocnemius muscles derived from tPA-MG53 and WT mice at 3–4-months age. b Quantification of the ratio of muscle weight to tibia length (TL) showed no significant differences between WT and tPA-MG53 mice. c Cross section IHC staining of soleus and EDL skeletal muscle from WT and tPA-MG53 mice. Green—MHCIIA to stain fast-twitch fiber. Red—MHCIIB another antibody to stain fast twitch fiber. Magenta—MHC I to stain slow twitch fiber. Purple—MHC IIx to stain fast twitch muscle fiber. d Fiber typing staining results were quantified. e 20 h after the first round of exercise training (10 m/min), mice were again subjected to running at 6, 8, 10, 12, 14, 16 m/min each for 3 min on the treadmill to test the capacity of recovery from muscle injury. The number of drop-outs were quantified. f tPA-MG53 and WT mice were subjected to voluntary wheel running for 7 days. *p < 0.05; **p < 0.001. g FDB fibers derived from WT (left) and tPA-MG53 mice (right) were loaded with Fura-2 AM; KCl-induced changes in intracellular Ca2+ were measured with 2 mM Ca2+ present in the extracellular solution. h Half-decay times of intracellular Ca2+ signaling were significantly longer in tPA-MG53 muscle compared with WT muscle (p = 0.0215). Error bar represents the standard deviation and p value was generated by t test
Fig. 5
Fig. 5
tPA-MG53 skeletal muscle showed enhanced regeneration capacity after cardiotoxin injury. a H/E staining of gastrocnemius muscle derived from KO, WT, and tPA-MG53 mice at 7 days post cardiotoxin injury. Enlarged pictures show different regenerative capacity of muscle fibers derived from WT, KO, and tPA-MG53 mice. b IHC staining of MG53 (red) and mouse IgG (green). IgG positive staining, mostly in KO muscle, indicates necrotic muscle fibers after cardiotoxin injury. c IHC staining with antibody against CD11b to show the different degrees of the presence of immune cells at the muscle injury sites (7 days post cardiotoxin treatment). d Quantification of muscle fiber size (left), IgG staining of necrotic fibers (middle) and number of cells positive for CD11b (right). ***p < 0.001, **p < 0.01. Error bar represents the standard deviation and p value was generated by t test
Fig. 6
Fig. 6
MG53 modulates muscle satellite cell (mSC) proliferation to contribute to muscle regeneration. a Outgrowth of mSCs from isolated EDL muscle fibers derived from adult wild-type, tPA-MG53 and KO mice. At day 5 after culture, there were fewer mSCs growing out from KO muscle fibers, as compared to those from wild type and tPA-MG53 muscle fibers. Incubation with exogenous rhMG53 (20 μg/ml) led to increased number of mSCs near the KO myofiber (KO + rhMG53). b Flow cytometry analysis with different cell lineage antibodies (Pax7 for mSCs, MyoD for myoblasts, and PDGFα for fibroblasts) to validate the identity of the cultured mSCs. c Immunofluorescent staining confirmed the high purity of muscle-fiber derived mSCs, as they are positive for Pax7 and negative for MyoD. d Statistical analysis demonstrate that the compromised proliferation of mSCs derived from KO muscle could be restored by incubation with rhMG53 (n = 3–6 per condition, *p < 0.05 **p < 0.01 as compared to mg53−/− fibers). Error bar represents the standard deviation and p value was generated by t test
Fig. 7
Fig. 7
rhMG53 protects against eccentric-contraction-induced acute and chronic muscle injury in mice. a Time-dependent effects of rhMG53 in protection against eccentric-contraction-induced acute muscle injury in mice. rhMG53 (2 mg/kg, i.v.) was administered at different times after muscle injury. b Dose-dependent effect of rhMG53 in protection against eccentric-contraction-induced acute muscle injury in mice. Various doses of rhMG53 were administered at 4 h post muscle injury. c 24 h after eccentric-contraction-induced muscle injury, rhMG53 (6 mg/kg) was administered subcutaneously. Mice were treated on a daily basis for 28 days. d Area under the curve (AUC) shows significant beneficial effects of rhMG53 in facilitation of recovery of contractile function after eccentric-contraction-induced muscle injury (p < 0.001, compared with saline control). e Blood glucose and triglyceride measurement did not show significant changes after 28 doses of 6 mg/kg (s.c., daily) in mice. Error bar represents the standard deviation and p value was generated by t test
Fig. 8
Fig. 8
Cross of tPA-MG53 mice with db/db mice did not alter the diabetic phenotype. a Growth patterns of WT and tPA-MG53 littermates; and db/db and db/db-tPA-MG53 littermates were followed during the period of 3–32 weeks, under normal diet conditions. b GTT tests were conducted with the four groups of mice at 18 weeks of age (left). ITT tests were conducted at 20 weeks of age. No significant differences were observed among the WT and tPA-MG53, and db/db and db/db-tPA-MG53 groups, respectively. n = 6 for WT, n = 5 for tPA-MG53, n = 6 for db/db, and n = 5 for db/db-tPA-MG53. c Echocardiogram shows similar ejection fraction and fractional shortening between db/db-tPA-MG53 and db/db littermates. d Western blot of soleus muscle derived from the four groups of mice. Expression levels of IRS-1, IR-β, and PPARα were probed with the respective antibodies. α-actinin serves as loading control. Error bar represents the standard deviation

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