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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 16  |  Issue : 3  |  Page : 284-289

High serum myostatin level suggests accelerated muscle senescence in active idiopathic inflammatory myositis


1 Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Rheumatology, All India Institute of Medical Sciences, Delhi, India
3 Department of Nephrology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Date of Submission20-Nov-2020
Date of Acceptance04-Mar-2021
Date of Web Publication21-Sep-2021

Correspondence Address:
Dr. Latika Gupta
Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow - 226 014, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/injr.injr_309_20

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  Abstract 


Introduction: Inflammation is the forerunner to fibrosis and premature aging in various systemic diseases. Hence, we hypothesized that idiopathic inflammatory myopathies (IIM) may exhibit accelerated senescence, and the serum myostatin (MSTN):follistatin system may be a reflection of early senescence events in the muscle.
Methods: Patients with IIM (ACR/EULAR criteria) were recruited (2017–2019) for comparison with healthy and disease controls (DCs). Those with active infection, pregnancy, renal dysfunction, or chronic kidney disease were excluded from the study. MSTN and follistatin were estimated in sera using ELISA (R&D systems, USA). Juvenile myositis and young adults (18–40 years) were subsequently analyzed separately. Nonparametric tests were used for paired and unpaired analysis. Results expressed as median and interquartile range.
Results: A total of 84 myositis (3 juvenile myositis, 40 DM, 30 PM, 11 overlap) patients (68 females) with median age 38 (27–47.0) years and median disease duration of 0.9 (2.3–5.1) years were included. Serum MSTN was lower in IIM than in healthy control (149.3 vs. 243.6 P < 0.0001) but higher in IIM as compared with DCs (149.3 vs. 85.11, P = 0.0174). MSTN levels were higher in active as compared with inactive myositis in young adults (189.6 vs. 115.8, P = 0.0349). Serum MSTN correlated with height (r = 0.3, P = 0.003) and weight (r = 0.2, P = 0.047) but not MMT8 or muscle enzymes. On follow-up, the serial MSTN estimation paralleled change in disease activity.
Conclusion: Elevated serum MSTN levels in active myositis raise the possibility of accelerated senescence in the inflamed muscle tissues which need further investigation.

Keywords: Dermatomyositis, Follistatin, myositis, myostatin, myostatin protein, senescence


How to cite this article:
Anuja AK, Bhadu D, Naveen R, Singh MK, Rai MK, Agarwal V, Gupta L. High serum myostatin level suggests accelerated muscle senescence in active idiopathic inflammatory myositis. Indian J Rheumatol 2021;16:284-9

How to cite this URL:
Anuja AK, Bhadu D, Naveen R, Singh MK, Rai MK, Agarwal V, Gupta L. High serum myostatin level suggests accelerated muscle senescence in active idiopathic inflammatory myositis. Indian J Rheumatol [serial online] 2021 [cited 2021 Dec 6];16:284-9. Available from: https://www.indianjrheumatol.com/text.asp?2021/16/3/284/324766




  Introduction Top


Inflammation is the forerunner for fibrosis and premature aging in various systemic diseases. Recent insights have highlighted the convergence of inflammatory pathways onto muscle metabolism as well.[1] MSTN endows skeletal muscle with high oxidative capacity, thus regulating the delicate balance between muscle mass, muscle force, energy metabolism, and endurance capacity.[2],[3]

In spite of the well-established fact that inflammatory myositis is an immune-mediated disease, much attention has been given to the role of nonimmune mechanisms of muscle dysfunction.[1] In the most successful animal model of inflammatory myositis (MHC I conditional upregulation in H + T + mice), Nagaraju et al. have clearly shown that muscle weakness occurs much before inflammatory infiltrates which are evident on muscle biopsy specimens.[4] Dysregulation of cellular bioenergetics (due to AMP deaminase-1 dysfunction) occurs early in the disease course and correlates with onset of weakness.[1] It seems plausible that a protein such as myostatin (MSTN), which is crucial to muscle development and metabolism, is altered in the disease as well.

MSTN, a member of the transforming growth factor-beta (TGF β) family of super peptides, is a myokine produced and released by myocytes.[5] The peptide undergoes three steps of cleavage to be secreted as a hormone into the circulation, which acts in an autocrine as well as paracrine fashion on the muscle to inhibit muscle growth and differentiation as depicted in [Figure 1]. These effects are mediated through downregulation of myogenic proliferation factors (myoD1, myf-5, and Pax-3), activation of profibrotic genes, and inhibition of autophagy through the mTOR pathway. MSTN null mice exhibit marked muscle hypertrophy and hyperplasia.[6] Monoclonal antibodies to MSTN have been successfully used to reverse weakness in mouse model of muscle dystrophy and are in clinical trials in humans.[7]
Figure 1: Myostatin and follistatin downstream signaling pathway

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MSTN is released as a propeptide and after undergoing cleavage to active form, its action of activin R2B is regulated by a glycoprotein known as follistatin.[5] The availability of new generation augmented ELISA targeting the active form of MSTN protein has enabled measurement of this circulating myokine in the serum.[8] MSTN:follistatin ratios have been seen to correlate with age as a reflection of fibrosis and aging of the muscle.[9] Vector-based recombinant therapies of follistatin have been successfully used in nonhuman primates for suppression of MSTN and salvage of the diseased muscle.[10],[11]

In most diseases, inflammation is the forerunner for fibrosis and premature aging. Hence, it seems plausible that inflammatory myopathies are no exception and may exhibit markers of senescence in excess of expected for the age. Hence, in this proposal, we studied the MSTN:follistatin system as a reflection of early senescence in the various subsets of idiopathic inflammatory myopathies (IIMs), namely juvenile myositis (JIIM, including juvenile dermatomyositis [JDM]), dermatomyositis (DM), polymyositis (PM), and overlap myositis (OM).


  Methods Top


After taking written informed consent, consecutive patients with IIM (ACR/EULAR criteria) prospectively evaluated for clinical features and laboratory data (including autoantibodies) in an institutional review board certified study (2017-41-IP-76) were screened to identify cases without an active ongoing infection, pregnancy, acute renal dysfunction, or chronic kidney disease.[12] Those with metabolic, degenerative, inherited, or other forms of myopathies were also excluded from the study [Figure 2]a.
Figure 2: (a) Flowchart depicting the methodology of the study. (b) MSA/MAA in the 84 patients

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Disease definitions for the types of idiopathic inflammatory myopathies

Dermatomyositis

As per ACR/EULAR criteria, in the absence of classic skin rashes, two or more of the nonclassic rashes were deemed necessary (V sign, shawl sign, holster sign, diffuse erythema, photosensitivity, malar rash, diffuse hyperpigmentation, and periungual capillary changes).

Polymyositis

Polymyositis was defined as per ACR/EULAR criteria.

Overlap myositis

OM was defined as those who fulfilled both, the Bohan-Peter criteria (B-P) and any one criteria for connective tissue disease, namely Systemic Lupus Collaborating Clinics criteria 2012 for systemic lupus erythematosus (SLE), Kahn criteria for mixed connective tissue disorder, ACR/EULAR criteria for SSc, American-European Consensus Group Sjogren's criteria, or undifferentiated connective tissue disorder (defined by the presence of two or more overlap features – Raynaud's phenomenon, Sclerodactyly, ILD, trigeminal neuropathy, puffy fingers, trigeminal neuropathy, esophageal dysmotility, antibodies-RNP, fibrillarin, PM/Scl, RNAP, Ku, or Th).

JIIM (Juvenile IIM)

Definitions as per adults, age of onset before 18 years. JPM, juvenile overlap myositis, JDM.

Samples of healthy and diseased controls were extracted from the biorepository as previously described.[13]

Clinical details

A standardized case record form was used to record clinical and laboratory variables.[12],[13],[14],[15] Clinical data and sera of patients and healthy controls (HCs) were retrieved from the database and biobank, respectively.[13],[14],[15] Case details were supplanted with standard outcome measures, as described by the International Myositis Assessment and Clinical Studies Group.[16] MDAAT activity score greater than or equal to one defined active disease.

For participants with myositis within 6 months of the first enrolment into the study (called the inception cohort), sera obtained at 6 months of therapy were also retrieved. A disease duration of ≤1 year was classified as early IIM.[17] A polyphasic disease course was defined as previously described.[14] We followed the STROBE'S checklist for reporting results.[18]

Controls

Disease controls

Seven SLE patients without renal involvement or myositis, one HIV with myositis, and one Duchenne's muscular dystrophy patients were included as disease control (DC).

Healthy controls

Nine healthy adults without any comorbidities were included as HC.

Laboratory assays

Plasma concentration of MSTN and follistatin in fasting blood sample was be determined by ELISA as per the manufacture's instructions of Quantikine1 (GDF-8/MSTN Immunoassay, R&D systems Inc.) and Follistatin (R&D).

Sera from all the patients were also investigated for the presence of MSAs/MAAs by Line immunoassay (G4 panel, Euro-Immune, Lubeck, Germany) and antinuclear antibodies using the immune fluorescence assay (IFA, Nova-lite, Inova, CA, USA), according to the manufacturer's instructions. Samples were diluted to 1 in 100 for the latter.

Statistics

Adults and children were analyzed separately. Mann–Whitney is used for scale variables and Chi-square/Fisher's exact for categorical variables. Descriptive statistics alone were used for each autoantibody subgroup when sample size in any analytic group was <5. Data are expressed as median and interquartile range. P < 0.05 is deemed as statistically significant, and all reported values were two sided. Statistical analysis was done using GraphPad Prism © version 7.0 for Mac.


  Results Top


Baseline characteristics

84 myositis (3 JIIM, 40 DM, 30 PM, and 11 OM) patients (16 males and 68 females) with median age 38 (27–47) years and disease duration 0.9 (2.3–5.1) years were included along with 9 HC (7 males, age: 31 [30–32] years) and 9 DC (7 SLE, 1 HIV myositis, 1 Duchenne's muscular dystrophy, 2 males, age: 27 [20–36] years) as shown in [Table 1]. Since MSTN levels are dependent on the muscle mass and age of the individual, further analysis in active and inactive IIM was limited to age group 18–40 years. Of a total of 87 patients retrieved from the database, 3 were excluded as they had raised creatinine (>1.5 mg/dl). MSTN in older set (n = 35, age >40) was 143 (62–205) pg/ml as compared with 171 (82–256) pg/ml in those aged 18–40 years (P = 0.37). MSA/MAA is as shown in [Figure 2]b.
Table 1: Demographics of patients

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Serum myostatin in idiopathic inflammatory myopathies as compared with healthy and disease controls

Serum MSTN was lower in IIM than in HC [149.3 vs. 243.6 P = 0.02, [Figure 3]a but higher in IIM as compared with DCs [149.3 vs. 85.11 P = 0.012 [Figure 3]a. Serum MSTN was comparable between juvenile (184 [79–257] pg/ml) and adult (144 [64–241] pg/ml) myositis and in the various subsets of adult myositis (DM 138 [70–239], PM 186 [112–251], OM 120 [44–195], and JDM 186 [63–186] pg/ml) [Figure 3]b and [Figure 3]c. MSTN levels in those (n = 3) excluded due to renal disease were 53 (36–126) pg/ml as compared with 149 (69–244) pg/ml in the 84 patients.
Figure 3: (a) Serum myostatin levels in idiopathic inflammatory myopathies as compared with healthy controls and disease controls. (b) Levels in juvenile myositis as compared with adult idiopathic inflammatory myopathies (c) and in various subsets of idiopathic inflammatory myopathies. (d and e) Serum myostatin levels in active and inactive disease in age 18–40 and >18 years. (f) Myostatin levels on follow-up in 3 patients

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Serum myostatin in active versus inactive idiopathic inflammatory myopathies

MSTN levels were higher in active (n = 34) as compared with inactive (n = 50) myositis in all adults (207.2 vs. 110.4, P = 0.015) as well as young adults (18–40, inactive 16 and active 9) (189.6 vs. 115.8, P = 0.028) [Figure 3]d and [Figure 3]e.

Effect of body composition on myostatin levels

Serum MSTN did not correlate with weight (r = 2 0.2, P = 0.038), body mass index (BMI) (r = 2 0.2, P = 0.05), age, disease duration, MMT8, or level of muscle enzymes [Figure 4]a, [Figure 4]b, [Figure 4]c.
Figure 4: Correlation of serum myostatin with body mass index, weight, and height (a-c)

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Serum follistatin

Although follistatin was lower in IIM than HC (198.4 vs. 243.6, P ≤ 0.0001), neither follistatin nor MSTN: follistatin ratios differed between subsets, and in active versus inactive disease [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]d.
Figure 5: (a) Serum follistatin levels in idiopathic inflammatory myopathies as compared with healthy controls and disease controls. (b) Levels in juvenile and adult idiopathic inflammatory myopathies (c) and in various subsets of idiopathic inflammatory myopathies. (d) Levels in active versus inactive myositis aged >18 years

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Myostatin values upon follow-up

On follow-up (n = 3) for 2 months, the serial MSTN estimation paralleled change in disease activity [Figure 3]f.


  Discussion Top


MSTN is a negative regulator of muscle growth and differentiation. It is also speculated to induce myofibroblast transformation into fibroblasts in myopathies leading to loss of muscle strength. Thus, an upregulation of MSTN in IIM may suggest slow muscle regeneration and accelerated fibrosis.[19] Follistatin is an important antagonist of MSTN, so the ratio of follistatin and MSTN may be reflective of the eventual outcome.[20]

Here, in a large cohort of various subsets of IIM, we found that MSTN levels were elevated in active IIM as compared to inactive IIM. Changes in myostatin levels on follow-up paralleled disease activity. However, MSTN levels were lower than HCs as expected, more so in younger individuals.

MSTN is linked to insulin resistance and obesity in many studies.[21] This could also explain the insulin resistance and metabolic syndromes accompanying myositis patients with high BMI. However, in our study, MSTN did not correlate with BMI and body weight.

Our results are supportive of previous observations by Vernerová et al., wherein decreased MSTN in IIM was attributed a compensatory downregulation of immunosenescence pathways by the body in the face of ongoing muscle disease.[20] This is supported by the positive correlation with MMT in both the studies, suggested lower MSTN when the patients had more severe disease. On the contrary, Follistatin levels were not reflective of active or inactive disease in the current study.

MSTN is a myokine that undergoes renal excretion. Previously, a weak negative correlation with glomerular filtration rate is proposed to be a marker of sarcopenia.[22] However, in our population, which possibly already had sarcopenia, the levels were higher in those with an underlying acute or chronic kidney disease. Whether this was due to an exaggerated sarcopenia in these individuals or due to renal underexcretion of the molecule merits further exploration. The muscle power of this group was no different from the rest, favouring renal underexcretion.

MSTN signals through the pathways such as mTOR-glycolysis and TGF-β and promote the expression of several genes involved in glucose metabolism.[3],[23] These muscle regeneration pathways have potential and can act as a window of opportunity to intervene with pathways specific inhibition.[24] Hence, the convergence of MSTN onto mTOR, TGF-β, and muscle regeneration pathways can suggest these as plausible targets, pending further validation.[25],[26]

Although the results between the various subsets group of IIM do not have a significant difference, the few numbers of OM in the young adult group may limit comparisons. Moreover, a marginal effect of glucocorticoid usage on MSTN levels in vitro studies based on myofibroblast cultures suggests a plausible confounding effect in active disease.[27],[28] We have not explored the relation of MSTN levels with cumulative glucocorticoid exposure, which may provide greater insight into this. Furthermore, we have not explored the effects of common disease modifying drugs such as methotrexate in the study.

This is the first study to have comparison of MSTN levels in a prospective cohort of various subsets of IIM, including juvenile IIM. Small sample size of young adults was the major limitation of our study. Further assessments of the relative contributions of downstream pathways (mTOR, TGF-β, Fstn, and Act) together with quantification of muscle mass may provide greater insight into the MSTN pathway as a potential therapeutic target to prevent accelerated muscle senescence in active IIM.


  Conclusion Top


Thus, to conclude, elevated serum MSTN levels in active myositis raise the possibility of accelerated senescence in the inflamed muscle tissues which merit further investigation.

Financial support and sponsorship

This study was financially supported by IRA research grant 2019.

Conflicts of interest

There are no conflicts of interest.



 
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