Muscle Wasting Aggravates Rheumatoid Arthritis in Elderly Patients as a Mediator

Background: Aging leads to loss of muscle mass causing functional limitation and reduced quality of life. Myopenia is a new universal term for the presence of clinically relevant muscle wasting. However, little is known about the prevalence of myopenia in elderly rheumatoid arthritis (RA) patients and its influence on RA pathogenesis and disease characteristics. We aimed to explore the characteristics of muscle mass and distribution and their clinical significance. Methods: Consecutive RA patients were recruited and clinical data including disease activity (DAS28-CRP), physical function (health assessment questionnaire disability index, HAQ-DI) and radiographic indicators (modified Sharp score) were collected. The muscle mass and distribution were assessed by bioelectric impedance analysis. Myopenia was defined as appendicular skeletal muscle mass index (ASMI) ≤7.0kg/m2 (men) and ≤5.7kg/m2 (women). Results: Among 643 RA patients recruited, there were 165 (25.7%) elderly patients (age≥60 years) with mean age 65.1±4.5 years. Compared with young patients (age<60 years), elderly RA patients had significantly higher DAS28-CRP (median 3.4 vs. 3.2), HAQ-DI (0.38 vs. 0.13) and modified total Sharp score (mTSS, 16 vs. 9), as well as higher proportion of myopenia (54.5% vs. 41.4%, all P<0.01). Elderly RA patients with myopenia (n=90, 14.0%) had significantly higher DAS28-CRP (3.6 vs. 3.0), HAQ-DI (0.50 vs. 0.12) and mTSS (21 vs. 7) than those in young RA patients without myopenia (n=280, 43.5%), and had higher mTSS (21 vs. 10) than those in elderly RA patients without myopenia (n=75, 11.7%, all P<0.0083). Multiple linear regression analysis after adjustment showed that both age (10 years as a unit, β=0.065~0.108) and ASMI (β= -0.369~ -0.220, Spearman’s rank order correlation test showed that age was positively correlated with DAS28-CRP (r = 0.127), HAQ-DI (r = 0.225) and mTSS (r = 0.203, all P < 0.001), while ASMI was negatively correlated with DAS28-CRP (r= -0.131), HAQ-DI (r= -0.195) and mTSS (r= -0.305, all P < 0.001). Linear regression analysis was used to identify the association of age and ASMI with RA disease characteristics. Univariate linear regression analysis showed that age was positively correlated with DAS28-CRP (β = 0.157), HAQ-DI (β = 0.082) and mTSS (β = 0.090), while ASMI was negatively correlated with DAS28-CRP (β= -0.244), HAQ-DI (β= -0.117) and mTSS (β= -0.197, all P < 0.01).

Conclusion: Our data firstly reveal that half of elderly RA patients manifest myopenia which aggravates the whole disease including disease activity, physical dysfunction and joint destruction as a mediator of age. Myopenia, a neglected complication in elderly RA should be emphasized.

Background
Rheumatoid arthritis (RA) is one of the most prevalent chronic inflammatory diseases leading to joint damage and disability [1]. The incidence of RA varies from 0.1-1.9% worldwide and increases with age [2,3]. The cumulative lifetime risk of developing RA is less than 1% before 50 years old, but greatly increases at approximately 60 years old [4].
As life expectancy increases, there were more than 900 million elderly people (defined as ≥ 60 years old) in the world in 2015 [5]. In China, the number of elderly people was over 240 million in 2017 [6]. The estimated prevalence of RA in China is 0.42% and there were more than 5 million RA patients in 2013 [7]. According to a nationwide, multicenter, prospective Chinese Registry of Rheumatoid Arthritis (CREDIT), there were 32.7% elderly patients among the total 13210 patients in 2017, which predicts at least 1.6 million elderly RA patients in China now [8]. However, elderly RA patients are often excluded from clinical trials for their possible co-morbidities or other factors which might affect the results of intervention. Insufficient clinical data challenged the evidence-based management of elderly RA patients.
Aging is a multidimensional process comprising changes in different systems, such as altering innate immune system to become more active, but making the adaptive immune system declined [3]. These changes resulted in a special clinical manifestation of RA in elderly patients who are characterized by higher level of systemic inflammation and poor function [9]. Recent evidence showed that aging might also lead to loss of muscle mass which caused functional limitation and reduced quality of life in the elderly [10]. Myopenia is a new universal term for the presence of clinically relevant muscle wasting which might be caused by diseases such as muscular dystrophies, aging, persistent infection, chronic obstructive pulmonary disease, chronic kidney disease or cancers [11]. Benefit from the wide use of bioelectric impedance analysis (BIA) [12], the mass and distribution of muscle are available for the accurate assessment in daily clinical practice. Recently we reported 45.1% Chinese RA patients with myopenia who manifested high disease activity as well as severe joint destruction [13]. However, the prevalence of myopenia in elderly RA patients and its influence on RA pathogenesis and disease characteristics have not yet been elucidated. Here, we further investigated the characteristics of muscle mass and distribution in elderly RA patients, aiming to explore their impact on RA disease characteristics.

Patients and groups
Consecutive Chinese patients with RA who fulfilled the 1987 revised criteria of the American College of Rheumatology (ACR) [14] or the 2010 ACR/European League Against Rheumatism (EULAR) criteria for the classification of RA [15]  and SYSEC-KY-KS-012). All patients signed informed consent.
All RA patients were classified into young (age < 60 years) and elderly RA groups (age ≥ 60 years), young onset (onset age < 60 years) and elderly onset RA groups (onset age ≥ 60 years) according to age and onset age respectively.

Radiographic Assessment
Conventional radiographs of bilateral hands and wrists (anteroposterior view) of all patients were performed at enrollment and assessed according to Sharp/van der Heijdemodified Sharp score including modified total Sharp score (mTSS), joint erosion (JE) and joint space narrowing (JSN) subscores as we described previously [16]. The mean intraclass correlation coefficient (ICC) for reliability and inter-observer agreement was 0.953.

Muscle Assessment
The mass and distribution of muscle in trunk and appendicular extremities were assessed by BIA using an InBody 230 device (Biospace Co., Shanghai, China) as we described previously [13]. Appendicular skeletal muscle mass index (ASMI) was defined by appendicular skeletal muscle mass/height 2 (kg/m 2 ). Myopenia was defined by ASMI ≤ Two independent samples t-Test or the Mann-Whitney test was used for comparison between two independent groups. Analysis of Variance (ANOVA) or Kruskal-Wallis analysis of variance on ranks were used among four groups according to distributions. Categorical variables were presented as numbers and percentages. Chi-square test or Fisher's exact test was used to compare categorical variables. Bonferroni correction was used for multiple comparisons in subgroups. Correlations of age, ASMI and clinical indicators were analyzed by Spearman's rank order correlation test. All significance tests were two-tailed and were conducted at the 5% significance level.
Univariate and multiple linear regression analysis was used to identify the association between age (10 years as a unit) and ASMI (independent variables) with RA disease characteristics (dependent variables) including disease activity (DAS28-CRP), physical function (HAQ-DI) and joint destruction (mTSS), and adjusted for the confounders including gender, disease duration, active smoking, BMI, rheumatoid factor (RF) status, anti-cyclic citrullinated peptide antibody (ACPA) status, previous medications (categorized by treatment naïve, glucocorticoids, csDMARDs and bDMARDs therapy due to the number of cases), comorbidities (including hypertension, diabetes, dyslipidemia, fatty liver and heart disease) with or without DAS28-CRP. In fitting these multifactorial models, HAQ-DI was normalized by a natural square root transformation while mTSS was normalized by a lg(X + 1) transformation to make sure the models.
Mediation analysis is widely used to quantify how an exposure or intervention affects outcome [18]. It was performed to identify the effect of ASMI (mediator) on the association between age (10 years as a unit, independent variable) and RA disease characteristics (dependent variables) including disease activity (DAS28-CRP), physical function HAQ-DI and joint destruction (mTSS). Confounders mentioned above were adjusted in two models due to the number of factors. Mediation analysis was conducted on SPSS utilizing PROCESS macro (Version 3.3) provided by Andrew F. Hayes [19]. The bootstrap method was used as it was free from assumptions regarding the shape of the sampling distribution of the indirect effect and had better control on type I errors [20].
The number of bootstrap samples was 10,000. Bootstrap bias-corrected 95% confidence intervals (CI) was calculated and when they didn't contain zero, they were considered significant.

Demographic characteristics of all RA patients
There were 643 RA patients recruited with 82.3% female, mean age 49.7 ± 12.9 years and median disease duration 48 (IQR 21,108) months. There were 31 (4.8%) early RA patients (short disease duration) and 439 (68.3%) with long disease duration. There were no significant differences in muscle mass and distribution, prevalence of myopenia between elderly RA patients with young onset and elderly onset.  Table 2).    Table 4).

M e d i a t i o n a n a l y s i s o f A S M I o n t h e a s s o c i a t i o n b e t w e e n a g e a n d R A d i s e a s e c h a r a c t e r i s t i c s
Mediation analysis was performed to identify the effect of ASMI on the association between age and DAS28-CRP. Adjusted for confounding factors including gender, disease duration, BMI, active smoking, RF status, ACPA status, previous medications, age had total effect and direct effect on DAS28-CRP (β = 0.128, P = 0.002 and β = 0.089, P = 0.031). The path from age to DAS28-CRP through mediation of ASMI was statistically significant (P 0.05). Further adjusted for additional DAS28-CRP, age also had total effects and direct effects on HAQ-DI (β = 0.046, P 0.001 and β = 0.034, P = 0.002) and mTSS (β = 0.076, P 0.001 and β = 0.052, P = 0.004), while the path from age to HAQ-DI or mTSS through mediation of ASMI were statistically significant (both P 0.05). These results indicated that ASMI partially mediated the association between age and DAS28-CRP, HAQ-DI or mTSS, which were further confirmed by mediation analysis after adjustment of comorbidities instead of previous medication (Fig. 2).

Discussion
This is the first report about the characteristics of muscle mass and distribution in a largescale study of elderly RA patients and their impact on RA disease characteristics.Half of elderly Chinese RA patients (54.5%) manifested myopenia who had the most severe disease of RA as higher disease activity with more than 4 folds of physical dysfunction (HAQ-DI) and 3 folds of joint destruction (mTSS), indicating a new phenotype of RA.
Myopenia might aggravate the whole disease of RA as a mediator of age, which imply that myopenia should be emphasized as an important complication in RA especially in elderly patients.
Remarkable changes of immune systems with aging result in immunosenescence. During this process, aging activates innate immune system resulting in increased systemic inflammatory indicators including ESR and CRP, as well as inflammatory cytokines such as TNF-α and IL-6 [3]. A new term named inflamm-aging was developed in 2000 for the condition that high levels of proinflammatory markers express in cells and tissues of older organisms [21]. Local and systemic inflammation not only cause impaired anabolism and increased protein breakdown of myofiber, but also play a critical role on the onset of muscle wasting [22]. In clinical studies, muscle wasting was found in 20% Italian RA patients and 49% Japanese RA patients [23,24]. In our previous study, there were 45.1% Chinese RA patients with myopenia which was more than 3 folds higher than that in controls of white-collar employees [13]. Longitudinal study showed that inhibition of IL-6 by tocilizumab, an IL-6 receptor blocker, increased muscle mass in RA patients [25]. In our present study, elderly RA patients had higher disease activity indicators as well as higher proportion of myopenia compared with young patients, which confirmed the impacts of inflammation on muscle wasting in RA patients. On the other hand, a brand-new finding showed that IL-17 and/or TNFα leaded to dysfunction of human myoblasts and increased their secretion of IL-6, indicating muscle cell dysfunction could promote inflammation [26]. We revealed that ASMI partially mediated the association between age and DAS28-CRP, which might be a clinical evidence that inflamm-aging affects not only joints, but also muscle which in turn aggravates inflammation in RA.
Structural and electrophysiological changes in skeletal muscle are frequently found in humans over 60 years of age and followed by a progressive age-related decline [10].
Human manifests decline in muscle mass and strength of about 1% per year from the age of around 40 years [27]. Low muscle mass combined with low muscle strength and physical performance in the elderly people were academically recognized as a new independent disease called sarcopenia, which highlighted the relationship of ageassociated muscle wasting and physical limitation in the elderly people [28][29][30]. Muscle wasting is also associated with joint dysfunction in RA patients. Clinical study from Jon T.
Giles et al showed that the mean HAQ score was 0.81 units lower in RA patients with the highest quartile of appendicular lean mass than those with the lowest quartile [31], while Hanh-Hung Dao et al found similar results that HAQ score was negatively correlated with appendicular lean mass in RA patients [32]. In our study, negative correlations of age and ASMI as well as ASMI and HAQ-DI were both significant after adjusted for confounding factors including disease activity (DAS28-CRP). Further mediation analysis showed ASMI partially mediated the association between age and HAQ-DI, which supported the impact of age-associated muscle wasting on physical dysfunction in elderly RA patients.
Numerous evidences showed that older RA patients had more severe joint destruction [33][34][35]. Recent study including five longitudinal cohorts showed that increased age was associated with more severe joint destruction in RA patients [36]. Aging disturbs the homoeostasis of skeletal muscle and the cross-talk between skeletal muscle and bone has become a research hotspot in the field of aging these years [37,38]. Skeletal muscle functions in an endocrine manner by secreting various myokines such as irisin, myostatin, osteocalcin and so on, which are involved in osteoclastogenesis [39]. Irisin, a myokine produced from skeletal muscle after exercise, can increase osteoblast differentiation and suppresses osteoclast differentiation [40], while myostatin, as a negative regulator of muscle growth and regeneration, can enhance bone degradation by RANKL-induced osteoclastogenesis [41]. To find out how aging influence the cross-talk between skeletal muscle and bone from a clinical perspective, we found that half of elderly RA patients (54.5%) manifested myopenia who had the worst joint destruction than others. After adjusted for confounding factors especially disease duration and disease activity (DAS28-CRP), mediation analysis revealed that ASMI partially mediated the association between age and mTSS, which suggested that myopenia might aggregate joint destruction through the cross-talk of muscle and bone as a result of aging in RA patients.   confidence intervals were showed within parentheses. Significances are shown in bold. β, regression coefficient. *P < 0.05, **P < 0.01, ***P < 0.001.