CO-LOCALIZATION OF MACROPHAGE INHIBITORY FACTOR AND NIX IN SKELETAL MUSCLE OF THE AGED MALE INTERLEUKIN 10 NULL MOUSE

Abstract

Chronic inflammation is associated with muscle weakness and frailty in older adults. The antagonistic cross-talk between macrophage migration inhibitory factor (Mif), an anti-apoptotic cytokine and NIP3-like protein X (Nix), a pro-apoptotic mitochondrial protein, may play a role in mitochondrial free radical homeostasis and inflammatory myopathies. We examined Nix-Mif interaction in inflammation and aging using young and old, IL-10tm/tm (a rodent model of chronic inflammation) and C57BL/6 mice. In this study, we observed that Nix and Mif were co-localized in skeletal muscles of aged and inflamed mice. We show an inflammation- and age-related association between Nix and Mif gene expression, with the strongest positive correlation observed in old IL-10tm/tm skeletal muscles. The IL-10tm/tm skeletal muscles also had the highest levels of oxidative stress damage. These observations suggest that Nix-Mif cross-talk may play a role in the interface between chronic inflammation and oxidative stress in aging skeletal muscles.

Introduction

Frailty in older adults is highly associated with a host of adverse health outcomes including falls, functional decline, disability and early mortality (1, 2). While the etiology of frailty remains unknown, there is substantial evidence supporting the association of chronic inflammation characteristic in frailty with skeletal myopathy (sarcopenia and muscle weakness), fatigue, and unintentional weight loss (3–6). Thus, better characterization of the molecular underpinning of inflammatory myopathies may help to elucidate frailty biology.

Macrophage migration inhibitory factor (Mif) may play a role in the development and progression of inflammatory myopathies in various muscle types (smooth muscle, cardiomyocytes, and skeletal muscles) (7–10). Mif is an antiapoptotic, proproliferative and chemotactic cytokine (11) that may exert some of these effects via functionally antagonizing the apoptotic effects of the mitochondrial protein NIP3-like protein X (Nix) (12). However, the mechanistic nature of this interaction is poorly understood in the context of aging biology. In this study we used the homozygous interleukine10 null (B6.129P2-Il10tm1Cgn/J (IL-10tm/tm)) frail mouse to study the interface between Nix and Mif in the context of aging and chronic inflammation. The IL-10tm/tm mouse has been proposed as a model to study the biology linking chronic inflammation, aging and late-life decline given its propensity to develop age-related elevated serum inflammatory cytokines (e.g. interleukin 6, tumor necrosis factor α), muscle weakness, and higher mortality compared to C57BL/6 (B6) controls (13, 14). While the biology that links genetically-induced chronic inflammation to accelerated late-life decline in the IL-10tm/tm mouse is not known, abnormal mitochondrial energy production and ATP kinetics (15) and alterations in apoptosis and mitochondrial function (13) in the skeletal muscles may play an important role. Given this evidence, we hypothesized that mitochondrial alterations in the skeletal muscles of IL-10tm/tm mouse are precipitated by age- and inflammation-associated effects on Nix and Mif interaction. In order to test this hypothesis, we sought to identify in the quadriceps femoris muscles of male IL-10tm/tm and B6 mice, age- and inflammation-associated differences in Nix and Mif expression, Nix-Mif co-localization and nitrotyrosine (ONOO--) a marker of oxidative stress.

Methods

Animals

Male IL-10tm/tm and B6 mice (Jackson Laboratory, Bar Harbor, ME; National Institute on Aging, Bethesda, MD) were housed in specific pathogen free barrier conditions until the appropriate age was reached and then sacrificed. A cross-sectional study design was utilized to compare differences in quadriceps femoris muscle biology between groups (N=3–6) of young (5–7 months-old) and old (21–26 months-old) mice. All studies were approved by the IACUC at Johns Hopkins University School of Medicine.

Immunofluorescence confocal microscopy

Co-localization of Nix and Mif proteins in skeletal muscles were determined by immunofluorescence laser scanning confocal microscopy. Quadriceps femoris muscles were embedded in Tissue-Tek O.C.T. Compound (Sakura) and multiple thin sections (10 μm) were cut using a cryostat (Microm). The muscle sections were fixed with 4% paraformaldehyde for 15 minutes at RT, blocked with 5% BSA/0.3% TritonX-100/PBS for one hour at RT, incubated with primary antibodies Nix (Cat#sc-28240, Santa Cruz Biotech) and Mif (Invitrogen) overnight at 4°C, and then incubated with secondary AlexFluor antibody (Invitrogen) at RT for 1 hour. Slides were mounted with Vectashield Hard Set with Dapi (Vector Laboratories). All images were taken with a Zeiss LSM 700 microscope.

Protein extraction and western blot

As described previously (16). Briefly, proteins were extracted from flash frozen quadriceps femoris muscles using T-PER (Thermo Scientific) with protease (Complete Mini, Roche) and phosphatase (PhosStop, Roche) inhibitors. Equal concentrations of proteins were electrophoresed using Bis-Tris gels (Invitrogen) and transferred onto nitrocellulose membranes which were incubated with primary antibodies overnight at 4°C. Primary antibodies used include Nix (Cat#sc-28240, Santa Cruz Biotech), Mif (Invitrogen), nitrotyrosine (Millipore) and Actin (Sigma). HRP-conjugated secondary antibodies were used to detect bands (Amersham). Quantitative Western blot analyses were performed using ImageJ (National Institutes of Health).

Real time PCR

Total cellular RNA from quadriceps femoris muscles was isolated using NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany) and cDNA was synthesized at 37°C for 60 minutes. Real time qPCR to determine Nix and Mif expression was performed using TaqMan gene expression assay mix (Applied Biosystems) after reverse transcription of RNA with the high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA). Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the threshold cycle for amplification as 2^−ΔΔC_T), where as ΔΔC_T = ΔC_T,Control − Δ C_T,Target.

Statistical analysis

One-way ANOVA with Tukey’s post-hoc test or Kruskal-Wallis nonparametric analysis with a Dunnett’s post-hoc test were used to determine differences among groups. When 2 groups were compared, an unpaired, 2-tailed Student’s t-test or a Wilcoxon rank-sum test was used.

Results

To study the impact of aging and inflammation on Nix and Mif proteins, we compared young and old B6 mice to age- and sex-matched IL-10tm/tm mice. The old IL-10tm/tm mice captured the combined effects of age and chronic inflammation when compared to B6 controls.

We used laser scanning confocal microscopy to study the distribution and localization of Nix and Mif proteins in skeletal muscles of our mouse cohorts. As shown in figure 1, Nix and Mif co-localized in skeletal muscles of our mice, with strongest signal seen in the aged and inflamed mice (Aged IL-10tm/tm). To determine if the high co-localization observed in aged IL10 was driven by changes in expression of Nix and Mif, we quantified changes in the mRNA levels of Nix and Mif in individual mice. Our data demonstrate a significantly high positive correlation between Nix and Mif mRNA transcripts in our aged and inflamed mice (Fig 2). This association was again most pronounced in old IL-10tm/tm mice (Table 1).

Figure 1.

Nix and Mif in Aged and inflamed skeletal muscle. Immunofluorescence analyses of skeletal muscle cryosections using an antibody against NIX (red) and MIF (green) in aged C57bl/6 (top) and IL10tm/tm (bottom). Scale bar: 20 μm

Figure 2.

Correlation of NIX and MIF genes in aged IL10tm/tm

Table 1.

Spearman correlation of NIX and MIF in mouse cohorts showing overall positive correlation between NIX and MIF in all groups with the correlation being the strongest in IL-10tm/tm

	Spearman Rank Correlation	P-value
Young C57bl/6	0.236	0.511
Old C57bl/6	0.661	0.038
Young IL10tm/tm	0.831	0.003
Old IL10tm/tm	0.976	<0.001

At the protein level, inflammation (IL-10tm/tm genotype) was associated with increased skeletal muscle expression of Nix (young B6, 0.05 ± 0.02 AU vs. young IL-10tm/tm, 2.4± 0.3 AU, P<0.001; old B6, 0.4 ± 0.08 AU vs. old IL-10tm/tm, 2.3 ± 0.3 AU, P<0.001; Fig 3). Mif levels were slightly higher in IL-10tm/tm, but was not statistically significant (old B6, 0.2 ± 0.06 AU vs. old IL-10tm/tm, 1.1 ± 0.3 AU; Fig 3). Age was associated with increased Nix expression in B6 (young B6, 0.05 ± 0.02 AU vs. old B6, 0.4 ± 0.08 AU, P<0.05) but not in IL-10tm/tm (young IL-10tm/tm, 2.4 ± 0.3 AU vs. old IL-10tm/tm, 2.3± 0.3 AU)

Figure 3.

Western blot analyses of skeletal muscle protein extracts using antibodies against NIX, MIF and Peroxynitrite (ONOO--). Actin was used as a loading control. Relative expression was calculated for the Western blots displayed in in arbitrary units (AU)

Data are means ± SEM (n=3–5 animals) *p<0.05; **p<0.01; ***p>0.001.

Inflammation (IL-10tm/tm genotype) was associated with increased nitrotyrosine (ONOO--) expression in young (young B6, 0.1 ± 0.03 AU vs. young IL-10tm/tm, 4.4 ± 0.2 AU, P<0.0001; Fig 3) and old (old B6, 0.8 ± 0.2 AU vs. old IL-10tm/tm, 4.1 ± 0.4 AU, P<0.0001) mice.

Taken together, these data suggest age- and inflammation-associated differences in Nix and Mif expression and Nix-Mif interaction with particularly pronounced changes observed in old IL-10tm/tm mice.

Discussion

Prior studies have confirmed the presence of Nix and Mif in skeletal muscles and suggested a functional antagonism between these two functionally important proteins (12). Both Nix and Mif are involved in the development and progression of myopathies (7–10, 16, 17). In our present study, we provided evidence of direct interaction between Nix and Mif in skeletal muscles that is enhanced with aging. These changes are also particularly enhanced in IL-10tm/tm mice regardless of age, compared to age-matched B6 controls, suggesting a more prominent role for inflammation in driving this interaction. Given that the IL-10tm/tm mouse demonstrates key features of frailty including early onset muscle weakness (13) chronic inflammation (13, 14), and ATP kinetics impairment, future studies utilizing this mouse as an in vivo model to explore molecular mechanisms connecting the dynamic process of mitochondrial oxidative stress, chronic inflammation, loss of muscle mass, and frailty are merited.

Both Nix and Mif have been implicated in various pathological conditions. Aberrant Nix signaling is known to mediate apoptotic cardiomyopathy (17). The blockade of Mif protects mice against septic shock and has been suggested to play a role in pathological processes of skeletal muscle. Mechanistically, prior reports have shown that Mif inhibits mitochondria-dependent death pathways and prevents release of cytochrome c from the mitochondria and subsequent activation of the critical effector caspase-3. In contrast, Nix binds to mitochondrial membranes and engages direct molecular interactions with microtubule-associated protein-1 light chain 3 (LC3), ultimately leading to mitochondrial degradation and clearance (18). Although the functional consequences of this cross talk between Nix and Mif in aged-organisms are not known, it is tempting to speculate that mitochondrial homeostasis and consequently altered mitophagy (autophagy-mediated mitochondrial turnover) may be operant in the accelerated decline in age-related muscle weakness, abnormalities in energy generation, and frailty previously reported in the IL-10tm/tm mice (13). Moreover, because both young and old IL-10tm/tm mice have a more pronounced effect on expression of both Nix and Mif, it is possible that the Nix-Mif interaction may be a factor in influencing myopathy and late-life decline observed in chronic inflammatory conditions such as frailty.

The elevated levels of nitrotyrosine in the skeletal muscles of old IL-10tm/tm mouse suggest the presence of increased oxidative stress. This increased oxidative stress, which appears to be associated with aging and inflammation, maybe the result of impaired mitochondrial homeostasis in IL-10tm/tm mouse. While many previous studies have shown that a combination of mitochondrial and autophagy dysfunction could contribute to age-associated degenerative and neuromuscular diseases (18–24) others have shown that mitochondrial oxidative stress many be beneficial (25, 26). Interestingly, the increase in oxidative stress in IL-10tm/tm mouse is accompanied by evidence of increased mitochondrial death signaling (Nix) and and apoptosis inhibition (Mif). Thus, these concurrent changes may underscore a compensatory response in skeletal muscle cells to maintain cellular homeostasis in the setting of oxidative stress. Future mechanistic studies focusing on autophagy flux and mitochondrial function are needed to validate and enhance our present findings.

The biology of aging, chronic inflammation and mitochondrial dysfunction are likely to have multiple interacting pathways. The Nix-Mif cross-talk may be a key interface between mitochondrial homeostasis and oxidative stress in aging skeletal muscles (16). Because the IL-10tm/tm mouse demonstrates key features of frailty including early onset myopathy (13), chronic inflammation (13, 14) and impaired energy production, future studies using this mouse as an in vivo model to explore molecular mechanisms linking mitochondrial turnover and energy production, sarcopenia, and inflammatory myopathies are merited.

The limitations to this exploratory study include a relatively small sample size and the observational nature of the study. Perhaps more importantly, the study is designed to ascertain associations, the effects of Nix-Mif interaction on autophagy, apoptosis or muscle homeostasis were not analyzed. Larger studies are required to further dissect the impact of the cross talk between Nix and Mif proteins.