Muscle Damage in Systemic Sclerosis and CXCL10: The Potential Therapeutic Role of PDE5 Inhibition

Abstract

Skeletal muscle damage is a common clinical manifestation of systemic sclerosis (SSc). C-X-C chemokine ligand 10 (CXCL10) is involved in myopathy and cardiomyopathy development and is associated with a more severe SSc prognosis. Interestingly, the phosphodiesterase type 5 inhibitor (PDE5i) sildenafil reduces CXCL10 sera levels of patients with diabetic cardiomyopathy and in cardiomyocytes. Here, we analyzed the levels of CXCL10 in the sera of 116 SSc vs. 35 healthy subjects and explored differences in 17 SSc patients on stable treatment with sildenafil. CXCL10 sera levels were three-fold higher in SSc vs. healthy controls, independent of subset and antibody positivity. Sildenafil treatment was associated with lower CXCL10 sera levels. Serum CXCL10 strongly correlated with the clinical severity of muscle involvement and with creatine kinase (CK) serum concentration, suggesting a potential involvement in muscle damage in SSc. In vitro, sildenafil dose-dependently reduced CXCL10 release by activated myocytes and impaired cytokine-induced Signal transducer and activator of transcription 1 (STAT1), Nuclear factor-κB (NFκB) and c-Jun N-terminal kinase (JNK) phosphorylation. This was also seen in cardiomyocytes. Sildenafil-induced CXCL10 inhibition at the systemic and human muscle cell level supports the hypothesis that PDE5i could be a potential therapeutic therapy to prevent and treat muscle damage in SSc.

Keywords: CXCL10; inflammation; myocytes; sildenafil; systemic sclerosis.

Figure 1

Circulating level of CXCL10 is higher in systemic sclerosis (SSc) patients than healthy subjects. Treatment with sildenafil significantly reduced CXCL10 serum level in SSc subjects. (A) CXCL10 serum level was significantly elevated in SSc patients without sildenafil (n = 99) in their therapeutic regimen vs. sildenafil treated-SSc patients (n = 17) and healthy subjects (n = 35) (633.07 ± 183.02 pg/mL vs. 455.21 ± 211.8 pg/mL and 197.5 ± 14.89 pg/mL, respectively, * p < 0.05 and *** p < 0.001). (B) CXCL10 was analyzed in the sera of SSc patients divided in different subgroups: Limited (LcSSc) (n = 72) (575.21 ± 8.49 pg/mL), diffuse (DcSSc) (n = 37) (640.73 ± 186.33 pg/mL), anti-topoisomerase I (ScL70) positive (n = 18) (324.22 ± 34.10 pg/mL), anti-centromere (ACA) positive (n = 51) (370.24 ± 39.81 pg/mL) and ScL70-ACA double negative (n = 38) (1062.74 ± 435.36 pg/mL). All the subgroups analyzed showed CXCL10 serum levels significantly higher than healthy subjects (** p < 0.01 and *** p < 0.001, respectively). No significant differences within SSc subgroups were noted. Data are expressed as CXCL10 serum level, in pg/mL (mean ± standard error of the mean (SE)).

Figure 2

CXCL10 serum level positively correlates with an above normal range of creatine kinase (CK) blood level in SSc patients. The scatterplot has the CK blood level in μmol/L on the horizontal (X) axis, and the CXCL10 serum level, in pg/mL, on the vertical (Y) axis. CK < 180μ mol/L is considered the cut-off of normal range. Each individual is identified by a single point (dot) on 180 mol/L; each square symbolizes SSc–myositis patient with CK > 180 μmol/L (above normal range). SSc patients with a CK > 180 μmol/L showed higher levels of circulating CXCL10. The relationship between CK blood level and serum CXCL10 levels in SSc–myositis patients is strong, linear, and positive with a Pearson’s correlation coefficient of +0.97 (p < 0.001). No significant correlation is observed in SSc patients with CK within normal range (R² = 0.02, p = 0.245).

Figure 3

Cytokine-induced CXCL10 release in muscle cells is significantly diminished by sildenafil. Effect of sildenafil STAT 1, JNK and NF-kB phosphorylation in human myocytes (Hfsmc), dermal fibroblasts (Hdfbs) and cardiomyocytes (Hfcm). (A) Incubation with increasing doses of sildenafil (1 × 10⁻⁷, 2.5 × 10⁻⁷, 5 × 10⁻⁷, 1 × 10⁻⁶, 2.5 × 10⁻⁶, 5 × 10⁻⁶, 1 × 10⁻⁵, 2.5 × 10⁻⁵ M) for 24 h significantly albeit not strictly dose-dependently reduced CXCL10 protein release by Hfsmc (upper left panel) stimulated with interferon (IFN)γ (1000 U/mL) + tumor necrosis factor (TNFα) (10 ng/mL) starting from 2.5 × 10⁻⁷ M of sildenafil dose (54.61 ± 19.05 % of CXCL10 secretion * p < 0.05). Maximal inhibitory effect was at 2.5 × 10⁻⁵ M of sildenafil concentration (36.55 ± 6.61 % of CXCL10 secretion, ** p < 0.01). Data are obtained from three different experiments using different cell preparations. 24 h treatment with IFNγ (1000 U/mL) + TNFα (10 ng/mL) significantly upregulated CXCL10 mRNA expression in Hfsmc and Hdfbs; sildenafil (1 μM) did not exert any significantly change on CXCL10-cytokines induced gene level in both cell types (upper and lower right panels). CXCL10 mRNA expression is determined by RT-qPCR and is expressed as fold increase vs. IFNγ+TNFα-induced expression taken as 1 (mean ± SE). Data are obtained from three different experiments using three different cell preparations. (B) Western blot analysis in Hfsmc demonstrated that IFNγ+TNFα significantly increased STAT 1, JNK and NF-kB phosphorylation (** p < 0.01 vs. c) and the treatment with sildenafil significantly reduced all of these phosphorylation (### p < 0.001 and ## p < 0.01 vs. IFNγ+TNFα, respectively). (C) Western blot analysis in human healthy dermal fibroblasts revealed that IFNγ+TNFα significantly increased STAT 1, JNK and NF-kB phosphorylation (** p < 0.01 and * p < 0.05 vs. c, respectively) but the treatment with sildenafil did not affect pathway activation. (D) Western blot analysis in human fetal cardiomyocytes (Hfcm) confirmed the results obtained in muscle cells and showed that IFNγ+TNFα significantly increased STAT 1, JNK and NF-kB phosphorylation (** p < 0.01 and * p < 0.05 vs. c, respectively) and the treatment with sildenafil significantly reduced all these path phosphorylation (# p < 0.01 and ## p < 0.01 vs. IFNγ+TNFα, respectively). Figure 3, panel B, C and D: The densitometric analysis of band intensities is reported under each panel. Results are derived from three to five experiments, using distinct cell preparations. Data are expressed as ratio phosphorylated/β-actin fold increase vs. IFNγ+TNFα, taken as 1 (mean ± standard error of the mean (SE)).

Figure 4

Cytokine-induced CXCL10 secretion, mRNA expression and STAT1, JNK and NF-kB phosphorylation do not change in human SSc fibroblasts after sildenafil. (A) Secretion and gene expression were evaluated in SSc human dermal fibroblasts challenged with IFNγ (1000 U/mL) + TNFα (10 ng/mL) and with/without sildenafil scalar doses (1 × 10⁻⁷, 2.5 × 10⁻⁷, 5 × 10⁻⁷, 1 × 10⁻⁶, 2.5 × 10⁻⁶, 5 × 10⁻⁶, 1 × 10⁻⁵, 2.5 × 10⁻⁵ M) or fixed dose (1 × 10⁻⁶ M), respectively, for 24 h. Sildenafil neither essentially reduced CXCL10 secretion in SSc fibroblasts (left panel) nor reduced CXCL10 mRNA expression, significantly upregulated after inflammatory stimuli (right panel). (B) Western blot analysis documented that sildenafil did not affect the basal level of STAT1, JNK and NF-kB phosphorylation in SSc human dermal fibroblasts. At variance with STAT1, both JNK and NF-kB basal phosphorylation showed the trend to be higher in SSc vs. healthy cells (* p < 0.05). Densitometric analysis of band intensities is depicted as relative density (arbitrary units) under each panel and normalized against optical density of STAT1, NF-kB and JNK total protein, respectively. Results are derived from three different experiments, using distinct cell preparations.

Figure 5

Sildenafil does not exert any effect onto the cell viability of human skeletal muscle cells (Hfsmc), normal and SSc fibroblasts. A, B and C depict cell viability in Hfsmc, healthy and SSc human dermal fibroblasts, respectively, as evaluated with an MTT test after 24 h treatment with 0.25; 1; 2.5; 25 μM of sildenafil. Results are reported as change in absorbance at 560 nm. Results are derived from three different experiments, using distinct cell preparations.