Inflammation-Linked Muscle Atrophy in Limb Girdle Muscular Dystrophy R1 (LGMDR1): Insights into Disease Mechanisms

Figures

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  Figure 1. Expression of TRIM63 and FBXO32 genes in LGMDR1 patients by qRT-PCR. (A) Column bar graph showing significant upregulation of TRIM63 gene expression in LGMDR1 patients (p = 0.0442, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 5). (B) Column bar graph showing significant upregulation of FBXO32 gene expression in LGMDR1 patients (p = 0.0261, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 5).

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  Figure 2. Expression of MuRF1 and Atrogin-1 proteins in LGMDR1 patients by Western blotting. (A) MuRF1 protein expression by Western blot, (B) Atrogin-1 protein expression by Western blot, C, control; P1-P3, individual LGMDR1 patient samples. (C) Column bar graph showing significant upregulation of MuRF1 protein expression in LGMDR1 patients (p = 0.0351, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 4). (D) Column bar graph showing significant upregulation of Atrogin-1 protein expression in LGMDR1 patients (p = 0.0021, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 4).

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  Figure 3. Expression of TNF, IL1B, and IL6 genes in LGMDR1 patients by qRT-PCR. (A) Column bar graph showing significant upregulation of TNF gene expression in LGMDR1 patients (p = 0.0208, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 5). (B) Column bar graph showing significant upregulation of IL1B gene expression in LGMDR1 patients (p = 0.0333, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 5). (C) Column bar graph showing significant upregulation of IL6 gene expression in LGMDR1 patients (p = 0.0043, Mann–Whitney test). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 5).

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  Figure 4. Expression of TNF-α and IL-1β cytokines in LGMDR1 patients by ELISA. (A) Column bar graph showing significant upregulation of TNF-α cytokine in LGMDR1 patients (p = 0.0021, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 4). (B) Column bar graph showing upregulation of IL-1β cytokine in LGMDR1 patients (nsp = 0.3051, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 4).

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  Figure 5. Expression of RELA, FOXO1, and FOXO3 genes in LGMDR1 patients by qRT-PCR. (A) Column bar graph showing significant upregulation of RELA gene expression in LGMDR1 patients (p = 0.0238, Mann–Whitney test). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 3). (B) Column bar graph showing significant upregulation of FOXO1 gene expression in LGMDR1 patients (p = 0.0255, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 5). (C) Column bar graph showing significant upregulation of FOXO3 gene expression in LGMDR1 patients (p = 0.0175, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 6; controls, n = 5).

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  Figure 6. Expression of NFκB and IκBα proteins in LGMDR1 patients by Western blotting. (A) Phospho-NFκB (P65) and NFκB (P65) protein expression in LGMDR1 patients by Western blot. (B) Phospho-IκBα and IκBα protein expression in LGMDR1 patients by Western blot. C, control; P1, P3, P5, individual LGMDR1 patient samples (C) Column bar graph showing significant upregulation of NFκB protein expression in LGMDR1 patients (p = 0.0009, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 4; controls, n = 2). (D) Column bar graph showing upregulation of IκBα protein expression in LGMDR1 patients (p = 0.0492, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 4; controls, n = 3).

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  Figure 7. Expression of FOXO1 and AKT proteins in LGMDR1 patients by Western blotting. (A) Phospho-FOXO1 and FOXO1 protein expression by Western blot. (B) Phospho-AKT and AKT protein expression by Western blot. C, control; P2, individual LGMDR1 patient sample (C) Column bar graph showing decreased phosphorylation of FOXO1 protein in LGMDR1 patients (p = 0.0314, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 2; controls, n = 2). (D) Column bar graph showing significantly decreased phosphorylation of AKT protein in LGMDR1 patients (p = 0.0362, unpaired t-test with Welch’s correction). Data are mean ± SEM and shown as fold change relative to control (LGMDR1, n = 2; controls, n = 2).

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  Figure 8. Overview of inflammation-induced muscle atrophy in LGMDR1. Inflammation may play an essential role in driving muscle atrophy in LGMDR1 patients. The current study showed a significant increase in the expression of pro-inflammatory cytokines—TNF-α, IL-1β, and IL-6 in muscle biopsies of LGMDR1 patients, as measured by qRT-PCR. Among these, only TNF-α expression was significantly increased in muscle tissue (qRT-PCR) and serum samples (by ELISA) of these patients, compared to controls. However, IL-1β level was not statistically significant in these patients. Additionally, IL-6 level was not significantly detectable in serum samples of these patients. This may be because of a lack of correlation between mRNA and protein abundance due to post-transcriptional regulation. Thus, the data suggest that TNF-α is a muscle-wasting factor in LGMDR1 patients. Blue indicates the NFκB pathway, pink indicates the AKT/FOXO signaling pathway, “P” denotes phosphorylation, and “p-” indicates the phosphorylated form of the indicated protein.