Activation of PDGF Signaling in the Adult Muscle Stem Cell Niche in Patients With Type 2 Diabetes Mellitus

Abstract

Context: Type 2 diabetes mellitus (T2D) negatively affects muscle mass and function throughout life. Whether adult muscle stem cells contribute to the decrease in muscle health is not clear and insights into the stem cell niche are difficult to obtain.

Objective: To establish the upstream signaling pathway of microRNA (miR)-501, a marker of activated myogenic progenitor cells, and interrogate this pathway in muscle biopsies from patients with T2D.

Methods: Analysis of primary muscle cell cultures from mice and 4 normoglycemic humans and muscle biopsies from 7 patients with T2D and 7 normoglycemic controls using gene expression, information on histone methylation, peptide screening, and promoter assays.

Results: miR-501 shares the promoter of its host gene, isoform 2 of chloride voltage-gated channel 5 (CLCN5-2), and miR-501 expression increases during muscle cell differentiation. We identify platelet-derived growth factor (PDGF) as an upstream regulator of CLCN5-2 and miR-501 via Janus kinase/signal transducer and activator of transcription. Skeletal muscle biopsies from patients with T2D revealed upregulation of PDGF (1.62-fold, P = .002), CLCN5-2 (2.85-fold, P = .03), and miR-501 (1.73-fold, P = .02) compared with normoglycemic controls. In addition, we observed a positive correlation of PDGF and miR-501 in human skeletal muscle (r = 0.542, P = .045, n = 14).

Conclusions: We conclude that paracrine signaling in the adult muscle stem cells niche is activated in T2D. Expression analysis of the PDGF-miR-501 signaling pathway could represent a powerful tool to classify patients in clinical trials that aim to improve muscle health and glucose homeostasis in patients with diabetes.

Keywords: PDGF; microRNA; skeletal muscle; type 2 diabetes mellitus.

Figure 1.

Regulation of Clcn5-2 and miR-501 during muscle cell differentiation. A-C) Expression of Pax7, Myh3, Clcn5-2, and miR-501 in C2C12 (A), mouse primary myoblasts (B), and human primary myoblasts (C) during differentiation; n = 3-4. (D) Schematic representation of ENCODE H3K4me3 ChIP-seq data at the 5′-UTR of the mouse Clcn5 gene in C57BL/6 kidney (1), C2C12 myoblasts (2), and C2C12 differentiated for 60 hours (3), as well as the human CLCN5-2 gene in adult (22 years) human skeletal muscle myoblasts (4). All data are plotted as mean ± SEM; qPCR values were normalized to snoRNA234 (mmu-miR-501), U6 small nuclear RNA (hsa-miR-501) or 18S ribosomal RNA. Significance was evaluated vs growth medium (GM) by 1-way analysis of variance with multiple comparisons test; *P ≤ .05; **P ≤ .01; ***P ≤ .001.

Figure 2.

PDGF signaling regulates expression levels of miR-501 in muscle cells. (A-C) Clcn5-2 and miR-501 expression in C2C12 myoblasts (A), mouse primary myoblasts (B), and human primary myoblasts (C) after treatment with recombinant proteins for 2 × 24 hours; n = 3-5. (D) Clcn5-2 expression after treatment with PDGF and inhibitors against STAT3 (Nifuroxazide), JAK3 (VX-509), FGFR1 (PD173074), MEK1/2 (PD184352), PI3Kα/δ/β (LY294002), Rac GTPase (NSC23766), or PLC-γ (U73122); n = 4. (E-G) Clcn5 and miR-501 expression in C2C12 myoblasts (E), mouse primary myoblasts (F), and human primary myoblasts (G) after treatment with PDGF and inhibition of STAT3 signaling (Nifuroxazide); n = 3-4. (H, I) Effect of PDGF inhibitor Crenolanib in mouse primary myoblasts on miR-501 expression (H) and p-Stat3 as determined by immunoblot and quantified relative to γ-tubulin (I); n = 3. (J-L) Clcn5-2 and miR-501 expression in C2C12 myoblasts (J), and mouse primary myoblasts (K) upon overexpression of STAT3; n = 4. (L) miR-501 promoter activity upon overexpression of STAT3; n = 3. All data are plotted as mean ± SEM; qPCR values were normalized to snoRNA234 (mmu-miR-501), U6 small nuclear RNA (hsa-miR-501), or 18S ribosomal RNA. All data are plotted as mean ± SEM. Significance was evaluated vs control by t-test (A, B, C: miRNA) and 1-way analysis of variance with the multiple comparisons test (A, B, C: mRNA; D, E, F, H, I, J, K); *P ≤ .05; **P ≤ .01; ***P ≤ .001.

Figure 3.

PDGF treatment during myogenic differentiation affects gene expression profile. (A) mouse and human primary myoblasts were cultured in growth medium with added PDGF-AA for 2 × 24 hours prior to differentiation, during which cell culture medium ± PDGF was exchanged every 2 days. Gene expression was assessed at indicated time points (last four arrows). (B) Light microscopy of mouse (top) and human (bottom) primary myofibers at day 6 of differentiation ± PDGF. Scale bar = 200 μm. (C, D) Pax7 and Myh3 expression in mouse primary myoblasts (C; n = 4 independent cell cultures), and human primary myoblasts (D; n = 3 independent cell cultures). All data are plotted as mean ± SEM; qPCR values were normalized to 18S ribosomal RNA. All data are plotted as mean ± SEM. Significance was evaluated vs control 2-way analysis of variance with the multiple comparisons test; *P ≤ .05; **P ≤ .01; ***P ≤ .001.

Figure 4.

miR-501 levels are not altered in aged compared with young human skeletal muscle. (A) Expression levels of PDGF, CLCN5-2, pri-miR-501, miR-501, and IGF-1; n = 5 (young) vs 6 (aged). (B) Immunofluorescence (wheat germ agglutinin) in aged human muscle and quantification of average fiber size; n = 5 vs 6. All data are shown as mean ± SEM; qPCR data was normalized to U6 small nuclear RNA (hsa-miR-501) or 18S ribosomal RNA. Significance was evaluated by the t-test; **P < .01.

Figure 5.

miR-501 levels are increased in skeletal muscle from humans with type 2 diabetes. (A) Expression levels of PDGF, CLCN5-2, pri-miR-501, miR-501, GLUT4, and PAX7 in muscle from subjects with T2D and control; n = 7. (B) Correlation between miR-501 and PDGF levels as shown in A; n = 7. All data are shown as mean ± SEM; qPCR data were normalized to U6 small nuclear RNA (hsa-miR-501) or 18S ribosomal RNA. Significance was evaluated by the t-test (A) and Pearson correlation (B); *P < .05, **P < .01.

Figure 6.

Graphical representation of the regulation of miR-501 in myogenic progenitor cells by differentiation and PDGF signaling and its activation in T2D.