Deletion of metal transporter Zip14 (Slc39a14) produces skeletal muscle wasting, endotoxemia, Mef2c activation and induction of miR-675 and Hspb7

Abstract

Skeletal muscle represents the largest pool of body zinc, however, little is known about muscle zinc homeostasis or muscle-specific zinc functions. Zip14 (Slc39a14) was the most highly expressed zinc transporter in skeletal muscle of mice in response to LPS-induced inflammation. We compared metabolic parameters of skeletal muscle from global Zip14 knockout (KO) and wild-type mice (WT). At basal steady state Zip14 KO mice exhibited a phenotype that included muscle wasting and metabolic endotoxemia. Microarray and qPCR analysis of gastrocnemius muscle RNA revealed that ablation of Zip14 produced increased muscle p-Mef2c, Hspb7 and miR-675-5p expression and increased p38 activation. ChIP assays showed enhanced binding of NF-[Formula: see text] to the Mef2c promoter. In contrast, LPS-induced systemic inflammation enhanced Zip14-dependent zinc uptake by muscle, increased expression of Atrogin1 and MuRF1 and markedly reduced MyoD. These signatures of muscle atrophy and cachexia were not influenced by Zip14 ablation, however. LPS-induced miR-675-3p and -5p expression was Zip14-dependent. Collectively, these results with an integrative model are consistent with a Zip14 function in skeletal muscle at steady state that supports myogenesis through suppression of metabolic endotoxemia and that Zip14 ablation coincides with sustained activity of phosphorylated components of signaling pathways including p-Mef2c, which causes Hspb7-dependent muscle wasting.

Figure 1

Expression of Zip and ZnT transcripts in skeletal muscle during acute inflammation induced with lipopolysaccharide (endotoxin). (A) Relative abundance of Zip mRNAs and ZnT mRNAs in gastrocnemius muscle of WT mice at 18 h after LPS. (B) Serum IL-6 concentrations at 18 h after LPS. (C) Serum zinc concentrations of male and female mice at 0–18 h after LPS. The LPS dose was 2 mg/kg (i.p.). Values are means $\pm$ SEM, n = 3–4 mice per treatment group. *P < 0.05; **P < 0.01; ***P < 0.001. Solid bars are WT mice; shaded bars are LPS-treated WT mice. nd = not detectable.

Figure 2

Metabolic endotoxemia at steady state and Influence of acute inflammation induced by LPS on skeletal muscle of wild type and Zip14 knockout mice. (A) Induction of Zip14 mRNA, 3–48 h after LPS. (B) Western analysis of induction of muscle Zip14 protein 0–18 h after LPS. Each lane is pooled sample from n = 4 per group. Blots were cut horizontally at the appropriate molecular mass and incubated with the appropriate antibody for the target protein and show contiguous lanes. The blots are representative of multiple experiments. (C) Muscle Zn concentration in WT and Zip14 KO mice 18 h after LPS. The LPS dose was 2 mg/kg (i.p.). (D) Uptake of orally administered 65Zn into muscle in WT and Zip14 KO mice. (E) Serum endotoxin levels. (F) Serum IL-6 concentrations. Values are means $\pm$ SEM, n = 4. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Zip14 knockout mice exhibit skeletal muscle wasting at steady state. Representative images of hind legs of WT and Zip14 KO female (A) and male (B) mice showing reduced length and mass and representative excised gastrocnemius muscle from both genotypes. The mice used were 12–16 wks of age. Ratio of muscle weight to body weight for female (C) and male (D) mice of both genotypes. H$\&$E stained sections of these muscles from female (E) and male (F) mice. Magnification is 40x (E) and 20x (F), respectively. Interstitial area of muscle from the H$\&$E stained images (G, female mice; H, male mice; n = 3) Numerical data are means $\pm$ SEM. *P < 0.05, ****P < 0.0001.

Figure 4

Genome-wide transcriptome comparisons using microarrays identify differentially expressed genes in skeletal muscle of wild type and Zip14 KO mice at steady state. (A) Volcano plot of differentially expressed genes. Total RNA was isolated from individual gastrocnemius muscle tissue of WT and Zip14 knockout mice and microarray analysis was performed. Treatment groups had n = 3–4 mice. A total of 76 transcripts were designated as differentially expressed between the genotypes at a FC $\ge$ +2 or $\le -2$. Data are FC vs. probability expressed as a ratio of KO/WT. (B) Relative mRNA levels for Mef2c, Hspb7, MyoD and miR-675-5p as subsequently measured by qPCR. Values are means $\pm$ SEM, n = 4 mice per group. **P < 0.01; ***P < 0.001. (C) Western analysis of Hsbp7 and Mef2c protein levels. Each lane represents a lysate from one mouse. Blots were cut horizontally at the appropriate molecular mass and incubated with the appropriate antibody for the target protein and show contiguous lanes. The blots are representative of multiple experiments.

Figure 5

Comparison of specific pathway activation in skeletal muscle of wild type and Zip14 KO mice at steady state. Western analysis of activation of p38, p-p38 and NF-$\kappa \beta$. Each lane represents a lysate from one mouse. Blots were cut horizontally at the appropriate molecular mass and incubated with the appropriate antibody for the target protein and show contiguous lanes. The blots are representative of multiple experiments.

Figure 6

Elevation of haptoglobin expression in skeletal muscle as influenced by Zip14 ablation in mice at steady state. (A) Abundance of Hp mRNA from the individual qPCR analysis. ChIP analysis of the Hp promoter performed with DNA from muscle using antibodies for (B) STAT3 and (C) C/EBP. Values are means $\pm$ SEM, n = 4 mice per group. ***P < 0.001.

Figure 7

Comparison of Mef2c expression and promoter activation in skeletal muscle from wild type and Zip14 KO mice at steady state and during acute inflammation as induced by LPS. (A) Levels of Mef2c mRNA as measured by qPCR. (B) ChIP analysis of Mef2c promoter binding of NF-$\kappa \beta$ using muscle DNA. Values are means $\pm$ SEM, n = 4 per genotype. ***P < 0.001. The LPS dose was 2 mg/kg (i.p.) 18 h before sacrifice.

Figure 8

Transcript abundance of Atrogin1, MuRF1, MyoD, Metallothionein 1, miR-675-3p, and miR-675-5p and Atrogin1 and MuRF1 protein in skeletal muscle as influenced by Zip14 ablation and acute inflammation induced by LPS. (A, B, E–G) Relative transcript abundances as measured by qPCR. (C,D) Relative protein abundance was measured by western analysis. Each lane represents a lysate from one mouse. The LPS dose was 2 mg/kg (i.p.) given 18 h before sacrifice. Values for mRNA levels are means $\pm$ SEM, n = 4 mice per group. ***P < 0.001.

Figure 9

Proposed systems summary of the influence of Zip14 ablation and metabolic endotoxemia of pathways involved in muscle wasting in mice. Global deletion of Zip14 leads to diminished intestinal barrier function and leakage of endotoxins from intestinal microbiota into the systemic circulation creating metabolic endotoxemia. Inflammation-regulated pathways activate transcription factors including NF-$\kappa \beta$ and Mef2c in skeletal muscle of the Zip14 knockout mice creating a localized zinc deficiency leading to enhanced production of acute phase proteins and Hspb7, an autophagic protein, thus resulting in muscle wasting. White lines represent inhibitory influence of zinc ions in muscle with functioning Zip14-mediated zinc transport.