Deletion of muscle GRP94 impairs both muscle and body growth by inhibiting local IGF production

Abstract

Insulin-like growth factors (IGFs) are critical for development and growth of skeletal muscles, but because several tissues produce IGFs, it is not clear which source is necessary or sufficient for muscle growth. Because it is critical for production of both IGF-I and IGF-II, we ablated glucose-regulated protein 94 (GRP94) in murine striated muscle to test the necessity of local IGFs for normal muscle growth. These mice exhibited smaller skeletal muscles with diminished IGF contents but with normal contractile function and no apparent endoplasmic reticulum stress response. This result shows that muscles rely on GRP94 primarily to support local production of IGFs, a pool that is necessary for normal muscle growth. In addition, body weights were ∼30% smaller than those of littermate controls, and circulating IGF-I also decreased significantly, yet glucose homeostasis was maintained with little disruption to the growth hormone pathway. The growth defect was complemented on administration of recombinant IGF-I. Thus, unlike liver production of IGF-I, muscle IGF-I is necessary not only locally but also globally for whole-body growth.

Figure 1.

Muscle-specific deletion of GRP94. A) Schematic representation of the GRP94 gene showing the loxP sites flanking exon 1, whose deletion removes 900 bp of DNA. B) Example of PCR assays used to genotype mouse tissues. When the WT allele is amplified with primers a (5′ to the gene) and b (within exon 2), it yields a 1320-bp product from nondeleted tissues (liver and kidney), but only a 420-bp product if the floxed gene segment is deleted. C) IHC of tissue sections for GRP94 expression. The detecting antibody is mAb 9G10, developed with an HRP reaction. D) Quantitative assessment of GRP94 depletion by Western blot analysis using extracts from the muscles of WT (MCK-Cre−) or mutant (MCK-Cre+). GRP94 is detected with mAb 9G10. Percentage depletion was calculated as the ratio of GRP94 in the Cre+ and Cre− muscle in each pair, normalized to 14-3-3 (loading control). MCK-Cre is not active in liver, only in skeletal and cardiac muscles. EDL, extensor digitorum longus; sol, soleus, TA, tibialis anterior; gast, gastrocnemius; quad, quadriceps.

Figure 2.

Effect of GRP94 deletion on production of muscle IGF. A) IGF-I contents of extracts of tibialis anterior and gastrocnemius muscles from the indicated ages, and genders were determined by ELISA. □, WT mice; ●, mGRP94^−/− mice. Each point represents an individual mouse. The 4-wk-old cohort consisted of 8 males and 5 females. B) Skeletal muscle IGF-I level correlates with the level of GRP94 protein. The values of tissue IGF-I from individual 8-wk mGRP94^−/− females (●) and mGRP94^+/− females (▲) were plotted as a function of the amount of GRP94 protein from the same samples, estimated by Western blots of the harvested TA or gastrocnemius muscles. The nondepleted samples were from WT littermates (□). C) Quantitation of IGF-I secreted by primary myoblasts. Cultures were set up in triplicate as described in Materials and Methods, supernatants were collected at the indicated time points, and their IGF-I contents were measured by ELISA. Shown is a representative experiment out of two independent repetitions, with cultures from 3 mice per genotype. *P < 0.05 vs. corresponding WT; unpaired t test. D) Quantitation of IGF-II secreted by primary myoblasts (as in C).

Figure 3.

Size distribution of fiber types. A, B) Transverse sections of gastrocnemius muscles from 8-wk-old WT (A) and mGRP94^−/− (B) animals were double-stained with anti-laminin (red), to delineate the fiber circumference, and anti-MHC 1/β isoform (green). C) Histogram of size distributions of MHC1/β-positive fibers from WT (□) or mutant (■) muscles, determined from images as in A and B. Plots represent percentage of fibers in each size bin. Fibers are significantly smaller in mGRP94^−/− muscles compared with those in WT fibers (Mann-Whitney test). n = 4 muscles/genotype, with >500 muscle fibers sampled to generate the size distributions. D–F) Sections and histograms (as in A–C) for MHC2A-positive fibers. G–I) Sections and histograms (as in A–C) for MHC2B-positive fibers. J, M) Histograms of fiber distributions of soleus muscles from 4-wk-old (J) and 8-wk-old (M) animals show that mGRP94^−/− muscles have smaller muscle fibers. n = 4-5 for each age and genotype. K, N) Fiber type proportions for soleus muscles from 4-wk-old (K) and 8-wk-old (N) animals. L, O) Fiber numbers in 4-wk-old (L) and 8-wk-old (O) animals. Numbers of fibers are lower in soleus muscles from mGRP94^−/− mice, losing predominantly MHC 2A fibers.

Figure 4.

Changes in the ER of GRP94-depleted muscles. A) Immunoblotting of muscle for luminal proteins shows no evidence of global ER stress. GRP94 levels are significantly reduced in muscles of mGRP94^−/− mice, causing a compensatory increase BiP/GRP78 and PDIA6, but no change in calreticulin. Data are shown as means ± se for n = 6 samples/ genotype, *P < 0.05 vs. WT; unpaired t test. B) XBP-1 splicing assay to monitor ER stress. Positive and negative controls were generated from cDNAs of 3T3 cells in the presence or absence of thapsigargin (Tg), respectively, which cause splicing (S) of XBP-1. Neither muscle nor liver samples from WT and mGRP94^−/− mice show significant XBP-1 splicing, supporting the lack of ER stress. U, unspliced. C) Calsequestrin 1 (CSQ1) levels do not differ between muscles from n = 5 matched pairs of WT and mGRP94^−/− mice. Ratios are plotted as a function of the GRP94 deletion in the same mice, determined by blotting with anti-GRP94 antibody. D) GRP94 deficiency in the muscle does not lead to Ca²⁺-dependent cleavage of talin. In neither WT nor mutant muscles does talin display appreciable cleavage by calpain. As a positive control, NIH 3T3 cells were treated with the indicated concentrations of the Ca²⁺ ionophore A23187, to increase artificially free Ca²⁺ and activate calpain cleavage of talin (band marked with *). Blots of total protein extracts were probed with anti-talin monoclonal 8D4. Ratio of the cleavage product to full-length talin is provided below each lane.

Figure 5.

Growth of WT and mGRP94^−/− mice. A, B) Male (A) and female (B) weight growth curves based on 16 mGRP94^−/− and 19 WT males and 14 mGRP94^−/− and 12 WT females. Error bars are sd. The differences between mutant and WT mice in the weight growth curves are statistically significant at P < 0.05 (unpaired t test). C, D) Male (C) and female (D) body length growth curve. Graphs are based on 9 mGRP94^−/− and 8 WT males and 14 mGRP94^−/− and 12 WT females. □, WT mice; ●, mGRP94^−/− mice. E) Example of an mGRP94^−/− mouse (left) and a WT littermate (right) at 8 wk. F) Restoration of growth by exogenous IGF-I. Histogram depicts the growth rate of mGRP94^−/− (Mut) and WT females between 4 and 8 wk, when either injected daily with recombinant IGF-I (IPLEX) or saline or not injected (none). Note that mGRP94^−/− mice respond even better than WT mice to administration of IGF-I. n = 5 females/condition, except Mut + saline, for which only 1 animal was available (4 mutant mice did not survive the full course of injections) *P < 0.05; unpaired t test.

Figure 6.

Effect of GRP94 deletion on serum IGF-I levels. A) Serum IGF-I levels in 8-wk-old male or female mice. ■, WT mice; ●, mGRP94^−/− mice. Each point represents an individual mouse, whose IGF-I was measured by ELISA. Horizontal dashed lines are the means of each group of values. Mean serum values for mGRP94^−/− mice are significantly lower than serum IGF-I values for sex-matched WT mice by unpaired t test. B) Serum IGF-I levels as a function of GRP94 deletion in the muscles. Each point is the IGF-I value determined by ELISA from an individual mouse and the corresponding GRP94 deletion determined by immunoblotting. ▲, mice heterozygous for both Cre and loxP. C, D) Body weight of 8-wk-old mGRP94^−/− mice as a function of their serum IGF-I levels. Each point is an individual animal and shows a high correlation between body weight and circulating IGF-I for both males (C) and females (D). E, F) Comparison of ALS, IGFBP1, IGFBP3, and IGF-I levels in sera from WT and mGRP94^−/− mice. E) Immunoblot. F) Quantification of data. IGFBP1 is elevated in the mGRP94^−/− serum, whereas there is no significant change in the levels of IGFALS or IGFBP3. IGF-I levels are diminished in the mGRP94^−/− serum, with a marked decrease in both mature and pro-IGF-I.

Figure 7.

Metabolic consequences of GRP94 depletion. A) Levels of growth hormone in the blood of 4- and 8-wk-old mice. □, WT mice. ●, mGRP94^−/− mice. There was no statistical difference between GH levels in mGRP94^−/− mice and in WT mice. B) Relative expression of muscle Igf1, Igf2, and myostatin and liver Igf1, Igf2, Igfbp1, Igfbp3, and Igfals compared with that of WT controls by qRT-PCR. n = 4–6 samples/genotype from 8-wk-old mice were used for analysis. There were no differences in expression in muscle samples, and only Igfbp1 and Igfals expression was increased in the livers of mGRP94^−/− mice. Data are presented as means ± se of the fold change in expression. *P < 0.05; unpaired t test. C) Fasting blood glucose levels measured from 8-wk-old WT and mGRP94^−/− mice (n=6). D) Glucose tolerance curves performed on the same mice as in C. Differences are not statistically significant. E) Circulating insulin levels in 4- and 8-wk-old WT and mGRP94^−/− mice. There is a 2-fold increase in insulin at 8 wk of age in the mGRP94^−/− mice. Data represent means ± se for n = 10–12 samples/age and genotype. *P < 0.05 vs. age-matched WT; unpaired t test.