Neuregulin (NRG-1β) Is Pro-Myogenic and Anti-Cachectic in Respiratory Muscles of Post-Myocardial Infarcted Swine

Figures

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  Figure 1. Low and high doses of GGF2 alter intercostal skeletal muscle transcription. (a) Hierarchical clustering of 581 genes significantly differentially expressed between GGF2-treated and untreated post-MI swine. Columns represent individual samples for each group, and rows represent individual probes/genes. Red, blue, and gray represent the highest, lowest, and medial fluorescent signal values, respectively. (b) Venn diagram showing numbers of genes differentially expressed in post-MI swine treated with GGF2 at low (teal circle, 311 genes) and high doses (orange circle, 512 genes) compared to untreated control animals.

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  Figure 2. Dose-like response genes altered in GGF-2 treated pigs post-MI. Dot plots show relative counts as reads per kilobase per transcript, per million mapped reads (RPKM) for genes exhibiting fold-differences for GGF2 versus untreated pigs (y-axis). Individual samples are indicated on the x-axis and by color. Black represents untreated (Unt), teal represents low-dose (0.67 mg/kg/day), and high dose (2 mg/kg/day) is shown in orange.

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  Figure 3. Functional categorization of GGF2-altered genes. (a) Bar chart showing functions enriched in differentially expressed gene lists from GGF-2 treated versus untreated LV tissues, identified using Ingenuity Pathway Analysis software. Functions are listed on the y-axis, and the x-axis indicates the number of differentially expressed genes in each category. Teal and orange represent low and high doses of GGF2, respectively. Enrichment p-value is indicated on each bar. (b) Pie chart showing top gene ontology categories for GGF-2 altered genes manually grouped into three generalized associative categories: development (blue), metabolic (red), and stress response (green). Uncharacterized and poorly characterized transcripts were excluded.

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  Figure 4. Genes commonly altered in cardiac and intercostal skeletal muscle encode functionally associated proteins. A vector graphic network created using STRING network version 11.5 with K means clustering of proteins encoded by differentially expressed genes commonly altered in both skeletal muscle and cardiac tissues of GGF2-treated post-MI pigs is shown. Full gene names with functions are provided in Table 1, except for NRG1 (neuregulin 1) and its receptors (ERBB2-4). Bubbles represent individual proteins, and lines represent associations between proteins. Line thickness indicates edge confidence, low (0.150), medium (0.400), high (0.700) and highest (0.900). Line shape indicates the predicted mode of action. Each color represents an individual cluster.

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  Figure 5. GGF2 reversibly alters myopathic genes identified through meta-analysis of repository gene expression data. (a) Bar chart showing numbers of genes (x-axis) significantly altered in various myopathies (study details listed in Table 2) that were reversibly expressed in GGF2-treated post-MI pigs. Species indicated by an icon at end of bar. Color of bar indicates directionality in listed experimental conditions relative to respective controls. Abbreviations are T1DM = (Type I Diabetes Model), PCOS = polycystic ovary syndrome, MS, T2DM = Type II diabetes mellitus, MD2 = myotonic dystrophy type II, COPD = chronic obstructive pulmonary disease, ISCU = iron-sulfur cluster scaffold homolog myopathy, and TMD = tibial muscular dystrophy. (b) Line graphs comparing alterations in four genes after denervation in an animal model of cachexia (left panel, GSE52676) and the same genes reversibly altered in GGF2-treated pigs (right panel). Log2 fold difference (Experiment vs. Control) is shown on the y axis, and time (in days) or dose (in ng/mL) is labeled on the x-axis. Colored lines correspond to genes, as labeled. LRRN1 = Leucine Rich Repeat Neuronal 1, SPP2 = Secreted Phosphoprotein 2, DUSP26 =…

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  Figure 6. NRG-1β treatment of cultured myoblasts enhances differentiation. (a) C2C12 cells were cultured in differentiation media (DMEM + 2% horse serum) for 5 days in the absence (control) or presence of 10 ng/mL of recombinant neuregulin-1β (NRG, 10 ng/mL) or with both NRG and 0.2 μM of the ErbB2 receptor inhibitor TAK164. (b) Western blots of lysates from C2C12 cells grown in differentiation media for 5 days in the absence (control, CTR) or presence of recombinant NRG-1β (NRG) or fibroblast growth factor (FGF). Full blots provided as Supplementary Figures S8–S14. Bar graphs, grouped by day, show results of quantification by densitometry for the indicated proteins (n = 2, n = 3 for p-FAK). Bar colors indicate treatment type: CTRL = white, NRG-1β = black, and FGF = gray. Asterisks indicate statistical significance, * p value < 0.05), ** p value < 0.005), *** p < 0.0005).

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  Figure 7. Depiction of inferred relationships of altered genes in GGF2-treated post-MI pigs. Transcripts that were up-regulated (highlighted in red) or down-regulated (blue) in intercostal muscle of neuregulin-treated pigs are italicized. Proteins identified as altered or activated based on Western blot analyses are also indicated. Bold indicates protein level inference based on functional analyses as described in the text. Known effects of neuregulin (NRG) on cell transitions based on published in vitro studies are indicated in red boxes with arrows (for promoting) or in blue (for inhibiting). Transcript abbreviations are official gene (italic) or protein symbols, and p- indicates phosphorylation (determined by Western blotting of lysates from NRG-treated myoblasts). MSC = mesenchymal stem cell; mir-29 = microRNA 29. Commissioned illustrations by Andrew Celso Gutierrez.