Slc39a2-Mediated Zinc Homeostasis Modulates Innate Immune Signaling in Phenylephrine-Induced Cardiomyocyte Hypertrophy

Figures

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  Figure 1. Abnormal expression of Slc39a2 is associated with zinc depletion in phenylephrine (PE)-treated cardiomyocytes. (A) Representative images (left) and quantification data (right) of Zincpyr1 staining in neonatal rat ventricular myocytes (NRVMs) with or without phenylephrine (PE; 50 μM) treatment. Scale bar, 200μm. ***P < 0.001 vs. Control. n = 3. (B) qRT-PCR analysis for Slc39a family members (1-14) in PE-induced cardiomyocyte hypertrophy. ***P < 0.001 vs. Control. n = 3. (C) Immunoblots (left) and quantification data (right) of SLC39A2 in NRVMs with or without PE treatment. n = 3. (D) Validation of knockdown efficacy of siSlc39a2. **P < 0.01 vs. siNeg (negative control siRNA). n = 3. (E) Impact of Slc39a2 knockdown on Zinc contents in NRVMs. ***P < 0.001 vs. siNeg. n = 3.

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  Figure 2. Slc39a2 deficiency exacerbates PE-induced cardiomyocyte hypertrophy. (A) Representative images of WGA staining showing the impact of Slc39a2 knockdown on PE-induced cell size enlargement. Scale bar, 100μm. (B) Averaged cell size measured by WGA staining. **P < 0.01 vs. siNeg; ###P < 0.001 vs. Control. n = 3. (C) Counts of NRVMs with fragmented cell size range. (D–F) Impact of Slc39a2 on PE-induced hypertrophy markers, including Nppa (D), Nppb (E), and Myh7 (F). *P < 0.05, ***P < 0.001 vs. siNeg; ###P < 0.001 vs. Control. n = 3. (G) qRT-PCR analysis for Nppa and Nppb with and without ZnCl2 administration (10μM) in PE-treated NRVMs. n = 3. (H) Impact of zinc supplementation on NRVMs with or without Slc39a2 knockdown after PE treatment. *P < 0.05, ***P < 0.001 vs. siNeg; ###P < 0.001 vs. siSlc39a2. n = 3.

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  Figure 3. Slc39a2 overexpression increases zinc uptake and alleviates PE-induced cardiomyocyte hypertrophy. (A) Overexpression of Slc39a2 by adenovirus (Ad-Slc39a2). ***P < 0.001 vs. Ad-Vector. n = 3. (B) Representative images (left) and quantification data (right) showing the impact of Slc39a2 overexpression on Zinpry-1 staining in NRVMs with or without PE treatment. *P < 0.05 vs. Ad-Vector. n = 3. (C,D) Impact of Slc39a2 overexpression on PE-induced expressions of Nppa (C) and Nppb (D). ***P < 0.001 vs. Ad-Vector; ##P < 0.01, ###P < 0.001 vs. Control. n = 3.

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  Figure 4. Slc39a2 deficiency potentiates the transcriptome reprogramming induced by PE. (A) Principal component analysis (PCA) from the RNA-seq data showing the overall impact of Slc39a2 knockdown on the transriptome of NRVMs with or without PE treatment. (B) Volcano mapping showing the number of up- and down-regulated genes (≥ 2 folds) by Slc39a2 knockdown. (C) Venn map showing the relationship of differentially expressed genes among 3 comparisons (siNeg PE vs. siNeg; siSlc39a2 vs. siNeg; siSlc39a2 PE vs. siNeg PE). (D) Heatmap showing the impact of siSlc39a2 on PE-induced transcriptome reprogramming. (E,F) Gene Ontology (GO) analyses of up-regulated (E) and down-regulated (F) genes by siSlc39a2 measured by Metascape.

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  Figure 5. Slc39a2 deficiency activates innate immune signaling during cardiomyocyte hypertrophy. (A,B) Gene set enrichment analysis (GSEA) identified NOD-like receptor (A) and TOLL-like receptor (B) pathways to be significantly enriched by Slc39a2 knockdown. (C,D) KEGG analysis showing the siSlc39a2-affected genes involved in NOD-like receptor (C) and TOLL-like receptor (D) pathways. (E,F) GSEA identified NFκB and IRF as key factors involved in siSlc39a2-mediated transcriptome reprogramming.

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  Figure 6. Slc39a2 deficiency enhances IκBα degradation and NFκB/STAT3 phosphorylation. (A) Immunoblots of phosphorylated STAT3, total STAT3, phosphorylated P65, total P65, IκBα, and GAPDH in siNeg- or siSlc39a2-transfected NRVMs with and without PE treatment. (B-D) Quantification data of p-STAT3/STAT3 ratio (B), p-P65/P65 ratio (C), and IκBα/GAPDH ratio (D). *P < 0.05, **P < 0.01 vs. siNeg; ##P < 0.01, ###P < 0.001 vs. Control. n = 3. (E) Impact of TPCA-1, an inhibitor of NFκB (2μM), on Nppa and Nppb expressions in NRVMs with or without Slc39a2 knockdown after PE treatment. n = 3.

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  Figure 7. Interplay between SLC39A2 and IRF7 during cardiomyocyte hypertrophy. (A) Reads of IRF family genes (Irf1-9) from RNA-seq data. n = 3. (B) Validation of Irf7 expression by qRT-PCR. ***P < 0.001 vs. siNeg; #P < 0.05, ###P < 0.001 vs. Control. n = 3. (C) Immunoblots (upper) and quantification data (lower) for IRF7 expression at protein level in NRVMs with or without Slc39a2 knockdown and PE treatment. ***P < 0.001 vs. siNeg; ##P < 0.01 vs. Control. n = 3. (D,E) Validation of knockdown efficacy siSlc39a2 (D) and siIrf7 (E) in PE-treated NRVMs. Data were normalized to control group (dotted line). ***P < 0.001 vs. siNeg; ###P < 0.001 vs. Control. n = 3. (F,G) Impact of combined knockdown of Slc39a2 and Irf7 on hypertrophy markers, Nppa (F) and Nppb (G). **P < 0.01 vs. siNeg. n = 3.