The transcriptional profile of iron deficiency in patients with heart failure: Heme-sparing and reduced immune processes

Abstract

Aims: Iron deficiency (ID) is highly prevalent in patients with heart failure (HF) and associated with morbidity and poor prognosis, but pathophysiological mechanisms are unknown. We aimed to identify novel biological pathways affected by ID.

Methods and results: We studied 881 patients with HF from the BIOSTAT-CHF cohort. ID was defined as a transferrin saturation <20%. Transcriptome profiling was performed in whole blood. Identified targets were validated in a human in vitro stem cell-derived cardiomyocyte ID model utilizing deferoxamine as iron chelator. ID was identified in 554 (62.9%) patients, and 89 differentially expressed genes between ID and non-ID were identified, of which 60 were up- and 29 were downregulated. Upregulated genes were overrepresented in pathways of erythrocyte development and homeostasis. Heme biosynthetic processes were confirmed as relatively upregulated in ID, while iron-sulfur cluster assembly was downregulated. Downregulated processes further included natural killer cell and lymphocyte mediated immunity. In agreement with patient data, cardiomyocyte iron depletion significantly induced the expression of two genes (SIAH2 and CLIC4), which could be normalized upon iron supplementation. Both SIAH2 and CLIC4 are associated with increased mortality in patients with HF (hazard ratio 2.40, 95% confidence interval 1.86-3.11, p < 0.001 hazard ratio 1.78, 95% confidence interval 1.53-2.07, p < 0.001, respectively).

Conclusion: Iron deficiency is associated with the preservation of heme-related processes at the cost of iron-sulfur clusters. Immune processes are downregulated, uncovering another high energy demand system affected. SIAH2 and CLIC4 might be modifiable factors in the relation between ID and impaired prognosis.

Keywords: Heart failure; Heme; Iron deficiency; Iron–sulfur; Transcriptome profiling.

Figure 1

Volcano plot showing differentially expressed genes between patients with and without iron deficiency. Each dot represents one of the 17 900 genes quantified in the whole blood microarray in 881 patients. On the x‐axis, the log2‐fold change in gene expression is depicted (positive log2‐fold change is higher biomarker expression in patients with iron deficiency; negative log2‐fold change is lower biomarker expression in patients with iron deficiency), while the y‐axis shows the magnitude of the biomarker expression difference as –log10 of the p‐value. Green dots indicate biomarkers that are not significantly different, orange dots represent biomarkers that are significantly different with a log2‐fold change between −0.4 and 0.4, purple dots represent biomarkers that are significantly different and have a log2‐fold change of ≥0.4 or ≤−0.4. Significance was tested using a logistic multivariable model adjusted for multiple testing based on the Benjamini–Hochberg method.

Figure 2

Significantly up‐ or downregulated gene expression networks in patients with iron deficiency as compared to no iron deficiency.

Figure 3

Gene set enrichment analyses for heme and iron–sulfur associated pathways. Barcode plot, each vertical line depicts a single gene, with the red lines representing genes of the heme pathway (153 genes) and the blue lines representing genes of the iron–sulfur cluster pathway (24 genes). In case a gene is located to the left (blue area) it is downregulated while it is upregulated when it appears on the rights (red area). It can be observed that blue genes (iron–sulfur cluster related) are downregulated and thus cluster mainly to the left, while the opposite is true for the red genes (heme related), which cluster to the right.

Figure 4

Gene expression in human cardiomyocytes of upregulated genes in whole blood. Gene expression levels relative to control conditions (ctrl) for iron‐deficient cardiomyocytes (iron deficient) after 4 days of exposure to deferoxamine and iron supplemented cardiomyocytes (transferrin treated) who were first subjected to 4 days of deferoxamine and subsequently treated with transferrin bound iron for 3 days. Biological replicates were 14 ctrl, 12 iron deficient and 8 transferrin treated, 2 technical replicates were performed. *p < 0.05, statistical test is the one‐way ANOVA with a post hoc Tukey's honestly significant difference test. CISD2, CDGSH iron–sulfur domain 2; CLIC2, chloride intracellular channel protein 2; CLIC4, chloride intracellular channel protein 4; CTNNAL1, alpha‐catulin; SIAH2, E3 ubiquitin‐protein ligase siah2; SLC22A4, solute carrier family 22; TMOD1, tropomodulin 1.