Reciprocal transcriptional regulation of metabolic and signaling pathways correlates with disease severity in heart failure

Abstract

Background: Systolic heart failure (HF) is a complex systemic syndrome that can result from a wide variety of clinical conditions and gene mutations. Despite phenotypic similarities, characterized by ventricular dilatation and reduced contractility, the extent of common and divergent gene expression between different forms of HF remains a matter of intense debate.

Methods and results: Using a meta-analysis of 28 experimental (mouse, rat, dog) and human HF microarray studies, we demonstrate that gene expression changes are characterized by a coordinated and reciprocal regulation of major metabolic and signaling pathways. In response to a wide variety of stressors in animal models of HF, including ischemia, pressure overload, tachypacing, chronic isoproterenol infusion, Chagas disease, and transgenic mouse models, major metabolic pathways are invariably downregulated, whereas cell signaling pathways are upregulated. In contrast to this uniform transcriptional pattern that recapitulates a fetal gene expression program in experimental animal models of HF, human HF microarray studies displayed a greater heterogeneity, with some studies even showing upregulation of metabolic and downregulation of signaling pathways in end-stage human hearts. These discrepant results between animal and human studies are due to a number of factors, prominently cardiac disease and variable exposure to cold cardioplegic solution in nonfailing human samples, which can downregulate transcripts involved in oxidative phosphorylation (OXPHOS), thus mimicking gene expression patterns observed in failing samples. Additionally, β-blockers and ACE inhibitor use in end-stage human HF was associated with higher levels of myocardial OXPHOS transcripts, thus partially reversing the fetal gene expression pattern. In human failing samples, downregulation of metabolism was associated with hemodynamic markers of disease severity.

Conclusions: Irrespective of the etiology, gene expression in failing myocardium is characterized by downregulation of metabolic transcripts and concomitant upregulation of cell signaling pathways. Gene expression changes along this metabolic-signaling axis in mammalian myocardium are a consistent feature in the heterogeneous transcriptional response observed in phenotypically similar models of HF.

Figure 1. A) KEGG pathway analysis of heart failure (HF)

Transcripts up- and downregulated in HF compared to non-failing (NF) ventricular myocardium are represented by white and black columns, respectively and displayed on the y-axis as percentage of the total number of transcripts per given KEGG pathway. Four different microarray studies of canine tachypacing-induced HF microarray studies are presented in the top panel (studies #15–18), four different HF models in rodents are shown in the middle graph (studies #8, #11, #12, #24), and four different microarray studies of human HF are displayed in the bottom panel (studies #1–4). A detailed description of these studies is given in Supplemental Table 1. B) When the net regulation, i.e. the number of up- minus downregulated genes within a study expressed as percentage of genes per given KEGG pathway (this corresponds to white minus black columns of Figure 1a), was compared between TCA-cycle, fatty acid metabolism and OXPHOS, a positive correlation was found (upper panels, p<0.05 for all correlations), while JAK-STAT-signaling and OXPHOS show a negative correlation (lower right panel). The correlation plots include all 28 animal and human HF microarray studies listed in Supplemental Table 1.

Figure 1. A) KEGG pathway analysis of heart failure (HF)

Transcripts up- and downregulated in HF compared to non-failing (NF) ventricular myocardium are represented by white and black columns, respectively and displayed on the y-axis as percentage of the total number of transcripts per given KEGG pathway. Four different microarray studies of canine tachypacing-induced HF microarray studies are presented in the top panel (studies #15–18), four different HF models in rodents are shown in the middle graph (studies #8, #11, #12, #24), and four different microarray studies of human HF are displayed in the bottom panel (studies #1–4). A detailed description of these studies is given in Supplemental Table 1. B) When the net regulation, i.e. the number of up- minus downregulated genes within a study expressed as percentage of genes per given KEGG pathway (this corresponds to white minus black columns of Figure 1a), was compared between TCA-cycle, fatty acid metabolism and OXPHOS, a positive correlation was found (upper panels, p<0.05 for all correlations), while JAK-STAT-signaling and OXPHOS show a negative correlation (lower right panel). The correlation plots include all 28 animal and human HF microarray studies listed in Supplemental Table 1.

Figure 2. Comprehensive analysis of 160 KEGG pathways expressed in 48 different myocardial gene expression datasets

The net expression of a KEGG pathway (number of up- minus downregulated genes within a study in relation to total number of genes per given KEGG pathway, see also Figure 1), is color-coded with yellow and blue representing low and high expression of the pathway, respectively. The net expression of 160 different KEGG pathways is depicted on the y-axis and sorted according to their similarity of gene expression to “oxidative phosphorylation” which is represented by the first row (labeled OXPHOS). 48 different microarray studies, described in detail in Supplemental Table 1, are shown on the x-axis: Samples #1–#28 are from datasets comparing non-failing vs. failing myocardial samples. Within these heart failure samples, samples from the four different species were grouped together. For samples #29–#48, we grouped samples according to the pathophysiology (e.g. regional differences, high vs. low OXPHOS, etc.) and then by species. Across a wide range of diverse myocardial gene expression datasets, metabolic and biosynthesis pathways were consistently positively correlated to each other and negatively correlated with the expression of cell signaling pathways.

Figure 3. Correlation of OXPHOS gene expression and clinical parameters in human HF

The analysis utilizes the largest publicly available microarray dataset of human HF (GSE5406, dataset #3). A and B) In 210 human HF samples, pro-BNP (NPPB) and OXPHOS expression (average expression of all genes comprised in the KEGG pathway of oxidative phosphorylation) displayed a high variability, independent of the etiology of HF (NF = non-failing, NICM = non-ischemic cardiomyopathy, ICM = ischemic cardiomyopathy). Boxplots delineate the median value as well as the 25^th and 75^th percentiles. Raw data for NPPB and OXPHOS are plotted as diamonds next to the boxplots. C) Patients on mechanical circulatory support (IABP, intra-aortic balloon pump) had lower myocardial expression of OXPHOS genes compared to more hemodynamically stable patients. Likewise, patients with a rapidly progressive course of HF (less than one month between clinical onset of HF and cardiac transplantation) and a higher pulmonary capillary wedge pressure (PCWP) had significantly lower OXPHOS expression compared to patients with a longer clinical course and lower PCWP, respectively. In contrast, use of beta-blockers and ACE-inhibitors was associated with higher myocardial OXPHOS levels. The number of patients is indicated on the respective columns.

Figure 4. A) Effect of hypothermia and cardioplegic solution on myocardial gene expression in rats

Expression of eight transcripts was measured with SYBR-green semiquantitative RT-PCR in control hearts that were immediately snap-frozen after harvest or perfused with St. Thomas Hospital cardioplegic solution and stored at 4°C for 4–10 hours. Significant downregulation of four OXPHOS transcripts (PGC1α, NDUFA5, NDUFB5, COX5A) was noted in left ventricular myocardium perfused with cold cardioplegic solution (p<0.05, unpaired t-test, n=4 hearts each group), while there was no statistically significant change in mRNA expression for transcripts involved in signal transduction pathways (MAPK1, MAPK14, JAK1, IL6). B) Concordant regulation of pathways in early and late HF. Characteristic HF-induced gene expression changes develop as early as 3 days after initiation of tachypacing in dogs (left panel) or 6 hours after RV pressure overload induced by experimental pulmonary embolism (PE) in rats.

Figure 5. Graphical representations of protein interactions

A query was conducted with 90 transcripts consistently regulated in HF (listed in Supplemental Table 3) using the Unified Human Interactome database (UniHI4). Interacting partners of the list of query proteins are displayed if they have an expression in heart tissue of more than 1000 units (expression summaries for 44,775 transcripts were derived utilizing the MAS5 algorithm by Affymetrix). The display was restricted to direct interactions (yellow proteins) between query proteins (red and blue for up- and downregulated transcripts, respectively). Network analysis revealed that 18 out of 90 transcripts formed a network with multiple interconnected nodes (hubs) between up- and downregulated genes. Importantly, many of the predicted interaction partners, were also part of the common HF genes listed in Supplemental Table 3.