RNA SEQ Analysis Indicates that the AE3 Cl-/HCO3- Exchanger Contributes to Active Transport-Mediated CO2 Disposal in Heart

Abstract

Loss of the AE3 Cl^-/HCO₃^- exchanger (Slc4a3) in mice causes an impaired cardiac force-frequency response and heart failure under some conditions but the mechanisms are not known. To better understand the functions of AE3, we performed RNA Seq analysis of AE3-null and wild-type mouse hearts and evaluated the data with respect to three hypotheses (CO₂ disposal, facilitation of Na⁺-loading, and recovery from an alkaline load) that have been proposed for its physiological functions. Gene Ontology and PubMatrix analyses of differentially expressed genes revealed a hypoxia response and changes in vasodilation and angiogenesis genes that strongly support the CO₂ disposal hypothesis. Differential expression of energy metabolism genes, which indicated increased glucose utilization and decreased fatty acid utilization, were consistent with adaptive responses to perturbations of O₂/CO₂ balance in AE3-null myocytes. Given that the myocardium is an obligate aerobic tissue and consumes large amounts of O₂, the data suggest that loss of AE3, which has the potential to extrude CO₂ in the form of HCO₃^-, impairs O₂/CO₂ balance in cardiac myocytes. These results support a model in which the AE3 Cl^-/HCO₃^- exchanger, coupled with parallel Cl^- and H⁺-extrusion mechanisms and extracellular carbonic anhydrase, is responsible for active transport-mediated disposal of CO₂.

Figure 1

Differential expression of genes involved in hypoxia responses and vasodilation. Relevant genes were identified by Gene Ontology analyses and/or by PubMatrix analyses of genes with an FDR < 0.05. RPKM (Reads Per Kilobase of transcript per Million mapped reads) values for WT (black bars) and AE3-null (white bars) hearts are shown. The genes shown are a subset of 143 genes in Supplementary Table S2 and in a subset of 158 signaling genes in Supplementary Table S3; values are means ± SE; n = 4 for each genotype; *p ≤ 0.01 vs WT controls.

Figure 2

Differential expression of genes involved in angiogenesis. Relevant genes were identified by Gene Ontology analyses and/or by PubMatrix analyses of genes with an FDR < 0.05. RPKM values for WT (black bars) and AE3-null (white bars) hearts are shown. The genes shown are a subset of 143 genes in Supplementary Table S2; values are means ± SE; n = 4 for each genotype; *p ≤ 0.01 vs WT controls.

Figure 3

Differential expression of genes involved in energy metabolism. Genes encoding proteins that function in (A) regulation of energy metabolism, (B) glucose metabolism, (C) fatty acid metabolism, and (D) regulation of ATP and substrate utilization were identified by Gene Ontology analyses and/or by PubMatrix analyses of genes with an FDR < 0.05. RPKM values for WT (black bars) and AE3-null (white bars) hearts are shown. Genes encoding proteins that are regulated by Akt are indicated (Akt). The genes shown are a subset of 142 genes in Supplementary Table S4; values are means ± SE; n = 4 for each genotype; *p ≤ 0.01 vs WT controls.

Figure 4

Differential expression of genes involved in membrane excitability and cardiac conduction. Genes relevant to these categories were identified by Gene Ontology analyses. RPKM values for WT (black bars) and AE3-null (white bars) hearts are shown. See Supplementary Information for detailed explanations and references for individual genes. The genes shown are a subset of 104 genes presented in Supplementary Table S5 and 84 transporter, pump, and channel genes in Supplementary Table S6; values are means ± SE; n = 4 for each genotype; *p ≤ 0.01 vs WT controls, except Kcne1 (p = 0.015).

Figure 5

Differential expression of genes encoding sarcomere and sarcomeric cytoskeletal proteins. Genes relevant to these categories were identified by Gene Ontology analyses. RPKM values for WT (black bars) and AE3-null (white bars) hearts are shown. See Supplementary Information for detailed explanations and references for individual genes. The genes shown are a subset of 116 genes presented in Supplementary Table S7. Values are means ± SE; n = 4 for each genotype; *p ≤ 0.01 vs WT controls.

Figure 6

Differential expression of genes with potential for adaptation via Na⁺-loading or regulation of pH_i. Genes in the top row have the potential to contribute to increased contractility via regulation of Na⁺-loading. Genes in the second row are affected by or involved in intracellular acid-base homeostasis. RPKM values for WT (black bars) and AE3-null (white bars) hearts are shown. Values are means ± SE; n = 4 for each genotype; *p ≤ 0.01 vs WT controls, except Car14 (p = 0.019), which encodes CA XIV, an AE3-interacting protein.

Figure 7

Model for the role of the AE3 Cl⁻/HCO₃ ⁻ exchanger in transport-mediated CO₂ disposal. Oxygen entering the myocyte is rapidly converted to CO₂ in mitochondria. CO₂ venting from mitochondria is facilitated by CA-mediated conversion of CO₂ to HCO₃ ⁻ and H⁺, with H⁺ buffered by histidyl dipeptides (HDP) and other components, thereby effectively blocking the back reaction by keeping the concentration of free H⁺ low. CO₂ disposal is proposed to be mediated by a combination of HCO₃ ⁻ extrusion by AE3, Cl⁻ recycling via Cl⁻ channel activity, H⁺-extrusion via HVCN1 during each depolarization, and extracellular carbonic anhydrase (CA) activity to generate CO₂.