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.

NBCe1 Na+-HCO3- cotransporter ablation causes reduced apoptosis following cardiac ischemia-reperfusion injury in vivo

“…Because they have opposing activities with respect to HCO 3 - fluxes, it is of interest to compare the results of ablating NBCe1, the predominant HCO 3 - -uptake mechanism in heart, with the results of ablating the AE3 ( Slc4a3 ) Cl - /HCO 3 - exchanger. Both the very high mRNA expression levels of AE3[ 7 ] and its predominant role in recovery of myocytes from an alkaline load[ 33 ] demonstrate that it is the predominant HCO 3 - -extrusion mechanism in the heart. In humans, heterozygous mutations in AE3 contribute to heart disease[ 34 ].…”

“…To determine the degree of knockdown of NBCe1 mRNA in KO ( Slc4a4 flx/flx(Cre) ) relative to WT ( Slc4a4 flx/flx ) hearts, quantitative reverse transcriptase-PCR analysis (qRT-PCR) was performed using an ABI 7300 Real Time PCR system as described previously[ 7 ]. cDNA was prepared from mRNA isolated from whole heart of 4 mo old male mice ( n = 6 for each genotype) and was PCR amplified using a forward primer (5’-TTCAGGCTCTCTCTGCGATT-3’) from coding exon 11 and reverse primer from coding exon 12 (5’-CTCAAGATGGTAAGCGGTTGA-3’).…”

“…For Gene Ontology (GO) analyses, we used the GOrilla program[ 24 ] ( ). As discussed previously[ 7 , 25 ], two analysis options are available: (1) Two Unranked Lists, in which a target list of genes with a specific range of P values is compared against the list of all genes analyzed; and (2) a Single Rank List, with the entire gene set ranked according to P values, thereby avoiding the use of an arbitrary cutoff of P values. Both analyses were performed.…”

“…The Cl - /HCO 3 - exchangers extrude HCO 3 - in exchange for Cl - and therefore serve as acid-loading mechanisms[ 1 , 2 ]. In addition, they facilitate Na + -loading by operating in concert with the Na + -dependent acid extruders[ 6 ] and have the potential to contribute to CO 2 disposal[ 7 ]. The sarcolemmal Na + /H + exchanger and Na + -HCO 3 - cotransporters are activated by intracellular acidification and serve to alkalinize the cell by extruding H + or bringing in HCO 3 [ 1 , 2 ].…”

“…Two Na + /HCO 3 - cotransporters, one electrogenic (NBCe1) and one electroneutral (NBCn1), are expressed in mammalian hearts. Based on RNA seq data[ 5 , 7 ], NBCe1 mRNA expression in mouse heart is about double that of NBCn1 and data available in the EMBL-EBI Expression Atlas (https:// ) shows that this is also the case in the human heart. NBCe1 has been localized to the lateral sarcolemma, intercalated disc, and t-tubules of cardiomyocytes[ 1 ], and it has been suggested that the presence of NBCe1 in the t-tubule may contribute to electrical events involved in excitation-contraction coupling[ 1 ].…”