Circulating mitochondrial genes detect acute cardiac allograft rejection: Role of the mitochondrial calcium uniporter complex

Abstract

Acute rejection after heart transplantation increases the risk of chronic dysfunction. Disturbances in mitochondrial function may play a contributory role, however, the relationship between histological signs of rejection in the human transplanted heart and expression levels of circulating mitochondrial genes, such as the mitochondrial Ca²⁺ uniporter (MCU) complex, remains unexplored. We conducted an RNA-sequencing analysis to identify altered mitochondrial genes in serum and to evaluate their diagnostic accuracy for rejection episodes. We included 40 consecutive samples from transplant recipients undergoing routine endomyocardial biopsies. In total, 112 mitochondrial genes were identified in the serum of posttransplant patients, of which 28 were differentially expressed in patients with acute rejection (p < .05). Considering the receiver operating characteristic analysis with an area under the curve (AUC) >0.900 to discriminate patients with moderate or severe degrees of rejection, we found that the MCU system showed a strong capability for detection: MCU (AUC = 0.944, p < .0001), MCU/MCUR1 ratio (AUC = 0.972, p < .0001), MCU/MCUB ratio (AUC = 0.970, p < .0001), and MCU/MICU1 ratio (AUC = 0.970, p < .0001). Mitochondrial alterations are reflected in peripheral blood and are capable of discriminating between patients with allograft rejection and those not experiencing rejection with excellent accuracy. The dysregulation of the MCU complex was found to be the most relevant finding.

Keywords: biomarker; cell death: apoptosis; genomics; heart biology; heart transplantation/cardiology; molecular biology; rejection: acute; translational research/science.

Figure 1

Circulating MCU complex genes are differentially expressed between normal and rejected heart allografts. Values were obtained by Illumina HiSeq 2500 sequencing. Differential expression levels for molecules involved in mitochondrial calcium uptake (MCU, MCUR1, MCUB, and MICU1) and their respective ratios (MCU/MCUR1, MCU/MCUB, and MCU/MICU1) in each group of patients (nonrejection, rejection grade 1R and grade ≥2R) are displayed. Bars indicate protein expression levels ±SEM (standard error of the mean). Au, arbitrary units; *p < .05 and **p < .01 against the nonrejection group

Figure 2

Circulating mitochondrial genes are differentially expressed between normal and rejected heart allografts. Values obtained by Illumina HiSeq 2500 sequencing. Differential expression levels for molecules involved in intramitochondrial protein transport (TIMM50, ABCB10), metabolism and energy production (SDHA, DHRS7, NUDFA10, UQCRFS1, FMC1, IDH2, GMAP, ALDH2, DTYMK, NT5M, MRPL33, TFB1M, LARS2), pseudogene (MRPS31P2), transcription, stress response, and apoptosis (TBRG4, KANK1, AIFM2, PGAM5, BNIP3, APOPT1, CASP9, PRKN) in each group of patients (nonrejection, rejection grade 1R and grade ≥2R) are displayed. Bars indicate protein expression levels ±SEM (standard error of the mean). Au, arbitrary units; *p < .05 and **p < .01 against the nonrejection group

Figure 3

Relative expression levels of altered circulating mitochondrial genes in rejection grade ≥2R. Controls values were set to 1. FC units represent the fold change over the control RNA relative expression levels (nonrejection group). Bars indicate FC ±SEM (standard error of the mean) (A). Hierarchical clustering heat map based on the relative expression level of genes between normal and rejected heart allografts. The hierarchical clustering heat map analyses show the separation of the ≥2R rejection group and nonrejection group for circulating mitochondrial genes. Genes are listed in the columns and samples in the rows. Colors depict the relative expression level of each molecule with blue being the lowest and yellow the highest (B). *p < .05 and **p < .01 against the nonrejection group [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Receiver operating characteristic (ROC) curve of circulating mitochondrial genes for the detection of cardiac allograft rejection