Circulating long non-coding RNAs detection after heart transplantation and its accuracy in the diagnosis of acute cardiac rejection.

Long non-coding RNAs (lncRNAs) are closely implicated in biological processes and diseases with high inflammatory components. These molecules exhibit significant temporal and tissue specificity. However, the expression and function of lncRNAs have not been studied in patients after heart transplantation. Thus, we aimed to identify circulating lncRNAs in these patients and evaluate their diagnostic capacity as potential biomarkers for the non-invasive detection of acute cellular rejection (ACR). For them, we performed a transcriptomic study based on ncRNA-seq technology to detect lncRNAs in serum samples, matched to routine endomyocardial biopsies, from patients without rejection episode (0R, n = 12) and with mild (1R, n = 16) or moderate-severe (≥ 2R, n = 12) ACR. We identified 11,062 circulating lncRNAs in the serum of patients after heart transplantation. Moreover, 6 lncRNAs showed statistically significant expression when the different ACR grades were compared. Among them, AC008105.3, AC006525.1, AC011455.8, AL359220.1, and AC025279.1 had relevant diagnostic capacity for detection of ≥ 2R (AUC of 0.850 to 1.000) and 1R (AUC of 0.750 to 0.854) grades, along with high specificity and positive predictive values (≥ 83%). In addition, AL359220.1 and AC025279.1 were independent predictors for the presence of moderate-severe ACR (odds ratio = 31.132, p < 0.01 and C statistic = 0.939, p < 0.0001; odds ratio = 18.693, p < 0.05 and C statistic = 0.902, p < 0.001; respectively). In conclusion, we describe, for the first time, circulating lncRNAs after heart transplantation as potential candidates for non-invasive detection of ACR. AL359220.1 and AC025279.1 showed excellent diagnostic capability correlating with the severity episode and were strong independent predictors of rejection.

Alterations in Mitochondrial Oxidative Phosphorylation System: Relationship of Complex V and Cardiac Dysfunction in Human Heart Failure.

Heart failure (HF) is a disease related to bioenergetic mitochondrial abnormalities. However, the whole status of molecules involved in the oxidative phosphorylation system (OXPHOS) is unknown. Therefore, we analyzed the OXPHOS transcriptome of human cardiac tissue by RNA-seq analyses (mRNA n = 36; ncRNA n = 30) in HF patients (ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM)) and control subjects. We detected 28 altered genes in these patients, highlighting greater deregulation in ICM. Specifically, we found a general overexpression of complex V (ATP synthase) elements, among them, ATP5I (ICM, FC = 2.04; p < 0.01), ATP5MJ (ICM, FC = 1.33, p < 0.05), and ATP5IF1 (ICM, FC = 1.81; p < 0.001), which presented a significant correlation with established echocardiographic parameters of cardiac remodeling and ventricular function as follows: left ventricular end-systolic (p < 0.01) and end-diastolic (p < 0.01) diameters, and ejection fraction (p < 0.05). We also detected an increase in ATP5IF1 protein levels (ICM, FC = 1.75; p < 0.01) and alterations in the microRNA expression levels of miR-208b-3p (ICM, FC = -1.44, p < 0.001), miR-483-3p (ICM, FC = 1.37, p < 0.01), regulators of ATP5I. Therefore, we observed the deregulation of the OXPHOS transcriptome in ICM patients, highlighting the overexpression of complex V and its relationship with cardiac remodeling and function.

Plasma fibroblast activation protein is decreased in acute heart failure despite cardiac tissue upregulation.

BACKGROUND: Cardiac fibroblast activation protein (FAP) has an emerging role in heart failure (HF). A paradoxical reduction in its levels in pathological conditions associated with acute processes has been observed. We aimed to identify FAP cardiac tissue expression and its relationship with the main cardiac fibrosis-related signaling pathways, and to compare plasma FAP levels in acute and chronic HF patients. METHODS: Transcriptomic changes were assessed via mRNA/ncRNA-seq in left ventricle tissue from HF patients (n = 57) and controls (n = 10). Western blotting and immunohistochemistry were used to explore FAP protein levels and localization in cardiac tissue. ELISA was performed to examine plasma FAP levels in acute HF (n = 48), chronic HF (n = 15) and control samples (n = 7). RESULTS: FAP overexpression in cardiac tissue is related to the expression of molecules directly involved in cardiac fibrosis, such as POSTN, THBS4, MFAP5, COL1A2 and COL3A1 (P < 0.001), and is directly and inversely related to pro- and antifibrotic microRNAs, respectively. The observed FAP overexpression is not reflected in plasma. Circulating FAP levels were lower in acute HF patients than in controls (P < 0.05), while chronic HF patients did not show significant changes. The clinical variables analyzed, such as functional class or etiology, do not affect plasma FAP concentrations. CONCLUSIONS: We determined that in HF cardiac tissue, FAP is related to the main cardiac fibrosis signaling pathways as well as to pro- and antifibrotic microRNAs. Additionally, an acute phase of HF decreases plasma FAP levels despite the upregulation observed in cardiac tissue and regardless of other clinical conditions.

Combining Serum miR-144-3p and miR-652-3p as Potential Biomarkers for the Early Diagnosis and Stratification of Acute Cellular Rejection in Heart Transplantation Patients.

BACKGROUND: There is a dire need for specific, noninvasive biomarkers that can accurately detect cardiac acute cellular rejection (ACR) early. Previously, we described miR-144-3p as an excellent candidate for detecting grade ≥2R ACR. Now, we investigated the combination of miR-144-3p with miR-652-3p, other differentially expressed serum miRNA we previously described, to improve diagnostic accuracy mainly in mild rejection to avoid reaching severe stages. METHODS: We selected miR-652-3p from a preliminary RNA-seq study to be validated by reverse transcription-quantitative polymerase chain reaction on 212 consecutive serum samples from transplantation recipients undergoing routine endomyocardial biopsies to subsequently combine them with miR-144-3p results and investigate their diagnostic capability. RESULTS: We confirmed the miR-652-3p overexpression (P < 0.0001) and its capability to discriminate between patients with and without ACR of any grade (P < 0.0001). The combined serum levels of miR-144-3p and miR-652-3p were significantly higher in patients with rejection regardless of posttransplantation time (P < 0.0001). This combination resulted in a diagnostic efficacy for 1R (area under the curve = 0.794) and ≥2R (area under the curve = 0.892; P < 0.0001) that was superior to each biomarker alone. Furthermore, it was a strong independent predictor of ACR for 1R (odds ratio of 10.950; P < 0.0001) and ≥2R (odds ratio of 14.289; P < 0.01). CONCLUSIONS: We demonstrated that an appropriate combination of blood-based biomarkers could exhibit greater efficiency for cardiac rejection diagnosis. The combined detection of abnormal expression of miR-144-3p and miR-652-3p in the serum of ACR patients can improve the diagnostic sensitivity of rejection at an early stage and contribute to increasing the diagnostic accuracy, mainly in the lower rejection grades.

Altered MicroRNA Maturation in Ischemic Hearts: Implication of Hypoxia on XPO5 and DICER1 Dysregulation and RedoximiR State.

Ischemic cardiomyopathy (ICM) is associated with abnormal microRNA expression levels that involve an altered gene expression profile. However, little is known about the underlying causes of microRNA disruption in ICM and whether microRNA maturation is compromised. Therefore, we focused on microRNA maturation defects analysis and the implication of the microRNA biogenesis pathway and redox-sensitive microRNAs (redoximiRs). Transcriptomic changes were investigated via ncRNA-seq (ICM, n = 22; controls, n = 8) and mRNA-seq (ICM, n = 13; control, n = 10). The effect of hypoxia on the biogenesis of microRNAs was evaluated in the AC16 cell line. ICM patients showed a reduction in microRNA maturation compared to control (4.30 ± 0.94 au vs. 5.34 ± 1.07 au, p ˂ 0.05), accompanied by a deregulation of the microRNA biogenesis pathway: a decrease in pre-microRNA export (XPO5, FC = -1.38, p ˂ 0.05) and cytoplasmic processing (DICER, FC = -1.32, p ˂ 0.01). Both processes were regulated by hypoxia in AC16 cells (XPO5, FC = -1.65; DICER1, FC = -1.55; p ˂ 0.01; Exportin-5, FC = -1.81; Dicer, FC = -1.15; p ˂ 0.05). Patients displayed deregulation of several redoximiRs, highlighting miR-122-5p (FC = -2.41, p ˂ 0.001), which maintained a good correlation with the ejection fraction (r = 0.681, p ˂ 0.01). We evidenced a decrease in microRNA maturation mainly linked to a decrease in XPO5-mediated pre-microRNA export and DICER1-mediated processing, together with a general effect of hypoxia through deregulation of biogenesis pathway and the redoximiRs.

Alpha-cardiac Actin Serum Expression Levels Detect Acute Cellular Rejection in Heart Transplant Patients.

BACKGROUND: Given the central role of sarcomeric dysfunction in cardiomyocyte biology and sarcomere alterations described in endomyocardial biopsies of transplant patients with rejection, we hypothesized that the serum expression levels of genes encoding sarcomeric proteins were altered in acute cellular rejection (ACR). The aim of this study is to identify altered sarcomere-related molecules in serum and to evaluate their diagnostic accuracy for detecting rejection episodes. METHODS: Serum samples from transplant recipients undergoing routine endomyocardial biopsies were included in an RNA sequencing analysis (n = 40). Protein concentrations of alpha-cardiac actin were determined using a specific enzyme-linked immunoassay (n = 80). RESULTS: We identified 17 sarcomeric genes differentially expressed in patients with clinically relevant rejection (grade ≥2R ACR). A receiver operating characteristic curve was done to assess their accuracy for ACR detection and found that 6 relevant actins, myosins, and other sarcomere-related genes showed great diagnostic capacity with an area under the curve (AUC) > 0.800. Specifically, the gene encoding alpha-cardiac actin ( ACTC1 ) showed the best results (AUC = 1.000, P < 0.0001). We determine ACTC1 protein levels in a larger patient cohort, corroborating its overexpression and obtaining a significant diagnostic capacity for clinically relevant rejection (AUC = 0.702, P < 0.05). CONCLUSIONS: Sarcomeric alterations are reflected in peripheral blood of patients with allograft rejection. Because of their precision to detect ACR, we propose sarcomere ACTC1 serum expression levels as potential candidate for to be included in the development of molecular panel testing for noninvasive ACR detection.

Cardiac Sodium/Hydrogen Exchanger (NHE11) as a Novel Potential Target for SGLT2i in Heart Failure: A Preliminary Study.

Despite the reduction of cardiovascular events, including the risk of death, associated with sodium/glucose cotransporter 2 inhibitors (SGLT2i), their basic action remains unclear. Sodium/hydrogen exchanger (NHE) has been proposed as the mechanism of action, but there are controversies related to its function and expression in heart failure (HF). We hypothesized that sodium transported-related molecules could be altered in HF and modulated through SGLT2i. Transcriptome alterations in genes involved in sodium transport in HF were investigated in human heart samples by RNA-sequencing. NHE11 and NHE1 protein levels were determined by ELISA; the effect of empagliflozin on NHE11 and NHE1 mRNA levels in rats' left ventricular tissues was studied through RT-qPCR. We highlighted the overexpression of SLC9C2 and SCL9A1 sodium transport genes and the increase of the proteins that encode them (NHE11 and NHE1). NHE11 levels were correlated with left ventricular diameters, so we studied the effect of SGLT2i on its expression, observing that NHE11 mRNA levels were reduced in treated rats. We showed alterations in several sodium transports and reinforced the importance of these channels in HF progression. We described upregulation in NHE11 and NHE1, but only NHE11 correlated with human cardiac dysfunction, and its levels were reduced after treatment with empagliflozin. These results propose NHE11 as a potential target of SGLT2i in cardiac tissue.

DNMT3B System Dysregulation Contributes to the Hypomethylated State in Ischaemic Human Hearts.

A controversial understanding of the state of the DNA methylation machinery exists in ischaemic cardiomyopathy (ICM). Moreover, its relationship to other epigenetic alterations is incomplete. Therefore, we carried out an in-depth study of the DNA methylation process in human cardiac tissue. We showed a dysregulation of the DNA methylation machinery accordingly with the genome-wide hypomethylation that we observed: specifically, an overexpression of main genes involved in the elimination of methyl groups (TET1, SMUG1), and underexpression of molecules implicated in the maintenance of methylation (MBD2, UHRF1). By contrast, we found DNMT3B upregulation, a key molecule in the addition of methyl residues in DNA, and an underexpression of miR-133a-3p, an inhibitor of DNMT3B transcription. However, we found many relevant alterations that would counteract the upregulation observed, such as the overexpression of TRAF6, responsible for Dnmt3b degradation. Furthermore, we showed that molecules regulating Dnmts activity were altered; specifically, SAM/SAH ratio reduction. All these results are in concordance with the Dnmts normal function that we show. Our analysis revealed genome-wide hypomethylation along with dysregulation in the mechanisms of addition, elimination and maintenance of methyl groups in the DNA of ICM. We describe relevant alterations in the DNMT3B system, which promote a normal Dnmt3b function despite its upregulation.

Electron Microscopy Reveals Evidence of Perinuclear Clustering of Mitochondria in Cardiac Biopsy-Proven Allograft Rejection.

Acute cellular rejection is a major complication in heart transplantation. We focus on the analysis of new ultrastructural findings in cardiac biopsy rejection based on mitochondrial intracellular organization. This study includes heart transplanted patients from a single center who were referred for endomyocardial biopsies as a scheduled routine screening. Participants were divided into two groups: patients transplanted without allograft rejection (Grade 0R), and patients with biopsy-proven allograft rejection (Grade ≥ 2R). Using electronic microscopy, we detected a significant increase in the volume density of mitochondria (p < 0.0001) and dense bodies (p < 0.01) in the rejection group. The most relevant finding was the presence of local accumulations of mitochondria close to the nuclear envelope, pressing and molding the morphology of this membrane in all rejection samples (100%). We identified this perinuclear clustering of mitochondria phenomenon in a 68 ± 27% of the total cardiac nucleus observed from rejection samples. We did not observe this phenomenon in any non-rejection samples, reflecting excellent sensitivity and specificity. We have identified a specific phenomenon affecting the architecture of the nuclear membrane-perinuclear clustering of mitochondria-in endomyocardial biopsies from patients with cardiac rejection. This ultrastructural approach might complement and improve the diagnosis of rejection.

Implication of Sphingolipid Metabolism Gene Dysregulation and Cardiac Sphingosine-1-Phosphate Accumulation in Heart Failure.

Disturbances in sphingolipid metabolism lead to biological function dysregulation in many diseases, but it has not been described in heart failure (HF). Sphingosine-1-phosphate (S1P) levels have not ever been measured in the myocardium. Therefore, we analyze the gene dysregulation of human cardiac tissue by mRNA-seq (n = 36) and ncRNA-seq (n = 50). We observed most major changes in the expression of genes belonging to de novo and salvage pathways, and the tight gene regulation by their miRNAs is largely dysregulated in HF. We verified using ELISA (n = 41) that ceramide and S1P accumulate in HF cardiac tissue, with an increase in the ceramide/S1P ratio of 57% in HF. Additionally, changes in left ventricular mass and diameters are directly related to CERS1 expression and inversely related to S1P levels. Altogether, we define changes in the main components of the sphingolipid metabolism pathways in HF, mainly de novo and salvage, which lead to an increase in ceramide and S1P in cardiac tissue, as well as an increase in the ceramide/S1P ratio in HF patients. Therapeutic gene modulation focused on restoring ceramide levels or reversing the ceramide/S1P ratio could be a potential therapy to be explored for HF patients.

Diagnostic value of serum miR-144-3p for the detection of acute cellular rejection in heart transplant patients.

BACKGROUND: The development of noninvasive approaches for the early diagnosis of acute cellular rejection (ACR), an important complication of cardiac transplantation, is of great importance in clinical practice. We conducted a nontargeted transcriptomic study focused on identifying serum miRNAs to evaluate their diagnostic accuracy for detecting rejection episodes. METHODS: We included consecutive serum samples from transplant recipients undergoing routine endomyocardial biopsies. In the discovery phase (n = 40), an RNA sequencing analysis (Illumina HiSeq 2500 sequencer) was performed. We focused on the validation of miR-144-3p in a larger patient cohort (n = 212), selected based on the criteria of higher accuracy for ACR detection. ACR was assessed according to the International Society for Heart and Lung Transplantation. RESULTS: In the discovery phase, 26 altered miRNAs were identified as potential markers for detecting ACR. miR-144-3p showed the best results, it was the only molecule with an AUC greater than 0.95 to detect Grade ≥2R ACR and it showed significant differences in its levels when we compared Grade 1R ACR with the nonrejection group. In the validation phase, we confirmed this finding, and it had an excellent diagnostic capacity for clinically relevant rejection (Grade ≥2R AUC = 0.801, p < 0.0001), detecting mild rejection (Grade 1R AUC = 0.631, p < 0.01) and was an independent predictor for the presence of ACR (odds ratio of 14.538, p < 0.01). CONCLUSIONS: ACR is associated with the differential expression of specific serum miRNAs that correlate with the severity of the episode. Circulating miR-144-3p is a candidate noninvasive ACR biomarker that could contribute to improving the surveillance of cardiac transplanted patients.

Relationships of Telomere Homeostasis with Oxidative Stress and Cardiac Dysfunction in Human Ischaemic Hearts.

Although the roles of telomeres and oxidative stress in ischaemic cardiomyopathy (ICM) are known, mechanisms of telomere homeostasis and their relationship with oxidative stress are incompletely understood. We performed two RNA-seq analyses (mRNA n = 23; ncRNA n = 30) and protein validation on left ventricles of explanted hearts from ICM and control subjects. We observed dysregulation of the shelterin and cohesin complexes, which was related to an increase in the response to cellular oxidative stress. Moreover, we found alterations at mRNA level in the mechanisms of telomeric DNA repair. Specifically, increased RAD51D mRNA levels were correlated with left ventricular diameters. RAD51D protein levels were unaltered, however, and were inversely corelated with the miR-103a-3p upregulation. We also observed the overexpression of lncRNAs (TERRA and GUARDIN) involved in telomere protection in response to stress and alterations in their regulatory molecules. Expression of the TERRA transcription factor ATF7 was correlated with superoxide dismutase 1 expression and left ventricular diameters. The levels of GUARDIN and its transcription factor FOSL2 were correlated with those of catalase. Therefore, we showed specific alterations in the mechanisms of telomeric DNA repair and protection, and these alterations are related to an increase in the response mechanisms to oxidative stress and cardiac dysfunction in ICM.

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

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.