Heart failure (HF) is a complex disease entity with high clinical impact, poorly understood pathophysiology and scantly known miRNA-mediated epigenetic regulation. We have analysed miRNA patterns in patients with chronic HF (cHF) and a sex- and age-matched reference group and pursued an in silico system biology analysis to discern pathways involved in cHF pathophysiology. Twenty-eight miRNAs were identified in cHF that were up-regulated in the reference group, and eight of them were validated by RT-qPCR. In silico analysis of predicted targets by STRING protein-protein interaction networks revealed eight cluster networks (involving seven of the identified miRNAs) enriched in pathways related to cell cycle, Ras, chemokine, PI3K-AKT and TGF-β signaling. By ROC curve analysis, combined probabilities of these seven miRNAs (let-7a-5p, miR-107, miR-125a-5p, miR-139-5p, miR-150-5p, miR-30b-5p and miR-342-3p; clusters 1–4 [C:1–4]), discriminated between HF with preserved ejection fraction (HFpEF) and HF with reduced ejection fraction (HFrEF), and ischaemic and non-ischaemic aetiology. A combination of miR-107, miR-139-5p and miR-150-5p, involved in clusters 5 and 7 (C:5+7), discriminated HFpEF from HFrEF. Pathway enrichment analysis of miRNAs present in C:1–4 (let-7a-5p, miR-125a-5p, miR-30b-5p and miR-342-3p) revealed pathways related to HF pathogenesis. In conclusion, we have identified a differential signature of down-regulated miRNAs in the plasma of HF patients and propose novel cellular mechanisms involved in cHF pathogenesis.

BACKGROUND: Cardiovascular disease causes >50% of deaths in patients with advanced chronic kidney disease (CKD). Clinical studies suggest that kidney-derived factors contribute to cardiovascular disease development in CKD, independently of comorbidities. However, to date, no kidney-specific humoral risk factor that triggers direct cardiotoxicity has been identified. In this cross-sectional study, we investigate how, in patients with CKD, circulating extracellular vesicles (EVs) facilitate pathological kidney-heart communication, thereby causing cardiotoxicity, impairing cardiac function, and contributing to heart failure progression. METHODS: We investigated the function of EVs from patients with CKD and adenine diet–induced CKD mice on cardiomyocyte and cardiac contractility. microRNA (miRNA) cargo of EVs was identified by small RNA sequencing and quantitative reverse transcription polymerase chain reaction, and their cardiotoxicity was tested by using miRNA mimics. Tissue and cellular origin of CKD-EV-miRNAs were determined from their corresponding primary miRNA expressions in mice. RESULTS: EVs from plasma of patients with CKD, but not from healthy controls, were cardiotoxic; they significantly induced apoptosis both in vitro and in vivo and impaired contractility of adult rat primary cardiomyocytes in vitro. Likewise, EVs from both plasma and kidneys of CKD mice were cardiotoxic. Pharmacologically depleting circulating EVs in CKD mice significantly recovered cardiac function and ameliorated heart failure, improvements that suggest CKD-EVs play a causal role in heart failure pathogenesis. Both human and mouse CKD-EVs were enriched in distinct miRNAs compared with control EVs. CKD-EV-miRNA mimics were cardiotoxic, impairing contractility and downregulating contractile gene expression in human induced pluripotent stem cell–derived cardiomyocytes. It is interesting that levels of endogenous primary miRNAs corresponding to circulating CKD-EV-miRNAs were significantly higher in CKD kidney tissues, specifically in CD45 ^-ve CD31 ^-ve renal cells, but not in CKD hearts, CKD livers, or CKD-peripheral blood mononuclear cells, a result that indicates CKD-EV-miRNAs originate renally. It is remarkable that CKD-EV-miRNA levels correlated with established markers of cardiac injury, thus uncovering the presence of subclinical heart disease and demonstrating heterogeneity in reno-cardiac disease. CONCLUSIONS: Collectively, our human subject and mouse studies show that circulating CKD-EVs, carrying distinct renal-derived miRNAs, mediate the molecular crosstalk that contributes to the pathogenesis of heart failure in CKD. Consequently, CKD-EVs hold promise as diagnostic and prognostic biomarkers for early disease detection and as targets for novel therapeutic interventions in chronic reno-cardiac disease.

Obesity is associated with metabolic disorders such as insulin resistance and type 2 diabetes mellitus (T2DM), further increasing an already heightened cardiovascular risk. Here, amongst obese class III bariatric surgery patients, we have investigated the effect of T2DM in serum and in two, same patient, adipose tissue (AT) depots through proteomic profile expression analyses. Serum and AT samples from subcutaneous (SAT) and visceral (VAT) fat were collected during bariatric surgery. Bead-based targeted multiplex assay systems were used to simultaneously detect and quantify multiple targets in serum samples (targeted proteomics) and analyze changes in adipokine serum composition. AT samples were assessed through an untargeted proteomics approach. Through a systems biology analysis of the proteomic data, information on the affected biological pathways was acquired. In obese class III individuals, the presence of T2DM induced a significantly higher systemic release of ghrelin, GLP-1, glucagon, MMP3, BAFF, chitinase 3-like 1, TNF-R1 and TNF-R2, and a lower systemic release of IL-8. SAT and VAT proteomes belonging to the same patient showed significant differences in local protein content. While the proteins upregulated in VAT were indicative of metabolic dysregulation, SAT protein upregulation suggested adequate endocrine regulation. The presence of T2DM significantly affected VAT protein composition through the upregulation of dysregulating metabolic pathways, but SAT protein composition was not significantly modified. Our results show that T2DM induces metabolic dysregulation in obese individuals with changes in systemic marker levels and impairment of proteostasis in VAT but not in SAT.

Using proteomics, we previously found that serum levels of glycosylated (Glyc) forms of apolipoprotein J (ApoJ), a cytoprotective and anti-oxidant protein, decrease in the early phase of acute myocardial infarction (AMI). We aimed to investigate: (i) ApoJ-Glyc intracellular distribution and secretion during ischaemia; (ii) the early changes in circulating ApoJ-Glyc during AMI; and (iii) associations between ApoJ-Glyc and residual ischaemic risk post-AMI. Glycosylated apolipoprotein J was investigated in: (i) cells from different organ/tissue origin; (ii) a pig model of AMI; (iii) de novo AMI patients (n = 38) at admission within the first 6 h of chest pain onset and without troponin T elevation at presentation (early AMI); (iv) ST-elevation myocardial infarction patients (n = 212) who were followed up for 6 months; and (v) a control group without any overt cardiovascular disease (n = 144). Inducing simulated ischaemia in isolated cardiac cells resulted in an increased intracellular accumulation of non-glycosylated ApoJ forms. A significant decrease in ApoJ-Glyc circulating levels was seen 15 min after ischaemia onset in pigs. Glycosylated apolipoprotein J levels showed a 45% decrease in early AMI patients compared with non-ischaemic patients (P < 0.0001), discriminating the presence of the ischaemic event (area under the curve: 0.934; P < 0.0001). ST-elevation myocardial infarction patients with lower ApoJ-Glyc levels at admission showed a higher rate of recurrent ischaemic events and mortality after 6-month follow-up (P = 0.008). These results indicate that ischaemia induces an intracellular accumulation of non-glycosylated ApoJ and a reduction in ApoJ-Glyc secretion. Glycosylated apolipoprotein J circulating levels are reduced very early after ischaemia onset. Its continuous decrease indicates a worsening in the evolution of the cardiac event, likely identifying patients with sustained ischaemia after AMI. Glycosylated apolipoprotein J as a novel marker of ischaemia. Under ischaemic conditions there is a decrease in the secretion of glycosylated apolipoprotein J. The quantification of specific glycosylated apolipoprotein J variants with a novel immunoassay discriminates the presence of a cardiac ischaemic event, indicating that glycosylated apolipoprotein J could be a novel marker of cardiac ischaemia.

Infarct size is the major risk predictor for developing heart failure after an acute myocardial infarction (AMI). The discovery of the conditioning phenomena (i.e., repetitive brief cycles of ischemia applied either before or after a prolonged ischemic insult) has highlighted the existence of endogenous protective mechanisms of the heart potentially limiting infarct size after revascularization. However, most cardioprotective strategies, aiming at infarct size reduction, have failed in clinical studies. Thus, cardioprotection is an unmet clinical need. In the present study, we took a network-assisted systems biology approach to explore the mitochondrial proteomic signature of the myocardium after ischemia, ischemia with direct revascularization, and ischemia with re-establishment of blood flow by post-conditioning in a swine model of AMI. Furthermore, network extension with the ENCODE project human regulatory data allowed the prediction of potential transcription factors at play in the response to post-conditioning of the myocardium. Collectively, our results identify cardiac metabolism as a driver of cardioprotection, highlighting a dual role for post-conditioning promoting metabolic reprogramming of the myocardium, and a protective response mediated by VDAC2 and DJ-1 in the mitochondria.