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Small extracellular vesicles (EVs) and their cargo are an important component of cell-to-cell communication in cardiac disease. Allogeneic adipose derived stem cells (ADSCs) are thought to be a potential approach for cardiac regenerative therapy in ischemic heart disease. The SCIENCE study investigated the effect of ADSCs administered via intramyocardial injection on cardiac function in patients with ischemic heart disease. The aim of this substudy, based on samples from 15 patients, was to explore small EV miRNA dynamics after treatment with ADSCs compared to a placebo. Small EVs were isolated at several timepoints after the percutaneous intramyocardial application of ADSCs. No significant effect of ADSC treatment on small EV concentration was detected. After 12 months, the expression of miR-126 decreased significantly in ADSC patients, but not in the placebo-treated group. However, all cardiac miRNAs correlated with plasma cardiac biomarkers. In line with the overall negative results of the SCIENCE study, with the exception of one miR, we did not detect any significant regulation of small EV miRNAs in this patient collective.

We have designed translational animal models to investigate cardiac profibrotic gene signatures. Domestic pigs were treated with cardiotoxic drugs (doxorubicin, DOX, n = 5 or Myocet ® , MYO, n = 5) to induce replacement fibrosis via cardiotoxicity. Reactive interstitial fibrosis was triggered by LV pressure overload by artificial isthmus stenosis with stepwise developing myocardial hypertrophy and final fibrosis (Hyper, n = 3) or by LV volume overload in the adverse remodeled LV after myocardial infarction (RemoLV, n = 3). Sham interventions served as controls and healthy animals (Control, n = 3) served as a reference in sequencing study. Myocardial samples from the LV of each group were subjected to RNA sequencing. RNA-seq analysis revealed a clear distinction between the transcriptomes of myocardial fibrosis (MF) models. Cardiotoxic drugs activated the TNF-alpha and adrenergic signaling pathways. Pressure or volume overload led to the activation of FoxO pathway. Significant upregulation of pathway components enabled the identification of potential drug candidates used for the treatment of heart failure, such as ACE inhibitors, ARB, ß-blockers, statins and diuretics specific to the distinct MF models. We identified candidate drugs in the groups of channel blockers, thiostrepton that targets the FOXM1-regulated ACE conversion to ACE2, tyrosine kinases or peroxisome proliferator-activated receptor inhibitors. Our study identified different gene targets involved in the development of distinct preclinical MF protocols enabling tailoring expression signature-based approach for the treatment of MF.

Circular RNAs (circRNAs) are crucial in gene regulatory networks and disease development, yet circRNA expression in myocardial infarction (MI) is poorly understood. Here, we harvested myocardium samples from domestic pigs 3 days after closed-chest reperfused MI or sham surgery. Cardiac circRNAs were identified by RNA-sequencing of rRNA-depleted RNA from infarcted and healthy myocardium tissue samples. Bioinformatics analysis was performed using the CIRIfull and KNIFE algorithms, and circRNAs identified with both algorithms were subjected to differential expression (DE) analysis and validation by qPCR. Circ-RCAN2 and circ-C12orf29 expressions were significantly downregulated in infarcted tissue compared to healthy pig heart. Sanger sequencing was performed to identify the backsplice junctions of circular transcripts. Finally, we compared the expressions of circ-C12orf29 and circ-RCAN2 between porcine cardiac progenitor cells (pCPCs) that were incubated in a hypoxia chamber for different time periods versus normoxic pCPCs. Circ-C12orf29 did not show significant DE in vitro, whereas circ-RCAN2 exhibited significant ischemia-time-dependent upregulation in hypoxic pCPCs. Overall, our results revealed novel cardiac circRNAs with DE patterns in pCPCs, and in infarcted and healthy myocardium. Circ-RCAN2 exhibited differential regulation by myocardial infarction in vivo and by hypoxia in vitro. These results will improve our understanding of circRNA regulation during acute MI.

Clusterin exerts anti-inflammatory, cytoprotective and anti-apoptotic effects. Both an increase and decrease of clusterin in acute myocardial infarction (AMI) has been reported. We aimed to clarify the role of clusterin as a systemic biomarker in AMI. AMI was induced by percutaneous left anterior artery (LAD) occlusion for 90 min followed by reperfusion in 24 pigs. Contrast ventriculography was performed after reperfusion to assess left ventricular ejection fraction (LVEF), left ventricular end diastolic volume (LVEDV) and left ventricular end systolic volume (LVESV) and additional cMRI + late enhancement to measure infarct size and LV functions at day 3 and week 6 post-MI. Blood samples were collected at prespecified timepoints. Plasma clusterin and other biomarkers (cTnT, NT-proBNP, neprilysin, NGAL, ET-1, osteopontin, miR21, miR29) were measured by ELISA and qPCR. Gene expression profiles of infarcted and remote region 3 h ( n = 5) and 3 days ( n = 5) after AMI onset were analysed by RNA-sequencing. AMI led to an increase in LVEDV and LVESV during 6-week, with concomitant elevation of NT-proBNP 3-weeks after AMI. Plasma clusterin levels were increased immediately after AMI and returned to normal levels until 3-weeks. Plasma NGAL, ET-1 and miR29 was significantly elevated at 3 weeks follow-up, miR21 increased after reperfusion and at 3 weeks post-AMI, while circulating neprilysin levels did not change. Elevated plasma clusterin levels 120 min after AMI onset suggest that clusterin might be an additional early biomarker of myocardial ischemia., Competing Interests: The authors declare no conflict of interest.

We investigated the antiarrhythmic effects of ischemic preconditioning (IPC) and postconditioning (PostC) by intracardiac electrocardiogram (ECG) and measured circulating microRNAs (miRs) that are related to cardiac conduction. Domestic pigs underwent 90-min. percutaneous occlusion of the mid left anterior coronary artery, followed by reperfusion. The animals were divided into three groups: acute myocardial infarction (AMI, n = 7), ischemic preconditioning-acute myocardial infarction (IPC-AMI) ( n = 9), or AMI-PostC ( n = 5). IPC was induced by three 5-min. episodes of repetitive ischemia/reperfusion cycles (rI/R) before AMI. PostC was induced by six 30-s rI/R immediately after induction of reperfusion 90 min after occlusion. Before the angiographic procedure, a NOGA endocardial mapping catheter was placed again the distal anterior ventricular endocardium to record the intracardiac electrogram (R-amplitude, ST-Elevation, ST-area under the curve (AUC), QRS width, and corrected QT time (QTc)) during the entire procedure. An arrhythmia score was calculated. Cardiac MRI was performed after one-month. IPC led to significantly lower ST-elevation, heart rate, and arrhythmia score during ischemia. PostC induced a rapid recovery of R-amplitude, decrease in QTc, and lower arrhythmia score during reperfusion. Slightly higher levels of miR-26 and miR-133 were observed in AMI compared to groups IPC-AMI and AMI-PostC. Significantly lower levels of miR-1, miR-208, and miR-328 were measured in the AMI-PostC group as compared to animals in group AMI and IPC-AMI. The arrhythmia score was not significantly associated with miRNA plasma levels. Cardiac MRI showed significantly smaller infarct size in the IPC-AMI group when compared to the AMI and AMI-PostC groups. Thus, IPC led to better left ventricular ejection fraction at one-month and it exerted antiarrhythmic effects during ischemia, whereas PostC exhibited antiarrhythmic properties after reperfusion, with significant downregulaton of ischemia-related miRNAs.

Although the application of cardioprotective ischaemia/reperfusion (I/R) stimuli after myocardial infarction (MI) is a promising concept for salvaging the myocardium, translation to a clinical scenario has not fulfilled expectations. We have previously shown that in pigs, ischaemic postconditioning (IPostC) reduces myocardial oedema and microvascular obstruction (MVO), without influencing myocardial infarct size. In the present study, we analyzed the mechanisms underlying the IPostC-induced microvascular protection by transcriptomic analysis, followed by pathway analysis. Closed-chest reperfused MI was induced by 90 min percutaneous balloon occlusion of the left anterior descending coronary artery, followed by balloon deflation in anaesthetised pigs. Animals were randomised to IPostC ( n = 8), MI (non-conditioned, n = 8), or Control (sham-operated, n = 4) groups. After three hours or three days follow-up, myocardial tissue samples were harvested and subjected to RNA-seq analysis. Although the transcriptome analysis revealed similar expression between IPostC and MI in transcripts involved in cardioprotective pathways, we identified gene expression changes responding to IPostC at the three days follow-up. Focal adhesion signaling, downregulated genes participating in cardiomyopathy and activation of blood cells may have critical consequences for microvascular protection. Specific analyses of the gene subsets enriched in the endothelium of the infarcted area, revealed strong deregulation of transcriptional functional clusters, DNA processing, replication and repair, cell proliferation, and focal adhesion, suggesting sustentative function in the endothelial cell layer protection and integrity. The spatial and time-dependent transcriptome analysis of porcine myocardium supports a protective effect of IPostC on coronary microvasculature post-MI., Competing Interests: P.F. is the founder and CEO of Pharmahungary, a group of R&D companies. The other authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.