Chemical and mechanical activation of resident cardiac macrophages in the living myocardial slice ex vivo model.

Resident cardiac macrophages (rcMACs) are among the most abundant immune cells in the heart. Plasticity and activation are hallmarks of rcMACs in response to changes in the microenvironment, which is essential for in vitro experimentation. The in vivo investigation is confounded by the infiltration of other cells hindering direct studies of rcMACs. As a tool to investigate rcMACs, we applied the ex vivo model of living myocardial slices (LMS). LMS are ultrathin ex vivo multicellular cardiac preparations in which the circulatory network is interrupted. The absence of infiltration in this model enables the investigation of the rcMACs response to immunomodulatory and mechanical stimulations. Such conditions were generated by applying interferon-gamma (IFN-γ) or interleukine-4 (IL-4) and altering the preload of cultured LMS, respectively. The immunomodulatory stimulation of the LMS induced alterations of the gene expression pattern without affecting tissue contractility. Following 24 h culture, low input RNA sequencing of rcMACs isolated from LMS was used for gene ontology analysis. Reducing the tissue stretch (unloading) of LMS altered the gene ontology clusters of isolated rcMACs with intermediate semantic similarity to IFN-γ triggered reaction. Through the overlap of genes affected by IFN-γ and unloading, we identified Allograft inflammatory factor 1 (AIF-1) as a potential marker gene for inflammation of rcMACs as significantly altered in whole immunomodulated LMS. MicroRNAs associated with the transcriptomic changes of rcMACs in unloaded LMS were identified in silico. Here, we demonstrate the approach of LMS to understand load-triggered cardiac inflammation and, thus, identify potential translationally important therapeutic targets.

A practical guide for investigating cardiac physiology using living myocardial slices.

Ex vivo multicellular preparations are essential tools to study tissue physiology. Among them, the recent methodological and technological developments in living myocardial slices (LMS) are attracting increasing interest by the cardiac research field. Despite this, this research model remains poorly perceived and utilized by most research laboratories. Here, we provide a practical guide on how to use LMS to interrogate multiple aspects of cardiac function, structure and biochemistry. We discuss issues that should be considered to conduct successful experiments, including experimental design, sample preparation, data collection and analysis. We describe how laboratory setups can be adapted to accommodate and interrogate this multicellular research model. These adaptations can often be achieved at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software, which is essential to broaden the utilization of this method. We will also highlight how current measurements can be improved to further enhance data quality and reliability to ensure inter-laboratory reproducibility. Finally, we summarize the most promising biomedical applications and envision how living myocardial slices can lead to further breakthroughs.

Resident cardiac macrophages: crucial modulators of cardiac (patho)physiology.

Resident cardiac macrophages (rcMacs) are integral components of the myocardium where they have key roles for tissue homeostasis and in response to inflammation, tissue injury and remodelling. In this review, we summarize the current knowledge and limitations associated with the rcMacs studies. We describe their specific role and contribution in various processes such as electrical conduction, efferocytosis, inflammation, tissue development, remodelling and regeneration in both the healthy and the disease state. We also outline research challenges and technical complications associated with rcMac research. Recent technological developments and contemporary immunological techniques are now offering new opportunities to investigate the separate contribution of rcMac in respect to recruited monocytes and other cardiac cells. Finally, we discuss new therapeutic strategies, such as drugs or non-coding RNAs, which can influence rcMac phenotype and their response to inflammation. These novel approaches will allow for a deeper understanding of this cardiac endogenous cell type and might lead to the development of more specific and effective therapeutic strategies to boost the heart's intrinsic reparative capacity.

Leptin Expression and Gene Methylation Patterns in Alcohol-Dependent Patients with Ethyltoxic Cirrhosis-Normalization After Liver Transplantation and Implications for Future Research.

AIMS: Liver transplantation is the only curative treatment available for patients with end-stage alcoholic liver disease. As different studies showed a significant association between leptin plasma levels, gene methylation patterns and the extent of craving in alcohol-dependent patients, we investigated the effect of liver transplantation on leptin expression and promoter methylation. SHORT SUMMARY: The present study shows that in alcohol-dependent patients with liver cirrhosis leptin is significantly higher before liver transplantation and decreases significantly after transplantation. Alcohol-dependent patients on the waiting list had significantly higher leptin promoter methylation values than patients who underwent liver transplantation for other reasons than alcoholic liver disease. METHODS: Only plasma of 118 and peripheral blood mononuclear cells of 121 patients were used: healthy controls (C, n = 24/22), alcohol-dependent patients without ethyltoxic liver cirrhosis (AD, n = 24/22), patients after liver transplantation for other reasons than ethyltoxic liver cirrhosis (C-Tx, n = 18/21), alcohol-dependent patients suffering from ethyltoxic liver cirrhosis on the transplantation waiting list (Pre-Tx, n = 30/28) and patients with prior ethyltoxic liver cirrhosis after liver transplantation (Post-Tx, n = 22/28). RESULTS: Leptin protein was significantly elevated in the pre-transplantation cohort when compared to post-transplantation and alcohol-dependent cohorts. Furthermore, leptin promoter methylation was higher in ethyltoxic patients before transplantation compared to non-ethyltoxic patients after transplantation, but not when compared to ethyltoxic patients after transplantation. C-Tx had lower methylation values than all other groups except for Post-Tx. CONCLUSIONS: Our study outlines the role of leptin protein levels as a marker for AD-related liver damage, contrasting it from AD without severe liver damage. With regard to the results of the methylation analysis, inflammation of the liver appears to cause mechanisms of leptin regulation to deviate from transcriptional regulation. Our data also suggest that leptin regulation is altered in ethyltoxic liver disease when compared to liver cirrhosis caused by other pathologies.

[Non-coding RNA : Innovative regulators with therapeutic perspective]. / Nichtkodierende RNA : Innovative Regulatoren mit therapeutischer Perspektive.

As a result of the Human Genome Project it became evident that only 1-3% of all gene transcripts encode proteins. The vast majority of gene transcripts are in fact characterized as non-coding RNAs (ncRNAs). These ncRNAs have a huge impact on diverse physiological and pathological mechanisms within an organism. In particular, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), which are differentiated by their size and function, are involved in the regulation and development of many illnesses. In the context of heart and cardiovascular diseases numerous ncRNAs have also already been described in some detail. As these molecules represent therapeutic target structures, ncRNAs provide a completely new level for the discovery of promising therapeutic approaches. Many approaches have already been developed aimed at influencing the expression levels of specific ncRNAs in order to induce beneficial effects on pathological processes. In fact, first medications based on miRNAs have already achieved approval. Additionally, ncRNAs contained in plasma can serve as new non-invasive diagnostic markers for the detection of diseases.

The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions.

The 13th Banff Conference on Allograft Pathology was held in Vancouver, British Columbia, Canada from October 5 to 10, 2015. The cardiac session was devoted to current diagnostic issues in heart transplantation with a focus on antibody-mediated rejection (AMR) and small vessel arteriopathy. Specific topics included the strengths and limitations of the current rejection grading system, the central role of microvascular injury in AMR and approaches to semiquantitative assessment of histopathologic and immunophenotypic indicators, the role of AMR in the development of cardiac allograft vasculopathy, the important role of serologic antibody detection in the management of transplant recipients, and the potential application of new molecular approaches to the elucidation of the pathophysiology of AMR and potential for improving the current diagnostic system. Herein we summarize the key points from the presentations, the comprehensive, open and wide-ranging multidisciplinary discussion that was generated, and considerations for future endeavors.

microRNA-206 correlates with left ventricular function after transcatheter aortic valve implantation.

Transcatheter aortic valve implantation (TAVI) is the method of choice in patients with high risk or contraindications for conventional aortic valve replacement. However, it is not well understood which parameters predict the overall cardiac function postprocedurally. miRNAs are small noncoding RNA molecules that repress gene expression by different mechanisms and can also be detected in the blood. Recent studies have shown that miRNAs detected in the blood may serve as sensitive and specific biomarkers in various diseases; therefore, we examined the levels of different microRNAs in the serum of patients undergoing TAVI. We thereby intended to find potential predictors for cardiac function after TAVI. Serum from patients with aortic valve disease was obtained at five different points: before the TAVI procedure, at days 1 and 3 after the TAVI procedure, and the day of dischargement and after a period of 3 mo. We next performed quantitative real-time PCRs to examine the samples for changes in the level of miRNAs previously described as cardiac enriched. Our results show that the level of miR-206 in the serum of patients after TAVI correlated negatively with the left ventricular ejection fraction of individual patients. We found left ventricular function to be better in patients with lower levels of miR-206 after implantation of the new valve. A decrease in the serum level of miR-206 may be linked to changes in cardiac function of patients after TAVI. Further studies are necessary to test the miRNA for its potential value as a prognostic marker. NEW & NOTEWORTHY This study is the first to investigate novel miRNA-based biomarkers within the context of transcatheter aortic valve implantation. miRNA-206 proved to correlate inversely with the postprocedural left ventricular ejection fraction of patients.

RNA Profiling in Human and Murine Transplanted Hearts: Identification and Validation of Therapeutic Targets for Acute Cardiac and Renal Allograft Rejection.

Acute cellular rejection (ACR) is the adverse response of the recipient's immune system against the allogeneic graft. Using human surveillance endomyocardial biopsies (EMBs) manifesting ACR and murine allogeneic grafts, we profiled implicated microRNAs (miRs) and mRNAs. MiR profiling showed that miR-21, -142-3p, -142-5p, -146a, -146b, -155, -222, -223, and -494 increased during ACR in humans and mice, whereas miR-149-5p decreased. mRNA profiling revealed 70 common differentially regulated transcripts, all involved in immune signaling and immune-related diseases. Interestingly, 33 of 70 transcripts function downstream of IL-6 and its transcription factor spleen focus forming virus proviral integration oncogene (SPI1), an established target of miR-155, the most upregulated miR in human EMBs manifesting rejection. In a mouse model of cardiac transplantation, miR-155 absence and pharmacological inhibition attenuated ACR, demonstrating the causal involvement and therapeutic potential of miRs. Finally, we corroborated our miR signature in acute cellular renal allograft rejection, suggesting a nonorgan specific signature of acute rejection. We concluded that miR and mRNA profiling in human and murine ACR revealed the shared significant dysregulation of immune genes. Inflammatory miRs, for example miR-155, and transcripts, in particular those related to the IL-6 pathway, are promising therapeutic targets to prevent acute allograft rejection.

MicroRNA-based therapy in cardiology.

The pathogenic role of noncoding microRNA (miR, miRNA) has been demonstrated for several disease conditions in the heart. The underlying molecular mechanisms have been deciphered for numerous miRs that are deregulated as a result of cardiac stress. Innovative therapeutic strategies based on antifibrotic, antihypertrophic, or proangiogenic effects of miRNAs are being currently developed to improve the function of the failing heart. Identifying a safe and efficient miR-based strategy remains challenging, yet these novel approaches offer enormous potential for the treatments for heart failure. In this review we highlight the latest development in the cardiac miRNA field.

Microvesicles as novel biomarkers and therapeutic targets in transplantation medicine.

Microvesicles (MVs) including exosomes are emerging new biomarkers and potential regulators of inflammation and immunological processes. Such particles contain proteins and genetic information including DNA and microRNAs that may be of importance for cell/cell communication. However, their role during and after organ transplantation and immunomodulatory effects is only in its beginning of understanding. We here, in brief, introduce generation and biological importance of MVs, describe their (patho)physiological roles and their potential use as future biomarkers and therapeutic agents in transplantation medicine. Circulating MVs may have a great potential to detect possible immune rejections and MV modulation may emerge as a therapeutic approach in organ rejection therapy.

Dissecting the transcriptome in cardiovascular disease.

The human transcriptome comprises a complex network of coding and non-coding RNAs implicated in a myriad of biological functions. Non-coding RNAs exhibit highly organized spatial and temporal expression patterns and are emerging as critical regulators of differentiation, homeostasis, and pathological states, including in the cardiovascular system. This review defines the current knowledge gaps, unmet methodological needs, and describes the challenges in dissecting and understanding the role and regulation of the non-coding transcriptome in cardiovascular disease. These challenges include poor annotation of the non-coding genome, determination of the cellular distribution of transcripts, assessment of the role of RNA processing and identification of cell-type specific changes in cardiovascular physiology and disease. We highlight similarities and differences in the hurdles associated with the analysis of the non-coding and protein-coding transcriptomes. In addition, we discuss how the lack of consensus and absence of standardized methods affect reproducibility of data. These shortcomings should be defeated in order to make significant scientific progress and foster the development of clinically applicable non-coding RNA-based therapeutic strategies to lessen the burden of cardiovascular disease.

Identification of cardiovascular microRNA targetomes.

MicroRNAs (miRNAs) are strong post-transcriptional regulators targeting multiple targets. Endogenously transcribed, miRNAs specifically bind to complementary sequences of mRNAs and repress their expression thus govern control of cellular signaling pathways. An altered miRNA expression is causally related to cardiovascular disease. Identification of miRNA-dependent pathways is therefore an important aim to develop new therapeutic approaches. To understand miRNA function in various cardiovascular cells, the identification of individual miRNA target genes is of utmost importance. Indeed, the biological function of a miRNA is dependent on the availability of potential targets in a cell. We here summarize and discuss current challenging approaches to identify miRNA targetomes which will help to understand miRNA function in cardiac homeostasis and disease.

Urinary miR-210 as a mediator of acute T-cell mediated rejection in renal allograft recipients.

MicroRNAs (miRNAs) are small ribonucleotides regulating gene expression. Circulating miRNAs are remarkably stable in the blood. We tested whether miRNAs are also detectable in urine and may serve as new predictors of outcome in renal transplant patients with acute rejection. We profiled urinary miRNAs of stable transplant patients and transplant patients with acute rejection. The miR-10a, miR-10b and miR-210 were strongly deregulated in urine of the patients with acute rejection. We confirmed these data in urine of a validation cohort of 62 patients with acute rejection, 19 control transplant patients without rejection and 13 stable transplant patients with urinary tract infection by quantitative RT-PCR. The miR-10b and miR-210 were downregulated and miR-10a upregulated in patients with acute rejection compared to controls. Only miR-210 differed between patients with acute rejection when compared to stable transplant patients with urinary tract infection or transplant patients before/after rejection. Low miR-210 levels were associated with higher decline in GFR 1 year after transplantation. Selected miRNAs are strongly altered in urine of the patients with acute renal allograft rejection. The miR-210 levels identify patients with acute rejection and predict long-term kidney function. Urinary miR-210 may thus serve as a novel biomarker of acute kidney rejection.

Circulating long-non coding RNAs as biomarkers of left ventricular diastolic function and remodelling in patients with well-controlled type 2 diabetes.

Contractile dysfunction is underdiagnosed in early stages of diabetic cardiomyopathy. We evaluated the potential of circulating long non-coding RNAs (lncRNAs) as biomarkers of subclinical cardiac abnormalities in type 2 diabetes. Forty-eight men with well-controlled type 2 diabetes and 12 healthy age-matched volunteers were enrolled in the study. Left ventricular (LV) parameters were measured by magnetic resonance imaging. A panel of lncRNAs was quantified in serum by RT-qPCR. No differences in expression levels of lncRNAs were observed between type 2 diabetes patients and healthy volunteers. In patients with type 2 diabetes, long intergenic non-coding RNA predicting cardiac remodeling (LIPCAR) was inversely associated with diastolic function, measured as E/A peak flow (P < 0.050 for all linear models). LIPCAR was positively associated with grade I diastolic dysfunction (P < 0.050 for all logistic models). Myocardial infarction-associated transcript (MIAT) and smooth muscle and endothelial cell-enriched migration/differentiation-associated long noncoding RNA (SENCR) were directly associated with LV mass to LV end-diastolic volume ratio, a marker of cardiac remodelling (P < 0.050 for all linear models). These findings were validated in a sample of 30 patients with well-controlled type 2 diabetes. LncRNAs are independent predictors of diastolic function and remodelling in patients with type 2 diabetes.

Endothelial progenitor cells as potential drug targets.

Endothelial progenitor cells (EPC) are bone marrow derived cells with the potential to differentiate into mature functional endothelial cells. First clinical trials have been performed investigating the effects of EPC transplantation into cardiac ischemic areas after myocardial infarction, in patients with peripheral atherovascular disease or on endothelialisation of artificial heart valves. Next to EPC transplantation, the pharmacological mobilisation and functional modification of EPC may also play a major role in future therapies. Studies have raised the concern that patients with coronary heart disease or severe heart failure may suffer from decreased amounts and impaired function of peripheral circulating EPC. Drug induced mobilization of EPC and normalization of EPC function may therefore improve prognosis of certain cardiovascular diseases. The underlying molecular events of a disturbed mobilisation, differentiation, homing and/or function of EPC are not well understood. In the present review we will highlight the current knowledge of the role of EPC dysfunction in various cardiovascular diseases and focus on potential causally related molecular mechanisms, which might be novel drug targets.

Reprogramming of gene expression in cultured cardiomyocytes and in explanted hearts by the myosin ATPase inhibitor butanedione monoxime.

BACKGROUND: Butanedione monoxime (BDM) is a reversible myosin ATPase inhibitor. Its use in transplantation medicine may be of benefit in the preservation of hearts. As little is known about its ability to prevent stress and metabolic deregulation, we wanted to investigate the genomic response in cultured cardiomyocytes and explanted, preserved hearts at the transcriptional level. METHODS: We thus investigated the gene expression of the transcription factors GATA-4, Nkx2.5, MEF-2c, and Oct-1 and of the downstream target genes atrial and brain natriuretic peptide, alpha- and beta-myosin heavy chain, alpha-cardiac actin, and alpha-skeletal actin. Additionally, lactate dehydrogenase and creatine kinase enzyme activities were measured as markers for membrane integrity and metabolic deregulation of cardiomyocytes. RESULTS: In untreated cardiomyocyte cultures, expression of GATA-4 and Nkx2.5 was increased 7- and 4-fold, 72 hr after isolation, but the gene expression of MEF-2c and Oct-1 was reduced to 10% and 70%, at day 3 in culture. We show atrial natriuretic peptide and brain natriuretic peptide gene expression to be maximal 24 and 72 hr after isolation, the level being 3- and 2-fold, when compared with freshly isolated cells. The gene expression of alpha- and beta-myosin heavy chain was reduced to approximately 30% at day 3 in culture and similar observations were made for alpha-cardiac and alpha-skeletal actin, which declined to approximately 20% and 10% of control values, 72 hr after isolation. BDM prevented at the transcriptional level enhanced expression of markers for stress and metabolic deregulation, and the activities of lactate dehydrogenase and creatine kinase were highly significantly reduced. Similar results were obtained when explanted hearts were stored in BDM-containing organ preservation solution. CONCLUSIONS: Preservation of metabolic function in donor organs is of critical importance in transplantation medicine, and we show gene markers for stress and metabolic deregulation in cultures of cardiomyocytes and explanted hearts to be significantly reduced by BDM. Reprogramming of gene expression of nuclear transcription factors and downstream target genes may prolong the acceptable storage time between explantation and transplantation.

Cytochrome P450 mono-oxygenase gene expression and protein activity in cultures of adult cardiomyocytes of the rat.

There are a substantial number of drugs acting either directly or indirectly on the heart, but surprisingly, little is known about the metabolic capacity of heart muscle cells. We therefore investigated the gene expression and protein activity of cytochrome P450 isozymes in cultures of adult cardiomyocytes of the rat. Semi-quantitative CYP gene expression pattern suggests CYP1A1 and CYP2B1/2 to be key players in cardiomyocytes and upon treatment with Aroclor 1254 approximate 4 fold inductions could be observed for both gene families, when compared with appropriate controls. The mRNA expression of most genes was sustained for prolonged periods of time, e.g. up to 120 h in culture and in the case of the CYP3A1 gene an approximate 10 fold induction was observed at the higher Aroclor 1254 dose level (10 microM) in 24 h old cultures. The constitutively expressed genes, e.g. CYP2C11 and CYP2E1 are expressed throughout the entire culture period (5 days) and did not respond to Aroclor 1254 treatment. CYP4A1 was mainly expressed in freshly isolated cardiomyocytes of control animals and its expression declined rapidly in culture. There was good agreement between gene expression and translated protein activity using 7-ethoxyresorufin and testosterone as substrates. The data reported herein should foster the routine use of freshly isolated and cultivated cardiomyocytes for drug profiling and toxicity studies.

Induction of nuclear transcription factors, cytochrome P450 monooxygenases, and glutathione S-transferase alpha gene expression in Aroclor 1254-treated rat hepatocyte cultures.

Aroclor 1254 is a complex mixture of polychlorinated biphenyls and is well known for its potency to induce drug-metabolising enzymes, but little is known about its ability to modulate gene expression of transcription factors, which code for proteins that bind to the regulatory elements of DNA and facilitate transcriptional activation. We therefore investigated the gene expression of the liver-specific transcription factors CCAAT/enhancer-binding protein alpha (c/EBPalpha), hepatic nuclear factor (HNF) 1 and 4, and major cytochrome P450 (CYP) isozymes in addition to glutathione S-transferase alpha 2 (GSTA-2) in cultures of primary rat hepatocytes. We found highly significant and dose-dependent increases of c/EBPalpha (up to 62-fold), HNF-1 (up to 7-fold), HNF-4 (up to 8-fold), and 50- and 4-fold inductions of GSTA-2 and CYP monooxygenases, respectively. Based on the ethoxyresorufin-O-deethylase assay, the gene expression and enzyme activity for CYP1A1 were in good agreement, but for other CYP isozymes similar correlations could not be obtained. In conclusion, the simultaneous induction of liver-specific TFs and of several detoxifying enzymes may point to a coordinate genomic response in cultures of rat hepatocytes upon treatment with Aroclor 1254.