Interaction of Cardiovascular Nonmodifiable Risk Factors, Comorbidities and Comedications With Ischemia/Reperfusion Injury and Cardioprotection by Pharmacological Treatments and Ischemic Conditioning.

Preconditioning, postconditioning, and remote conditioning of the myocardium enhance the ability of the heart to withstand a prolonged ischemia/reperfusion insult and the potential to provide novel therapeutic paradigms for cardioprotection. While many signaling pathways leading to endogenous cardioprotection have been elucidated in experimental studies over the past 30 years, no cardioprotective drug is on the market yet for that indication. One likely major reason for this failure to translate cardioprotection into patient benefit is the lack of rigorous and systematic preclinical evaluation of promising cardioprotective therapies prior to their clinical evaluation, since ischemic heart disease in humans is a complex disorder caused by or associated with cardiovascular risk factors and comorbidities. These risk factors and comorbidities induce fundamental alterations in cellular signaling cascades that affect the development of ischemia/reperfusion injury and responses to cardioprotective interventions. Moreover, some of the medications used to treat these comorbidities may impact on cardioprotection by again modifying cellular signaling pathways. The aim of this article is to review the recent evidence that cardiovascular risk factors as well as comorbidities and their medications may modify the response to cardioprotective interventions. We emphasize the critical need for taking into account the presence of cardiovascular risk factors as well as comorbidities and their concomitant medications when designing preclinical studies for the identification and validation of cardioprotective drug targets and clinical studies. This will hopefully maximize the success rate of developing rational approaches to effective cardioprotective therapies for the majority of patients with multiple comorbidities. SIGNIFICANCE STATEMENT: Ischemic heart disease is a major cause of mortality; however, there are still no cardioprotective drugs on the market. Most studies on cardioprotection have been undertaken in animal models of ischemia/reperfusion in the absence of comorbidities; however, ischemic heart disease develops with other systemic disorders (e.g., hypertension, hyperlipidemia, diabetes, atherosclerosis). Here we focus on the preclinical and clinical evidence showing how these comorbidities and their routine medications affect ischemia/reperfusion injury and interfere with cardioprotective strategies.

Heart failure pharmacotherapy and cancer: pathways and pre-clinical/clinical evidence.

Heart failure (HF) patients have a significantly higher risk of new-onset cancer and cancer-associated mortality, compared to subjects free of HF. While both the prevention and treatment of new-onset HF in patients with cancer have been investigated extensively, less is known about the prevention and treatment of new-onset cancer in patients with HF, and whether and how guideline-directed medical therapy (GDMT) for HF should be modified when cancer is diagnosed in HF patients. The purpose of this review is to elaborate and discuss the effects of pillar HF pharmacotherapies, as well as digoxin and diuretics on cancer, and to identify areas for further research and novel therapeutic strategies. To this end, in this review, (i) proposed effects and mechanisms of action of guideline-directed HF drugs on cancer derived from pre-clinical data will be described, (ii) the evidence from both observational studies and randomized controlled trials on the effects of guideline-directed medical therapy on cancer incidence and cancer-related outcomes, as synthetized by meta-analyses will be reviewed, and (iii) considerations for future pre-clinical and clinical investigations will be provided.

Sodium-glucose cotransporter 2 inhibitors and the cancer patient: from diabetes to cardioprotection and beyond.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i), a new drug class initially designed and approved for treatment of diabetes mellitus, have been shown to exert pleiotropic metabolic and direct cardioprotective and nephroprotective effects that extend beyond their glucose-lowering action. These properties prompted their use in two frequently intertwined conditions, heart failure and chronic kidney disease. Their unique mechanism of action makes SGLT2i an attractive option also to lower the rate of cardiac events and improve overall survival of oncological patients with preexisting cardiovascular risk and/or candidate to receive cardiotoxic therapies. This review will cover biological foundations and clinical evidence for SGLT2i modulating myocardial function and metabolism, with a focus on their possible use as cardioprotective agents in the cardio-oncology settings. Furthermore, we will explore recently emerged SGLT2i effects on hematopoiesis and immune system, carrying the potential of attenuating tumor growth and chemotherapy-induced cytopenias.

Cellular Alterations in Immune Checkpoint Inhibitor Therapy-Related Cardiac Dysfunction.

PURPOSE OF REVIEW: Immune checkpoint inhibitor (ICI) therapy has emerged as a pivotal advancement in cancer treatment, but the widespread adoption has given rise to a growing number of reports detailing significant cardiovascular toxicity. This review concentrates on elucidating the mechanisms behind ICI-related cardiovascular complications, emphasizing preclinical and mechanistic data. RECENT FINDINGS: Accumulating evidence indicates a more significant role of immune checkpoints in maintaining cardiac integrity than previously understood, and new key scientific data are available to improve our understanding of ICI-related cardiovascular toxicity, including hidden cardiotoxicity. New avenues for innovative concepts are hypothesized, and opportunities to leverage the knowledge from ICI-therapy for pioneering approaches in related scientific domains can be derived from the latest scientific projects. Cardiotoxicity from ICI therapy is a paramount challenge for cardio-oncology. Understanding the underlying effects builds the foundation for tailored cardioprotective approaches in the growing collective at risk for severe cardiovascular complications.

Circulating cardiomyocyte-derived extracellular vesicles reflect cardiac injury during systemic inflammatory response syndrome in mice.

The release of extracellular vesicles (EVs) is increased under cellular stress and cardiomyocyte damaging conditions. However, whether the cardiomyocyte-derived EVs eventually reach the systemic circulation and whether their number in the bloodstream reflects cardiac injury, remains unknown. Wild type C57B/6 and conditional transgenic mice expressing green fluorescent protein (GFP) by cardiomyocytes were studied in lipopolysaccharide (LPS)-induced systemic inflammatory response syndrome (SIRS). EVs were separated both from platelet-free plasma and from the conditioned medium of isolated cardiomyocytes of the left ventricular wall. Size distribution and concentration of the released particles were determined by Nanoparticle Tracking Analysis. The presence of GFP + cardiomyocyte-derived circulating EVs was monitored by flow cytometry and cardiac function was assessed by echocardiography. In LPS-treated mice, systemic inflammation and the consequent cardiomyopathy were verified by elevated plasma levels of TNFα, GDF-15, and cardiac troponin I, and by a decrease in the ejection fraction. Furthermore, we demonstrated elevated levels of circulating small- and medium-sized EVs in the LPS-injected mice. Importantly, we detected GFP+ cardiomyocyte-derived EVs in the circulation of control mice, and the number of these circulating GFP+ vesicles increased significantly upon intraperitoneal LPS administration (P = 0.029). The cardiomyocyte-derived GFP+ EVs were also positive for intravesicular troponin I (cTnI) and muscle-associated glycogen phosphorylase (PYGM). This is the first direct demonstration that cardiomyocyte-derived EVs are present in the circulation and that the increased number of cardiac-derived EVs in the blood reflects cardiac injury in LPS-induced systemic inflammation (SIRS).

Sex Difference in Cardioprotection against Acute Myocardial Infarction in MAO-B Knockout Mice In Vivo.

The cardiomyocyte-specific knockout (KO) of monoamine oxidase (MAO)-B, an enzyme involved in the formation of reactive oxygen species (ROS), reduced myocardial ischemia/reperfusion (I/R) injury in vitro. Because sex hormones have a strong impact on MAO metabolic pathways, we analyzed the myocardial infarct size (IS) following I/R in female and male MAO-B KO mice in vivo. METHOD AND RESULTS: To induce the deletion of MAO-B, MAO-B KO mice (Myh6 Cre+/MAO-Bfl/fl) and wild-type (WT, Cre-negative MAO-Bfl/fl littermates) were fed with tamoxifen for 2 weeks followed by 10 weeks of normal mice chow. Myocardial infarction (assessed by TTC staining and expressed as a percentage of the area at risk as determined by Evans blue staining)) was induced by 45 min coronary occlusion followed by 120 min of reperfusion. RESULTS: The mortality following I/R was higher in male compared to female mice, with the lowest mortality found in MAO-B KO female mice. IS was significantly higher in male WT mice compared to female WT mice. MAO-B KO reduced IS in male mice but had no further impact on IS in female MAO-B KO mice. Interestingly, there was no difference in the plasma estradiol levels among the groups. CONCLUSION: The cardiomyocyte-specific knockout of MAO-B protects male mice against acute myocardial infarction but had no effect on the infarct size in female mice.

Effects of Bempedoic Acid in Acute Myocardial Infarction in Rats: No Cardioprotection and No Hidden Cardiotoxicity.

Lipid-lowering drugs have been shown to have cardioprotective effects but may have hidden cardiotoxic properties. Therefore, here we aimed to investigate if chronic treatment with the novel lipid-lowering drug bempedoic acid (BA) exerts hidden cardiotoxic and/or cardioprotective effects in a rat model of acute myocardial infarction (AMI). Wistar rats were orally treated with BA or its vehicle for 28 days, anesthetized and randomized to three different groups (vehicle + ischemia/reperfusion (I/R), BA + I/R, and positive control vehicle + ischemic preconditioning (IPC)) and subjected to cardiac 30 min ischemia and 120 min reperfusion. IPC was performed by 3 × 5 min I/R cycles before ischemia. Myocardial function, area at risk, infarct size and arrhythmias were analyzed. Chronic BA pretreatment did not influence cardiac function or infarct size as compared to the vehicle group, while the positive control IPC significantly reduced the infarct size. The incidence of reperfusion-induced arrhythmias was significantly reduced by BA and IPC. This is the first demonstration that BA treatment does not show cardioprotective effect although moderately reduces the incidence of reperfusion-induced arrhythmias. Furthermore, BA does not show hidden cardiotoxic effect in rats with AMI, showing its safety in the ischemic/reperfused heart.

Inflammasome Activity in the Skeletal Muscle and Heart of Rodent Models for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is characterized by wasting of muscles that leads to difficulty moving and premature death, mainly from heart failure. Glucocorticoids are applied in the management of the disease, supporting the hypothesis that inflammation may be driver as well as target. However, the inflammatory mechanisms during progression of cardiac and skeletal muscle dysfunction are still not well characterized. Our objective was to characterize the inflammasomes in myocardial and skeletal muscle in rodent models of DMD. Gastrocnemius and heart samples were collected from mdx mice and DMDmdx rats (3 and 9-10 months). Inflammasome sensors and effectors were assessed by immunoblotting. Histology was used to assess leukocyte infiltration and fibrosis. In gastrocnemius, a tendency towards elevation of gasdermin D irrespective of the age of the animal was observed. The adaptor protein was elevated in the mdx mouse skeletal muscle and heart. Increased cleavage of the cytokines was observed in the skeletal muscle of the DMDmdx rats. Sensor or cytokine expression was not changed in the tissue samples of the mdx mice. In conclusion, inflammatory responses are distinct between the skeletal muscle and heart in relevant models of DMD. Inflammation tends to decrease over time, supporting the clinical observations that the efficacy of anti-inflammatory therapies might be more prominent in the early stage.

YB-1 Is a Novel Target for the Inhibition of α-Adrenergic-Induced Hypertrophy.

Cardiac hypertrophy resulting from sympathetic nervous system activation triggers the development of heart failure. The transcription factor Y-box binding protein 1 (YB-1) can interact with transcription factors involved in cardiac hypertrophy and may thereby interfere with the hypertrophy growth process. Therefore, the question arises as to whether YB-1 influences cardiomyocyte hypertrophy and might thereby influence the development of heart failure. YB-1 expression is downregulated in human heart biopsies of patients with ischemic cardiomyopathy (n = 8), leading to heart failure. To study the impact of reduced YB-1 in cardiac cells, we performed small interfering RNA (siRNA) experiments in H9C2 cells as well as in adult cardiomyocytes (CMs) of rats. The specificity of YB-1 siRNA was analyzed by a miRNA-like off-target prediction assay identifying potential genes. Testing three high-scoring genes by transfecting cardiac cells with YB-1 siRNA did not result in downregulation of these genes in contrast to YB-1, whose downregulation increased hypertrophic growth. Hypertrophic growth was mediated by PI3K under PE stimulation, as well by downregulation with YB-1 siRNA. On the other hand, overexpression of YB-1 in CMs, caused by infection with an adenovirus encoding YB-1 (AdYB-1), prevented hypertrophic growth under α-adrenergic stimulation with phenylephrine (PE), but not under stimulation with growth differentiation factor 15 (GDF15; n = 10-16). An adenovirus encoding the green fluorescent protein (AdGFP) served as the control. YB-1 overexpression enhanced the mRNA expression of the Gq inhibitor regulator of G-protein signaling 2 (RGS2) under PE stimulation (n = 6), potentially explaining its inhibitory effect on PE-induced hypertrophic growth. This study shows that YB-1 protects cardiomyocytes against PE-induced hypertrophic growth. Like in human end-stage heart failure, YB-1 downregulation may cause the heart to lose its protection against hypertrophic stimuli and progress to heart failure. Therefore, the transcription factor YB-1 is a pivotal signaling molecule, providing perspectives for therapeutic approaches.

Droplet Digital PCR Is a Novel Screening Method Identifying Potential Cardiac G-Protein-Coupled Receptors as Candidate Pharmacological Targets in a Rat Model of Pressure-Overload-Induced Cardiac Dysfunction.

The identification of novel drug targets is needed to improve the outcomes of heart failure (HF). G-protein-coupled receptors (GPCRs) represent the largest family of targets for already approved drugs, thus providing an opportunity for drug repurposing. Here, we aimed (i) to investigate the differential expressions of 288 cardiac GPCRs via droplet digital PCR (ddPCR) and bulk RNA sequencing (RNAseq) in a rat model of left ventricular pressure-overload; (ii) to compare RNAseq findings with those of ddPCR; and (iii) to screen and test for novel, translatable GPCR drug targets in HF. Male Wistar rats subjected to transverse aortic constriction (TAC, n = 5) showed significant systolic dysfunction vs. sham operated animals (SHAM, n = 5) via echocardiography. In TAC vs. SHAM hearts, RNAseq identified 69, and ddPCR identified 27 significantly differentially expressed GPCR mRNAs, 8 of which were identified using both methods, thus showing a correlation between the two methods. Of these, Prostaglandin-F2α-receptor (Ptgfr) was further investigated and localized on cardiomyocytes and fibroblasts in murine hearts via RNA-Scope. Antagonizing Ptgfr via AL-8810 reverted angiotensin-II-induced cardiomyocyte hypertrophy in vitro. In conclusion, using ddPCR as a novel screening method, we were able to identify GPCR targets in HF. We also show that the antagonism of Ptgfr could be a novel target in HF by alleviating cardiomyocyte hypertrophy.

Systematic transcriptomic and phenotypic characterization of human and murine cardiac myocyte cell lines and primary cardiomyocytes reveals serious limitations and low resemblances to adult cardiac phenotype.

BACKGROUND: Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner. METHODS AND RESULTS: Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation. CONCLUSION: Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes.

Efficient link prediction in the protein-protein interaction network using topological information in a generative adversarial network machine learning model.

BACKGROUND: The investigation of possible interactions between two proteins in intracellular signaling is an expensive and laborious procedure in the wet-lab, therefore, several in silico approaches have been implemented to narrow down the candidates for future experimental validations. Reformulating the problem in the field of network theory, the set of proteins can be represented as the nodes of a network, while the interactions between them as the edges. The resulting protein-protein interaction (PPI) network enables the use of link prediction techniques in order to discover new probable connections. Therefore, here we aimed to offer a novel approach to the link prediction task in PPI networks, utilizing a generative machine learning model. RESULTS: We created a tool that consists of two modules, the data processing framework and the machine learning model. As data processing, we used a modified breadth-first search algorithm to traverse the network and extract induced subgraphs, which served as image-like input data for our model. As machine learning, an image-to-image translation inspired conditional generative adversarial network (cGAN) model utilizing Wasserstein distance-based loss improved with gradient penalty was used, taking the combined representation from the data processing as input, and training the generator to predict the probable unknown edges in the provided induced subgraphs. Our link prediction tool was evaluated on the protein-protein interaction networks of five different species from the STRING database by calculating the area under the receiver operating characteristic, the precision-recall curves and the normalized discounted cumulative gain (AUROC, AUPRC, NDCG, respectively). Test runs yielded the averaged results of AUROC = 0.915, AUPRC = 0.176 and NDCG = 0.763 on all investigated species. CONCLUSION: We developed a software for the purpose of link prediction in PPI networks utilizing machine learning. The evaluation of our software serves as the first demonstration that a cGAN model, conditioned on raw topological features of the PPI network, is an applicable solution for the PPI prediction problem without requiring often unavailable molecular node attributes. The corresponding scripts are available at https://github.com/semmelweis-pharmacology/ppi_pred .

Single Donor Infusion of S-Nitroso-Human-Serum-Albumin Attenuates Cardiac Isograft Fibrosis and Preserves Myocardial Micro-RNA-126-3p in a Murine Heterotopic Heart Transplant Model.

Objectives: Cold ischemia and subsequent reperfusion injury are non-immunologic cornerstones in the development of graft injury after heart transplantation. The nitric oxide donor S-nitroso-human-serum-albumin (S-NO-HSA) is known to attenuate myocardial ischemia-reperfusion (I/R)-injury. We assessed whether donor preservation with S-NO-HSA affects isograft injury and myocardial expression of GATA2 as well as miR-126-3p, which are considered protective against vascular and endothelial injury. Methods: Donor C57BL/6 mice received intravenous (0.1 µmol/kg/h) S-NO-HSA (n = 12), or 0.9% saline (control, n = 11) for 20 min. Donor hearts were stored in cold histidine-tryptophan-α-ketoglutarate-N solution for 12 h and underwent heterotopic, isogenic transplantation, except 5 hearts of each group, which were analysed immediately after preservation. Fibrosis was quantified and expression of GATA2 and miR-126-3p assessed by RT-qPCR after 60 days or immediately after preservation. Results: Fibrosis was significantly reduced in the S-NO-HSA group (6.47% ± 1.76 vs. 11.52% ± 2.16; p = 0.0023; 12 h-S-NO-HSA-hHTX vs. 12 h-control-hHTX). Expression of miR-126-3p was downregulated in all hearts after ischemia compared to native myocardium, but the effect was significantly attenuated when donors received S-NO-HSA (1 ± 0.27 vs. 0.33 ± 0.31; p = 0.0187; 12 h-S-NO-HSA-hHTX vs. 12 h-control-hHTX; normalized expression to U6 snRNA). Conclusion: Donor pre-treatment with S-NO-HSA lead to reduced fibrosis and preservation of myocardial miR-126-3p and GATA2 levels in murine cardiac isografts 60 days after transplantation.

Prognostically relevant periprocedural myocardial injury and infarction associated with percutaneous coronary interventions: a Consensus Document of the ESC Working Group on Cellular Biology of the Heart and European Association of Percutaneous Cardiovascular Interventions (EAPCI).

A substantial number of chronic coronary syndrome (CCS) patients undergoing percutaneous coronary intervention (PCI) experience periprocedural myocardial injury or infarction. Accurate diagnosis of these PCI-related complications is required to guide further management given that their occurrence may be associated with increased risk of major adverse cardiac events (MACE). Due to lack of scientific data, the cut-off thresholds of post-PCI cardiac troponin (cTn) elevation used for defining periprocedural myocardial injury and infarction, have been selected based on expert consensus opinions, and their prognostic relevance remains unclear. In this Consensus Document from the ESC Working Group on Cellular Biology of the Heart and European Association of Percutaneous Cardiovascular Interventions (EAPCI), we recommend, whenever possible, the measurement of baseline (pre-PCI) cTn and post-PCI cTn values in all CCS patients undergoing PCI. We confirm the prognostic relevance of the post-PCI cTn elevation >5× 99th percentile URL threshold used to define type 4a myocardial infarction (MI). In the absence of periprocedural angiographic flow-limiting complications or electrocardiogram (ECG) and imaging evidence of new myocardial ischaemia, we propose the same post-PCI cTn cut-off threshold (>5× 99th percentile URL) be used to define prognostically relevant 'major' periprocedural myocardial injury. As both type 4a MI and major periprocedural myocardial injury are strong independent predictors of all-cause mortality at 1 year post-PCI, they may be used as quality metrics and surrogate endpoints for clinical trials. Further research is needed to evaluate treatment strategies for reducing the risk of major periprocedural myocardial injury, type 4a MI, and MACE in CCS patients undergoing PCI.

Somatostatin Primes Endothelial Cells for Agonist-Induced Hyperpermeability and Angiogenesis In Vitro.

Somatostatin is an inhibitory peptide, which regulates the release of several hormones, and affects neurotransmission and cell proliferation via its five Gi protein-coupled receptors (SST1-5). Although its endocrine regulatory and anti-tumour effects have been thoroughly studied, little is known about its effect on the vascular system. The aim of the present study was to analyse the effects and potential mechanisms of somatostatin on endothelial barrier function. Cultured human umbilical vein endothelial cells (HUVECs) express mainly SST1 and SST5 receptors. Somatostatin did not affect the basal HUVEC permeability, but primed HUVEC monolayers for thrombin-induced hyperpermeability. Western blot data demonstrated that somatostatin activated the phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt) and p42/44 mitogen-activated protein kinase (MAPK) pathways by phosphorylation. The HUVEC barrier destabilizing effects were abrogated by pre-treating HUVECs with mitogen-activated protein kinase kinase/extracellular signal regulated kinase (MEK/ERK), but not the Akt inhibitor. Moreover, somatostatin pre-treatment amplified vascular endothelial growth factor (VEGF)-induced angiogenesis (3D spheroid formation) in HUVECs. In conclusion, the data demonstrate that HUVECs under quiescence conditions express SST1 and SST5 receptors. Moreover, somatostatin primes HUVECs for thrombin-induced hyperpermeability mainly via the activation of MEK/ERK signalling and promotes HUVEC proliferation and angiogenesis in vitro.

Myocardial Protection and Current Cancer Therapy: Two Opposite Targets with Inevitable Cost.

Myocardial protection against ischemia/reperfusion injury (IRI) is mediated by various ligands, activating different cellular signaling cascades. These include classical cytosolic mediators such as cyclic-GMP (c-GMP), various kinases such as Phosphatydilinositol-3- (PI3K), Protein Kinase B (Akt), Mitogen-Activated-Protein- (MAPK) and AMP-activated (AMPK) kinases, transcription factors such as signal transducer and activator of transcription 3 (STAT3) and bioactive molecules such as vascular endothelial growth factor (VEGF). Most of the aforementioned signaling molecules constitute targets of anticancer therapy; as they are also involved in carcinogenesis, most of the current anti-neoplastic drugs lead to concomitant weakening or even complete abrogation of myocardial cell tolerance to ischemic or oxidative stress. Furthermore, many anti-neoplastic drugs may directly induce cardiotoxicity via their pharmacological effects, or indirectly via their cardiovascular side effects. The combination of direct drug cardiotoxicity, indirect cardiovascular side effects and neutralization of the cardioprotective defense mechanisms of the heart by prolonged cancer treatment may induce long-term ventricular dysfunction, or even clinically manifested heart failure. We present a narrative review of three therapeutic interventions, namely VEGF, proteasome and Immune Checkpoint inhibitors, having opposing effects on the same intracellular signal cascades thereby affecting the heart. Moreover, we herein comment on the current guidelines for managing cardiotoxicity in the clinical setting and on the role of cardiovascular confounders in cardiotoxicity.

Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Deletion but Not Inhibition of Extracellular PCSK9 Reduces Infarct Sizes Ex Vivo but Not In Vivo.

Hypoxia upregulates PCSK9 expression in the heart, and PCSK9 affects the function of myocytes. This study aimed to investigate the impact of PCSK9 on reperfusion injury in rats and mice fed normal or high-fat diets. Either the genetic knockout of PCSK9 (mice) or the antagonism of circulating PCSK9 via Pep2-8 (mice and rats) was used. Isolated perfused hearts were exposed to 45 min of ischemia followed by 120 min of reperfusion. In vivo, mice were fed normal or high-fat diets (2% cholesterol) for eight weeks prior to coronary artery occlusion (45 min of ischemia) and reperfusion (120 min). Ischemia/reperfusion upregulates PCSK9 expression (rats and mice) and releases it into the perfusate. The inhibition of extracellular PCSK9 does not affect infarct sizes or functional recovery. However, genetic deletion largely reduces infarct size and improves post-ischemic recovery in mice ex vivo but not in vivo. A high-fat diet reduced the survival rate during ischemia and reperfusion, but in a PCSK9-independent manner that was associated with increased plasma matrix metalloproteinase (MMP)9 activity. PCSK9 deletion, but not the inhibition of extracellular PCSK9, reduces infarct sizes in ex vivo hearts, but this effect is overridden in vivo by factors such as MMP9.

PACAP-38 and PAC1 Receptor Alterations in Plasma and Cardiac Tissue Samples of Heart Failure Patients.

Pituitary adenylate cyclase activating polypeptide-38 (PACAP-38) is a multifunctional neuropeptide, which may play a role in cardioprotection. However, little is known about the presence of PACAP-38 in heart failure (HF) patients. The aim of our study was to measure the alterations of PACAP-38 like immunoreactivity (LI) in acute (n = 13) and chronic HF (n = 33) and to examine potential correlations between PACAP-38 and HF predictors (cytokines, NT-proBNP). Tissue PACAP-38 LI and PAC1 receptor levels were also investigated in heart tissue samples of patients with HF. Significantly higher plasma PACAP-38 LI was detected in patients with acute HF, while in chronic HF patients, a lower level of immunoreactivity was observed compared to healthy controls (n = 13). Strong negative correlation was identified between plasma PACAP-38 and NT-proBNP levels in chronic HF, as opposed to the positive connection seen in the acute HF group. Plasma IL-1 ß, IL-2 and IL-4 levels were significantly lower in chronic HF, and IL-10 was significantly higher in patients with acute HF. PACAP-38 levels of myocardial tissues were lower in all end-stage HF patients and lower PAC1 receptor levels were detected in the primary dilated cardiomyopathy group compared to the controls. We conclude that PACAP-38 and PAC1 expression correlates with some biomarkers of acute and chronic HF; therefore, further studies are necessary to explore whether PACAP could be a suitable prognostic biomarker in HF patients.

Disease- and headache-specific microRNA signatures and their predicted mRNA targets in peripheral blood mononuclear cells in migraineurs: role of inflammatory signalling and oxidative stress.

BACKGROUND: Migraine is a primary headache with genetic susceptibility, but the pathophysiological mechanisms are poorly understood, and it remains an unmet medical need. Earlier we demonstrated significant differences in the transcriptome of migraineurs' PBMCs (peripheral blood mononuclear cells), suggesting the role of neuroinflammation and mitochondrial dysfunctions. Post-transcriptional gene expression is regulated by miRNA (microRNA), a group of short non-coding RNAs that are emerging biomarkers, drug targets, or drugs. MiRNAs are emerging biomarkers and therapeutics; however, little is known about the miRNA transcriptome in migraine, and a systematic comparative analysis has not been performed so far in migraine patients. METHODS: We determined miRNA expression of migraineurs' PBMC during (ictal) and between (interictal) headaches compared to age- and sex-matched healthy volunteers. Small RNA sequencing was performed from the PBMC, and mRNA targets of miRNAs were predicted using a network theoretical approach by miRNAtarget.com™. Predicted miRNA targets were investigated by Gene Ontology enrichment analysis and validated by comparing network metrics to differentially expressed mRNA data. RESULTS: In the interictal PBMC samples 31 miRNAs were differentially expressed (DE) in comparison to healthy controls, including hsa-miR-5189-3p, hsa-miR-96-5p, hsa-miR-3613-5p, hsa-miR-99a-3p, hsa-miR-542-3p. During headache attacks, the top DE miRNAs as compared to the self-control samples in the interictal phase were hsa-miR-3202, hsa-miR-7855-5p, hsa-miR-6770-3p, hsa-miR-1538, and hsa-miR-409-5p. MiRNA-mRNA target prediction and pathway analysis indicated several mRNAs related to immune and inflammatory responses (toll-like receptor and cytokine receptor signalling), neuroinflammation and oxidative stress, also confirmed by mRNA transcriptomics. CONCLUSIONS: We provide here the first evidence for disease- and headache-specific miRNA signatures in the PBMC of migraineurs, which might help to identify novel targets for both prophylaxis and attack therapy.

Telomerase/myocardin expressing mesenchymal cells induce survival and cardiovascular markers in cardiac stromal cells undergoing ischaemia/reperfusion.

Cardiac stromal cells (CSCs) contain a pool of cells with supportive and paracrine functions. Various types of mesenchymal stromal cells (MSCs) can influence CSCs in the cardiac niche through their paracrine activity. Ischaemia/reperfusion (I/R) leads to cell death and reduction of the paracrine activity of CSCs. The forced co-expression of telomerase reverse transcriptase (TERT) and myocardin (MYOCD), known to potentiate anti-apoptotic, pro-survival and pro-angiogenic activities of MSCs isolated from the adipose tissue (AT-MSCs), may increase CSC survival, favouring their paracrine activities. We aimed at investigating the hypothesis that CSCs feature improved resistance to simulated I/R (SI/R) and increased commitment towards the cardiovascular lineage when preconditioned with conditioned media (CM) or extracellular vesicles (EV) released from AT-MSCs overexpressing TERT and MYOCD (T/M AT-MSCs). Murine CSCs were isolated with the cardiosphere (CSps) isolation technique. T/M AT-MSCs and their secretome improved spontaneous intracellular calcium changes and ryanodine receptor expression in aged CSps. The cytoprotective effect of AT-MSCs was tested in CSCs subjected to SI/R. SI/R induced cell death as compared to normoxia (28 ± 4 vs 10 ± 3%, P = .02). Pre-treatment with CM (15 ± 2, P = .02) or with the EV-enriched fraction (10 ± 1%, P = .02) obtained from mock-transduced AT-MSCs in normoxia reduced cell death after SI/R. The effect was more pronounced with CM (7 ± 1%, P = .01) or the EV-enriched fraction (2 ± 1%, P = .01) obtained from T/M AT-MSCs subjected to SI/R. In parallel, we observed lower expression of the apoptosis marker cleaved caspase-3 and higher expression of cardiac and vascular markers eNOS, sarcomeric α-actinin and cardiac actin. The T/M AT-MSCs secretome exerts a cytoprotective effect and promotes development of CSCs undergoing SI/R towards a cardiovascular phenotype.