Reduced circulating CD63+ extracellular vesicle levels associate with atherosclerosis in hypercholesterolaemic mice and humans.

AIMS: The association and co-isolation of low-density lipoproteins (LDL) and extracellular vesicles (EVs) have been shown in blood plasma. Here we explore this relationship to better understand the role of EVs in atherogenesis. METHODS AND RESULTS: Wild type (WT), PCSK9-/-, and LDLR-/- C57BL/6 mice were used in this study. Eleven week-old male mice were fed high-fat diet (HFD) for 12 weeks or kept on normal diet until old age (22-months). Cardiac function was assessed by ultrasound, cholesterol was quantified with a colorimetric kit and circulating EVs were measured using flow cytometry. Plaques were analysed post-mortem using Oil-Red-O staining of the aortic arch. EVs were measured from platelet free blood plasma samples of normal and hypercholesterolaemic clinical patients. Based on annexin V and CD63 staining, we found a significant increase in EV levels in LDLR-/- and PCSK9-/- mice after HFD, but CD81 showed no significant change in either group. There was no significant change in plaque formation after HFD. PCSK9-/- mice show a favourable cardiac function after HFD. Blood cholesterol levels progressively increased during HFD, with LDLR-/- mice showing high levels while PCSK9-/- were significantly lowered compared to WT animals. In mice at old age, similar cholesterol levels were observed as in young mice. In old age, LDLR-/- mice showed significantly increased plaques. At old age, ejection fraction was decreased in all groups of mice, as were CD63+ EVs. Similarly to mice, in patients with hypercholesterolaemia, CD63+ EVs were significantly depleted. CONCLUSIONS: This research demonstrates an inverse relationship between circulating EVs and cholesterol, making EVs a potential marker for cardiovascular disease (CVD). HFD causes reduced cardiac function, but atherosclerotic development is slowly progressing in hypercholesterolaemic models and only observed with old animals. These results also bring further evidence for the benefit of using of PCSK9 inhibitors as therapeutic agents in CVD.

Comparison of mouse models of heart failure with reduced ejection fraction.

AIMS: Heart failure with reduced ejection fraction (HFrEF) is a leading cause of death worldwide; thus, therapeutic improvements are needed. In vivo preclinical models are essential to identify molecular drug targets for future therapies. Transverse aortic constriction (TAC) is a well-established model of HFrEF; however, highly experienced personnel are needed for the surgery, and several weeks of follow-up are necessary to develop HFrEF. To this end, we aimed (i) to develop an easy-to-perform mouse model of HFrEF by treating Balb/c mice with angiotensin-II (Ang-II) for 2 weeks by minipump and (ii) to compare its cardiac phenotype and transcriptome to the well-established TAC model of HFrEF in C57BL/6J mice. METHODS: Mortality and gross pathological data, cardiac structural and functional characteristics assessed by echocardiography and immunohistochemistry and differential gene expression obtained by RNA-sequencing and gene-ontology analyses were used to characterize and compare the two models. To achieve statistical comparability between the two models, changes in treatment groups related to the corresponding control were compared (ΔTAC vs. ΔAng-II). RESULTS: Compared with the well-established TAC model, chronic Ang-II treatment of Balb/c mice shares similarities in cardiac systolic functional decline (left ventricular ejection fraction: -57.25 ± 7.17% vs. -43.68 ± 5.31% in ΔTAC vs. ΔAng-II; P = 0.1794) but shows a lesser degree of left ventricular dilation (left ventricular end-systolic volume: 190.81 ± 44.13 vs. 57.37 ± 10.18 mL in ΔTAC vs. ΔAng-II; P = 0.0252) and hypertrophy (cell surface area: 58.44 ± 6.1 vs. 10.24 ± 2.87 µm2 in ΔTAC vs. ΔAng-II; P < 0.001); nevertheless, transcriptomic changes in the two HFrEF models show strong correlation (Spearman's r = 0.727; P < 0.001). In return, Ang-II treatment in Balb/c mice needs significantly less procedural time [38 min, interquartile range (IQR): 31-46 min in TAC vs. 6 min, IQR: 6-7 min in Ang-II; P < 0.001] and surgical expertise, is less of an object for peri-procedural mortality (15.8% in TAC vs. 0% in Ang-II; P = 0.105) and needs significantly shorter follow-up for developing HFrEF. CONCLUSIONS: Here, we demonstrate for the first time that chronic Ang-II treatment of Balb/c mice is also a relevant, reliable but significantly easier-to-perform preclinical model to identify novel pathomechanisms and targets in future HFrEF research.

Molecular fingerprints of cardiovascular toxicities of immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy by unleashing the power of the immune system against malignant cells. However, their use is associated with a spectrum of adverse effects, including cardiovascular complications, which can pose significant clinical challenges. Several mechanisms contribute to cardiovascular toxicity associated with ICIs. First, the dysregulation of immune checkpoints, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein-1 (PD-1) and its ligand (PD-L1), and molecular mimicry with cardiac autoantigens, leads to immune-related adverse events, including myocarditis and vasculitis. These events result from the aberrant activation of T cells against self-antigens within the myocardium or vascular endothelium. Second, the disruption of immune homeostasis by ICIs can lead to autoimmune-mediated inflammation of cardiac tissues, manifesting as cardiac dysfunction and heart failure, arrhythmias, or pericarditis. Furthermore, the upregulation of inflammatory cytokines, particularly tumor necrosis factor-alpha, interferon-γ, interleukin-1ß, interleukin-6, and interleukin-17 contributes to cardiac and endothelial dysfunction, plaque destabilization, and thrombosis, exacerbating cardiovascular risk on the long term. Understanding the intricate mechanisms of cardiovascular side effects induced by ICIs is crucial for optimizing patient care and to ensure the safe and effective integration of immunotherapy into a broader range of cancer treatment protocols. The clinical implications of these mechanisms underscore the importance of vigilant monitoring and early detection of cardiovascular toxicity in patients receiving ICIs. Future use of these key pathological mediators as biomarkers may aid in prompt diagnosis of cardiotoxicity and will allow timely interventions.

Effect of hypercholesterolemia on circulating and cardiomyocyte-derived extracellular vesicles.

Hypercholesterolemia (HC) induces, propagates and exacerbates cardiovascular diseases via various mechanisms that are yet not properly understood. Extracellular vesicles (EVs) are involved in the pathomechanism of these diseases. To understand how circulating or cardiac-derived EVs could affect myocardial functions, we analyzed the metabolomic profile of circulating EVs, and we performed an in-depth analysis of cardiomyocyte (CM)-derived EVs in HC. Circulating EVs were isolated with Vezics technology from male Wistar rats fed with high-cholesterol or control chow. AC16 human CMs were treated with Remembrane HC supplement and EVs were isolated from cell culture supernatant. The biophysical properties and the protein composition of CM EVs were analyzed. THP1-ASC-GFP cells were treated with CM EVs, and monocyte activation was measured. HC diet reduced the amount of certain phosphatidylcholines in circulating EVs, independently of their plasma level. HC treatment significantly increased EV secretion of CMs and greatly modified CM EV proteome, enriching several proteins involved in tissue remodeling. Regardless of the treatment, CM EVs did not induce the activation of THP1 monocytes. In conclusion, HC strongly affects the metabolome of circulating EVs and dysregulates CM EVs, which might contribute to HC-induced cardiac derangements.

NASH triggers cardiometabolic HFpEF in aging mice.

Both heart failure with preserved ejection fraction (HFpEF) and non-alcoholic fatty liver disease (NAFLD) develop due to metabolic dysregulation, has similar risk factors (e.g., insulin resistance, systemic inflammation) and are unresolved clinical challenges. Therefore, the potential link between the two disease is important to study. We aimed to evaluate whether NASH is an independent factor of cardiac dysfunction and to investigate the age dependent effects of NASH on cardiac function. C57Bl/6 J middle aged (10 months old) and aged mice (24 months old) were fed either control or choline deficient (CDAA) diet for 8 weeks. Before termination, echocardiography was performed. Upon termination, organ samples were isolated for histological and molecular analysis. CDAA diet led to the development of NASH in both age groups, without inducing weight gain, allowing to study the direct effect of NASH on cardiac function. Mice with NASH developed hepatomegaly, fibrosis, and inflammation. Aged animals had increased heart weight. Conventional echocardiography revealed normal systolic function in all cohorts, while increased left ventricular volumes in aged mice. Two-dimensional speckle tracking echocardiography showed subtle systolic and diastolic deterioration in aged mice with NASH. Histologic analyses of cardiac samples showed increased cross-sectional area, pronounced fibrosis and Col1a1 gene expression, and elevated intracardiac CD68+ macrophage count with increased Il1b expression. Conventional echocardiography failed to reveal subtle change in myocardial function; however, 2D speckle tracking echocardiography was able to identify diastolic deterioration. NASH had greater impact on aged animals resulting in cardiac hypertrophy, fibrosis, and inflammation.

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.

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.

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.

Cardioprotective efficacy of limb remote ischaemic preconditioning in rats: discrepancy between a meta-analysis and a three-centre in vivo study.

AIMS: Remote ischaemic preconditioning (RIPC) is a robust cardioprotective intervention in preclinical studies. To establish a working and efficacious RIPC protocol in our laboratories, we performed randomized, blinded in vivo studies in three study centres in rats, with various RIPC protocols. To verify that our experimental settings are in good alignment with in vivo rat studies showing cardioprotection by limb RIPC, we performed a systematic review and meta-analysis. In addition, we investigated the importance of different study parameters. METHODS AND RESULTS: Male Wistar rats were subjected to 20-45 min cardiac ischaemia followed by 120 min reperfusion with or without preceding RIPC by 3 or 4 × 5-5 min occlusion/reperfusion of one or two femoral vessels by clamping, tourniquet, or pressure cuff. RIPC did not reduce infarct size (IS), microvascular obstruction, or arrhythmias at any study centres. Systematic review and meta-analysis focusing on in vivo rat models of myocardial ischaemia/reperfusion injury with limb RIPC showed that RIPC reduces IS by 21.28% on average. In addition, the systematic review showed methodological heterogeneity and insufficient reporting of study parameters in a high proportion of studies. CONCLUSION: We report for the first time the lack of cardioprotection by RIPC in rats, assessed in individually randomized, blinded in vivo studies, involving three study centres, using different RIPC protocols. These results are in discrepancy with the meta-analysis of similar in vivo rat studies; however, no specific methodological reason could be identified by the systematic review, probably due to the overall insufficient reporting of several study parameters that did not improve over the past two decades. These results urge for publication of more well-designed and well-reported studies, irrespective of the outcome, which are required for preclinical reproducibility, and the development of clinically translatable cardioprotective interventions.

IL-1ß neutralization prevents diastolic dysfunction development, but lacks hepatoprotective effect in an aged mouse model of NASH.

Interleukin-1ß (IL-1ß) is a key mediator of non-alcoholic steatohepatitis (NASH), a chronic liver disease, and of systemic inflammation-driven aging. IL-1ß contributes to cardio-metabolic decline, and may promote hepatic oncogenic transformation. Therefore, IL-1ß is a potential therapeutic target in these pathologies. We aimed to investigate the hepatic and cardiac effects of an IL-1ß targeting monoclonal antibody in an aged mouse model of NASH. 24 months old male C57Bl/6J mice were fed with control or choline deficient (CDAA) diet and were treated with isotype control or anti-IL-1ß Mab for 8 weeks. Cardiac functions were assessed by conventional-and 2D speckle tracking echocardiography. Liver samples were analyzed by immunohistochemistry and qRT-PCR. Echocardiography revealed improved cardiac diastolic function in anti-IL-1ß treated mice with NASH. Marked hepatic fibrosis developed in CDAA-fed group, but IL-1ß inhibition affected fibrosis only at transcriptomic level. Hepatic inflammation was not affected by the IL-1ß inhibitor. PCNA staining revealed intensive hepatocyte proliferation in CDAA-fed animals, which was not influenced by neutralization of IL-1ß. IL-1ß inhibition increased hepatic expression of Pd-1 and Ctla4, while Pd-l1 expression increased in NASH. In conclusion, IL-1ß inhibition improved cardiac diastolic function, but did not ameliorate features of NASH; moreover, even promoted hepatic immune checkpoint expression, with concomitant NASH-related hepatocellular proliferation.

Characterization of immune checkpoint inhibitor-induced cardiotoxicity reveals interleukin-17A as a driver of cardiac dysfunction after anti-PD-1 treatment.

BACKGROUND AND PURPOSE: Immune checkpoint inhibitors (ICI), such as anti-PD-1 monoclonal antibodies, have revolutionized cancer therapy by enhancing the cytotoxic effects of T-cells against tumours. However, enhanced T-cell activity also may cause myocarditis and cardiotoxicity. Our understanding of the mechanisms of ICI-induced cardiotoxicity is limited. Here, we aimed to investigate the effect of PD-1 inhibition on cardiac function and explore the molecular mechanisms of ICI-induced cardiotoxicity. EXPERIMENTAL APPROACH: C57BL6/J and BALB/c mice were treated with isotype control or anti-PD-1 antibody. Echocardiography was used to assess cardiac function. Cardiac transcriptomic changes were investigated by bulk RNA sequencing. Inflammatory changes were assessed by qRT-PCR and immunohistochemistry in heart, thymus, and spleen of the animals. In follow-up experiments, anti-CD4 and anti-IL-17A antibodies were used along with PD-1 blockade in C57BL/6J mice. KEY RESULTS: Anti-PD-1 treatment led to cardiac dysfunction and left ventricular dilation in C57BL/6J mice, with increased nitrosative stress. Only mild inflammation was observed in the heart. However, PD-1 inhibition resulted in enhanced thymic inflammatory signalling, where Il17a increased most prominently. In BALB/c mice, cardiac dysfunction was not evident, and thymic inflammatory activation was more balanced. Inhibition of IL-17A prevented anti-PD-1-induced cardiac dysfunction in C57BL6/J mice. Comparing myocardial transcriptomic changes in C57BL/6J and BALB/c mice, differentially regulated genes (Dmd, Ass1, Chrm2, Nfkbia, Stat3, Gsk3b, Cxcl9, Fxyd2, and Ldb3) were revealed, related to cardiac structure, signalling, and inflammation. CONCLUSIONS: PD-1 blockade induces cardiac dysfunction in mice with increased IL-17 signalling in the thymus. Pharmacological inhibition of IL-17A treatment prevents ICI-induced cardiac dysfunction.

Rosiglitazone Does Not Show Major Hidden Cardiotoxicity in Models of Ischemia/Reperfusion but Abolishes Ischemic Preconditioning-Induced Antiarrhythmic Effects in Rats In Vivo.

Clinical observations are highly inconsistent with the use of the antidiabetic rosiglitazone regarding its associated increased risk of myocardial infarction. This may be due to its hidden cardiotoxic properties that have only become evident during post-marketing studies. Therefore, we aimed to investigate the hidden cardiotoxicity of rosiglitazone in ischemia/reperfusion (I/R) injury models. Rats were treated orally with either 0.8 mg/kg/day rosiglitazone or vehicle for 28 days and subjected to I/R with or without cardioprotective ischemic preconditioning (IPC). Rosiglitazone did not affect mortality, arrhythmia score, or infarct size during I/R. However, rosiglitazone abolished the antiarrhythmic effects of IPC. To investigate the direct effect of rosiglitazone on cardiomyocytes, we utilized adult rat cardiomyocytes (ARCMs), AC16, and differentiated AC16 (diffAC16) human cardiac cell lines. These were subjected to simulated I/R in the presence of rosiglitazone. Rosiglitazone improved cell survival of ARCMs at 0.3 µM. At 0.1 and 0.3 µM, rosiglitazone improved cell survival of AC16s but not that of diffAC16s. This is the first demonstration that chronic administration of rosiglitazone does not result in major hidden cardiotoxic effects in myocardial I/R injury models. However, the inhibition of the antiarrhythmic effects of IPC may have some clinical relevance that needs to be further explored.

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in Göttingen Minipigs and Landrace Pigs.

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an unmet clinical need for prevention and therapy of post-myocardial infarction heart failure (post-MI HF). Clinically relevant pig models of post-MI HF are prerequisites for final proof-of-concept studies before entering into clinical trials in drug and medical device development. Here we aimed to characterize a closed-chest porcine model of post-MI HF in adult Göttingen minipigs with long-term follow-up including serial cardiac magnetic resonance imaging (CMRI) and to compare it with the commonly used Landrace pig model. MI was induced by intraluminal balloon occlusion of the left anterior descending coronary artery for 120 min in Göttingen minipigs and for 90 min in Landrace pigs, followed by reperfusion. CMRI was performed to assess cardiac morphology and function at baseline in both breeds and at 3 and 6 months in Göttingen minipigs and at 2 months in Landrace pigs, respectively. Scar sizes were comparable in the two breeds, but MI resulted in a significant decrease of left ventricular ejection fraction (LVEF) only in Göttingen minipigs, while Landrace pigs did not show a reduction of LVEF. Right ventricular (RV) ejection fraction increased in both breeds despite the negligible RV scar sizes. In contrast to the significant increase of left ventricular end-diastolic (LVED) mass in Landrace pigs at 2 months, Göttingen minipigs showed a slight increase in LVED mass only at 6 months. In summary, this is the first characterization of post-MI HF in Göttingen minipigs in comparison to Landrace pigs, showing that the Göttingen minipig model reflects post-MI HF parameters comparable to the human pathology. We conclude that the Göttingen minipig model is superior to the Landrace pig model to study the development of post-MI HF.

Hidden Cardiotoxicity of Rofecoxib Can be Revealed in Experimental Models of Ischemia/Reperfusion.

Cardiac adverse effects are among the leading causes of the discontinuation of clinical trials and the withdrawal of drugs from the market. The novel concept of 'hidden cardiotoxicity' is defined as cardiotoxicity of a drug that manifests in the diseased (e.g. ischemic/reperfused), but not in the healthy heart or as a drug-induced deterioration of cardiac stress adaptation (e.g. ischemic conditioning). Here, we aimed to test if the cardiotoxicity of a selective COX-2 inhibitor rofecoxib that was revealed during its clinical use, i.e., increased occurrence of proarrhythmic and thrombotic events, could have been revealed in early phases of drug development by using preclinical models of ischemia/reperfusion (I/R) injury. Rats that were treated with rofecoxib or vehicle for four weeks were subjected to 30 min. coronary artery occlusion and 120 min. reperfusion with or without cardioprotection that is induced by ischemic preconditioning (IPC). Rofecoxib increased overall the arrhythmias including ventricular fibrillation (VF) during I/R. The proarrhythmic effect of rofecoxib during I/R was not observed in the IPC group. Rofecoxib prolonged the action potential duration (APD) in isolated papillary muscles, which was not seen in the simulated IPC group. Interestingly, while showing hidden cardiotoxicity manifested as a proarrhythmic effect during I/R, rofecoxib decreased the infarct size and increased the survival of adult rat cardiac myocytes that were subjected to simulated I/R injury. This is the first demonstration that rofecoxib increased acute mortality due to its proarrhythmic effect via increased APD during I/R. Rofecoxib did not interfere with the cardiprotective effect of IPC; moreover, IPC was able to protect against rofecoxib-induced hidden cardiotoxicity. These results show that cardiac safety testing with simple preclinical models of I/R injury uncovers hidden cardiotoxicity of rofecoxib and might reveal the hidden cardiotoxicity of other drugs.