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.

European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) SLE classification criteria item performance.

BACKGROUND/OBJECTIVES: The European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) 2019 classification criteria for systemic lupus erythematosus system showed high specificity, while attaining also high sensitivity. We hereby analysed the performance of the individual criteria items and their contribution to the overall performance of the criteria. METHODS: We combined the EULAR/ACR derivation and validation cohorts for a total of 1197 systemic lupus erythematosus (SLE) and n=1074 non-SLE patients with a variety of conditions mimicking SLE, such as other autoimmune diseases, and calculated the sensitivity and specificity for antinuclear antibodies (ANA) and the 23 specific criteria items. We also tested performance omitting the EULAR/ACR criteria attribution rule, which defines that items are only counted if not more likely explained by a cause other than SLE. RESULTS: Positive ANA, the new entry criterion, was 99.5% sensitive, but only 19.4% specific, against a non-SLE population that included other inflammatory rheumatic, infectious, malignant and metabolic diseases. The specific criteria items were highly variable in sensitivity (from 0.42% for delirium and 1.84% for psychosis to 75.6% for antibodies to double-stranded DNA), but their specificity was uniformly high, with low C3 or C4 (83.0%) and leucopenia <4.000/mm³ (83.8%) at the lowest end. Unexplained fever was 95.3% specific in this cohort. Applying the attribution rule improved specificity, particularly for joint involvement. CONCLUSIONS: Changing the position of the highly sensitive, non-specific ANA to an entry criterion and the attribution rule resulted in a specificity of >80% for all items, explaining the higher overall specificity of the criteria set.

Helium Conditioning Increases Cardiac Fibroblast Migration Which Effect Is Not Propagated via Soluble Factors or Extracellular Vesicles.

Helium inhalation induces cardioprotection against ischemia/reperfusion injury, the cellular mechanism of which remains not fully elucidated. Extracellular vesicles (EVs) are cell-derived, nano-sized membrane vesicles which play a role in cardioprotective mechanisms, but their function in helium conditioning (HeC) has not been studied so far. We hypothesized that HeC induces fibroblast-mediated cardioprotection via EVs. We isolated neonatal rat cardiac fibroblasts (NRCFs) and exposed them to glucose deprivation and HeC rendered by four cycles of 95% helium + 5% CO2 for 1 h, followed by 1 h under normoxic condition. After 40 h of HeC, NRCF activation was analyzed with a Western blot (WB) and migration assay. From the cell supernatant, medium extracellular vesicles (mEVs) were isolated with differential centrifugation and analyzed with WB and nanoparticle tracking analysis. The supernatant from HeC-treated NRCFs was transferred to naïve NRCFs or immortalized human umbilical vein endothelial cells (HUVEC-TERT2), and a migration and angiogenesis assay was performed. We found that HeC accelerated the migration of NRCFs and did not increase the expression of fibroblast activation markers. HeC tended to decrease mEV secretion of NRCFs, but the supernatant of HeC or the control NRCFs did not accelerate the migration of naïve NRCFs or affect the angiogenic potential of HUVEC-TERT2. In conclusion, HeC may contribute to cardioprotection by increasing fibroblast migration but not by releasing protective mEVs or soluble factors from cardiac fibroblasts.

Performance of the 2019 EULAR/ACR classification criteria for systemic lupus erythematosus in early disease, across sexes and ethnicities.

OBJECTIVES: The European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) 2019 Classification Criteria for systemic lupus erythematosus (SLE) have been validated with high sensitivity and specificity. We evaluated the performance of the new criteria with regard to disease duration, sex and race/ethnicity, and compared its performance against the Systemic Lupus International Collaborating Clinics (SLICC) 2012 and ACR 1982/1997 criteria. METHODS: Twenty-one SLE centres from 16 countries submitted SLE cases and mimicking controls to form the validation cohort. The sensitivity and specificity of the EULAR/ACR 2019, SLICC 2012 and ACR 1982/1997 criteria were evaluated. RESULTS: The cohort consisted of female (n=1098), male (n=172), Asian (n=118), black (n=68), Hispanic (n=124) and white (n=941) patients; with an SLE duration of 1 to <3 years (n=196) and ≥5 years (n=879). Among patients with 1 to <3 years disease duration, the EULAR/ACR criteria had better sensitivity than the ACR criteria (97% vs 81%). The EULAR/ACR criteria performed well in men (sensitivity 93%, specificity 96%) and women (sensitivity 97%, specificity 94%). Among women, the EULAR/ACR criteria had better sensitivity than the ACR criteria (97% vs 83%) and better specificity than the SLICC criteria (94% vs 82%). Among white patients, the EULAR/ACR criteria had better sensitivity than the ACR criteria (95% vs 83%) and better specificity than the SLICC criteria (94% vs 83%). The EULAR/ACR criteria performed well among black patients (sensitivity of 98%, specificity 100%), and had better sensitivity than the ACR criteria among Hispanic patients (100% vs 86%) and Asian patients (97% vs 77%). CONCLUSIONS: The EULAR/ACR 2019 criteria perform well among patients with early disease, men, women, white, black, Hispanic and Asian patients. These criteria have superior sensitivity than the ACR criteria and/or superior specificity than the SLICC criteria across many subgroups.

Cardiac miRNA Expression and their mRNA Targets in a Rat Model of Prediabetes.

Little is known about the mechanism of prediabetes-induced cardiac dysfunction. Therefore, we aimed to explore key molecular changes with transcriptomic and bioinformatics approaches in a prediabetes model showing heart failure with preserved ejection fraction phenotype. To induce prediabetes, Long-Evans rats were fed a high-fat diet for 21 weeks and treated with a single low-dose streptozotocin at week 4. Small RNA-sequencing, in silico microRNA (miRNA)-mRNA target prediction, Gene Ontology analysis, and target validation with qRT-PCR were performed in left ventricle samples. From the miRBase-annotated 752 mature miRNA sequences expression of 356 miRNAs was detectable. We identified two upregulated and three downregulated miRNAs in the prediabetic group. We predicted 445 mRNA targets of the five differentially expressed miRNAs and selected 11 mRNAs targeted by three differentially expressed miRNAs, out of which five mRNAs were selected for validation. Out of these five targets, downregulation of three mRNAs i.e., Juxtaposed with another zinc finger protein 1 (Jazf1); RAP2C, member of RAS oncogene family (Rap2c); and Zinc finger with KRAB and SCAN domains 1 (Zkscan1) were validated. This is the first demonstration that prediabetes alters cardiac miRNA expression profile. Predicted targets of differentially expressed miRNAs include Jazf1, Zkscan1, and Rap2c mRNAs. These transcriptomic changes may contribute to the diastolic dysfunction and may serve as drug targets.

Decorin Protects Cardiac Myocytes against Simulated Ischemia/Reperfusion Injury.

Search for new cardioprotective therapies is of great importance since no cardioprotective drugs are available on the market. In line with this need, several natural biomolecules have been extensively tested for their potential cardioprotective effects. Previously, we have shown that biglycan, a member of a diverse group of small leucine-rich proteoglycans, enhanced the expression of cardioprotective genes and decreased ischemia/reperfusion-induced cardiomyocyte death via a TLR-4 dependent mechanism. Therefore, in the present study we aimed to test whether decorin, a small leucine-rich proteoglycan closely related to biglycan, could exert cardiocytoprotection and to reveal possible downstream signaling pathways. Methods: Primary cardiomyocytes isolated from neonatal and adult rat hearts were treated with 0 (Vehicle), 1, 3, 10, 30 and 100 nM decorin as 20 h pretreatment and maintained throughout simulated ischemia and reperfusion (SI/R). In separate experiments, to test the mechanism of decorin-induced cardio protection, 3 nM decorin was applied in combination with inhibitors of known survival pathways, that is, the NOS inhibitor L-NAME, the PKG inhibitor KT-5823 and the TLR-4 inhibitor TAK-242, respectively. mRNA expression changes were measured after SI/R injury. Results: Cell viability of both neonatal and adult cardiomyocytes was significantly decreased due to SI/R injury. Decorin at 1, 3 and 10 nM concentrations significantly increased the survival of both neonatal and adult myocytes after SI/R. At 3nM (the most pronounced protective concentration), it had no effect on apoptotic rate of neonatal cardiac myocytes. No one of the inhibitors of survival pathways (L-NAME, KT-5823, TAK-242) influenced the cardiocytoprotective effect of decorin. MYND-type containing 19 (Zmynd19) and eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1) were significantly upregulated due to the decorin treatment. In conclusion, this is the first demonstration that decorin exerts a direct cardiocytoprotective effect possibly independent of NO-cGMP-PKG and TLR-4 dependent survival signaling.

2019 European League Against Rheumatism/American College of Rheumatology classification criteria for systemic lupus erythematosus.

OBJECTIVE: To develop new classification criteria for systemic lupus erythematosus (SLE) jointly supported by the European League Against Rheumatism (EULAR) and the American College of Rheumatology (ACR). METHODS: This international initiative had four phases. (1) Evaluation of antinuclear antibody (ANA) as an entry criterion through systematic review and meta-regression of the literature and criteria generation through an international Delphi exercise, an early patient cohort and a patient survey. (2) Criteria reduction by Delphi and nominal group technique exercises. (3) Criteria definition and weighting based on criterion performance and on results of a multi-criteria decision analysis. (4) Refinement of weights and threshold scores in a new derivation cohort of 1001 subjects and validation compared with previous criteria in a new validation cohort of 1270 subjects. RESULTS: The 2019 EULAR/ACR classification criteria for SLE include positive ANA at least once as obligatory entry criterion; followed by additive weighted criteria grouped in seven clinical (constitutional, haematological, neuropsychiatric, mucocutaneous, serosal, musculoskeletal, renal) and three immunological (antiphospholipid antibodies, complement proteins, SLE-specific antibodies) domains, and weighted from 2 to 10. Patients accumulating ≥10 points are classified. In the validation cohort, the new criteria had a sensitivity of 96.1% and specificity of 93.4%, compared with 82.8% sensitivity and 93.4% specificity of the ACR 1997 and 96.7% sensitivity and 83.7% specificity of the Systemic Lupus International Collaborating Clinics 2012 criteria. CONCLUSION: These new classification criteria were developed using rigorous methodology with multidisciplinary and international input, and have excellent sensitivity and specificity. Use of ANA entry criterion, hierarchically clustered and weighted criteria reflect current thinking about SLE and provide an improved foundation for SLE research.

L-Alpha-glycerylphosphorylcholine can be cytoprotective or cytotoxic in neonatal rat cardiac myocytes: a double-edged sword phenomenon.

L-Alpha-glycerylphosphorylcholine (GPC) is a widely used food supplement. GPC has been shown to exert beneficial effects in several organs; however, the cardiac effects of GPC have yet to be investigated. The aim of the present study was therefore to map out the effects of GPC on cardiac myocytes, with or without ischemia-reperfusion insult. Neonatal rat cardiac myocytes were treated with GPC at 1, 10, 80, and 100 µM concentrations for 15 min, 3 h, or 24 h, respectively. Cell viability by calcein assay and the degree of oxidative stress by DHE (superoxide level) and H2DCF (total ROS accumulation) staining were measured. In separate experiments, cardiomyocytes were pre-treated with the optimal concentration of GPC for 3 h and then cells were exposed to 4 h of simulated ischemia followed by 2 h of reperfusion (SI/R). Cell viability was measured at the end of the SI/R protocol. In normoxic conditions, the 15-min and the 3-h GPC treatment did not affect cell viability, total ROS, and superoxide levels. Under SI/R conditions, the 3-h GPC treatment protected the cardiac myocytes from SI/R-induced cell death and did not alter the level of oxidative stress. The 24-h GPC treatment in normoxic conditions resulted in significant cell death and increased oxidative stress at each concentration. Here we provide the first evidence for the cytoprotective effect of short-term GPC treatment. However, long-term administration of GPC may exert cytotoxicity in a wide concentration range in cardiac myocytes. These results may draw attention to a comprehensive cardiac safety protocol for the testing of GPC.

Minimization of metabolic cost of transport predicts changes in gait mechanics over a range of ankle-foot orthosis stiffnesses in individuals with bilateral plantar flexor weakness.

Neuromuscular disorders often lead to ankle plantar flexor muscle weakness, which impairs ankle push-off power and forward propulsion during gait. To improve walking speed and reduce metabolic cost of transport (mCoT), patients with plantar flexor weakness are provided dorsal-leaf spring ankle-foot orthoses (AFOs). It is widely believed that mCoT during gait depends on the AFO stiffness and an optimal AFO stiffness that minimizes mCoT exists. The biomechanics behind why and how an optimal stiffness exists and benefits individuals with plantar flexor weakness are not well understood. We hypothesized that the AFO would reduce the required support moment and, hence, metabolic cost contributions of the ankle plantar flexor and knee extensor muscles during stance, and reduce hip flexor metabolic cost to initiate swing. To test these hypotheses, we generated neuromusculoskeletal simulations to represent gait of an individual with bilateral plantar flexor weakness wearing an AFO with varying stiffness. Predictions were based on the objective of minimizing mCoT, loading rates at impact and head accelerations at each stiffness level, and the motor patterns were determined via dynamic optimization. The predictive gait simulation results were compared to experimental data from subjects with bilateral plantar flexor weakness walking with varying AFO stiffness. Our simulations demonstrated that reductions in mCoT with increasing stiffness were attributed to reductions in quadriceps metabolic cost during midstance. Increases in mCoT above optimum stiffness were attributed to the increasing metabolic cost of both hip flexor and hamstrings muscles. The insights gained from our predictive gait simulations could inform clinicians on the prescription of personalized AFOs. With further model individualization, simulations based on mCoT minimization may sufficiently predict adaptations to an AFO in individuals with plantar flexor weakness.

Encapsulation protocol for fecal microbiota transplantation.

Introduction: Clostridioides difficile infections (CDI) continue to pose a challenge for clinicians. Fecal microbiota transplantation (FMT) is an effective treatment option in CDI. Furthermore, recent and ongoing studies suggest potential benefits of FMT in other diseases as well. Methods: We would like to present a novel protocol for encapsulation of lyophilized fecal material. Our method provides with better compliance as well as improved flexibility, storage and safety. Results: FMT was conducted in 28 patients with an overall success rate of 82,14% using apsules containing lyophilized stool. 16 of patients were given capsules with lessened bacteria counts. The success rate in this group was 93,75%. Discussion: The results highlight the still unanswered questions about the mechanism of action and contribute to a wider use of FMT in the clinical praxis and in research.

Cardioprotective microRNAs (protectomiRs) in a pig model of acute myocardial infarction and cardioprotection by ischaemic conditioning: MiR-450a.

BACKGROUND AND PURPOSE: Cardioprotective miRNAs (protectomiRs) are promising therapeutic tools. Here, we aimed to identify protectomiRs in a translational porcine model of acute myocardial infarction (AMI) and to validate their cardiocytoprotective effect. EXPERIMENTAL APPROACH: ProtectomiR candidates were selected after systematic analysis of miRNA expression changes in cardiac tissue samples from a closed-chest AMI model in pigs subjected to sham operation, AMI and ischaemic preconditioning, postconditioning or remote preconditioning, respectively. Cross-species orthologue protectomiR candidates were validated in simulated ischaemia-reperfusion injury (sI/R) model of isolated rat ocardiomyocytes and in human AC16 cells as well. For miR-450a, we performed target prediction and analysed the potential mechanisms of action by GO enrichment and KEGG pathway analysis. KEY RESULTS: Out of the 220 detected miRNAs, four were up-regulated and 10 were down-regulated due to all three conditionings versus AMI. MiR-450a and miR-451 mimics at 25 nM were protective in rat cardiomyocytes, and miR-450a showed protection in human cardiomyocytes as well. MiR-450a has 3987 predicted mRNA targets in pigs, 4279 in rats and 8328 in humans. Of these, 607 genes are expressed in all three species. A total of 421 common enriched GO terms were identified in all three species, whereas KEGG pathway analysis revealed 13 common pathways. CONCLUSION AND IMPLICATIONS: This is the first demonstration that miR-450a is associated with cardioprotection by ischaemic conditioning in a clinically relevant porcine model and shows cardiocytoprotective effect in human cardiomyocytes, making it a promising drug candidate. The mechanism of action of miR-450a involves multiple cardioprotective pathways.

Migraine as a Disease Associated with Dysbiosis and Possible Therapy with Fecal Microbiota Transplantation.

Migraine is a painful neurological condition characterized by severe pain on one or both sides of the head. It may be linked to changes in the gut microbiota, which are influenced by antibiotic use and other factors. Dysbiosis, which develops and persists as a result of earlier antibiotic therapy, changes the composition of the intestinal flora, and can lead to the development of various diseases such as metabolic disorders, obesity, hematological malignancies, neurological or behavioral disorders, and migraine. Metabolites produced by the gut microbiome have been shown to influence the gut-brain axis. The use of probiotics as a dietary supplement may reduce the number and severity of migraine episodes. Dietary strategies can affect the course of migraines and are a valuable tool for improving migraine management. With fecal microbiota transplantation, gut microbial restoration is more effective and more durable. Changes after fecal microbiota transplantation were studied in detail, and many data help us to interpret the successful interventions. The microbiological alteration of the gut microflora can lead to normalization of the inflammatory mediators, the serotonin pathway, and influence the frequency and intensity of migraine pain.

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.

Significance of autoantibody assays in systemic lupus erythematosus / Az autoantitest-vizsgálatok jelentosége szisztémás lupus erythematosusban

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by pathological immune complexes and various autoantibodies. Detectable pathological autoantibodies produced by plasma cells differentiated from B cells play a significant role in the establishment of clinical diagnosis, classification, and differential diagnosis as well as assessing the disease activity during patients' follow-up. Autoantibody determination is very important in

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.

Full-scale use of glycogen-accumulating organisms for excess biological carbon removal.

The purpose of this study has been to verify the efficient full-scale applicability of glycogen-accumulating organisms (GAOs) for excess biological carbon removal, that is, for removing more carbon substrate than the amount of available nutrients would allow in the conventional activated sludge process of microbial growth. This aims to cost-effectively overcome the problem of viscous bulking occurring in a fully aerated system, with nutrient deficiency. Analytical data measured at the wastewater treatment plant of the Balatonboglár (BB) winery in Balatonboglár, Hungary, containing consecutive unaerated and aerated activated sludge basins, reflected a high performance with efficient carbon removal and good sludge settling, without dosing any external nutrient source to the severely nitrogen- and phosphorous-deficient influent. Supplementary laboratory-scale batch experiments and microbiological tests verified the abundance of GAOs in the activated sludge system and elucidated their role in efficient excess biological carbon removal.

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.

Cardiac dysfunction in cancer patients: beyond direct cardiomyocyte damage of anticancer drugs: novel cardio-oncology insights from the joint 2019 meeting of the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart.

In western countries, cardiovascular (CV) disease and cancer are the leading causes of death in the ageing population. Recent epidemiological data suggest that cancer is more frequent in patients with prevalent or incident CV disease, in particular, heart failure (HF). Indeed, there is a tight link in terms of shared risk factors and mechanisms between HF and cancer. HF induced by anticancer therapies has been extensively studied, primarily focusing on the toxic effects that anti-tumour treatments exert on cardiomyocytes. In this Cardio-Oncology update, members of the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart discuss novel evidence interconnecting cardiac dysfunction and cancer via pathways in which cardiomyocytes may be involved but are not central. In particular, the multiple roles of cardiac stromal cells (endothelial cells and fibroblasts) and inflammatory cells are highlighted. Also, the gut microbiota is depicted as a new player at the crossroads between HF and cancer. Finally, the role of non-coding RNAs in Cardio-Oncology is also addressed. All these insights are expected to fuel additional research efforts in the field of Cardio-Oncology.

A Comorbidity Model of Myocardial Ischemia/Reperfusion Injury and Hypercholesterolemia in Rat Cardiac Myocyte Cultures.

INTRODUCTION: The use of comorbidity models is crucial in cardioprotective drug development. Hypercholesterolemia causes endothelial and myocardial dysfunction, as well as aggravates ischemia/reperfusion (I/R)-induced myocardial injury. Endogenous cardioprotective mechanisms against I/R are impaired in hyperlipidemic and hyperglycemic in vivo animal models. Therefore, our aim was to develop a medium throughput comorbidity cell-based test system of myocardial I/R injury, hypercholesterolemia and hyperglycemia that mimics comorbidity conditions. METHODS: Cardiac myocytes isolated from neonatal or adult rat hearts were cultured in control or in three different hypercholesterolemic media with increasing cholesterol content (hiChol) or hiChol + hyperglycemic medium, respectively. Each group was then subjected to simulated ischemia/reperfusion (SI/R) or corresponding normoxic condition, respectively. Cholesterol uptake was tested by Filipin staining in neonatal cardiac myocytes. Cell viability, total cell count and oxidative stress, i.e., total reactive oxygen species (ROS) and superoxide level were measured by fluorescent assays. RESULTS: Neonatal cardiac myocytes took up cholesterol from the different hiChol media at a concentration-dependent manner. In normoxia, viability of hiChol neonatal cardiac myocytes was not significantly changed, however, superoxide levels were increased as compared to vehicle. After SI/R, the viability of hiChol neonatal cardiac myocytes was decreased and total ROS level was increased as compared to vehicle. HiChol combined with hyperglycemia further aggravated cell death and oxidative stress in normoxic as well as in SI/R conditions. Viability of hiChol adult cardiac myocytes was significantly decreased and superoxide level was increased in normoxia and these changes were further aggravated by SI/R. HiChol combined with hyperglycemia further aggravated cell death, however level of oxidative stress increased only in normoxic condition. CONCLUSION: HiChol rat cardiac myocytes showed reduction of cell viability and increased oxidative stress, which were further aggravated by SI/R and with additional hyperglycemia. This is the first demonstration that the combination of the current hypercholesterolemic medium and SI/R in cardiac myocytes mimics the cardiac pathology of the comorbid heart with I/R and hypercholesterolemia.

2019 European League Against Rheumatism/American College of Rheumatology Classification Criteria for Systemic Lupus Erythematosus.

OBJECTIVE: To develop new classification criteria for systemic lupus erythematosus (SLE) jointly supported by the European League Against Rheumatism (EULAR) and the American College of Rheumatology (ACR). METHODS: This international initiative had four phases. 1) Evaluation of antinuclear antibody (ANA) as an entry criterion through systematic review and meta-regression of the literature and criteria generation through an international Delphi exercise, an early patient cohort, and a patient survey. 2) Criteria reduction by Delphi and nominal group technique exercises. 3) Criteria definition and weighting based on criterion performance and on results of a multi-criteria decision analysis. 4) Refinement of weights and threshold scores in a new derivation cohort of 1,001 subjects and validation compared with previous criteria in a new validation cohort of 1,270 subjects. RESULTS: The 2019 EULAR/ACR classification criteria for SLE include positive ANA at least once as obligatory entry criterion; followed by additive weighted criteria grouped in 7 clinical (constitutional, hematologic, neuropsychiatric, mucocutaneous, serosal, musculoskeletal, renal) and 3 immunologic (antiphospholipid antibodies, complement proteins, SLE-specific antibodies) domains, and weighted from 2 to 10. Patients accumulating ≥10 points are classified. In the validation cohort, the new criteria had a sensitivity of 96.1% and specificity of 93.4%, compared with 82.8% sensitivity and 93.4% specificity of the ACR 1997 and 96.7% sensitivity and 83.7% specificity of the Systemic Lupus International Collaborating Clinics 2012 criteria. CONCLUSION: These new classification criteria were developed using rigorous methodology with multidisciplinary and international input, and have excellent sensitivity and specificity. Use of ANA entry criterion, hierarchically clustered, and weighted criteria reflects current thinking about SLE and provides an improved foundation for SLE research.