bluuebunny/crossref_metadata_2025 huggingface dataset:
Relevance of necroptosis in the hearts subjected to acute versus chronic ischemia/reperfusion injury
Authors: Horvath C , Szobi A , Young M , Jarabicova I , Hrdlicka J , Neckar J , Lewis M , Kolar F , Ravingerova T , Suleiman MS , Adameova A
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): Slovak Research and Development Agency

Medical Research Council Grant

Necroptosis, a necrosis-like programmed cell death modality dependent on the activity of receptor-interacting protein kinase 3 (RIP3) and mixed lineage domain kinase domain-like pseudokinase (MLKL), plays a prominent role in mediating myocardial ischemia/reperfusion injury. However, the extent to which necroptosis contributes to such damage under short and long reperfusion has not been evaluated in detail. In Wistar rat hearts, subjected to global 30-min ischemia followed by an acute 10-min reperfusion period, with compromised cardiac function, no changes in the protein expression of the main necroptotic markers (pThr231/Ser232-RIP3, MLKL) were found. Likewise, the non-canonical pathways of necroptosis involving Ca2+/calmodulin dependent protein kinase II–mitochondrial permeability transition pore (CaMKII–mPTP) or phosphoglycerate mutase 5–dynamin-related protein 1 (PGAM5–Drp1) axes were unlikely affected by such short reperfusion. In contrast, hearts subjected to global 30-min ischemia followed by a prolonged 40-min reperfusion period exhibited worsened hemodynamic parameters what was accompanied by the increased levels of RIP3, pSer229-RIP3 and MLKL. Moreover, this reperfusion period induced MLKL translocation to the plasma membrane, indicating necroptosis execution with resultant very likely cell disruption. Similarly, activated necroptosis, evidenced by the higher levels of proteins of the canonical pathway, has been suggested to contribute to the pathogenesis of post-infarction heart failure (30-min ischemia, 42-day reperfusion). Collectively, these findings suggest that short reperfusion seems to be insufficient to induce necroptosis in the heart and the molecular mechanisms being activated during the longer reperfusion phase are needed to promote necroptotic cell dying. Therefore, inhibition of necroptosis might represent a cardioprotective strategy in the settings of chronic, but not acute myocardial ischemia/reperfusion injury.
Searching for cardioprotective microRNA families by bioinformatics analysis of cross-species transcriptomic datasets
Authors: Agg B , Benczik B , Kemendi BV , Makkos A , Petervari M , Varga ZV , Ferdinandy P
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This study was supported by the National Research, Development and Innovation Office of Hungary (NKFIA; NVKP-16-1-2016-0017 National Heart Program) and the Thematic Excellence Programme (2020-4.1.1.-TKP2020) of the Ministry for Innovation and Technology in Hungary, within the framework of the Therapeutic Development and Molecular Biology thematic programmes of the Semmelweis University.

Introduction

There is a growing body of evidence that changes in cardiac microRNA expression pattern plays an important role in cardioprotective cellular signaling.

Purpose

Our aim was to identify evolutionarily conserved cardioprotective microRNA (protectomiR) families with a high probability of translation into human clinical practice.

Methods

Global cardiac microRNA expression profiles were measured in both a rat and a porcine model of myocardial infarction with or without ischemic preconditioning and postconditioning by microarray and high-throughput polymerase chain reaction, respectively. A software developed by our team was used to identify cross-species microRNA families (i.e. microRNAs with identic seed sequence) with cardioprotective expression patterns. By selecting experimentally validated human target genes from the miRTarBase database we built a microRNA family-target interaction network by the miRNAtarget.com software. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the selected target genes.

Results

Due to potential patenting, microRNAs and microRNA families were anonymized. We identified 7 microRNA families, showing cardioprotective expression pattern. Based on the analysis of the microRNA family-target network, expression change of the target hubs were predicted to be consistent with previously described changes in pathways with high relevance in cardioprotection (e.g. positive regulation of TGF-beta, HIF-1, PI3K-Akt signaling pathways).

Conclusion

By an unbiased transcriptomics and bioinformatics based approach we successfully identified microRNA families with robust cross-species protectomiR expression pattern and targets involved in well-established cardioprotective pathways. These cardioprotective microRNA families are of high translational value for human clinical application.
Implication of Orai1 in the diastolic calcium dysregulation during Ischemia and Reperfusion in cardiomyocytes
Authors: Falcon Boyano D , Mayoral Gonzalez I , Calderon Sanchez E , Ordonez A , Smani T
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación

Background

It is well established that abnormalities in the regulation of the intracellular concentration of Ca2+ ([Ca2+]i) occurrs in diseased heart. Ischemia/Reperfusion (I/R) syndrome is characterized by [Ca2+]i overload that might be associated with the increase of left ventricular pressure, which might trigger diastolic dysfunction. Several studies have demonstrated that Orai1/2/3 isoforms of the Store Operated Calcium Channels (SOCC) and TRPC channels play a role in cardiac pathologies. Previously, we demonstrated that the expression of Orai1 increased after I/R although their role in the diastolic [Ca2+]i regulation is unknown.

Objective

The aim of this study was to examine the role of SOCC in the diastolic [Ca2+] after I/R in cardiomyocytes and the activation of transcription factors by I/R.

Materials and Methods

The experiments were performed in adult (ARVM) and neonatal rat ventricular cardiomyocytes (NRVM) and in a rat model of myocardial I/R. Ca2+ studies were realized using microfluorimetric technique in FURA-2AM loaded cardiomyocytes. To evoke [Ca2+]i transients, ARVM were field stimulated at 0.5Hz and NRVMs at 1Hz. Immunofluorescence was used to investigated CREB activation using a specific phospho-CREB antibody and protein expression was analysed by Western Blot. Orai1 expression was assessed in rat samples and in ventricular biopsies of patients with ischemic heart failure.

Results

We found that field stimulation of ARVM from I/R animal showed significant increase in the diastolic [Ca2+]i as compare to AVRM isolated from sham rats. Also, NRVM subjected to simulated protocol of I/R presented similar increase in the diastolic [Ca2+]i. These responses correlated with significant increase in the expression of Orai1 in both cardiomyocytes. Interestingly, NRVM and ARVM preincubation with SYNTA-66 and GSK-7975A to block SOCC successfully reduced the increase in the diastolic [Ca2+]I induced by IR. Moreover, we found that I/R promoted CREB activation through PKA activation, but not though EPAC2 or ERK1/2. Finally, we found that Orai1 is overexpressed in ventricle biopsies of patient with heart failure from ischemic origin.

Conclusions

Our results confirms that I/R induced an upregulation of Orai1 which was associated with diastolic dysfunction, suggesting that Orai1 plays a role in the I/R induced diastolic [Ca2+]i increase. Moreover, our data demonstrated the activation of the transcription factor CREB in cardiomyocytes after I/R, known to activate a plethora of genes involved in the adverse cardiac remodelling.
Inflammatory mediators to predict left adverse ventricular remodelling in revascularized STEMI patients
Authors: Bevilacqua E , Galeano-Otero I , Guerrero-Marquez FJ , Falcon- Boyano D , Baron-Esquivias G , Smani T , Ordonez-Fernandez A , Del Toro R
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Institute of Carlos III the Spanish Ministry of Economy and Competitiveness

Background

Primary percutaneous coronary intervention (PPCI) in patients with ST-segment elevation myocardial infarction (STEMI) improves the survival of patients. Nevertheless, some patients develop left ventricular adverse remodeling (LVAR). The main objective of this study is to find reliable prognostic biomarkers for patients developing LVAR, focusing in the role of pro-inflammatory mediators, including cell populations and secreted molecules.

Methods

We assessed the level of pro-inflammatory cell subsets, before and after the revascularization, 1 and 6 months after the PPCI, using flow cytometry. We also performed miRNAs microarray in isolated peripheral blood mononuclear cells (PBMCs), and examined the levels of 27 cytokines in patients’ serum by multiplex ELISA.

Results

We observed that levels of pro-inflammatory monocytes were increased soon after PPCI, in patients who afterward developed LVAR. Furthermore, we detected significant increase in the concentration of secreted cytokines such as IL-1b, IL-17, IFN-g and VEGF in LVAR patients post-PPCI. Moreover, specific miRNAs levels regulating these cytokines secretion, also correlated with LVAR. Multivariate regression analysis and ROC curves indicated that these inflammatory mediators were good biomarkers to correlate with the appearance of LVAR in patients. In addition, we found that the combined analysis of these biomarkers increased the specificity and sensibility to identify patients with LVAR.

Conclusion

Our data suggest that the combined analysis of these inflammatory axes and related molecules, could reliably predict LVAR in STEMI patients.
E2F7/8 is involved in cardiomyocyte polyploidy but does not affect myocardial reperfusion injury recovery
Authors: Fang J , Van Der Geest JSA , Yao B , Yang Q , Chirico N , Brans MA , Roefs MT , Vader P , De Jager SCA , De Bruin A , Vink A , Van Mil A , Schiffelers RM , Lei Z , Sluijter JPG
Published: June 2022

Funding Acknowledgements

Type of funding sources: Other. Main funding source(s): CSC fellowship

Background

Polyploidy cells consist of more than two complete sets of homologous chromosomes. Although a characteristic feature of cardiomyocytes and observed in all mammalian species, its molecular mechanism and biological functions are still unknown. Cardiomyocytes polyploidy in rodents occurs mainly through incomplete cytokinesis and increases with age. Studies have demonstrated that E2F7/8 transcription factors are key regulators of polyploidy in the liver and pancreas, however, it remains unclear if E2F7/8 control the generation of polyploidy cardiomyocytes and what the functional consequence is post-myocardial infarction (MI).

Methods

By using a tamoxifen inducible Cre/LoxP approach in new-born mice, we deleted E2F7/8 transcription factors ubiquitously and evaluated the biological significance of postnatal E2F7/8 loss. Mice underwent myocardial ischemia reperfusion injury (IRI) and heart function was assessed by 4D-echocardiography. Cardiomyocyte nucleus polyploidy was measured by FACS and microscope.

Results

Deficiency of E2F7/8 significantly suppress cardiomyocyte mononucleated and multinucleated polyploidy, as well as dramatically decreased hepatocytes polyploidy. E2F7/8 defect also led to a decrease in cardiac stress related marker lever such as ANP, BNP, MMP2, β-MHC/α-MHC and an increase in CD31 expression level. Surprisingly, E2F7/8 deletion did not have impact on cardiac function and dimensions post-IRI.

Conclusion

In summary, we identified that E2F7/8 activity is involved in the cellular polyploidy in the heart but did not affect myocardial function after myocardial injury.
Implication of caveolae in the pathophysiology of human acute myocardial infarction: a histological study
Authors: Rios Navarro C , Cantos-Amores G , Ortega M , Gavara J , Marcos-Garces V , De Dios E , Perez-Sole N , Chorro FJ , Bodi V , Ruiz-Sauri A
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work has been funded by “Instituto de Salud Carlos III” and “Fondos Europeos de Desarrollo Regional FEDER” (Exp. PI20/00637) as well as by “Generalitat Valenciana” (Exp. PROMETEO/2021/008).

Introduction

Caveolae are lipid invaginations present in the membrane of most mammalian cells. They are mainly made up of three proteins: caveolin (Cav)-1, 2, and 3 and are involved in signal transduction and ion channels. Since caveolae regulate different signalling pathways to promote cardiac protection, the aim of this study is to compare the expression of Cav-1, 2, and 3 in the human myocardium of control subjects and in patients with myocardial infarction (MI).

Materials

Myocardial samples from human autopsies of 4 controls and 4 MI patients with more than 6 months of evolution were isolated. The infarction and control area were identified by haematoxylin-eosin and Masson’s trichromic stainings. The presence of Cav-1, 2 and 3 was detected by immunochemistry. Afterwards, 5 photographs were taken for each antibody and sample and the presence of each protein was morphometrically quantified using the image analyzer Image-Pro Plus. These results were also corroborated by immunofluorescence.

Results

A constitutive presence of Cav-1, 2, and 3 was observed in the myocardium of control patients, being Cav-1 and 3 higher expressed than Cav-2. Cav-2 and 3 were mainly found in cardiomyocytes, while Cav-1 was detected not only in cardiac muscle cells but also in endothelial cells. Comparing caveolae expression between the peri-infarct region of MI patients and controls, a significant reduction in the expression of the three proteins was observed by immunochemistry. Indeed, the decrease in Cav-2 and 3 could be also detected by immunofluorescence. Lastly, Cav-1 was more expressed in cardiomyocytes than in endothelial cells, but unlike muscle cells, its expression was not diminished after MI.

Conclusions

The presence of caveolin decreases in the myocardial tissue after MI. Further studies are needed to confirm the cardioprotective role of caveolae post-AMI and their use as potential therapeutic target.
Identification of cardioprotective miRNAs (protectomiRs) in a porcine model of acute myocardial infarction and cardioprotection by ischemic conditioning
Authors: Nagy RN , Makkos A , Baranyai T , Giricz Z , Kiss B , Puskas LG , Farago N , Lukovic D , Gyongyosi M , Gorbe A , Ferdinandy P
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research, Development and Innovation Office of Hungary (NKFIA; NVKP-16-1-2016-0017 National Heart Program and OTKA-FK 134751)

MTA-SE System Pharmacology Research Group, Department of Pharmacology and Pharmacotherapy, Semmelweis University, H-1089 Budapest, Hungary

Introduction

Changes in the expression profile of microRNAs contribute to cardioprotective signaling. We have previously identified cardioprotective microRNAs, termed protectomiRs, by a systematic analysis of microRNA expression pattern in myocardial infarction and cardioprotection induced by ischemic conditioning in rats. ProtectomiRs may be important therapeutics for cardioprotection.

Purpose

We aimed to identify protectomiRs in a translational porcine model of reperfused acute myocardial infarction (AMI) and cardioprotection, and validate their cardiocytoprotective effect.

Methods

We used cardiac tissue samples from our previous study in closed-chest AMI model in domestic pigs. Pigs were subjected to sham operation (Sham), ischemia/reperfusion to induce AMI (AMI) or preconditioning (IPreC), postconditioning (IPostC), and remote perconditioning (RIPerC). Tissue samples were collected from the infarcted region of the left ventricles. MiRNA expression pattern was detected by high-throughput qRT-PCR. Potential protectomiRs were selected by systematic comparison of significant expression changes due to different conditioning stimuli vs. AMI. To validate the cardiocytoprotective effect of potential protectomiRs, isolated rat cardiomyocytes were transfected with specific miRNA mimics or inhibitors (antagomiRs) of the selected protectomiRs, and the survival of cells was measured after simulated ischemia/reperfusion injury.

Results

Expression of 221 miRNAs was assessed. Expression of 57 microRNAs were changed by IPreC, 54 by IPostC and 68 by RIPerC  as compared to AMI (min. 1,5×log2 fold-change, -log10p>1,31 vs. AMI). Expression of 14 microRNAs changed significantly due to all three conditionings vs. AMI (10 miRNAs were downregulated and 4 upregulated). Rat homologs of these 14 protectomiR candidates were identified and 13 showed 100% sequence homology with the original pig miRNAs. The selected miRNAs (9 antagomiRs and 4 miRNA mimics) were transfected in isolated rat cardiomyocytes. Modulation of 2 mimics of these miRNAs significantly improved the survival of cells after ischemia/reperfusion injury (due to intellectual property protection, we do not disclose the protectomiRs here).

Conclusion

Here we identified 2 miRNAs in a clinically relevant porcine model of AMI that can be potential therapeutic protectomiRs.
Cardioprotection by limb remote ischemic preconditioning in rats: discrepancy between meta-analysis and a three-center in vivo study
Authors: Sayour N , Brenner GB , Makkos A , Kovacshazi CS , Gergely TG , Tian H , Zenkl V , Bencsik P , Heinen A , Schulz R , Baxter GF , Zuurbier CJ , Voko Z , Ferdinandy P , Giricz Z
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Heart Program funded by the National Research, Development and Innovation Office of Hungary

Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the Therapeutic development thematic programme of the Semmelweis University

Purpose and aims

Despite promising preclinical results on cardioprotection by remote ischemic preconditioning (RIPC), large-scale clinical trials show no reduction in infarct size (IS). This translational gap might be due to unknown confounding factors and lack of sufficient high-quality in vivo preclinical proof-of-concept studies. Therefore, here we performed randomized, blinded in vivo studies in three study centers using the most often reported methodological settings, as well as a systematic review and meta-analysis of RIPC studies.

Methods and Results

Male Wistar rats were subjected to 20 to 45 min cardiac ischemia 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 IS, microvascular obstruction, or arrhythmias at any study centers. Systematic review and meta-analysis focusing on in vivo rat models of myocardial ischemia/reperfusion injury with limb RIPC showed that RIPC reduces IS by 21% on average; however, a tendency for publication bias towards positive results was found. In addition, our systematic review showed methodological heterogeneity and insufficient reporting quality 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 multiple study centers. Although unanimously showing cardioprotection by RIPC, the majority of reviewed studies were inadequately reported, and under-publication of neutral studies is plausible. Methodological confounding factors leading to the discrepancy between meta-analysis and the present studies cannot be identified; insufficient reporting quality, heterogeneity of the applied settings, and selection bias against studies with neutral outcome may contribute to this discrepancy. More preclinical studies with adequate quality control, together with publication of neutral studies, are required to support development of clinically translatable cardioprotective interventions.
Ischemic postconditioning activates cardiac MMP-2 and decreases biglycan level as well as miR-34a-5p and miR-195-5p expression in early reperfusion in a porcine infarction model
Authors: Szabados T , Gomori K , Ferdinandy P , Gyongyosi M , Gorbe A , Bencsik P
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Kooperatív Doktori Program Doktori Hallgatói Ösztöndíj (KDP-2020)

Hungarian Scientific Research Fund-OTKA-138223

Background

Matrix metalloproteinase-2 (MMP-2) has been shown to be activated during myocardial ischemia-reperfusion (IR) injury leading to degradation of cardiac contractile proteins. In previous investigation, ischemic postconditioning (IPoC) failed to decrease myocardial necrosis in porcine model of acute myocardial infarction (AMI). However, IPoC significantly reduced the severity of myocardial microvascular obstruction (MVO) compared to ischemic control. Therefore, our aim was to reveal if two major extracellular players of cardiac pathophysiology, MMP-2 and biglycan (BGN) are involved in the MVO reducing effect of IPoC in a porcine AMI model.

Methods

AMI was induced in female domestic pigs (25-35 kg) with balloon catheter occlusion of the left descending coronary artery for 90 min in ischemic control group. IPoC group underwent 6×30 s I/R after 90-min occlusion. At the end of the 3-hour or 3-day reperfusion, infarcted left ventricular tissue samples were taken to determine MMP-2 activity and BGN levels as well as cardiac microRNA (miRNA) expression.

Results

Cardiac MMP-2 activity showed significant increase in IPoC group as compared to ischemic control after 3h reperfusion but after 3d reperfusion no significant change was observed between the two groups. Cardiac BGN level in IPoC was significantly decreased as compared to ischemic control after 3h reperfusion, which difference was also abolished after 3d reperfusion due to the decrease of BGN level of ischemic control group. Ischemic postconditioning altered the expression of three miRNAs: miR-34a-5p, and miR-195-5p were downregulated, and miR-15b-5p was upregulated as compared to ischemic control.

Conclusions

IPoC decreased MVO and BGN level and increased MMP-2 activity in the infarcted left ventricle during early reperfusion, and altered the expression on 3 cardiac miRNAs. Further increase was found in MMP activities after 3 days in both groups, which affected BGN level only in ischemic control group. Altered expression of miRNAs and early decrease in cardiac BGN level may contribute to the beneficial effect of IPoC on MVO.
Blockers of Hyperpolarization-activated Cyclic Nucleotide-gated channels exhibit antimuscarinic properties in human atrial cardiomyocytes
Authors: Gencarelli M , Laurino A , Balducci V , Spinelli V , Cameli M , Pecori R , Vistoli G , Matucci R , Cerbai E , Sartiani L
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): M.I.U.R. PRIN 2017 to E.C and ECRF to L.S.

Introduction

Ivabradine (Iva), Zatebradine (Zat) and ZD7288 (ZD) are blockers of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, which play definite electrophysiological functions in cardiac pacemakers, working cardiomyocytes, peripheral and central neurons [1]. Among them, Iva is in clinical use as bradycardic agent for uncontrolled angina and heart failure with reduced ejection fraction. Accumulating evidence suggests Iva may have a more complex pharmacological profile, including targets different from HCN channels [2, 3]. Reports on experimental atrial fibrillation, detrusor contraction and bladder overactivity [4] led us to hypothesize that muscarinic receptors (mAChRs) may represent still unexplored targets of Iva and analogues. Present study was designed to identify the effect of Iva on acetylcholine-gated inward-rectifier K+-current (IKACh), the main effector of atrial mAChRs, and to assess whether Iva and analogues interact directly with human atrial and recombinant mAChRs.

Methods

Patch-clamp recordings were performed on human atrial myocytes (hAMs) isolated from atrial appendages of patients in sinus rhythm. Competition and kinetic radioligand binding assays were performed on hAM preparations and on mAChR(1,2,3) expressed in heterologous systems.

Results

Patch-clamp recordings on hAMs demonstrated Iva is able to suppress IKAch similarly to atropine, thus suggesting a functional antagonism of Iva on atrial mAChRs.  Competition binding assays on hAM membranes revealed Iva, Zat and ZD bind to mAChRs with affinity values close to those of muscarinic ligands carbachol (Cch) and gallamine (Gal). Competition binding assays on recombinant mAChR(1,2,3), which are functionally relevant in hAMs, showed all blockers are ligands for each single mAChR type with affinity values close to those of CCh and Gal. Finally, using dissociation binding assays on mAChR(2) lacking the allosteric binding site, we showed Iva and Zat behave as allosteric ligands with affinity values similar to Gal. Doking studies confirmed Iva and Zat, but not ZD, have the structural requirements necessary to interact with the allosteric and the orthosteric site of mAChR(2).

Conclusions

This study demonstrates Iva directly interacts with mAChR expressed in hAMs, showing antimuscarinic activity on IKAch. This property is likely dependent on a double interaction of Iva with the orthosteric and the allosteric site of mAChR(2). Zat, but not ZD, shows similar binding properties compared to Iva. Our results identify a novel pharmacological target for Iva and analogues in human atria, which may have important consequences for the function of the healthy and diseased atrium.
ProANP31-67 ameliorates adverse cardiac remodeling and improves systolic and diastolic functions in a preclinical model of cardiorenal syndrome
Authors: Silva GJJ , Parvan R , Shen X , Frisk M , Altara R , Strand ME , Rypdal KB , Lunde IG , Louch WE , Aronsen JM , Stenslokken K-O , Stokke MK , Cataliotti A
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): South-Eastern Norway Regional Health Authority (HSØ-RHF, Project No. 25674)

Background

The cardiac hormone proANP31-67, a linear fragment of the N-terminal Atrial Natriuretic Peptide, has known enhancing renal effects. More recently, we described the cardio protective effects of this hormone in a model of chronic hypertension. More specifically, independently of the blood pressure level, proANP31-67 improved diastolic function, attenuated cardiac fibrosis, and reduced hypertrophy.

Purpose

The current study was designed to assess the cardiorenal effects of proANP31-67 in a rodent model of hampered renal function, followed by cardiac injury produced by ischemia/reperfusion (I/R).

Methods

Right uninephrectomy (UNX) was performed in Wistar rats (n=28). Sixteen weeks after UNX, rats underwent cardiac I/R injury and randomly assigned to proANP31-67 (50 ng/kg/day s.c., n=15) or Vehicle (n=13) for four weeks post I/R. Echocardiographic examinations were performed at baseline (before UNX), 16 weeks after UNX, and four weeks after I/R. At the end of the study, cardiomyocytes were isolated and tissue samples were collected.

Results

Chronic UNX resulted in diastolic impairment (E/A: 1.47±0.08 at baseline vs 0.98±0.14 at 16 wks post UNX, p=0.0010). I/R further accentuated the development of the cardiorenal syndrome, and induced a mild systolic dysfunction in the placebo treated animals. However, four weeks of treatment with proANP31-67 preserved systolic function (EF: 62±3% placebo vs 74±2% proANP31-67, p<0.0001), and reverted the diastolic dysfunction (E/A: 0.72±0.15 placebo vs 1.24±0.11 proANP31-67, p=0.0134). ProANP31-67 ameliorated the adverse cardiac remodeling (i.e., reduction in the cardiomyocyte cross-sectional area and interstitial fibrosis), enhanced Ca2+ handling, and improved cardiomyocyte t-tubules´ structural changes compared to vehicle. At the cellular level, in vitro experiments demonstrated the direct effect of proANP31-67 on cardiomyocyte hypertrophy (assessed by [3H]-leucine incorporation) induced by endothelin 1 and angiotensin II.

Conclusion

ProANP31-67 has a direct cardiomyocyte protective effect, leading to an improvement in Ca2+ homeostasis and t-tubules´ structures and, prevents the development of systolic and diastolic dysfunction in a pre-clinical model of cardiorenal syndrome.
Towards advanced cardiac miRNA therapies through DNA-based nanostructures
Authors: Hernandez-Bellido N , Postigo A , Orrite C , Garrido-Huescar E , Garcia-Mendivil L , Pueyo E , Hernandez-Ainsa S , Ordovas L
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Proyecto MINECO-Ministerio de Ciencia e Innovación (Spain)- PID2019-105674RB-I00

Proyecto DGA- Diputación General de Aragón (Spain)- LMP128_21

Introduction

Cardiac microRNAs (miRNAs) have been found to be dysregulated in cardiac disease and aging both in animal models and humans. Thanks to their pleiotropic effects, miRNAs are spotlighted as promising therapeutic targets to treat cardiac conditions. However, their low stability and potential off-target effects in vivo difficult the development of effective and safe miRNA therapies. Nanotechnology enables the fabrication of biocompatible carriers to perform targeted delivery of both conventional and advanced therapies. In particular, nanostructures built with DNA (DNS) have been successfully developed for miRNA therapies in cancer, but they have not been applied in cardiac therapy yet [1].

Purpose

The main goal of this study is to demonstrate the capacity of DNS to deliver miRNA therapies in human cardiomyocytes.

Methods

Our group has identified MIR24-2 upregulated with age in the human left ventricle and miR24-2-5p to interact with genes related with the cardiac function, such as SERCA2 [2]. Different versions of DNS were designed and generated by self-assembly of DNA strands containing anti-miR24-2-5p capture segments. Physicochemical characterization of DNS was then conducted to determine their auto-assembling capacity, size, morphology and thermal stability. The characterization of the biological effect of the DNS was also conducted in vitro and in cell culture of HEK293 and human induced pluripotent stem cells (iPSC)-derived cardiomyocytes (iCM) to assess their cytotoxicity and internalization capacity.

Results

DNS were assembled correctly and reproducibly into carriers of around 30-50 nm. In vitro, the DNS showed thermal stability at physiological temperature, proper stability in serum and they disassembled specifically in the presence of the target miR24-2-5p sequence. In HEK293 and iCM cultures, the DNS showed lack of cytotoxicity demonstrating in vitro biocompatibility. DNS were efficiently internalized by HEK293, but the uptake capacity of DNS by iCM was remarkably lower.

Conclusions

We created and characterized biocompatible anti-miR-loaded DNS that effectively captured the target miR-24-2-5p in vitro. DNS internalized in iCM, but with lower efficiency than in HEK293 cells. Our results indicate that DNS are suitable candidates to carry miRNA therapies to human cardiac cells in vitro, but the observed cell-type specific differences suggest that future efforts need to be directed towards developing DNS functionalization capable of promoting efficient and specific carrier uptake in vivo by primary cardiac cells in vivo.
Cardiomyocyte fusion as a new mechanism contributing to pathological cardiac hypertrophy
Authors: Colliva A , Vodret S , Bongiovanni W , Zacchigna S
Published: June 2022

Funding Acknowledgements

Type of funding sources: None.

Introduction

Pathological cardiac hypertrophy is a maladaptive remodeling response, induced by multiple pathogenetic mechanisms, including genetic mutations and hypertension. Despite the high prevalence of this condition, the precise mechanisms driving its onset and progression remain elusive. Recent evidence indicates that homotypic cell fusion between cardiomyocytes occurs in the heart of lower vertebrates in response to injury, as well as in the rodent heart during the first week after birth, when cardiomyocyte size and ploidy increase. We hypothesized that homotypic fusion of cardiomyocytes could be triggered by pressure overload and contribute to cardiac hypertrophy.

Methods and Results

We exploited a genetic mouse model (MHC-CreER/mTmG mice) to genetically label individual cardiomyocytes by either red or green fluorescence. These mice express a tamoxifen inducible Cre recombinase (CreER) under the control of the myosin heavy chain 6 promoter (MHC). Controlled injection of tamoxifen resulted in a mosaic heart, composed by either red or green cardiomyocytes. In this genetic background, we performed transverse aortic constriction (TAC) to induce cardiac hypertrophy and analyzed cardiomyocyte size and fluorescence at one month.

The systematic analysis of more than 5000 cardiomyocytes from 6 independent experiments showed a significant increase in the number of yellow cardiomyocytes after TAC compared to sham hearts, consistent with cell-cell fusion events occurred between green and red cardiomyocytes. Moreover, these yellow hypertrophic cardiomyocytes displayed a higher rate of polynucleation, with many cells bearing three or more nuclei.

Conclusions and future perspective

Collectively, these data point to cardiomyocyte fusion as a new mechanism responsible for to the onset of cardiac hypertrophy upon pressure overload. We cannot exclude that yellow, polynucleated cardiomyocytes derived from the heterotypic fusion between a green cardiomyocyte and other red cell types. To rule out this possibility we are currently performing a new a set of experiments in which 100% of cardiomyocytes are labelled in green. In the case of homotypic fusion, we should not observe any yellow cells upon TAC. Proving that cardiomyocyte fusion contributes to cardiac hypertrophy in response to increased pressure load and dissecting the underlying molecular mechanisms will pave the way to novel strategies to both prevent and revert this highly prevalent pathological condition.
Identification of small-molecule ERBB4 agonists for the treatment of heart failure
Authors: Feyen E , Cools J , Van Fraeyenhove J , Tubeeckx M , De Winter H , Audenaert D , De Keulenaer GW , Segers VF
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Dehousse fellowship

Introduction

Although progress has been made in the treatment of heart failure, morbidity and mortality remain high, requiring new therapeutic targets. The neuregulin-1 (NRG1)/ERBB4 axis is cardioprotective and antifibrotic when activated in the myocardium, and therefore a possible target for therapy. Phase 2 and 3 clinical trials with NRG1 are ongoing, but require intravenous administration regimens, limiting applicability and efficacy.

Purpose

To develop small-molecule ERBB4 agonists with cardioprotective and antifibrotic properties.

Methods

A high-throughput screening (HTS) of 10,240 compounds was performed on a ERBB4/ERBB4 dimerization assay. Hit compounds were co-administered with NRG1 or fluorescently labeled NRG1 to determine competitive binding. Selectivity, receptor phosphorylation, cell proliferation and toxicity were determined using Luminex RTK phosphoprotein, ERBB2/ERBB3 dimerization, WST-1 colorimetric, and adenylate kinase assays. Antifibrotic effects were studied in vitro on TGF-β-induced collagen synthesis in human dermal and atrial fibroblasts, and in a mouse model of angiotensin II (AngII, 1000 ng/kg/min)-induced left ventricular (LV) myocardial fibrosis with selected compounds (83 µg/kg/h), administrated with osmotic minipumps (N=4–5/group). mRNA expression was evaluated after 7 days; LV myocardial fibrosis area, cardiomyocyte cross sectional area (CSA), echocardiographic parameters and heart- to bodyweight ratio (HW:BW) were analyzed at 28 days. Antiapoptotic effects were studied on rat atrial cardiomyocytes (AM) after hydrogen peroxide (H2O2)-induced cardiotoxicity.

Results

The HTS (Z’=0.7) resulted in 8 similar pyrimidine derivatives (EF-1–8) inducing ERBB4/ERBB4 dimerization (Emax 9–33% relative to NRG1, EC50 6E-6 to 2E-7 M). Competition assays indicate allosteric binding. The compounds also significantly potentiated NRG1-induced ERBB4 receptor dimerization up to 2.7 fold. Two compounds were excluded because of in vitro toxicity. The other 6 compounds were non-toxic and induced ERBB4, but neither ERBB1, ERBB2 or ERBB3 phosphorylation, nor tumor growth–inducing ERBB2/ERBB3 dimerization. Selected compounds showed significant dose-dependent antiapoptotic properties on H2O2-stimulated AM, and antifibrotic effects on human atrial and dermal fibroblasts. In vivo, compound EF-1 significantly decreased myocardial fibrosis (by 76±26%) and Col1a1, Col3a1 (-70±17%; -61±20%), and Nppa (-78±32%) mRNA expression, and significantly enhanced cardiomyocyte CSA (+24±8%). No differences were observed in cardiac function or HW:BW ratio.

Conclusion

We identified novel pyrimidine derivative small-molecule ERBB4 agonists with cardiomyocyte protective effects and antifibrotic properties in vitro and in AngII-induced myocardial fibrosis in vivo.
The development of systolic heart failure in case of pressure overload-induced left ventricular myocardial hypertrophy is associated with a unique microRNA expression profile in a rat model
Authors: Ruppert M , Korkmaz-Icoz S , Agg B , Sayour AA , Olah A , Nagy D , Benke K , Ferdinandy P , Merkely B , Szabo G , Radovits T
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): New National Excellence Program of the Ministry of Human Capacities

Introduction

Growing body of evidence suggests that distinct alterations in myocardial microRNA (miRNA) expression contribute to pressure overload (PO)-induced pathological cardiac remodeling. Nevertheless, it is still under intense investigation whether the changes in miRNA expression patterns are also associated with the decompensation of LV systolic function in case of PO-evoked LV hypertrophy (LVH). Hence, we aimed to characterize miRNA expression in PO-induced LVH with and without systolic heart failure (HF).

Methods

PO was evoked by abdominal aortic banding (AB) in male Sprague-Dawley rats. Age-matched, sham-operated animals served as controls. Functional and morphological alterations were assessed by echocardiography and histology. At the end of the experimental period, rats in the AB group were subcategorized based on ejection fraction [EF] into ABLVH (EF>40%) and ABHF groups (EF<40%). Global miRNA expression profiling was performed using next generation sequencing. Bioinformatics analysis was carried out to predict miRNA-target interactions. Expression of selected target genes was measured by qRT-PCR.

Results

Increased heart weight-to-tibial length, LV mass and fibrosis confirmed the development of pathological LVH in both the ABLVH and ABHF groups. Nevertheless, increased lung weight-to-tibial length, chamber dilatation and severely reduced EF was noted only in the ABHF and not in the ABLVH, when compared to the sham group. 50 miRNA showed different expression in the ABHF compared to the ABLVH group. Based on the altered gene expression profile, in silico bioinformatics analysis predicted several target genes. Among them, reduced mRNA expression level of Fmr1 (FMRP translational regulator 1), Zfpm2 (zinc finger protein, multitype 2), Wasl (WASP like actin nucleation promoting factor), Ets1 (ETS proto-oncogene 1) and Atg16l1 (Autophagy Related 16 Like 1) was confirmed in ABHF compared to ABLVH.

Conclusions

Decompensation of systolic function in PO-induced LVH is associated with unique miRNA profile leading to specific regulation of gene expression.
Calcium, contraction, and hemodynamic alterations induced by arrhythmogenic drugs in the zebrafish larvae
Authors: Salgado-Almario J , Vicente M , Molina Y , Martinez-Sielva A , Vincent P , Domingo B , Llopis J
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): MCIN/AEI/ 10.13039/501100011033 and “ERDF A way of making Europe” by the European Union

Background

Ca2+ ion is pivotal in excitation-contraction coupling. The dysregulation of Ca2+ fluxes is at the core of inherited and acquired arrhythmias. In recent years zebrafish have become an attractive vertebrate model to study heart development, cardiotoxicity, and heart diseases. We recently reported that the transgenic zebrafish line Tg(myl7:Twitch-4) expressing the ratiometric Ca2+ indicator Twitch-4 in the heart, allowed imaging Ca2+ transients (CaT) simultaneously with heart contraction in zebrafish larvae, thus maintaining the physiological mechano-electrical feedback.

Purpose

This study aimed to detect subtle disturbances in Ca2+ cycling, contractility, and hemodynamics induced by arrhythmogenic drugs in zebrafish larvae.

Methods

We have used the zebrafish transgenic line Tg(myl7:Twitch-4) and a custom software to acquire and analyze fluorescence emission ratio images of the heart. Larvae of 3- and 5-days post-fertilization (dpf) were incubated with arrhythmogenic drugs, and their effects on the systolic and diastolic Ca2+ and the CaT amplitude in the atrium and ventricle were determined. As the ventricular diameter was assessed in the same fluorescence images, a simultaneous, real-time, measurement of contraction correlated with Ca2+ was obtained. The systolic and diastolic areas, measured independently, were used to estimate hemodynamic parameters.

Results

Dofetilide caused time-dependent bradycardia and ultimately a 2:1 arrhythmia in 3 and 5 dpf larvae, with an increase in the CaT amplitude. the ejection fraction and the stroke volume increased, but the cardiac output decreased due to the low heart rate (2:1 arrhythmia). The T-type Ca2+ channel antagonist ML218 induced bradycardia and decreased systolic and diastolic Ca2+ levels in 3 dpf larvae, but the CaT amplitude did not change. Interestingly, some larvae (3 out of 14) displayed a progressive lengthening of the time between the start of atrial and ventricular CaT, analogous to increased PR interval of the electrocardiogram, until a ventricular systole failed. This is reminiscent of a second-degree atrioventricular block in humans. At higher ML218 concentration, a 2:1 arrhythmia was observed and increased ventricular CaT amplitude in all larvae.

Conclusions

We characterized Ca2+ changes induced by arrhythmogenic drugs and correlated them with contractility and hemodynamic parameters. The zebrafish line Tg(myl7:Twitch-4) allowed investigating drug-induced arrhythmias in this animal model with unprecedented detail. This transgenic line will serve to screen cardiotoxicity during drug development and to study conduction and rhythm defects in zebrafish, and their correlation with human disease.
Artificial light pollution and obesity as a risk factors of malignant cardiac arrhythmias - antiarrhythmogenic effect of omacor
Authors: Szeiffova Bacova B , Sykora M , Egan Benova T , Zurmanova J , Andelova K , Tribulova N
Published: June 2022

Funding Acknowledgements

Type of funding sources: None.

Light pollution disrupts circadian rhythms and increases the development of obesity, but their connection to cardiovascular disease, in particular to malignant arrhythmias, is not well known.

Herein, we investigated if female rats exposed to continuous light and high-sucrose diet had altered myocardial gene transcripts and/or protein expression which affects arrhythmogenesis. We then assessed if Omacor® supplementation benefitted affected rats.

In a first experiment, adult female spontaneously hypertensive (SHR) and normotensive Wistar rats (WR) were housed under standard 12 h/12 h light/dark cycles or exposed to 6-weeks continuous 300 lux light for 24 h. In a second experiment, adult female WR rats received sucrose diet (30% sucrose solution) continuously (24 h) performed for 8 weeks. Half the rats from both experiments were throughout the experiment treated with 200 mg/100g b.w. Omacor®.

Continuous light resulted in higher rat vulnerability to malignant ventricular fibrillation (VF). This was linked with myocardial connexin-43 (Cx43) down-regulation and deteriorated intercellular electrical coupling, due in part to increased pro-inflammatory NF-κB and iNOS transcripts and decreased sarcoplasmic reticulum Ca 2+ ATPase transcripts. Omacor® treatment increased the electrical threshold to induce the VF linked with amelioration of myocardial Cx43 mRNA and Cx43 protein levels and the suppression of NF-κB and iNOS.

8 weeks lasting intake of 30% sucrose solution downregulated Cx43 and PKCε signaling along with an upregulation of myocardial PKCδ and miR-30a rendering the heart prone to ventricular arrhythmias. There was a clear benefit of Omacor® supplementation due to their antiarrhythmic effects associated with the attenuation of myocardial Cx43, PKC, and miR-30a abnormalities.

This indicates that rat exposure to continuous light and high-sucrose result in deleterious cardiac alterations jeopardizing intercellular Cx43 channel-mediated electrical communication, thereby increasing the risk of malignant arrhythmias. The adverse effects were attenuated by treatment with Omacor®, thus supporting its potential benefit and the relevance of monitoring omega-3 index in human populations at risk.