Joint Annual Meeting 2019 of the Swiss Society of Cardiology and the Swiss Society of Cardiac Surgery
P50
GDF11 promotes increased sensitivity of the murine heart to ischemic injury
A. Akhmedov1, F. Montecucco2, G.G. Camici1, A. Schaub Clerigue1, D. Vdovenko1, S. Costantino1, N.R. Bonetti1, C. Diaz Canestro1, F. Paneni1, F. Mach3, T.F. Lüscher1,4
1Center for Molecular Cardiology, University of Zurich, Schlieren, Switzerland, 2Department of Internal Medicine, University of Genoa School of Medicine., Genoa, Italy, 3Division of Cardiology, Foundation for Medical Researches, University of Geneva, Geneva, Switzerland, 4Royal Brompton and Harefield Hospitals and Imperial College, London, United Kingdom
Introduction: Recent studies have implicated a role of TGFβ family members in aging and cardiovascular diseases. Growth Differentiation Factor 11 (GDF11) is a member of TGFβ superfamily with high homology to myostatin/GDF8. Interestingly, in mice its levels decline with age, whereas myostatin and TGFβ1 levels remain unchanged, suggesting involvement of GDF11 in aging. In addition, GDF11 has recently been shown to play a role in cardiac hypertrophy. However, not much is known about its role in the myocardium. The goal of the present study was to investigate whether restoring GDF11 levels of aged mice to the ones observed in young mice by injecting recombinant GDF11 into blood stream would rescue myocardial infarction and provide “youthful” characteristics to the old myocardium. Therefore, restoring GDF11 levels in aged mice to the ones observed in young mice would improve the myocardial infarction outcome and provide “youthful” characteristics to the old myocardium.
Methods: 12-14-week-old and 22-24-month-old C57BL/6 male mice were injected daily with either recombinant human GDF11 or vehicle for 30 days. Afterwards mice were subjected to 30 min of ischemia (I) followed by 24h of reperfusion (R). Infarct size was assessed morphologically.
Results: After I/R, both young and aged GDF11-injected mice developed markedly larger infarcts as compared to vehicle-treated group. This was further associated with increased post-ischemic levels of serum cardiac troponin I. In addition, both GDF11-injected groups showed accelerated cardiac cell death after I/R as has been assessed by TUNEL on heart cross sections. Of note, both ageing groups showed higher mortality during the GDF11 treatment. Finally, cardiac transcription factor Nkx2.5 and its co-factor Gata4 were less activated in both GDF11-treated groups.
Conclusions: In summary, present study showed that daily injections of GDF11 promote increased sensitivity of the heart to myocardial infarction. Such GDF11-associated cardiac phenotype is likely to be driven by the increased cell death in the injured myocardium together with impaired function of prosurvival cardiac pathways involving Nkx2.5. Thus, these results do not support proposed role of GDF11 as “rejuvenation” factor for the heart.
P51
Targeting the methyltransferase setd7 protects the heart against ischemic damage
S. Ambrosini1, F. Montecucco2, A. Akhmedov1, S. Mohammed1, T. Lüscher1,3, S. Costantino1, F. Paneni1
1Center for Molecular Cardiology, University of Zürich, Schlieren, Switzerland, 2University of Genoa, Genoa, Italy, 3Royal Brompton & Harefield Hospital, Imperial College, London, United Kingdom
Introduction: Despite significant advances in coronary revascularization, acute myocardial infarction remains the leading cause of heart failure and death worldwide. The Hippo pathway plays a crucial role in cell survival after myocardial ischemia. Upon cellular stress, the Hippo pathway is activated leading to cytosolic retention and degradation of the pro-survival transcription factor YAP. Post-translational modifications have shown to regulate YAP activity. The protein SET domain-containing lysine methyltransferase 7 (SETD7) is emerging as a pivotal modulator of protein functionality and gene expression.
Methods: Neonatal rat ventricular myocytes (NRVMs) were exposed to normal glucose levels or glucose deprivation (GD) for 15 h, in the presence of a selective SETD7 inhibitor [(R)-PFI-2]. Western blot and qPCR were employed to investigate the effects of energy stress on SETD7 and the Hippo pathway, while apoptosis was assessed by Caspase-3 activity assay. YAP localization was examined by confocal microscopy. SETD7 knockout (SETD7-/-) mice and wild-type (WT) littermates underwent 1 h of left anterior descending (LAD) coronary artery ligation followed by 24 h of reperfusion. Infarct size was assessed by TTC staining, whereas cardiac function was investigated at 24h by echocardiography.
Results: GD for 15h in NRVMs increased YAP phosphorylation, thus leading to its cytosolic retention, as assessed by confocal microscopy. Reduced YAP nuclear content was associated with downregulation of pro-survival YAP target genes, namely Ctgf and Fgf2. GD-induced YAP inactivation was associated with an increase in SETD7 expression. Interestingly, pharmacological inhibition of SETD7 by (R)-PFI-2 restored nuclear retention of YAP in GD-treated NRVMs, thus increasing cell survival. In line with our in vitro findings, SETD7-/- mice showed decreased infarct size as compared to WT littermates. Consistently, cardiac function, as assessed by ejection fraction, fractional shortening as well as by Tissue Doppler Imaging, was preserved in mice lacking SETD7.
Conclusions: Our findings suggest that - in conditions of myocardial ischemia - SETD7 triggers cardiomyocyte apoptosis via increased YAP methylation and subsequent reduction of YAP-dependent pro-survival genes. Pharmacological modulation of the Hippo pathway by SETD7 may represent a novel therapeutic approach to prevent myocardial damage in patients with ischemic heart disease.
P52
Circulating exosomes as biomarkers in patients after ST-elevation myocardial infarction
J. Burrello1,2, S. Bolis1, E. Caporali1, L. Grazioli1, A. Burrello3, L. Barile1, G. Vassalli1
1Laboratory of Cellular and Molecular Cardiology, Cardiocentro Ticino Foundation, Lugano, Switzerland, 2Division of Internal Medicine and Hypertension, Department of Medical Sciences, University of Turin, 3Department of Electronics and Telecommunications, Polytechnic University of Turin, Torino, Italy
Introduction: Circulating extracellular vesicles including exosomes are released from a variety of cells (e.g., endothelial cells, inflammatory cells, platelets, cardiac cells), reflecting their activation in response to stimuli. As such, changes in circulating vesicles in response to acute myocardial injury may have diagnostic relevance. The aim of the study was to develop a reliable prediction model for the diagnosis of STEMI based on surface markers expressed on circulating exosomes.
Method: Blood was collected from 17 patients after STEMI (< 4 hrs from pain onset) and 24 healthy subjects with documented absence of CAD (controls). Exosomes were isolated from sera using microbeads coated with antibodies directed against the exosome markers CD9, CD63 and CD81 and characterized by nanoparticle tracking analysis (NTA). In a first step, a multiplex FACS analysis including 37 surface markers of interest was performed on the sera from 6 STEMI patients and 6 controls. Using a linear discriminant analysis (LDA) model, 9 markers were identified as possible discriminants of STEMI patients vs. controls. These markers were then analyzed in the entire cohort. The LDA model was validated by a leave-one-out algorithm.
Results: Using NTA, total vesicle number in serum was significantly increased in STEMI patients vs. controls (1.7-fold increase; P=0.043). Vesicle size distribution was consistent with approximately 75% of vesicles being exosomes in both groups. The multiplex FACS analysis identified CD2, CD31, CD40, CD41b, CD42a, CD49e, CD62P, CD146, and HLA-ABC as potential discriminants between groups. Of note, some of these markers are platelet and/or endothelial markers. The LDA model including the above 9 markers correctly classified 38 of 41 patients (prediction accuracy 92.7%; sensitivity 94.1%; specificity 91.7%; accuracy at validation 83.3%).
Conclusion: Circulating exosomes are increased in patients after STEMI. Expression levels of 9 surface markers on serum vesicles allows the discrimination of STEMI patients and controls. We currently are validating these results in a larger cohort. Our data suggest that circulating exosomes may constitute useful diagnostic biomarkers of cardiac injury.
P53
Fatty acid-induced activation of merlin causes endothelial dysfunction: insights in patients with cardiometabolic disturbances
S. Costantino, S.A. Mohammed, A. Akhmedov, G.G. Camici, T.F. Luscher, F. Paneni
Center for Molecular Cardiology, University of Zurich, Schlieren, Switzerland
Introduction: Obesity is strongly associated with endothelial dysfunction and cardiovascular disease. Yet, the underlying mechanisms remain to be deciphered. Merlin, a protein encoded by the NF2 gene, is a scaffold-like protein implicated in various cellular processes, namely growth, differentiation and survival. Merlin is inactivated by Akt-dependent phosphorylation at Ser518, whereas its dephosphorylation by the myosin phosphatase target subunit 1 (MYPT-1) leads to an active conformation. Whether Merlin participates to obesity-related endothelial dysfunction is unknown.
Methods: Human aortic endothelial cells (HAECs, passages 5-6) - cultured in endothelial growth medium-2 (EGM-2) supplemented with 2% fetal bovine serum (FBS) - were exposed to palmitic acid (PA, 200 uM) or vehicle for 48 hours, a well-established in vitro model mimicking obesity. Gene silencing of Merlin was performed by small interfering RNA (siRNA). Gene and protein expression were assessed by real time PCR and Western blot, respectively. Cellular apoptosis was assessed by caspase-3 activity assay, whereas 3-nitrotirosine (3-NT) was employed to measure oxidative stress levels. Interaction of Merlin with endothelial proteins was investigated by co- immunoprecipitation.
Results: Exposure of HAECs to PA significantly decreased Merlin phosphorylation at Ser518. This finding was explained by a reduction of Akt phosphorylation at Ser473 and a concomitant increase of MYPT-1 phosphorylation at Thr696. We next asked whether PA-induced Merlin activation was involved in the modulation of proteins relevant to endothelial homeostasis. Pull-down experiments revealed that Merlin binds and activates Caveolin-1 (Cav-1), a pivotal repressor of endothelial nitric oxide synthase (eNOS) catalytic activity. Interestingly, Merlin knockdown in PA-treated HAECs prevented eNOS-caveolin-1 interaction, thus preserving eNOS functionality. Consistently, gene silencing of Merlin blunted PA-induced endothelial apoptosis and oxidative stress, as assessed by caspase-3 and 3-NT levels.
Conclusions: Targeting Merlin may contribute to rescue endothelial damage in the setting of cardiometabolic disorders.
P54
Transcatheter Aortic Root Replacement: a preliminary in-vitro feasibility study
E. Ferrari1, P. Tozzi2, D. Berdajs3, F. Maisano4, L. Von Segesser2
Objective: TAVR is widely used in elderly patients with aortic stenosis. However, some patients (25%) have dilated aortic roots that are not addressed with TAVR. The concept of the new TARR technique combines aortic valve replacement and aortic root replacement in a single-stage transcatheter procedure. We present a preliminary feasibility in-vitro test.
Methods: 3D-printed aortic roots based on pre-operative CT-scans of selected patients with root dilation were prepared. Hydrodynamic tests were performed on 3D printed models with original configuration. A new device integrating a trans-catheter valve, an aortic prosthesis and two conduits for coronary perfusion was assembled. The device was deployed inside the 3D-printed aortic root and hydrodynamic tests were performed to test coronary perfusion.
Results: Mean non-pulsatile coronary flows for the standard root at 60,80 and 100 mmHg were 1066, 1307, 1460 mL/min. for the left coronary and 146, 233, 233 mL/min for the right coronary. Mean coronary flows for the root with the deployed TARR device at 60, 80 and 100 mmHg were1053, 1306, 1502 mL/min for the left coronary artery and 110, 146, 162mL/min for the right coronaryartery.
Conclusions: This preliminary report shows that the TARR technique guarantees an adeguate coronary flow in 3D-printed aortic roots when the device is deployed. Further tests are required to cofirm these data.
P55
Devices and methods for transcatheter removal of mechanical heart valve leaflets
P.P. Heinisch1,2, R. Brecht3, D. Obrist4, T. Carrel1, A. Khoynezhad2
1Cardiovascular Surgery | Cardiac Surgery, Inselspital Bern, Berne, Switzerland, 2Division of Cardiothoracic Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, United States, 3Department of Anaesthesiology, University Hospital Regensburg, Regensburg, Germany, 4ARTORG Center for Biomedical Engineering Research, University of Bern, Berne, Switzerland
Introduction: Heart valve replacement with a mechanical or a biological prosthesis is the primary treatment option for patients with severe aortic valve disease. Redo-surgery for patients with prosthetic heart valves is associated with an increased morbidity and mortality rate. For patients with previous bioprosthetic heart valve, the valve-in-valve transcatheter procedure has become a feasible and safe alternative. However, this procedure cannot be offered to patients with mechanical heart valves (MHV).Our group developed a novel device to expand the availability of transcatheter valve-in-valve replacement for patients with MHV.
Methods: In order to perform a valve-in-valve procedure in a MHV, the stiff pyrolytic carbon leaflets have to be removed in order to place a new transcatheter valve. Our laboratory has invented a novel device and methodology for transcatheter removal of MHV leaflets (Figure P55-1). The transcatheter device consists of two parts: (1) a balloon for dislodging the MHV leaflets and (2) a retrieval device that will catch and transport the two valve leaflets safely. Additionally a valve-in-valve procedure is performed in the excisting mechanical valve annulus.
Results: The feasibility of the novel device was evaluated in a pressurised in-vitro model using various sizes of mechanical heart valves. A critical issue of the disassembly is to avoid breaking the leaflets. The positioning of the device was performed within 1.4±0.4 minutes.
The removal of the mechanical leaflets took 4.5±0.6 seconds. The implantation of the valved stent was completed after 1.3±0.4 minutes, and it remained in a stable position in the prosthetic annulus. Our preliminary data has shown that using a non-compliant balloon increases the force on the leaflet. This allowed for a faster disassembly, but there was a higher risk of fracturing at least one leaflet into multiple fragments. The safe retrieval of the valve fragments in their entirety is an important step of the procedure. Therefore, the direction of blood flow, as well as the angulation and size of the blood vessels, have to be considered.
Conclusions: This technology offers solutions to an unmet clinical need in the field of cardiovascular medicine. This novel device can obviate the need for risky open-heart surgery, thereby reducing morbidity and mortality, and providing an interventional option to patients with mechanical heart valve who are considered inoperable or high-risk for open-heart surgery.
Figure
P55-1: Device construction and concept of dislodging with retrieval of the valve leaflets.
P56
Human iPSC-derived cardiomyocytes as cell-based platform to study cardiac senescence
E. Lazzarini1, A.M. Lodrini2, S. Bolis1, G. Vassalli1, M. Rocchetti2, C. Altomare1, L. Barile1
1Laboratory of Cellular and Molecular Cardiology, Cardiocentro Ticino, Lugano, Switzerland, 2Biotechnology and Bioscience, University of Milan Bicocca, Milan, Italy
Introduction: Ageing of Cardiomyocytes (CM) involves a series of structural and functional adverse remodelling, including QTc prolongation and metabolic imbalance, which contributes to the insurgence of cardiovascular diseases, including heart failure. This study exploits the potential of human CMs derived from induced pluripotent stem cells (hiPSCs) as an in vitro model for cardiac senescence. Such platform will be exploited to characterize mechanisms involved in cardiac ageing and to screen anti-aging drugs. In particular, cardiac progenitor cells (CPC)-derived exosomes (EXO), which has been previously shown as a source of cardioprotective factors, will be tested as anti-senescence agent.
Methods: Patient-derived CPCs were reprogrammed into hiPSCs and subsequently differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs). Finally, senescence phenotype was induced through a specific protocol based on exposure to doxorubicin (DOX) at sub-lethal concentration. Senescence induction was highlighted by protein and gene expression analysis and senescence-associated b-gal assay. Electrical activity of hiPSC-CMs was evaluated recording extracellular field potentials through a multielectrode arrays. Metabolism was evaluated with specific biochemical assay and by gene expression analysis.
Results: DOX treatment in hiPSC-CMs induce a senescent phenotype, as confirmed by activation of p21 and p16 pathway and increase of senescence associated β-gal staining positivity, compared to untreated cells (CTRL). DOX treated hiPSCs-CMs showed prolonged QTc in comparison to CTRL and this effect was prevented by EXO treatment. Biochemical and gene expression analysis revealed a metabolic switch of DOX-CMs towards glycolysis, with a concomitant reduction in the b-oxidative metabolism which results in a decreased ATP/AMP ratios.
Conclusion: Preliminary results highlight how the hiPSC-CMs based cellular model recapitulates the phenotype of aged CMs, in particular concerning electrical and metabolic features. In addition, our preliminary findings suggest the potential cardioprotective role of EXO in reducing age-related modifications of CMs. Further exploiting of this platform will include evaluation of anti-aging drugs and to deepen the study of aging-involved molecular mechanisms.
P57
Endothelial SIRT6 exerts a beneficial role in cerebral ischemia/reperfusion injury by preserving blood-brain barrier integrity
L. Liberale1,2, A. Akhmedov1, N.R. Bonetti1, V. Nageswaran1, S. Costantino1, J. Pahla1, C.M. Matter1, F. Montecucco2, J.H. Beer1, F. Paneni1, T.F. Lüscher1, G.G. Camici1
1Center for Molecular Cardiology, University of Zurich, Schlieren, Switzerland, 2Department of Internal Medicine, University of Genoa, Genoa, Italy
Figure
P57-1.
Figure
P57-2.
Introduction: Therapeutic strategies for stroke are limited to the early reperfusion which can, on the other hand, worsen the brain damage trough ischemia/reperfusion (I/R) injury. The extent of post-stroke blood-brain barrier (BBB) impairment is associated with worsened outcome. Aging is a major risk factor for stroke and genes regulating lifespan also contribute to the determination of cerebral damage during I/R injury. Given the pivotal role of endothelial cells in BBB, we hypothesized that the endothelial-specific expression of the longevity gene SIRT6 may protect the BBB from ischemia/reperfusion damage thus having a beneficial role on stroke outcome.
Methods: Endothelial-specific SIRT6 knockout (eSIRT6-/-) mice and control littermates (CTRL) underwent transient middle cerebral artery occlusion (tMCAO). Primary human brain microvascular endothelial cells (HBMVECs) transfected with either SIRT6 (siSIRT6) or scrambled (siSCR) small interfering RNA were subjected to hypoxia/reoxygenation (H/R) and used as an in vitro BBB model. SIRT6 mRNA expression in monocytes from stroke patients was correlated with the short-term neurological outcome [ΔNIHSS% = (NIHSS discharge-NIHSS admission)/ NIHSS admission*100].
Results: eSIRT6-/- displayed higher infarct volumes and lower survival rate compared to WT mice. Furthermore, eSIRT6-/- also showed worse post-stroke neurological impairment. Analysis of brain sections revealed increased BBB damage and increased endothelial expression of cleaved caspase-3 in eSIRT6-/- animals (Fig. P57-1). In vitro, H/R reduced SIRT6 expression in HBMVECs. Mirroring the animal results, SIRT6 silencing impaired the barrier function of HBMVECs as siSIRT6-treated cells showed lower resistance values as compared to siSCR-treated ones 48 h after exposure to H/R. In line with this, SIRT6-silenced HBMVECs showed reduced viability, increased cleaved caspase-3 expression and reduced activation of the anti-apoptotic survival pathway Akt as compared to control cells after H/R. The direct interaction between SIRT6 and Akt was confirmed by co-immunoprecipitation. In ischemic stroke patients, SIRT6 expression was higher in those with short-term neurological improvement (ΔNIHSS% > 0) and negatively correlated with ΔNIHSS% (Fig. P57-2).
Conclusion: SIRT6 exerts a beneficial role in ischemic stroke by blunting I/R-induced endothelial apoptosis and BBB damage. The longevity gene SIRT6 may represent a novel therapeutic target for the treatment of ischemic stroke.
P58
The nuclear receptor corepressor 1 blocks CD36-mediated foam cell formation in atherogenesis
S. Oppi1, S. Nusser-Stein2, V. Marzolla3, E. Osto4, Z. Rancic5, T.F. Lüscher6, M.H. Oosterveer7
1Centre for Molecular Cardiology, 2University of Zurich, 3Univeristy of Zurich, Schlieren, 4ETH Zurich, 5University Hospital Zurich, 6University of Zurich, Zurich, Switzerland, 7Department of Pediatrics and Laboratory Medicine, University Medical Centre Groningen, Groningen, The Netherlands
Introduction. Nuclear receptors and their cofactors regulate the expression of various target genes in different tissue and organs to orchestrate downstream (patho) physiological processes. Although the function of several nuclear receptors in atherosclerosis has been studied, very little is known about the role of nuclear receptor cofactors in atherosclerosis. Given its important role to suppress inflammatory processes, we speculated that macrophage nuclear receptor corepressor 1 (NCOR1) plays a protective function in atherosclerosis development.
Method. To evaluate the contribution of macrophage NCOR1 in atherosclerosis we exposed 8-week-old male and female myeloid cell-specific Ncor1 knockout mice to a high high-cholesterol diet for 12 weeks and assess atherosclerosis development.
Results. Our findings demonstrate that the lack of macrophage Ncor1 leads to a severe atherosclerotic phenotype in both sexes. These mice show a higher content of plaques along the thoraco-abdominal aortae as well as at the aortic sinus, which were characterized by larger necrotic cores and thinner fibrous caps, a typical signature of unstable plaques. Moreover, we found that the pro-atherogenic effects of the Ncor1 deletion are mediated via derepression of peroxisome proliferator-activated receptor gamma (PPARγ) target genes in mouse and human macrophages, especially the enhanced expression of the CD36 scavenger receptor and the subsequent rise in oxLDL uptake. Interestingly, while the expression of NCOR1 is reduced, the PPARγ signature is increased in human atherosclerotic plaques, and this signature is further pronounced in ruptured compared to stable carotid plaques.
Conclusion. Our findings suggest that macrophage NCOR1 blocks the pro-atherogenic functions of PPARγ in atherosclerosis and prevents the disease development.
