Funding Source Research Articles

Role of miRNAs regulation of Orai1 and store operated Ca2+ entry in vascular restenosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación Background The main cause of restenosis is attributed to the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). Orai1 is one of the main molecular components of Store-Operated Calcium Entry (SOCE), playing a key role in VSMCs proliferation during neointima formation and vascular remodeling. Recent studies have demonstrated the implication of microRNAs (miRNAs) in VSMCs proliferation and migration, however, the role of these miRNAs as regulators of SOCE during vascular remodeling has not been described. Purpose This study aims to determine whether miRNAs regulate the intracellular Ca2+ handling, focusing on SOCE, during neointima formation. Methods Experiments were conducted in an animal rat model following the "Principles of laboratory animal care" and according to the Spanish law. The neointima development was assessed after balloon injury of carotid arteries and confirmed by haematoxylin and eosin staining, and αSMA staining of tissue sections up to 3 weeks after surgery. miRNAs array and RT-qPCR was used to examine the expression of miRNAs. Dual-Luciferase reporter assay was performed in A7R5 VSMCs line. Results Injured arteries showed significant increase in the expression of Orai1, as compared to non-injured arteries, indicating their possible participation in the neointima layer formation. Microarray of miRNAs analysis of the injured carotids compared to control carotids showed significant increase in the expression of miRNAs related to signalling pathways involving cell proliferation and migration, cell differentiation, or VSMCs contraction. Of these, we confirmed the significant increase of miR-18a-5p, miR-20a-5p, miR-20b-5p and miR-17-5p, which belong to widely studied miR-17-92 cluster involved in different cell proliferation. Moreover, we found that mimics of those miRNAs were able to regulate differentially the activity of the Orai1 promotor in A7R5 cell line, indicating that Orai1 can be targeted by miRNAs during in VSMCs. Conclusions In conclusion, our data confirms that Orai1 is upregulated during neointima formation associated with miRNAs dysregulation. Moreover, we found that Orai1-dependent SOCE can be finely regulated by miRNAs during restenosis.

The diabetic myocardial transcriptome reveals Erbb3 as a novel biomarker of incident heart failure

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): British Heart Foundation Background/Introduction People with diabetes mellitus (DM) are at high risk of developing heart failure, but the molecular basis of this is not well understood. Elucidating the mechanistic pathways may help to improve patient stratification and develop targeted therapies. Purpose We set out to define differentially expressed genes (DEGs) in diabetic myocardium and validate arising hits in their circulating protein form, with a view to informing biomarker development and improving understanding of diabetic heart disease. Methods Transcriptomic (RNA-seq) data from the Genotype-Tissue Expression (GTEx) project was used to compare gene expression in participants with (n=>100) versus without (n>300) DM (pooled type 1 and 2 diabetes mellitus), in both the right atrial (RA) and left ventricular (LV) myocardium; analyses were adjusted for technical, demographic and disease covariates. Where available, plasma proteomic data from the UK Biobank (UKB) cohort was used to validate DEGs from GTEx in their circulating protein form. Hits with directionally concordant differential expression in GTEx and UKB were further characterised as plasma proteins in UKB, including over 50,000 participants irrespective of DM status; this included analysis of incident heart failure, cardiovascular mortality and left ventricular function on cardiac magnetic resonance imaging. Results Pooled across the RA and LV, there were 64 DEGs associated with DM, with no overlap between the RA and LV hits. UKB plasma protein data was available for 6 of these. Only ERBB3 showed directional concordance with transcriptomic data, being lower in the myocardium and blood of people with DM. People in the lowest quartile of ERBB3 plasma protein had increased adjusted risk of incident heart failure and cardiovascular death than participants in all other quartiles. Lower concentrations of plasma ERBB3 protein were also associated with increased LV mass and impaired LV contractility. Conclusion Lower plasma ERBB3 concentration is more common in people with DM and is associated with LV dysfunction, incident heart failure and cardiovascular mortality. This highlights the potential of ERBB3 as a novel biomarker for individuals at risk of heart failure. Importantly, cancer therapies targeting ERBB3 and related family members can induce heart failure, and recombinant forms of the ERBB3 ligand neuregulin-1 appear to improve LV contractility, further suggesting a role of ERBB3 in the development of diabetic heart disease.

Modeling doxorubicin-induced cardiotoxicity with human myocardial tissue slices: insights on genetic susceptibility and cardioprotection

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): PSIDER and HARVEY (ZonMW) Introduction Cardiotoxicity poses a significant challenge in drug development, often leading to the withdrawal of approved drugs. To address this issue, there is a critical need for improved models to predict cardiac adverse effects. In line with the imperative for animal-free preclinical testing, this study aimed to develop a novel model using human myocardial tissue slices to get a deeper understanding and potential prevention of doxorubicin-Induced cardiotoxicity. Methods Human myocardial tissue slices of 300 μm thick were derived from end-stage heart failure patients explanted hearts, including those harbouring likely or pathogenic cardiovascular mutations (class 4 and 5) (N=3) and non-genetic cardiovascular etiology (N=3). Myocardial tissue slices were cultured under physiological mechanical and electrical conditions. Slices were exposed to doxorubicin (1 µM) over a 10-day culture period, and their responses were compared to DMSO-vehicle controls, by recording contractile force and Ca2+ transients. Additionally, a subset of slices was treated with a cardioprotective agent, dexrazoxane (100 µM), 1 day prior to and during doxorubicin exposure to assess potential cardioprotection. Results Doxorubicin-exposed slices with a pathogenic mutation exhibited reduced mechanical force, increased threshold potential, and impaired ability to follow pacing at higher frequencies. Additionally, aberrations in calcium handling, manifested as arrhythmia-like disconnection of muscle fibers, were notably observed in these slices. Instead, slices from patients without pathogenic mutations showed minimal cardiotoxicity when exposed to doxorubicin. Encouragingly, pretreatment with dexrazoxane demonstrated a protective effect against doxorubicin-induced cardiotoxicity in the genetically susceptible group. Our functional findings corroborated our structural data, highlighting the breakdown of sarcomere structure and cell-cell connections and increased DNA damage, particularly in patients harbouring a genetic cardiovascular mutation. Conclusion In summary, human myocardial tissue slices faithfully recapitulate doxorubicin-induced cardiotoxicity. Importantly, our data suggests the utility of employing genetic testing for known cardiovascular mutations as a valuable step before initiating doxorubicin treatment. Additionally, we demonstrate for the first time the in vitro cardioprotective potential of dexrazoxane in a human tissue model. These findings pave the way for using ex vivo myocardial tissue slices to evaluate cardiotoxicity and their value as a powerful tool for evaluating the efficacy and delivery approaches.

From genes to phenotype: investigating the link between smad2rs29725537:c>a and high take off in mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Junta de Andalucía, Ministerio de Ciencia e Innovación, Gobierno de España. Introduction and Purpose High take off (HTO) is a congenital anomaly with a prevalence of 0.36% that can cause severe ischemia and sudden cardiac death. The C57Bl/6 (B6) mouse strain shows around 60% incidence of HTO, whereas it is absent in the Balb/c (BC) strain. The aim of this study was to identify the genes responsible for the development of HTO. Methods Following an inheritance study, a genetic linkage study was performed in B6C mice using 134 microsatellites (MIT). After selection of Smad2 as a candidate gene, a genotype-phenotype study was performed by PCR-RFLP genotyping of 293 B6C mice. The process of coronariogenesis was examined in E13.5 and E14.5 mouse embryos of B6 (n = 27) and BC (n = 23) strains with specific markers for the vascular plexuses involved in coronariogenesis (PROX1 and ERG 1/2/3) and for SMAD2. Finally, the SMAD2 and pSMAD2 protein expression levels were analyzed by western blot in ventricle, liver, and kidney of B6C mice. Results The incidence of HTO in B6, BC and B6C mice were concordant with an inheritance pattern mediated by a dominant causal allele and dominant modifier in the BC strain that reduces the incidence of HTO. The genetic linkage study revealed the association of a region between markers MIT-49 and 106 on chromosome 18 with the presence of HTO. This region contained a total of 10 genes, of which Smad2 was selected as a candidate gene because of its functions in aortic root formation. Comparative sequence analysis of Smad2 between the B6 and BC mouse strains uncovered an intronic SNP (Smad2rs29725537:C>A or Smad2C>A) that predicts and error in the splicing of intron 10-11. A statistically significant association (p<0.05) between the Smad2C>A allele and the presence of HTO was found. pSMAD2 was detected in embryonic coronary primordia, the subepicardial aortic plexus (SAP) and the primary coronary plexus. Around 60% of B6 examined embryos showed an exacerbated SAP compared to the BC embryos. Additionally, we detected a differential expression of total SMAD2, but not of pSMAD2, in different organs according to the Smad2C>A allele genotype (C/C > A/C > A/A). Conclusions We conclude that: 1) Smad2C>A allelic variant is associated with HTO in mice, suggesting it as a major causal factor. 2) Smad2 altered expression may affect the establishment of the embryonic coronary primordia leading to a mispositioned ostium. 3) the Smad2C>A allele has a systemic effect on total SMAD2 expression without affecting its phosphorylation level, which suggest a compensatory mechanism of expression that maintain the normal activity of pSMAD2 in most tissues.

Erythrocyte aggregability, and fibrinogen during atrial fibrillation

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Academician N. Kipshidze University Clinic - Republican Hospital; IVANE BERITASHVILI CENTER OF EXPERIMENTAL BIOMEDICINE, Department of rheology and diagnostic analytical services. Introduction Despite all biomedical efforts, the prevalence of atrial fibrillation and its associated morbidity and mortality are increasing globally year after year. The disease's unfavorable epidemiological picture determined the reality of the project. There are many hypotheses to explain the pathogenesis of atrial fibrillation. All the hypotheses are based on mechanisms at local points and microwaves (in whole or in part). None of them consider the role of hemodynamics and hemorheology in the formation and progression of atrial fibrillation. It is also unknown how atrial fibrillation affects intracardiac macro- and microcirculation. Purpose The goals of this project were to determine how hemodynamics and hemorheology change in patients with atrial fibrillation. In order to gain a better understanding of the pathological processes underlying AF, we sought to access the relationships between hemodynamics, rheological properties,and the coagulation system of blood. Methods The Erythrocyte (RBC) Aggregation Index (EAI) was monitored in the development and validation cohorts (90 patients and 20 healthy individuals) using the textural analysis system (Tas-Plus, Leitz, Germany) and an innovative "Georgian technique" with the unique function of a quantitative assessment of the EAI. The method was developed by Georgian scientists and is well-known around the world for being direct, numerical, and precise. In all patients Coagulogram and 12-lead standard ECG were performed. Results According to the data obtained, the patients' rheological and coagulation system conditions are the same, and they are distinct from the control group. During the statistical processing of the total amount of fibrinogen (Fib), the patients were divided into three categories. The average Fib in Category I patients was 336.40 ±27,77 mg/dl, and the average EAI-RBC value was 32.8± 9.4. Category II: FIB: 373.00±22.65mg/dl; EAI-RBC: 39.8±13.8 and Category III: FIB:471.27±21.58 mg/dl; EAI-RBC: 42.6±10.4. The control group: FIB: 301±18,1 mg/dl, EAI-RBC: 25.6±1.29. Fibrinogen and the Index of Erythrocyte Aggregation were found to have a linear relationship. The higher the concentration of fibrinogen, the more severe the hemorheological disorders. It should be noted that the categories were chosen without taking into account the forms of atrial fibrillation. Conclusions The findings clearly show the existence of two parallel mechanisms in the body. These are the coagulation systems, characterized by dynamic equilibrium, and the rheology systems, which lack a physiologically balanced opposing system. Coagulation control does not accurately describe hemorheological parameters, which are one of the most powerful mechanisms for atrial fibrillation formation In order to better understand the diseases' occurrence and progression, the simultaneous studies of fibrinogen concentration and rheological parameters are especially important for both fundamental and practical medicine.

Mitochondrial players as novel therapeutic options in arrhythmogenic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Joint Project Südtirol- FWF (grant number 23623; Italy-Austria) and the Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy) Background Mitochondria are dynamic organelles that sense and respond to environmental, nutritional, and pathological changes. These organelles can smartly adapt to different stressful conditions, by changing the way they network or the energy sources they use. Over the past years, numerous disorders have been associated with mitochondrial dysfunction, including cardiomyopathies. Arrhythmogenic Cardiomyopathy (ACM) is a cardiac disease characterized by the progressive replacement of the myocardium with fibro-fatty tissue. The current treatment primarily focuses on managing the symptoms rather than addressing the underlying issue of tissue substitution. Purpose: Therefore, in this study, we explored the mitochondrial and metabolic phenotype in ACM and tested the efficacy of a mitochondria-targeting library to rescue the fibro-fatty phenotype in ACM Methods: Human primary cardiac stromal cells (CStCs) were cultured in adipogenic medium (AM), a condition used to model the ACM fibro-fatty phenotype. Mitochondria-related analyses of ultrastructure, membrane potential, network connection, respiratory capacity, ROS production and redox system were performed at day (d) d0, d3, d7, d15 of AM exposure. A mitochondria-targeting library (MedChem Express) was tested on ACM-CStCs exposed to AM for 7 days. The extent of lipid accumulation was determined using the neutral lipid staining dye BODIPY 493/503, cell numbers were measured using Hoechst counterstaining, and images acquired using a high-content imaging system Results: At d0, the mitochondrial network was more fragmented in ACM compared to healthy CStCs (CTR). This result was in line with the observation of a decrease in mitochondrial respiratory capacity and an increase in ROS production. In addition, AM exposure led to faster and higher lipid accumulation in ACM CStCs, which was associated with higher mitochondrial oxygen consumption rates observed in ACM CStCs after 7 days of AM exposure. When ACM CStCs were simultaneously exposed to AM and 407 mitochondrial-directed compounds, about 25% of the compounds demonstrated the ability to reduce lipid accumulation Conclusion: ACM CStCs demonstrate an increased risk for mitochondrial network impairment. Moreover, prolonged exposure of ACM CStCs cells to AM negatively affects lipid deposition as well as mitochondrial and metabolic parameters. This highlights the potential for using specific mitochondria-targeting compounds to reduce lipid accumulation.

Right ventricular outflow tract cardiomyocytes possess distinct calcium handling and contractile properties

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Rembrandt Research Grant 2021 Background The right ventricular outflow tract (RVOT) is the predominant site of origin of idiopathic ventricular tachycardia. Idiopathic ventricular tachycardias are typically sensitive to adenosine, consistent with triggered activity caused by delayed afterdepolarizations due to intracellular Ca2+ overload. The distinct properties of RVOT myocytes explaining increased susceptibility to Ca2+ overload and triggered activity are unknown. Purpose To identify distinct RVOT electrophysiological properties including Ca2+ handling and its molecular underpinnings. Methods and Results Experiments were performed using freshly isolated cardiomyocytes from the RVOT and right ventricular (RV) apex of adult rabbit hearts. To identify distinct RVOT electrophysiological properties, we employed the perforated patch clamp methodology and measured action potentials and L-type Ca2+ current. Action potentials of RVOT myocytes were significantly shorter compared to RV myocytes, particularly at lower pacing frequency. L-type Ca2+ current peak density was significantly lower in RVOT myocytes compared to RV myocytes (34.0 ± 9.1% at 0 mV). However, voltage-dependencies of L-type Ca2+ current activation and inactivation were not statistically different. Next we measured intracellular Ca2+ transients using the fluorescent Ca2+ indicator indo-1 and found that Ca2+ transient amplitudes were significantly lower in RVOT myocytes compared to RV myocytes. To investigate a relationship between RVOT myocyte Ca2+ handling and contractility, we performed unloaded sarcomere shortening measurements using the Cytocypher Multicell system. Shortening of relaxation time following increased pacing frequency (1-2 Hz) was significantly larger for RVOT myocytes compared to RV myocytes. To identify molecular factors explaining the distinct properties of RVOT myocytes, we performed RNA-seq analysis of differential gene expression using the DESeq2 method. RVOT and RV transcriptomic profiles consisted of 30 differentially expressed genes (adjusted p ≤ 0.05). One of genes significantly higher expressed by the RV was synaptotagmin (SYT1), a Ca2+ sensor and positive regulator of the cardiac L-type Ca2+ channel through inhibition of syntaxin 1A [Ref. 1, 2]. Conclusion RVOT cardiomyocytes possess distinct electrophysiological and contractile properties, reflected by shorter action potentials, lower Ca2+ transient amplitudes, and larger shortening of relaxation time upon increasing pacing frequency. The RVOT and RV have similar but distinct gene expression signatures. Future experiments will focus on determining the role of SYT1 and other genes in regulation of the L-type Ca2+ current and Ca2+ handling in RVOT myocytes.Distinct properties of RVOT myocytes.Transcriptomic profile of the RVOT.

Poor in vivo transduction of myocardium by lentiviral vectors is likely caused by a post-entry block of transduction

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Stichting LSH-TKI Introduction In cardiac gene therapy, two prototypic viral vectors are typically considered for long-term transgene expression, i.e. lentivirus (LV) and adeno-associated virus (AAV) vectors. Compared to AAV vectors, LV vectors have the advantages of a larger insert capacity and the ability to confer permanent transgene expression due to integration of the vector genome in the host cell’s chromosomal DNA . However, standard VSV G protein-pseudotyped LV vectors (VSV G-LV vectors) has shown suboptimal transduction of cardiac myocytes in vivo. Purpose In this study, we aimed to improve LV-mediated transduction of cardiomyocytes in vivo by evaluating LV vectors carrying various different envelopes and compared the most potent variants with AAV1 capsid-pseudotyped AAV2 vectors (AAV2/1 vectors). Methods We made a panel of LV vectors pseudotyped with envelope proteins from 76 different viruses carrying an expression cassette for enhanced green fluorescent protein (eGFP). Transduction efficiencies in neonatal rat ventricular myocytes (NRVMs), human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cardiomyocyte progenitor cells (CMPCs) were determined by fluorescence-activated cell sorting (FACS) as percentage eGFP-positive cells. Male NOD -SCID mice aged 3-6 months were injected directly into the left ventricular myocardium with eGFP-encoding VSV G-LV and AAV2/1 vectors at doses of 1.4 x 10^7 infectious units and 2.0 x 10^10 genome copies, respectively, or PBS as a control. Mice were sacrificed one and four weeks after surgery. Per heart, left ventricular and eGFP-positive area was determined from 40 – 50 slides and the ratio was used as a measure of transduction efficiency. Results Of the panel of pseudotyped LVs none was able to consistently outperform VSV G-LV vectors, which reached transduction efficiencies in vitro of 58% in NRVMs to 96% in hiPSC-CMs and 100% in CMPCs . In AAV2/1 vector-injected hearts, the eGFP expressing area in the left ventricle increased from 1.07% in week 1 to 5.42% in week 4. In contrast, eGFP expression in LV-injected hearts was 0.31% and 0.16% after 1 week and 4 weeks, respectively (figure 1). Conclusion In vitro screening of a panel of pseudotyped LV vectors showed standard VSV G-LV vectors as top performer. Despite encouraging in vitro results and previously reported findings, VSV G-LV vectors was unable to provide for robust in vivo transduction. The observation that none of the 76 different envelopes yielded a meaningful improvement of lentiviral transduction rates together with the discrepancy between in vitro and in vivo transduction efficiencies, suggest that a post-entry block of transduction likely represents an important component of the poor in vivo performance.Figure 1in vivo transduction

Proteomic and transcriptomics profiles of serum selenium concentrations in patients with heart failure are associated with immunoregulatory processes

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Research Council, through the Open Competition ENW-KLEIN grant Background Low selenium concentrations are associated with worse outcomes in patients with heart failure (HF). However, the underlying pathophysiologic mechanisms remain incompletely understood. Therefore, we aimed to contrast serum selenium concentrations to blood proteomic and transcriptomic profiles in patients with HF. Methods Circulating proteins, whole blood transcriptomics and serum selenium measurements in a cohort of 2328 patients with worsening HF (BIOSTAT-CHF) were utilized to assess underlying pathophysiology. Penalized linear regression and gene expression analysis were used to assess proteins and transcriptomics profiles based on selenium concentrations, respectively. String database v11.5 was used for enrichment analyses. Additionally analyses of the association between genes encoding selenoproteins in relation to serum selenium and all-cause mortality were performed. Potential causality was investigated by supplementing human peripheral blood mononuclear cells (PBMCs) with selenium and measuring the concentrations of 18 different cytokines. Results From 356 biomarkers and 20 clinical features, the penalized linear regression model yielded 44 variables with <5% marginal false discovery rate as predictors of serum selenium. Biomarkers associated positively with selenium concentrations included: epidermal growth factor receptor (EGFR), IFN-gamma-R1, CD4, GDF15, and IL10. Biomarkers associated negatively with low selenium concentrations included: PCSK9, SPON2, TNFSF13, FGF21 and PAI. In addition, 148 RNA transcripts were found differentially expressed between high and low Se status (Padj.<0.05; fold change<|0.25|). Enrichment analyses of the selected biomarkers and the selected RNA transcripts identified similar enriched processes, including regulation processes of leukocyte differentiation and activation, as well as cytokines production. The expression of two gene encoding selenoproteins (MSRB1 and GPX4) were strongly correlated with serum selenium after multiple corrections, while GPX4, SELENOK, and SELENOS were associated with prognosis. In the in-vitro setting, PBMCs supplemented with selenium showed significantly lower abundance of several (pro-)inflammatory cytokines. Conclusion These data suggest that immunoregulation is an important mechanism through which selenium might have beneficial roles in heart failure. The beneficial effects of higher serum selenium concentrations are likely because of global immunomodulatory effects on the abundance of cytokines. MSRB1 and GPX4 are interesting targets as potential modulators of this immunoregulatory effect of selenium and should be pursued in future research.Cytokines concentrations

Propafenone-induced connexin-43 protein accumulation and intracellular dynamics

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): China Scholarship Council Background Connexin-43 (Cx43) plays a pivotal role in intercellular communication through gap junctions. It is instrumental in propagating electrical signals and allowing ions to flow between neighboring cardiomyocytes. This synchronized electrical activity allows the heart to work as a functional syncytium. Any disruption or malfunction of Cx43 can lead to arrhythmias and other cardiac issues. Understanding the functions and regulation of Cx43 is vital in both basic research and clinical contexts. Propafenone, a class Ic antiarrhythmic drug, has shown promise in rhythm control; however, its precise impact on cardiac cellular physiology, particularly regarding Cx43, remains incompletely understood. Purpose To investigate propafenone’s effect on Cx43 expression, physiology, and underlying mechanisms in cell systems. Methods Used cell lines include human embryonic kidney HEK293 cells transfected with Cx43; differentiated murine embryonic carcinoma EPI7 cells with an epithelioid morphology and visceral endoderm-like END2 cells, endogenously expressing high amounts of functional Cx43. Cx43 protein expression levels were determined by Western blot analysis, whereas immunofluorescence (IF) imaged by a Total Internal Reflection Fluorescence (TIRF) microscope was used to assess the subcellular localization of Cx43 proteins. Dye injections were used to gain insight into the effects of propafenone on Cx43 metabolic coupling. Results and Conclusion Full length Cx43 protein levels were increased after treatment with propafenone and IF microscopy showed an intracellular accumulation of Cx43 protein, both on ectopically and endogenously expressed Cx43. Propafenone did not alter Cx43 half-life, in contrast to the lysosomal inhibitor chloroquine. Finally, gap-junctional coupling was decreased by chronic propafenone treatment. We conclude that the class 1c antiarrhythmic drug propanone increases Cx43 protein expression, resulting in its intracellular accumulation, as a side effect.

Role of Orai1 and Ca2+-stimulated adenylyl cyclase 8 in the adverse cardiac remodeling after myocardial infarction

Abstract 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) occurs in diseased heart such as under acute myocardial infarction (AMI). Recent studies have demonstrated that proteins related to the Store Operated Calcium Entry (SOCE), such as Orai1/2/3 and TRPC channels, play a role in the adverse cardiac remodelling. Previously, we demonstrated that the expression of Orai1 increased in heart subjected to ischemia and reperfusion (I/R) syndrome. However, the signalling pathway implicated in Orai1 regulation has not been addressed. Objective The aim of this study was to examine the molecular mechanism and signalling pathways involved in the of Orai1 upregulation after I/R in cardiomyocytes. Materials and Methods The experiments were performed in adult (ARVM) and neonatal rat ventricular cardiomyocytes (NRVM), in a rat model of myocardial I/R and in ventricular biopsies of patients with ischemic heart failure. Ca2+ studies were realized using microfluorimetric technique in FURA-2AM loaded cardiomyocytes. Immunofluorescence was used to investigate CREB activation using a specific phospho-CREB antibody and Orai1 and AC8 protein expression were analysed by Western Blot. This study was performed in accordance with the recommendations of the Royal Decree 53/2013 in agreement to the Directive 2010/63/EU of the European Parliament and approved by the local Ethics Committee on human Research and the Animal Research Committee. Results We found that under I/R diastolic level of [Ca2+]i was significantly increased in cardiac myocytes isolated from I/R hearts, which was inhibited by SOCE blockers. Moreover, we found that Orai1 and Ca2+-sensitive adenylyl cyclase 8 (AC8) are overexpressed in the heart isolated from I/R rat model, as compared to sham. Similarly, we found significant increase in the expression of Orai1 and AC8 in ventricle biopsies of post-infarction patients with HF. We also observed that I/R stimulated cAMP production, involving Orai1 and AC8 activation in rats, indicating that Ca2+ entry through Orai1 stimulated AC8. Moreover, I/R phosphorylated cAMP response element-binding protein (CREB) through PKA activation, but not via EPAC2 or ERK1/2. Interestingly, we found that CREB facilitated I/R-induced Orai1 upregulation and related exacerbated SOCE by its activation of Orai1 promoter under I/R. Finally, the intramyocardial administration of AAV9 holding AC8 shRNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i. Conclusions Our results show that the activation of Orai1/AC8/CREB axis plays a key role in the I/R-induced Orai1 upregulation, which participated in diastolic [Ca2+]i mishandling related to the adverse cardiac remodelling.

Ventricular hyperinnervation in a mouse model of catecholaminergic polymorphic ventricular tachycardia

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): European Society of Cardiology Basic Research Fellowship. Dutch Heart Foundation Dekker Grant Background Inherited arrhythmia syndromes are a leading cause of sudden cardiac death in young and otherwise healthy patients. They are often caused by mutations in genes that encode ion channels or transporters, leading to conditions such as Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). CPVT is caused by mutations in RYR2 (encoding ryanodine receptor 2, RyR2). In arrhythmia research, investigation into the effects of these mutations have exclusively been performed in cardiac cells. However, RyR2 is also present in neuronal tissue, including intracardiac neurons [1] (which modulate cardiac function). Moreover, patients often present with clinical signs of autonomic nervous system (ANS) dysfunction, and treatment strategies frequently target the ANS. Aim Our novel research line aims to investigate the neuronal phenotype induced by RyR2 mutations, using a CPVT mouse model to assess functional alterations as well as broader autonomic remodelling. Results Using immunocytochemistry, we reveal for the first time that RyR2 is also expressed in mouse stellate ganglia (crucial autonomic modulators of cardiac function). Through analysis of published RNA sequencing datasets, we find that RYR2 is more abundantly expressed in both stellate ganglion and intracardiac neurons than characteristic neuronal markers such as CHAT, SYN1 and SCN8A [2,3]. Functional patch clamp investigation of isolated stellate ganglion neurons have revealed two distinct populations based on their patterns of action potential (AP) firing, identifying both phasic-firing and tonic-firing type neurons. Investigation of an established CPVT mouse model (RyR2-R2474S) has thus far not revealed any differences in the AP firing frequency of isolated stellate ganglion neurons during sustained depolarising stimuli of various amplitudes. Assessment of calcium homeostasis and neurotransmitter release is currently ongoing. We have furthermore investigated broader autonomic remodelling using immunohistochemistry in mouse heart cryosections. Neuronal innervation density was assessed by staining 10 µm sections of mid-ventricular myocardium for the sympathetic neuron marker tyrosine hydroxylase (TH). Quantification of the TH-positive area revealed a significant increase in the percentage of sympathetic neuronal innervation in ventricular tissue of CPVT mice (5.7% wild-type vs 7.2% RyR2-R2474S, p=0.0253, N=6). Conclusions The neurons that modulate heart function (stellate ganglion and intracardiac neurons) abundantly express RyR2. The RyR2-R2474S mutation leads to significant sympathetic hyper-innervation of ventricular myocardium, which may have important consequences for neurotransmitter release and hence cardiac (electrical) function and arrhythmogenesis.

Exploring the role of air pollution on the development of atherosclerosis disorder in human endothelial cells

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): QU - NSPP Introduction Atherosclerosis, the main cause of cardiovascular and cerebrovascular diseases, is a global health threat. Rising cardiovascular disease prevalence, unexplained by traditional risk factors, prompts exploration of the impact of occupational exposure to environmental toxins as a risk factor. benzo[a]pyrene (BaP) is a polyaromatic compound that mediate its toxicity through activation of the Aryl hydrocarbon receptor (AhR). The molecular mechanisms of involvement of environmental toxins and the role of AhR/CYP1A pathway in atherosclerosis development remain not fully investigated. Purpose The current study explores the AhR pathway activation in endothelial cells upon exposure to environmental toxins in order to elucidate the molecular mechanisms of atherosclerotic cardiovascular diseases and plaque formation through investigating pro-inflammatory (CYP1A1, CYP1B1, TNF-a) and pro-apoptotic markers (BCL-2, Caspase-3). Methods EA. hy926 cell line underwent treatment with increasing concentrations of BaP (0-20 microM), as well as 10nm of TCDD, to confirm the results. DSMO was used as a control. Total RNA and protein were isolated to investigate mRNA expression and protein expression of AhR, CYP1A1, CYP1B1, as well as caspase-3, Bcl-2, and TNF-α using RT-PCR and Western Blot analysis. Statistical analysis was done using one-way ANOVA. Results The results showed that BaP induce significant dose-dependent upregulation of AhR and target genes (CYP1A1, CYP1B1). At 20uM, expression of AhR (3-fold), CYP1A1 (2.5-fold) and CYP1B1 (2-fold) was significantly high as compared to their respective controls and low doses. As for apoptotic markers, Bcl-2 was significantly downregulated at the highest concentrations 10uM and 20uM (0.7- and 0.6-fold, respectively). Caspase-3 showed no significant change but exhibited upregulation across all doses. TCDD 10nM significantly upregulated AhR (2.0-fold), CYP1A1 (1.5-fold), CYP1B1 (1.7-fold). Bcl-2 downregulated (0.6-fold), TNF-α significantly upregulated (1.8-fold) at 10nM. Caspase-3 increased 2-folds but was not statistically significant. Conclusion This study illustrated that AhR inducers, such as BaP, can accelerate the pathological progress of atherosclerosis by evoking oxidative stress and inflammation response molecules in human endothelial cells, demonstrated by the reduced expression of anti-apoptotic markers and the increased expression of inflammatory markers.

Leaky RyR2 channels as pro-arrhythmic trigger in the PKP2-deficient atrial myocardium

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): American Heart Association National Institutes of Health Introduction Atrial arrhythmias, like atrial fibrillation, occur in 20–40% of patients with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). Atrial fibrillation in ARVC patients is often associated with an increased risk of sustained ventricular arrhythmias and inappropriate ICD shocks. However, the pathophysiology behind the development of atrial arrhythmias in ARVC is far from clear. A genome-wide association study, based on UK-biobank data, revealed a link between atrial fibrillation and variants in the desmosomal gene plakophilin-2 (PKP2). Previous studies show that PKP2 is necessary to maintain the transcription of genes that control intracellular calcium (Ca2+) cycling and that loss of PKP2 expression in adult mouse ventricular tissue leads to disruption of intracellular Ca2+ homeostasis. In this study, we test the hypothesis that loss of PKP2 expression leads to pro-arrhythmic changes in atrial myocytes. Methods Experiments were carried out using a cardiac-specific, tamoxifen (TAM)-activated PKP2 knockout murine model (PKP2-cKO). Cre-negative, flox-positive, TAM-injected littermates were used as controls. We used RNA sequencing, quantitative imaging modalities, as well as biochemical and electrophysiological methods to study the functional and structural properties of atrial PKP2cKO tissue. Studies were carried out 21 days post-tamoxifen injection, a time point at which an arrhythmogenic cardiomyopathy of right ventricular predominance is manifest. Results In contrast to the previously-reported observations in PKP2cKO ventricular myocytes, atrial PKP2cKO myocytes presented no significant differences in Ca2+ transient dynamics, SR load and action potential duration. However, PKP2cKO atrial myocytes showed an increased frequency and amplitude of Ca2+ sparks, in combination with increased diastolic Ca2+ levels as well as an increase in cellular ROS levels. RNAseq data showed an under-representation of mRNA for Integrinα7, a stabilizer of Ryr2 that reduces its phosphorylation. Separate studies showed that PKP2cKO atrial myocytes present impaired relaxation and enhanced sarcomere shortening, a finding consistent with a downregulation in the expression of Myosin Binding Protein C. Isoproterenol (ISO) exposure further increased the frequency of Ca2+ sparks and the levels of diastolic Ca2+, and these effects were reversed by acute (30 minute) in vivo treatment with Ryanodex (medical grade Dantrolene), a known RyR blocker. Conclusion Loss of PKP2 expression affects cellular Ca2+ handling and electrophysiology differently in the atrial versus ventricular myocardium. We speculate that an increased probability of opening of Ryr2 channels, caused by ROS-induced RyR2 oxidation and/or Integrinα7-induced RyR2 phosphorylation, serve as pro-arrhythmic trigger in the PKP2-deficient atrial myocardium, that these effects can be amplified by a catecholaminergic input and that RyR blockers can dampen these pro-arrhythmic effects.

Targeting serotonin receptor 7 to improve survival after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Background Myocardial infarction (MI) is among the leading causes of death worldwide. Survival and long-term recovery are highly dependent on regeneration of the cardiac tissue, mediated by precisely regulated inflammatory processes and invading immune cells. The serotonergic system is known to regulate the functions of various cells of the immune system, and is also affected in several cardiovascular diseases. Purpose We investigated the role of the serotonin receptor 7 (5-HT7R) in regulation of the inflammatory response after MI and its contribution to cardiac outcome. Methods Permanent left ascending coronary artery ligation (LAD) in mice was used as a model for MI. We compared the impact of MI on heart function and tissue integrity in WT and 5-HT7R-KO mice. In addition, we performed a systemic treatment with the selective 5-HT7R agonist LP-211. Results Murine C57BL/6N hearts reveal an upregulation of 5-HT7R mRNA expression in the infarct region compared to remote myocardium and sham operated mice at 3, 7 and 14 days after MI. Immunohistochemistry demonstrates 5-HT7R expression mainly on CD68+, CD206+ and CD80+ macrophages but not on cardiomyocytes and fibroblasts. Systemic 5-HT7R-KO mice display normal cardiac function and dimensions at 3-4 months of age comparable to their wildtype (WT) littermates. In response to MI, 5-HT7R-KO mice show a higher decrease in left ventricular (LV) function (assessed by transthoracic echocardiography as fractional area change, FAC%) and increased signs of tissue inflammation 14 days post-MI, while the infarct size remains unchanged. Interestingly, systemic treatment with the selective 5-HT7R agonist LP-211 enhances survival rates 14d post MI, without improving cardiac functional output. Conclusion 5-HT7R expression is upregulated post-MI in heart-invading inflammatory cells, such as macrophages. Loss of systemic 5-HT7R may contribute to pronounced functional cardiac impairment 14 days after MI, while systemic activation of 5-HT7R shows positive effects on survival, possibly through mediated inflammatory signalling of immune cells. Therefore, 5-HT7R activity could represent a novel therapeutic target to dampen inflammation and improve cardiac repair.

Sex-specific plasma-proteome of incident coronary artery disease

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): This work was supported by a Leiden University Medical Center MD/PhD research grant to V.Q. Sier and Dr. R. Noordam is supported by an innovation grant from the Dutch Heart Foundation. Purpose The onset and symptomatology of coronary artery disease (CAD) are different for men and women, yet insights into sex-specific biological mechanisms causing CAD are limited. To increase insight in the onset of CAD in men and women, we performed a sex-specific proteome-wide association analysis using data from a large cohort of middle-aged participants from the UK Biobank. Methods Associations between 2,923 plasma proteins and CAD incidence (angina pectoris, acute myocardial ischemia, chronic myocardial ischemia) were examined in European-ancestry participants from the UK Biobank who were free of CAD at baseline. Plasma samples were collected at baseline (2006-2010), and CAD incidence was monitored continuously for a median (interquartile range) follow-up of 5,013 (4775-5251) days. Using Cox proportional hazard modelling, the associations between CAD incidence and plasma protein levels were examined, adjusted for age, sex, body mass index, socioeconomic status, type 2 diabetes mellitus, and cholesterol-lowering and insulin drug use at baseline. The Metascape portal was employed for pathway analyses to identify (sex-specific) biological pathways. Results In a total sample comprising 40,829 individuals (55% women, mean age (sd) 56.9 (8.10)), featuring 3,155 incident CAD cases, our sex-combined model demonstrated significant associations between 93 proteins and hazard for CAD. Pathway analyses identified early plasma proteomic changes related to cytokine-cytokine receptor interactions (p=3.42e-17), matrix remodelling (p=9.24e-14, p=1.08 e-9), regulation of innate and adaptive immune cells (p=1.92e-12, p=3.88e-9), and angiogenesis (p=5.34e-9). Stratification by sex revealed distinct sex-specific proteomic patterns linked to CAD incidence, with 10 female-specific and 49 male-specific significant proteins. Through interaction analyses, we demonstrated that 8 proteins were distinctly associated with a significant increased hazard in women, while 20 proteins were significantly associated with increased hazard for CAD in men. Pathway analyses identified a significant female-specific relation between CAD incidence and matrix remodelling (p=9.72e-15, p=3.19e-13), angiogenesis/hypoxia (p=1.41e-12), and inflammatory signalling (p=7.18e-10, p=8.01e-10) pathways, whereas male-specific top pathways related to inflammatory signalling (p=9.58e-25), matrix remodelling (p=1.22e-17), and immune cell response/recruitment (p=3.08e-16, p=8.34e-12). Conclusion Using large-scale association analyses of proteomics data with CAD, we identified sex-specific proteomic profiles. Distinct patterns in more angiogenic (women) and more immune cell response (men) profiles were found. These findings contribute novel insights to the development of CAD, potentially guiding future sex-specific research and clinical strategies for disease prevention.Proteome-wide analysis for CAD incidence

Maturing hiPSC-cardiomyocytes in cardiac microtissues for accurate preclinical in vitro modelling of long-QT syndrome type 1

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): reNEW - Novo Nordisk Foundation Center for Stem Cell Medicine Background Long-QT syndrome type 1 (LQT1), a cardiac arrhythmia often leading to sudden cardiac death, is due to mutations in KCNQ1 gene. Human iPSC-derived cardiomyocytes (hiPSC-CMs) have been widely used to model LQT1, promoting the concept of their use in preclinical studies. However, KCNQ1 locus is subjected to developmentally regulated genomic imprinting in the heart: in fetal cardiomyocytes the paternal allele is repressed, while both alleles become expressed after birth. Since hiPSC-CMs are typically immature fetal-like cells, residual imprinting may affect evaluation of KCNQ1 mutations in this system. Purpose This study wants to address the imprinting status of KCNQ1 in hiPSC-CMs and propose maturing hiPSC-CMs as a solution for reliable LQT1 modelling. Methods We derived hiPSC-CMs from a LQT1 patient carrying a heterozygous KCNQ1 mutation on the paternal allele and compared their electrical properties with the isogenic corrected hiPSC-CMs by patch clamp. We analysed KCNQ1 expression by ddPCR and DNA methylation. We used three-dimensional cardiac microtissues (MTs) composed by hiPSC-CMs, hiPSC-derived cardiac fibroblasts and endothelial cells to induce cardiomyocte maturation. Results LQT1 and isogenic corrected hiPSC-CMs showed no difference in action potential duration (APD). We hypothesized that residual KCNQ1 imprinting in hiPSC-CMs due to their immaturity was masking the phenotype. When we analyzed different hiPSC-CM lines, we found in all an unbalanced KCNQ1 allelic expression, with paternal allele accounting for 5-15% of the total. We then investigated whether maturing hiPSC-CMs could remove KCNQ1 imprinting. We included hiPSC-CMs in MTs, as this system previously showed to promote functional maturation and upregulation of KCNQ1. Here, we observed a substantial increased expression of the paternal allele and a reduction in the expression of KCNQ1OT1, the long non-coding RNA inducing paternal allele repression. LQT1 hiPSC-CMs dissociated from MTs showed prolonged APD compared to the corrected line. We also demonstrated that this was due to the reduction of the ionic current mediated by KCNQ1 (IKs) in LQT1 hiPSC-CMs compared to the corrected controls, thus revealing the mutation causative effect. Conclusions Our findings show that residual KCNQ1 imprinting in immature hiPSC-CMs can lead to underestimate (or overestimate) functional effects of pathological variants based on the parental allele that carries the mutation. This is overcome by maturation of hiPSC-CMs in tri-cellular cardiac microtissue which promotes KCNQ1 bi-allelic expression and allow dissecting mutation mechanisms. This study brings attention to the epigenetic regulation of hiPSC models and demonstrates the utility of using matured hiPSC-CMs as in vitro preclinical model for cardiac arrhythmias.

The effect of ageing in an experimental model of myocardial infarction in senescent mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Spanish Ministry of Science and Innovation (ISCIII) PI19/01714 and PI22/1083 co-financed by the European Regional Development Fund (ERDF), and by the Generalitat Valenciana. Introduction The murine experimental model of myocardial infarction allows the study of underlying molecular and cellular mechanisms in cardiac function. Specifically, intercellular communication mediated by small non-coding RNAs, microRNAs (miRNAs) which can modulate gene expression post-transcriptionally, is one of the fundamental processes in the investigation of this pathology that needs to be explored in animal models. The Senescence-Accelerated Mouse Prone (SAMP8) and its control strain SAM Resistant (SAMR1) have been established as an experimental model to study ageing-associated vascular dysfunction but scarcely used in epigenetic studies. Purpose To assess the suitability of the SAMR1/SAMP8 ageing model for the study of miRNAs expression following an acute myocardial infarction (AMI), and to determine the optimal time after AMI to measure circulating miRNA levels. Methods Six-month old male and female SAMR1 and SAMP8 (n = 6 per group) underwent AMI by ligation of the left anterior descending coronary artery and sham surgery. All interventions were performed under full anesthesia and analgesia. Animals were euthanized at different times (1h, 4h and 24h) and blood were collected. AMI was confirmed by immediate discoloration of the left ventricle, by ST segment elevation in the electrocardiogram, and by triphenyltetrazolium chloride staining of the non-ischemic areas of heart sections. RNA was obtained from non-hemolyzed serum. Circulating miRNA previously considered as possible AMI biomarkers, miR-1-3p, miR-133-3p, miR-208-3p and miR-499-5p, were measured by qRT-PCR. Results are shown as mean ± SEM of the relative expression (2-ΔΔCt), using U6 snRNA as endogenous control. p-values were calculated using ANOVA and statistical significance was considered when p<0.05. The investigation complies with ethical standards and was approved by the Ethics Committee for Animal Welfare of University of Valencia (2020/VSC/PEA/0128). Results miR-1-3p, miR-133-3p, miR-208-3p and miR-499-5p were significantly increased 4 hours after AMI when compared with sham group in both SAMR1 and SAMP8. It was noteworthy that only with the sham surgery all biomarkers exhibited a time-dependent decrease (p < 0.05). While miR-1-3p rose at 4 hours after AMI (6.46 ± 1.32, p<0.001), miR-133-3p, miR-208-3p and miR-499-5p showed a time-dependent upregulation, being maximal at 24 hours after AMI (p < 0.001 vs. sham group). When comparing samples from SAMR1 and SAMP8, the expression of the analyzed miRNAs was higher in SAMP8 4 hours after AMI (p < 0.05), to a greater extent than in SAMR1. Conclusion Through the use of the SAMR1/SAMP8 mice model, the impact of ageing on miRNA expression following an AMI was notably confirmed, particularly at 4 hours after AMI. Consequently, this study proposes the SAMR1/SAMP8 mouse model as suitable for studying the effects of both AMI and ageing on circulating miRNA expression.

Proteomic profiling reveals higher presence of glycolytic enzymes in vulnerable atherosclerotic lesions

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq PlaqOmics Background Our definition of vulnerable plaques is predominantly driven by histological analysis. The molecular mechanisms behind the vulnerable histological characteristics are not fully understood. Here we use mass spectrometry-based proteomics data, to identify molecular mechanisms linked to vulnerable plaques. Methods Atherosclerotic lesions from two hundred patients undergoing endarterectomy surgery were collected (149 male and 51 female plaques). From a total of two hundred plaque samples, extracellular matrix proteins were extracted and processed for untargeted proteomics analysis. The resulting protein levels were linked to existing histological characteristics, transcriptomic, and clinical data. Results We detected 1499 proteins in atherosclerotic lesions, which were mostly affiliated with biological processes including extracellular matrix organization, coagulation, and peptidase activity. Linear regression identified 240 proteins associated with plaque vulnerability index (a semi-quantitative score summarizing the presence of SMC cells, macrophages, plaque hemorrhage, and fat in the plaque). Among the proteins associated with high plaque vulnerability index we identified an overrepresentation of enzymes involved in glycolysis – including the rate limiting enzymes HK3 (beta =0.40 p =0.003) and PKM (beta =0.19 p =0.009). This was driven by macrophage numbers and fat content in the plaque. Furthermore, data from a validation cohort of 120 patients from the Medical University of Vienna, Austria, revealed that HK3 and PKM were associated with plaque progression (p =<0.001, p =0.017) and clinical symptoms (p = <0.001, p = <0.001). Conclusion Enzymes involved in the glycolysis process show higher presence in vulnerable plaques. This suggests an important role for energy metabolism in plaque vulnerability.

Cryopreservation of human adult ventricular tissue for the preparation of viable myocardial slices

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): REGeRNA Myocardial slices are ultra-thin slices of living adult myocardium which can be cultured. As a multicellular 3-D culture model, myocardial slices offer a novel opportunity to investigate how adult cardiomyocytes (CMs) interact with other cardiac cell types and the extracellular matrix, while providing long-term preservation of CMs cytoarchitecture and electrical phenotype. Additionally, myocardial slices are a new tool in drug discovery by allowing to test chronic treatments, as opposed to isolated CMs which are limited to acute timepoints. Despite their benefits over other cardiac preparations, myocardial slices also have several limitations, of which one is the quality of the slices. The main determinant of myocardial slice quality is the procedure by which the cardiac tissue block is handled. Current methods depend on the preparation of slices directly after tissue excision, therefore limiting the moment when the experiment is performed. We developed a protocol for the cryopreservation of human adult ventricular tissue allowing preparation of viable myocardial slices afterwards. Fresh and cryopreserved cardiac tissue from donor hearts was processed and evaluated for viability, tissue integrity, and inflammatory markers using molecular biology and histological assays. The tissue samples retained viability, biomass, and tissue and vascular integrity following cryopreservation compared to fresh tissue. Additionally, we compared myocardial slices obtained from fresh or cryopreserved tissue and cultured for 24 hours. Histologically, normal cytomorphological features and gap junctions between CMs were preserved in all slices. Comparable retention of tissue viability and structural integrity post cryopreservation (4-6 weeks) and culture (up to 7 days) was observed in cardiac samples from 3 additional donors. In summary, cold storage extends the time-interval between sample collection and slice preparation, allowing for more long-term, complex endpoints. Moreover, efficient banking of cryopreserved cardiac samples from various donors and disease states is critical for regular use of myocardial tissue slices in drug discovery and development. Additional research on the effects of cryopreservation on different cardiac cell types will allow to improve donor organ management to extend the donor pool and increase utilization rate.