Cardiac Slices Research Articles

Tyrosine Kinase Inhibitor Lenvatinib Causes Cardiotoxicity by Inducing Endoplasmic Reticulum Stress and Apoptosis through Activating ATF6, IRE1α and PERK Signaling Pathways.

Lenvatinib is a tyrosine kinase inhibitor that can improve progression-free survival in patients with thyroid cancer and hepatocellular carcinoma. However, it is limited by adverse cardiovascular events, including hypertension and cardiac dysfunction. Activation of endoplasmic reticulum stress is involved in cardiomyocyte apoptosis. This study aimed to confirm whether the cardiotoxicity of lenvatinib is associated with endoplasmic reticulum stress by targeting the activating transcription factor 6 (ATF6), inositol- requiring enzyme 1α (IRE1α) and protein kinase RNA-like ER kinase (PERK) signaling pathways. Male C57/BL6 mice were intragastric administration with 30 mg/kg/day lenvatinib. Electrocardiography (ECG) and echocardiography were used to detect arrhythmias and cardiac function. Neonatal rat cardiomyocytes were treated with lenvatinib for 48h. Cell counting kit (CCK8), 2´,7´-dichlorodihydrofluoresceine diacetate (H2DCFHDA), Hoechst 33258 and dihydrorhodamine 123 were respectively used for evaluating cell viability, the level of reactive oxygen species (ROS), nuclear morphological changes and mitochondrial membrane potential (MMP) level. Lenvatinib remarkably decreased the posterior wall thickness of left ventricle during diastole and systole but caused little decrease to the left ventricular ejection fraction (LVEF, %). Furthermore, lenvatinib greatly prolonged the corrected QT interval (QTc) and altered the morphology of cardiomyocytes. No dramatic difference in fibrosis was found in mouse cardiac slices. Lenvatinib upregulates apoptosis-related protein expression. In addition, lenvatinib increased ERS-related protein expression (GRP78, CHOP, and ATF6) and enhanced PERK phosphorylation. In neonatal rat cardiac myocytes, lenvatinib markedly decreased the viability of cardiomyocytes and induced apoptosis. Furthermore, ROS production increased and MMP decreased. Similar to the mice experiment, lenvatinib caused upregulation of apoptosis-related and ERS-related proteins and increased the phosphorylation levels of PERK and IRE1α. Lenvatinib-induced cardiotoxicity is associated with ERS-induced apoptosis by targeting the ATF6, IRE1α, and PERK signaling pathways.

Left Atrial Appendage Analysis from Echocardiographic Images: Relevance in Left Atrial Appendage Occlusion

Abstract Atrial fibrillation, a prevalent cardiac arrhythmia, disrupts the normal path of electrical signals within the heart. To address this issue, the left atrial appendage closure is often considered. This involves using a closure instrument named the Watchman to obstruct the left atrial appendage (LAA) ostium. The wide variety of LAA shapes, sizes, and forms in individuals makes selecting the correct Watchman size crucial. This study first isolates the LAA from echocardiographic images and then recommends the appropriate Watchman size. The 3-D echocardiographic images used in this study are from 32 male and female patients who underwent successful LAA closure at Kings College Hospital London over two years. For each patient, there are 208 cardiac echo slices in the imaging mode. This study presents an image processing-based model to separate LAA regions and extract relevant features. These features are then inputted into a classification platform in which a neural network determines the suitable Watchman size. The average and mode Hausdorff distances, calculated between the proposed method and the traditional manual calculation for the 501 analyzed LAA images, are approximately 0.2467 and 0.0587, respectively. Because of limited data, a 10-fold cross-validation was used to assess the classifier, yielding an accuracy of 74.07 %. The proposed model effectively isolated the LAA from the corresponding slices of the 3-D echocardiographic images. However, the classifier’s accuracy is not ideal because of insufficient data, which could be improved by expanding the database. This research’s outcomes could aid physicians in selecting the proper Watchman size.

The role of serotonin receptor 7 signalling in macrophages for the inflammatory response after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Background The hormone and neurotransmitter serotonin regulates multiple processes, including immune responses and inflammatory signalling. After a cardiac injury, such as myocardial infarction (MI), a precise regulation of the local inflammation is determining the healing process and therefore functional outcome. Immune cells invading the infarcted tissue can be critical modulators, and various immune cells, including macrophages, express different serotonin receptors. Purpose We characterized the importance of serotonin receptor 7 (5-HT7R) signalling in the regulation of macrophage function, which infiltrate the heart after MI and thus can determine the course of cardiac remodelling processes. Methods For investigation of 5-HT7R signalling on a cellular level, we used two in vitro models: primary murine bone marrow-derived macrophages (BMDM) and secondary human THP-1-derived macrophages. To validate the impact of macrophages on intact cardiac tissue, we pre-treated pro-inflammatory M1-like BMDMs with a selective 5-HT7R agonist and then co-cultured them with living cardiac rat slices for up to 48 h (ex vivo) followed by functional analysis. Results We detected 5-HT7R expression in both models during all stages of macrophage polarization at mRNA as well as protein levels. Pro-inflammatory THP-1-derived macrophages displayed significantly enhanced mRNA expression levels of IL-1β and CD80, while pro-inflammatory differentiated M1-like BMDMs showed increased levels of CD38 and TNFα. Pharmacological activation of 5-HT7R with LP-211 reduced phagocytic activity up to 4-fold in THP-1-derived pro-inflammatory macrophages, altered their cytokine secretion profile and inhibited their migration ability (recovered area in scratch assay 1.3 % after LP-211 treatment compared to 6.1 % in control). A reduction in phagocytic activity was also verified in pro-inflammatory BMDMs. Our results also indicate reduced ability of LP-211 treated M1-like BMDMs to infiltrate living cardiac slices 3, 12, 24 and 48 h after seeding. Conclusion 5-HT7R signalling affects the functional properties of pro-inflammatory macrophages, including phagocytic activity, motility and secretion profile. Targeted manipulation of 5-HT7R activity in macrophages represents thus a possibility to modulate their inflammatory profile, which might be a reasonable approach to guide recovery post MI.

Human cardiomyocyte derived extracellular vesicles regulate cardiac fibroblast activation through miR-24

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondazione Leonardo Introduction Cardiovascular diseases are associated with increase in interstitial fibrosis due to cardiac fibroblast (CF) activation into myofibroblast leading to the secretion and deposition of extracellular matrix (ECM). The intricate functional activation of these processes is not well understood, especially concerning the contribution of the microenvironment. One of the most important mechanisms of paracrine communication are due to Extracellular vesicles (EVs). EVs are cell-released nanoparticles carrying proteins, lipids, and nucleic acids, particularly miRs. To note, previous transcriptomic analysis of healthy human heart demonstrated that miR24-3p is one of the most abundant miRs [1], suggesting an important role associated with its expression. Aim of the work Here we aim to investigate the role of miR24-3p as regulator of cardiac fibroblast activation, focusing on healthy and pathological conditions. Materials and methods Bioinformatic analysis of open-data, single-cell RNA sequencing from heart tissue was conducted to assess the expression of miR24-3p and its specific targets in both healthy and infarcted myocardium. To explore the functional role of miR24-3p, loss- and gain-of-function experiments were performed by transfecting cardiac fibroblasts (CF) with specific antimiR or mimic oligonucleotides, respectively. In investigating the influence of cardiomyocytes (CM) on CF regulation, extracellular vesicles (EVs) from human-induced pluripotent stem cell-derived CM were isolated and utilized in in vitro experiments. The expression levels of miRNA24-3p were also evaluated in the plasma of healthy individuals and patients who had experienced myocardial infarction. Finally, cardiac slices from human tissue were employed as ex-vivo microtissues to functionally validate the in vitro findings. Results RealTime analysis of CF, CM and cardiac endothelial cells (EC) revealed a CM specific expression of miR24-3p, not only at cellular level but also in EVs. Upregulation of miR24-3p in CF was able to significantly reduce protein expression of FURIN, CYCLD1 and SMAD4, resulting in decrease of CF activation and ECM secretion. Interestingly, treatment of CF with CM-EVs recapitulate miR24-3p transfection, while EVs derived from hypoxia cultured CM (MI-like) does not. RNAScope and ddPCR analysis on human cardiac slice cultured under hypoxia conditions confirmed decrease in miR24-3p expression upon induction of stress. Ultimately, a significant decrease in miR24-3p in circulating serum EVs of MI patients’ was observed. Conclusions These data demonstrate the role of miR24-3p in CF regulation through the paracrine activity mediated by CM-EVs. Further analysis, including the use of a 3D human heart microtissue, will confirm the mechanism of cross-talk between CM and CF.

Dihydrotestosterone Augments the Angiogenic and Migratory Potential of Human Endothelial Progenitor Cells by an Androgen Receptor-Dependent Mechanism.

Endothelial progenitor cells (EPCs) play a critical role in cardiovascular regeneration. Enhancement of their native properties would be highly beneficial to ensuring the proper functioning of the cardiovascular system. As androgens have a positive effect on the cardiovascular system, we hypothesized that dihydrotestosterone (DHT) could also influence EPC-mediated repair processes. To evaluate this hypothesis, we investigated the effects of DHT on cultured human EPCs' proliferation, viability, morphology, migration, angiogenesis, gene and protein expression, and ability to integrate into cardiac tissue. The results showed that DHT at different concentrations had no cytotoxic effect on EPCs, significantly enhanced the cell proliferation and viability and induces fast, androgen-receptor-dependent formation of capillary-like structures. DHT treatment of EPCs regulated gene expression of androgen receptors and the genes and proteins involved in cell migration and angiogenesis. Importantly, DHT stimulation promoted EPC migration and the cells' ability to adhere and integrate into murine cardiac slices, suggesting it has a role in promoting tissue regeneration. Mass spectrometry analysis further highlighted the impact of DHT on EPCs' functioning. In conclusion, DHT increases the proliferation, migration, and androgen-receptor-dependent angiogenesis of EPCs; enhances the cells' secretion of key factors involved in angiogenesis; and significantly potentiates cellular integration into heart tissue. The data offer support for potential therapeutic applications of DHT in cardiovascular regeneration and repair processes.

Effects of electro-mechanical uncouplers, hormonal stimulation and pacing rate on the stability and function of cultured rabbit myocardial slices.

Introduction: Recent advances have enabled organotypic culture of beating human myocardial slices that are stable for weeks. However, human myocardial samples are rare, exhibit high variability and frequently originate from diseased hearts. Thus, there is a need to adapt long-term slice culture for animal myocardium. When applied to animal cardiac slices, studies in healthy or genetically modified myocardium will be possible. We present the culture of slices from rabbit hearts, which resemble the human heart in microstructure, electrophysiology and excitation-contraction coupling. Methods: Left ventricular myocardium from New Zealand White rabbits was cut using a vibratome and cultured in biomimetic chambers for up to 7days (d). Electro-mechanical uncoupling agents 2,3-butanedione monoxime (BDM) and cytochalasin D (CytoD) were added during initiation of culture and effects on myocyte survival were quantified. We investigated pacing rates (0.5Hz, 1Hz, and 2Hz) and hormonal supplements (cortisol, T3, catecholamines) at physiological plasma concentrations. T3 was buffered using BSA. Contractile force was recorded continuously. Glucose consumption and lactate production were measured. Whole-slice Ca2+ transients and action potentials were recorded. Effects of culture on microstructure were investigated with confocal microscopy and image analysis. Results: Protocols for human myocardial culture resulted in sustained contracture and myocyte death in rabbit slices within 24h, which could be prevented by transient application of a combination of BDM and CytoD. Cortisol stabilized contraction amplitude and kinetics in culture. T3 and catecholaminergic stimulation did not further improve stability. T3 and higher pacing rates increased metabolic rate and lactate production. T3 stabilized the response to β-adrenergic stimulation over 7d. Pacing rates above 1Hz resulted in progredient decline in contraction force. Image analysis revealed no changes in volume fractions of cardiomyocytes or measures of fibrosis over 7d. Ca2+ transient amplitudes and responsiveness to isoprenaline were comparable after 1d and 7d, while Ca2+ transient duration was prolonged after 7d in culture. Conclusions: A workflow for rabbit myocardial culture has been established, preserving function for up to 7d. This research underscores the importance of glucocorticoid signaling in maintaining tissue function and extending culture duration. Furthermore, BDM and CytoD appear to protect from tissue damage during the initiation phase of tissue culture.

Culturing Cardiac Tissue Slices Under Continuous Physiological Mechanical Stretches.

Testing drugs in vivo and in vitro have been essential elements for the discovery of new therapeutics. Due to the recent advances in in vitro cell culture models, such as human-induced pluripotent stem cell-derived cardiomyocytes and 3D multicell type organoid culture methods, the detection of adverse cardiac events prior to human clinical trials has improved. However, there are still numerous therapeutics whose adverse cardiac effects are not detected until human trials due to the inability of these cell cultures to fully model the complex multicellular organization of an intact human myocardium. Cardiac tissue slices are a possible alternative solution. Myocardial slices are a 300-micron thin snapshot of the myocardium, capturing a section of the adult heart in a 1×1cm section. Using a culture method that incorporates essential nutrients and electrical stimulation, tissue slices can be maintained in culture for 6days with full viability and functionality. With the addition of mechanical stimulation and humoral cues, tissue slices can be cultured for 12days. Here we provide detailed methods for how to culture cardiac tissue slices under continuous mechanical stimulation in the cardiac tissue culture model (CTCM) device. The CTCM incorporates four essential factors for maintaining tissue slices in culture for 12days: mechanical stimulation, electrical stimulation, nutrients, and humoral cues. The CTCM can also be used to model disease conditions, such as overstretch-induced cardiac hypertrophy. The versatility of the CTCM illustrates its potential to be a medium-throughput screening platform for personalized drug testing.

Modeling cardiac injury using human cardiac slices to test therapeutic approaches for cardiac regeneration and repair

Abstract Background Due to limited regenerative capacity of the adult human heart, myocardial infarction causes irreversible damage by inducing the death of cardiomyocytes and the formation of a fibrotic scar. These remodeling mechanisms cause loss of contractility and eventually heart failure ensues in a significant proportion of patients. Purpose There is a need for a reliable model that can replicate the adult cardiac pathophysiology in order to test treatments against cardiac remodeling. Here, we developed a cardiac tissue culture model that can simulate in vitro the molecular changes occurring after cardiac injury using human cultured cardiac slices. Methods We obtained cardiac slices by sectioning ventricular myocardium from the surgical waste material of adult patients undergoing valve surgery or Morrow myectomy. Cardiac slices were cultured at a liquid-air interface and were viable up to 7 days of culture, even in the absence of stimulation. During this time cardiac slices underwent mechanical and chemical treatments to induce ischemia and/or local injury. Results We found that following these treatments we were able to simulate cardiac remodeling in terms of cardiomyocyte death, activation of cardiac fibroblasts and induction of inflammation and oxidative stress. Following small molecule screening, we discovered that addition of compounds with anti-inflammatory properties (e.g. dexamethasone) or targeting the TGF-β pathway (e.g. SB-431542) into our culture medium helped in preserving the microscopic structure of the slices. Additionally, we found that treatment with these compounds protected cardiac slices against remodeling following ischemic injury. Conclusions In conclusion, our model can emulate cardiac pathophysiology in culture, providing us a reliable platform to test therapies for cardiac regeneration and repair in an adult, multicellular, 3-D environment.

Myosin activator omecamtiv mecarbil exhibits divergent inotropic and lusitropic effects in cardiac slices from patients with heart failure

Myosin activator omecamtiv mecarbil exhibits divergent inotropic and lusitropic effects in cardiac slices from patients with heart failure

Ryanodine Receptor Staining Identifies Viable Cardiomyocytes in Human and Rabbit Cardiac Tissue Slices.

In terms of preserving multicellularity and myocardial function in vitro, the cultivation of beating myocardial slices is an emerging technique in basic and translational cardiac research. It can be used, for example, for drug screening or to study pathomechanisms. Here, we describe staining for viable cardiomyocytes based on the immunofluorescence of ryanodine receptors (RyRs) in human and rabbit myocardial slices. Biomimetic chambers were used for culture and measurements of contractile force. Fixable fluorophore-conjugated dextran, entering cells with a permeable membrane, was used for death staining. RyRs, nuclei and the extracellular matrix, including the t-system, were additionally stained and analyzed by confocal microscopy and image processing. We found the mutual exclusion of the RyR and dextran signals in cultivated slices. T-System density and nucleus size were reduced in RyR-negative/dextran-positive myocytes. The fraction of RyR-positive myocytes and pixels correlated with the contractile force. In RyR-positive/dextran-positive myocytes, we found irregular RyR clusters and SERCA distribution patterns, confirmed by an altered power spectrum. We conclude that RyR immunofluorescence indicates viable cardiomyocytes in vibratome-cut myocardial slices, facilitating the detection and differential structural analysis of living vs. dead or dying myocytes. We suggest the loss of sarcoplasmic reticulum integrity as an early event during cardiomyocyte death.

Evaluation of convolutional neural networks for the detection of inter-breath-hold motion from a stack of cardiac short axis slice images

PurposeThis study aimed to develop and validate a deep learning-based method that detects inter-breath-hold motion from an estimated cardiac long axis image reconstructed from a stack of short axis cardiac cine images.MethodsCardiac cine magnetic resonance image data from all short axis slices and 2-/3-/4-chamber long axis slices were considered for the study. Data from 740 subjects were used for model development, and data from 491 subjects were used for testing. The method utilized the slice orientation information to calculate the intersection line of a short axis plane and a long axis plane. An estimated long axis image is shown along with a long axis image as a motion-free reference image, which enables visual assessment of the inter-breath-hold motion from the estimated long axis image. The estimated long axis image was labeled as either a motion-corrupted or a motion-free image. Deep convolutional neural network (CNN) models were developed and validated using the labeled data.ResultsThe method was fully automatic in obtaining long axis images reformatted from a 3D stack of short axis slices and predicting the presence/absence of inter-breath-hold motion. The deep CNN model with EfficientNet-B0 as a feature extractor was effective at motion detection with an area under the receiver operating characteristic (AUC) curve of 0.87 for the testing data.ConclusionThe proposed method can automatically assess inter-breath-hold motion in a stack of cardiac cine short axis slices. The method can help prospectively reacquire problematic short axis slices or retrospectively correct motion.

Development of new adeno-associated virus capsid variants for targeted gene delivery to human cardiomyocytes

Recombinant adeno-associated viruses (rAAVs) have emerged as one of the most promising gene therapy vectors that have been successfully used in pre-clinical models of heart disease. However, this has not translated well to humans due to species differences in rAAV transduction efficiency. As a result, the search for human cardiotropic capsids is a major contemporary challenge. We used a capsid-shuffled rAAV library to perform directed evolution in human iPSC-derived cardiomyocytes (hiPSC-CMs). Five candidates emerged, with four presenting high sequence identity to AAV6, while a fifth divergent variant was related to AAV3b. Functional analysis of the variants was performed invitro using hiPSC-CMs, cardiac organoids, human cardiac slices, non-human primate and porcine cardiac slices, as well as mouse heart and liver invivo. We showed that cell entry was not the best predictor of transgene expression efficiency. The novel variant rAAV.KK04 was the best-performing vector in human-based screening platforms, exceeding the benchmark rAAV6. None of the novel capsids demonstrate a significant transduction of liver invivo. The range of experimental models used revealed the value of testing for tropism differences under the conditions of human specificity, bona fide, myocardium and cell type of interest.

Abstract P1145: All-in-one AAV CRISPR/dCas9VPR Vectors Enable Endogenous Gene Activation In Cardiomyocytes

The transcriptional landscape of cardiomyocytes harbors the cellular adaptation upon stress, but remains out of scope for therapies preventing heart failure progression. CRISPR/Cas9 mediated endogenous gene activation (CRISPRa) using enzymatically inactive Cas9 (dCas9) fused to transcription activation domains (VPR) allows for precise and fine-tuned gene activity control when targeted to promoter or enhancer regions of interest via guide RNAs (gRNAs). Transgenic mouse models and human induced pluripotent stem cell (hiPSC) lines were established for proof-of-concept (PoC) gene activation studies in cardiomyocytes. However, the size of the commonly used Streptococcus pyogenes Cas9 exceeds the packaging limits of gene therapy relevant adeno-associated virus (AAV), hampering the therapeutic use of the technology. We aimed to engineer AAV vectors based on smaller, enzymatically inactive Cas9 variants fused to a minimalized VPR-transactivation domain co-expressing a single gRNA for all-in-one gene activation strategies. We rendered mini-Cas9s enzymatically inactive by mutating the evolutionary conserved RUVC and HNH regions and C-terminally attached a minimal VPR domain as well as a HA-tag. As PoC, we expressed mini-dCas9VPR under the CMV promoter in HEK293T cells and confirmed expression of the Cas9 variants via immunoblotting. When we targeted mini-dCas9VPR to the anti-hypertrophic transcription factor KLF15 promoter region or the PoC target POU5F1 promoter, we could confirm significant gene activation compared to non-transfected and non-targeted gRNA controls (KLF15: 3-fold change, POU5F1: 4-fold change, n ≤ 4). We next exchanged the CMV promoter by the TNNT2 promoter to allow cardiomyocyte-specific expression of mini-dCas9VPR. Upon targeting the KLF15 promoter with these vectors transfected in hiPSC-cardiomyocytes, we observed significantly elevated KLF15 mRNA levels compared to controls (1.5-fold change, n ≤ 4) and robust expression of the mini-CRISPRa system, respectively. In conclusion, we present novel vectors for AAV mediated cardiomyocyte-specific endogenous gene regulation which are currently tested in hiPSC-based models, patient-derived cardiac slices and for in vivo gene activation.

Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion

AimsArrhythmogenic cardiomyopathy (AC) is a severe heart disease predisposing to ventricular arrhythmias and sudden cardiac death caused by mutations affecting intercalated disc (ICD) proteins and aggravated by physical exercise. Recently, autoantibodies targeting ICD proteins, including the desmosomal cadherin desmoglein 2 (DSG2), were reported in AC patients and were considered relevant for disease development and progression, particularly in patients without underlying pathogenic mutations. However, it is unclear at present whether these autoantibodies are pathogenic and by which mechanisms show specificity for DSG2 and thus can be used as a diagnostic tool.Methods and ResultsIgG fractions were purified from 15 AC patients and 4 healthy controls. Immunostainings dissociation assays, atomic force microscopy (AFM), Western blot analysis and Triton X-100 assays were performed utilizing human heart left ventricle tissue, HL-1 cells and murine cardiac slices. Immunostainings revealed that autoantibodies against ICD proteins are prevalent in AC and most autoantibody fractions have catalytic properties and cleave the ICD adhesion molecules DSG2 and N-cadherin, thereby reducing cadherin interactions as revealed by AFM. Furthermore, most of the AC-IgG fractions causing loss of cardiomyocyte cohesion activated p38MAPK, which is known to contribute to a loss of desmosomal adhesion in different cell types, including cardiomyocytes. In addition, p38MAPK inhibition rescued the loss of cardiomyocyte cohesion induced by AC-IgGs.ConclusionOur study demonstrates that catalytic autoantibodies play a pathogenic role by cleaving ICD cadherins and thereby reducing cardiomyocyte cohesion by a mechanism involving p38MAPK activation. Finally, we conclude that DSG2 cleavage by autoantibodies could be used as a diagnostic tool for AC.

Extracellular flux analysis in intact cardiac tissue slices-A novel tool to investigate cardiac substrate metabolism in mouse myocardium.

Cardiac pathologies are accompanied by alterations in substrate metabolism, and extracellular flux analysis is a standard tool to investigate metabolic disturbances, especially in immortalized cell lines. However, preparations of primary cells, such as adult cardiomyocytes require enzymatic dissociation and cultivation affecting metabolism. Therefore, we developed a flux analyzer-based method for the assessment of substrate metabolism in intact vibratome-sliced mouse heart tissue. Oxygen consumption rates were determined using a Seahorse XFe24-analyzer and "islet capture plates." We demonstrate that tissue slices are suitable for extracellular flux analysis and metabolize both free fatty acids (FFA) and glucose/glutamine. Functional integrity of tissue slices was proven by optical mapping-based assessment of action potentials. In a proof-of-principle approach, the sensitivity of the method was tested by analyzing substrate metabolism in the remote myocardium after myocardial infarction (I/R). Here, I/R increased uncoupled OCR compared with sham animals indicating a stimulated metabolic capacity. This increase was caused by a higher glucose/glutamine metabolism, whereas FFA oxidation was unchanged. In conclusion, we describe a novel method to analyze cardiac substrate metabolism in intact cardiac tissue slices by extracellular flux analysis. The proof-of-principle experiment demonstrated that this approach has a sensitivity allowing the investigation of pathophysiologically relevant disturbances in cardiac substrate metabolism.

It takes two to tango: cardiac fibroblast-derived NO-induced cGMP enters cardiac myocytes and increases cAMP by inhibiting PDE3

The occurrence of NO/cGMP signalling in cardiac cells is a matter of debate. Recent measurements with a FRET-based cGMP indicator in isolated cardiac cells revealed NO-induced cGMP signals in cardiac fibroblasts while cardiomyocytes were devoid of these signals. In a fibroblast/myocyte co-culture model though, cGMP formed in fibroblasts in response to NO entered cardiomyocytes via gap junctions. Here, we demonstrate gap junction-mediated cGMP transfer from cardiac fibroblasts to myocytes in intact tissue. In living cardiac slices of mice with cardiomyocyte-specific expression of a FRET-based cGMP indicator (αMHC/cGi-500), NO-dependent cGMP signals were shown to occur in myocytes, to depend on gap junctions and to be degraded mainly by PDE3. Stimulation of NO-sensitive guanylyl cyclase enhanced Forskolin- and Isoproterenol-induced cAMP and phospholamban phosphorylation. Genetic inactivation of NO-GC in Tcf21-expressing cardiac fibroblasts abrogated the synergistic action of NO-GC stimulation on Iso-induced phospholamban phosphorylation, identifying fibroblasts as cGMP source and substantiating the necessity of cGMP-transfer to myocytes. In sum, NO-stimulated cGMP formed in cardiac fibroblasts enters cardiomyocytes in native tissue where it exerts an inhibitory effect on cAMP degradation by PDE3, thereby increasing cAMP and downstream effects in cardiomyocytes. Hence, enhancing β-receptor-induced contractile responses appears as one of NO/cGMP’s functions in the non-failing heart.

Vacuum-assisted tissue embedding for whole-heart imaging.

The use of combined optical imaging and tissue sectioning has potential for use in visualizing heart-wide fine structures at single-cell resolution. However, existing tissue preparation methods fail to generate ultrathin cavity-containing cardiac tissue slices with minimal deformation. This study developed an efficient vacuum-assisted tissue embedding method to prepare high-filled, agarose-embedded whole-heart tissue. Utilizing optimized vacuum parameters, we achieved 94% filled whole-heart tissue with the thinnest cut slice of 5 µm. We subsequently imaged a whole mouse heart sample using vibratome-integrated fluorescence micro-optical sectioning tomography (fMOST) with a voxel size of 0.32 µm × 0.32 µm × 1 µm. The imaging results indicated that the vacuum-assisted embedding method enabled whole-heart tissue to withstand long-term thin cutting while ensuring that slices were consistent and of high quality.

PO-04-208 CLINICAL APPLICATION OF ARTIFICIAL INTELLIGENCE IN PREDICTION OF RECURRENCE IN ATRIAL FIBRILLATION PATIENTS WITH CATHETER ABLATION

The atrial fibrillation (AF) recurrence is still a challenging issue after catheter ablation. It is important to identify which patients are intended to have a post-ablation recurrence before ablation for decision-making. We aimed to build AI models in PVCT images and clinical variables. We also combined deep learning in PVCT images and machine learning of clinical parameters by nonparametric statistical method or I-score to build an ensemble model to predict the post-ablation one-year AF recurrence. We retrospectively analyzed 518 PAF patients who have undergone catheter ablation of PAF (Table 1). PVCT geometric slices (2-3mm for each slice, 20-200 slices for each patient, a total of 36943 images of slices in 518 AF patients) were used in the deep learning process for the prediction of AF trigger origin as model 1. In 1 year follow-up, there was no AF recurrence in 388 (74.9%) patients (Group 1) and AF recurrence in 130 (25.1%) patients (Group 2). The deep learning process (EfficientNet) of cardiac CT geometric slices was applied for the prediction of AF recurrence. A total of 43 clinical variables were obtained before catheter ablation, including baseline characteristics, personal habits, medical histories, a stroke risk score of AF, laboratory values of blood tests, transthoracic echocardiographic parameters, left atrial volume calculated by 3D geometric reconstruction, and medications. Using different machine learning methods, the predictive performance of clinical variables using all variables or selected features by Nonparametric Statistics ranged from an AUC of 0.691 to 0.760. the predictive performance of clinical variables by I-Score was between AUC of 0.702 to 0.729 (Table 2). The best predictive performance was an AUC of 0.729 using I-Score rank 3 parameters (Table 3) and the CatBoost machine learning method. We combined the features of PVCT images and clinical variables. The ensemble model was able to promote the predictive performance up to an AUC of 0.722 (accuracy of 75.9%, sensitivity of 50.0%, and specificity of 83.7%) in the test set. Deep-learning AI using pre-ablation cardiac CT and clinical variables can be applied in the prediction of recurrence in AF patients receiving catheter ablation. Application of this model may identify patients with post-ablation AF recurrence.

Using cultured canine cardiac slices to model the autophagic flux with doxorubicin.

Chemotherapy-induced impairment of autophagy is implicated in cardiac toxicity induced by anti-cancer drugs. Imperfect translation from rodent models and lack of in vitro models of toxicity has limited investigation of autophagic flux dysregulation, preventing design of novel cardioprotective strategies based on autophagy control. Development of an adult heart tissue culture technique from a translational model will improve investigation of cardiac toxicity. We aimed to optimize a canine cardiac slice culture system for exploration of cancer therapy impact on intact cardiac tissue, creating a translatable model that maintains autophagy in culture and is amenable to autophagy modulation. Canine cardiac tissue slices (350 μm) were generated from left ventricular free wall collected from euthanized client-owned dogs (n = 7) free of cardiovascular disease at the Foster Hospital for Small Animals at Tufts University. Cell viability and apoptosis were quantified with MTT assay and TUNEL staining. Cardiac slices were challenged with doxorubicin and an autophagy activator (rapamycin) or inhibitor (chloroquine). Autophagic flux components (LC3, p62) were quantified by western blot. Cardiac slices retained high cell viability for >7 days in culture and basal levels of autophagic markers remained unchanged. Doxorubicin treatment resulted in perturbation of the autophagic flux and cell death, while rapamycin co-treatment restored normal autophagic flux and maintained cell survival. We developed an adult canine cardiac slice culture system appropriate for studying the effects of autophagic flux that may be applicable to drug toxicity evaluations.

Intermittent cold exposure upregulates regulators of cardiac mitochondrial biogenesis and function in mice.

Hypothermic conditions enhance the incidence of cardiovascular diseases due to increased blood pressure. Cold-induced adaptive thermogenesis increased mitochondrial biogenesis and function in skeletal muscles and adipocytes. Here, we studied the effect of intermittent cold exposure on the regulators of cardiac mitochondrial biogenesis, function, and its regulation by SIRT-3. Intermittent cold exposed mice hearts showed normal histopathology with increased mitochondrial antioxidant and metabolic function, as evidenced by an increase in the activity and expression of MnSOD and SDH. A substantial increase in mitochondrial DNA copy number and increase in the expression of PGC-1α and its downstream targets NRF-1 and Tfam indicated the possibility of enhanced cardiac mitochondrial biogenesis and function on intermittent cold exposure. Increased mitochondrial SIRT-3 level and decreased total protein lysine acetylation indicate increased sirtuin activity in cold exposed mice hearts. Ex vivo cold mimic using norepinephrine showed a significant increase in PGC-1α, NRF-1, and Tfam levels. AGK-7, a SIRT-3 inhibitor, reversed the norepinephrine-induced upregulation of PGC-1α and NRF-1, indicating the role ofSIRT-3 on the production of PGC-1α and NRF-1. Inhibition of PKA with KT5720 in norepinephrine treated cardiac tissue slices indicates the role of PKA in regulating the production of PGC-1α and NRF-1. In conclusion, intermittent cold exposure upregulated the regulators of mitochondrial biogenesis and function through PKA and SIRT-3 mediated pathway. Our results emphasize the role of intermittent cold-induced adaptive thermogenesis in overcoming chronic cold-induced cardiac damage.