Border Zone Of Myocardial Infarction Research Articles

Abstract 12784: Ischemic Cardiac Autophagy/Mitophagy is Regulated by TRPML1 and Mitochondrial Reactive Oxygen Species

Introduction: In myocardial infarction (MI), activated autophagy/mitophagy helps degrade cytoplasmic macromolecules/proteins/ organelles (especially damaged mitochondria), recycle breakdown products, and prevent oxidative damage and cell death. It had reported that reactive oxygen species (ROS) activates the transcription factor EB (TFEB) via a lysosomal transient receptor potential mucolipin 1 (TRPML1) Ca 2+ efflux. TFEB is thought to be the master regulator of lysosome biogenesis. Hypothesis: We investigated whether lysosomal TRPML1 was activated by ROS and contributed to ischemic cardiac autophagy/mitophagy. Methods: MI was induced in wild type C57BL/6J mice by ligated left anterior descending coronary artery. Mice were studied at three weeks after MI surgery. Results: The lysosomal-restricted TRPML1 Ca 2+ release channel was significantly upregulated by 84.8% in cardiac border zone (BZ) and 55.4% in a remote zone after MI. As expected, dephosphorylated TFEB (i.e., active TFEB), mitophagy marker Parkin, autophagy marker LC3II, and lysosome marker LAMP1 increased substantially after MI. Autophagy degradation was upregulated as measured by SQSTM1/p62. TRPML1 protein expression was reduced in a dose-dependent manner by TRPML1 shRNA AAV9 (AAV9 was injected 1 week after MI for two weeks). The protein levels of LC3II and LAMP1 significantly decreased in MI BZ after AAV9 transfection. Mitochondrial ROS scavenger (mitoTEMPO, 1 mg/kg/day, i.p.) treatment for two weeks (starting at 1 week after MI) significantly inhibited TRPML1, LAMP1, LC3II and Parkin expression in the MI BZ. Conversely, when TRPML1 was overexpressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), the protein levels of LC3II/Parkin were increased. Speaking to human relevance, TRPML1 was significantly increased in human patients with ischemic cardiomyopathy. Conclusion: Autophagy and mitophagy after MI are regulated by TRPML1 and mitochondrial ROS.

Abstract 13022: Myocardial Restoration and Functional Recovery Through Transplantation of Vascularized Cardiac Microtissues Derived From Human Induced Pluripotent Stem Cells in a Rat Model of Myocardial Infarction

Introduction: Cardiac regenerative therapy is acknowledged as a promising strategy for treating individuals suffering from severe heart failure. To address the current constraints of stem cell therapy, which primarily relies on indirect paracrine effects for functional improvement, the ideal goal would be to restore the damaged myocardium in the diseased heart. To achieve this, the transplantation of biomimetic heart tissues that closely resemble the natural structure of the heart is highly anticipated. Methods: We successfully generated 3-dimensional vascularized cardiac microtissues (VCMs) using human induced pluripotent stem cells (hiPSCs) through dynamic culture on thin cardiac tissue sheets (CTSs) composed of hiPSC-derived cardiomyocytes and vascular cells. These VCMs exhibited a robust vascular network comprising human vascular cells. Subsequently, we transplanted CTSs (n=6) or VCMs (n=6) onto an athymic rat model of myocardial infarction (MI), while a sham operation was performed on another group (n=6). To assess cardiac function, we employed cardiac magnetic resonance imaging. The survival of grafts, extension of fibrotic scars, neovascularization in the MI border zone, and the formation of a human vascular network within the engrafted region were evaluated through histopathology and light sheet fluorescence microscopy with tissue clearing techniques. Results: Functional analyses demonstrated that the VCM group exhibited the highest left ventricular ejection fraction (Sham: 36.5±0.9%, CTS: 43.5±2.9%, VCM: 55.2±2.5%, p < 0.01). Histopathological evaluation revealed that the VCM group had a larger engraftment area, along with the smallest scar extension and the highest vascular density in the myocardial infarction border zone. Furthermore, LSFM revealed the presence of vascular networks originating from human cells within the engrafted VCMs. Conclusions: The transplantation of hiPSC-derived VCMs demonstrated remarkable therapeutic potential in a rat model of MI, leading to the formation of a vascular network by human cells. This approach might hold great promise as an effective strategy for efficiently restoring the damaged myocardium in hearts afflicted with disease.

Abstract P3084: Neutrophil Expansion And Netosis Are Essential For Adverse Left Ventricular Remodeling And Dysfunction In Chronic Ischemic Heart Failure

Chronic inflammation contributes significantly to disease progression in heart failure (HF) with the expansion and adverse activation of macrophages, dendritic cells, and T cells in the failing heart. Neutrophils play a well-known role in the initial acute immune response to myocardial infarction (MI), however, whether and how they contribute to inflammation and adverse LV function and remodeling in chronic ischemic HF is unknown. We tested the hypothesis that activated neutrophils are required for tissue remodeling and disease progression in HF in mice studied 4 and 8 weeks after MI during established ischemic HF (vs sham). CD11b + Ly6G + neutrophils were expanded in peripheral blood (PB), heart, spleen and bone marrow 8 w post-MI. Neutrophils in the failing heart were localized to the MI border zone and augmented in remote zone myocardium by immunofluorescent staining. CXCL1 and CXCL5 expression was increased in the failing heart, consistent with chemotactic signals for myocardial neutrophil recruitment. Plasma from HF mice elicited greater chemotaxis in naïve neutrophils (vs naïve plasma) by time-lapse video microscopy. Further, PB neutrophils from HF mice exhibited significantly increased neutrophil extracellular trap (NET) production, with concomitant increase in plasma NETs (histone-DNA complexes) in HF mice. The direct pathological impact of NETs was demonstrated by intramyocardial injection of isolated NETs into naïve hearts mimicking HF with decreased EF, LV dilation, and fibrosis. Finally, the in vivo role of neutrophils in chronic HF (4 and 8 w post-MI) was determined via depletion studies using genetic (Ly6G-diptheria toxin receptor mice) and antibody (1A8) based approaches. Neutrophil depletion significantly improved LV EF and reduced end systolic and diastolic volumes. 1A8 treated HF mice also exhibited reduced remote zone fibrosis. The necessity of NETosis in HF development was confirmed using neutrophil and myeloid – specific PAD4 deletion where the decline in LV EF and increase in volumes in HF were attenuated. In conclusion , neutrophil expansion and NETosis are important contributors to chronic inflammation in HF and play an obligatory role in the development of LV dysfunction and remodeling in ischemic cardiomyopathy.

Heterogeneity of Repolarization and Cell-Cell Variability of Cardiomyocyte Remodeling Within the Myocardial Infarction Border Zone Contribute to Arrhythmia Susceptibility.

After myocardial infarction, the infarct border zone (BZ) is the dominant source of life-threatening arrhythmias, where fibrosis and abnormal repolarization create a substrate for reentry. We examined whether repolarization abnormalities are heterogeneous within the BZ in vivo and could be related to heterogeneous cardiomyocyte remodeling. Myocardial infarction was induced in domestic pigs by 120-minute ischemia-reperfusion injury. After 1 month, remodeling was assessed by magnetic resonance imaging, and electroanatomical mapping was performed to determine the spatial distribution of activation-recovery intervals. Cardiomyocytes were isolated and tissue samples collected from the BZ and remote regions. Optical recording allowed assessment of action potential duration (di-8-Anepps, stimulation at 1 Hz, 37 °C) of large cardiomyocyte populations while gene expression in cardiomyocytes was determined by single nuclear RNA sequencing. In vivo, activation-recovery intervals in the BZ tended to be longer than in remote with increased spatial heterogeneity evidenced by a greater local SD (3.5±1.3 ms versus remote: 2.0±0.5 ms, P=0.036, npigs=5). Increased activation-recovery interval heterogeneity correlated with enhanced arrhythmia susceptibility. Cellular population studies (ncells=635-862 cells per region) demonstrated greater heterogeneity of action potential duration in the BZ (SD, 105.9±17.0 ms versus remote: 73.9±8.6 ms; P=0.001; npigs=6), which correlated with heterogeneity of activation-recovery interval in vivo. Cell-cell gene expression heterogeneity in the BZ was evidenced by increased Euclidean distances between nuclei of the BZ (12.1 [9.2-15.0] versus 10.6 [7.5-11.6] in remote; P<0.0001). Differentially expressed genes characterizing BZ cardiomyocyte remodeling included hypertrophy-related and ion channel-related genes with high cell-cell variability of expression. These gene expression changes were driven by stress-responsive TFs (transcription factors). In addition, heterogeneity of left ventricular wall thickness was greater in the BZ than in remote. Heterogeneous cardiomyocyte remodeling in the BZ is driven by uniquely altered gene expression, related to heterogeneity in the local microenvironment, and translates to heterogeneous repolarization and arrhythmia vulnerability in vivo.

In vivo pulsed-field ablation in healthy vs. chronically infarcted ventricular myocardium: biophysical and histologic characterization.

Data on ventricular pulsed-field ablation (PFA) are sparse in the setting of chronic myocardial infarction (MI). The objective of this study was to compare the biophysical and histopathologic characteristics of PFA in healthy and MI swine ventricular myocardium. Myocardial infarction swine (n = 8) underwent coronary balloon occlusion and survived for 30 days. We then performed endocardial unipolar, biphasic PFA of the MI border zone and a dense scar with electroanatomic mapping and using an irrigated contact force (CF)-sensing catheter with the CENTAURI System (Galaxy Medical). Lesion and biophysical characteristics were compared with three controls: MI swine undergoing thermal ablation, MI swine undergoing no ablation, and healthy swine undergoing similar PFA applications that included linear lesion sets. Tissues were systematically assessed by gross pathology utilizing 2,3,5-triphenyl-2H-tetrazolium chloride staining and histologically with haematoxylin and eosin and trichrome. Pulsed-field ablation in healthy myocardium generated well-demarcated ellipsoid lesions (7.2 ± 2.1 mm depth) with contraction band necrosis and myocytolysis. Pulsed-field ablation in MI demonstrated slightly smaller lesions (depth 5.3 ± 1.9 mm, P = 0.0002), and lesions infiltrated into the irregular scar border, resulting in contraction band necrosis and myocytolysis of surviving myocytes and extending to the epicardial border of the scar. Coagulative necrosis was present in 75% of thermal ablation controls but only in 16% of PFA lesions. Linear PFA resulted in contiguous linear lesions with no gaps in gross pathology. Neither CF nor local R-wave amplitude reduction correlated with lesion size. Pulsed-field ablation of a heterogeneous chronic MI scar effectively ablates surviving myocytes within and beyond the scar, demonstrating promise for the clinical ablation of scar-mediated ventricular arrhythmias.

ONO-1301 enhances post-transplantation survival of human induced pluripotent stem cell-derived cardiac tissue sheet by promoting angiogenesis

Transplanting human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) tissue sheets effectively treat ischemic cardiomyopathy. Cardiac functional recovery relies on graft survival in which angiogenesis played an important part. ONO-1301 is a synthetic prostacyclin analog with proangiogenic effects. We hypothesized that transplantation of hiPSC-CM tissue sheets with slow-release ONO-1301 scaffold could promote hostgraft angiogenesis, enhance tissue survival and therapeutic effect. We developed hiPSC-CM tissue sheets with ONO-1301 slow-release scaffold and evaluated their morphology, gene expression, and effects on angiogenesis. Three tissue sheet layers were transplanted into a rat myocardial infarction (MI) model. Left ventricular ejection fraction, gene expression in the MI border zone, and angiogenesis effects were investigated 4 weeks after transplantation. In vitro assessment confirmed the slow-release of ONO-1301, and its pro-angiogenesis effects. In addition, in vivo data demonstrated that ONO-1301 administration positively correlated with graft survival. Cardiac tissue as thick as ∼900 μm was retained in the ONO (+) treated group. Additionally, left ventricular ejection fraction of the ONO (+) group was significantly enhanced, compared to ONO (-) group. The ONO (+) group also showed significantly improved interstitial fibrosis, higher capillary density, increased number of mature blood vessels, along with an enhanced supply of oxygen, and nutrients. Slow-release ONO-1301 scaffold provided an efficient delivery method for thick hiPSC-CM tissue. ONO-1301 promotes angiogenesis between the host and graft and improves nutritional and oxygen supply, thereby enhancing the survival of transplanted cells, effectively improving ejection fraction, and therapeutic effects.

Reduced sarcoplasmic reticulum Ca2+ pump activity is antiarrhythmic in ischemic cardiomyopathy

We have described an arrhythmic mechanism seen only in cardiomyopathy that involves increased mitochondrial Ca2+ handling and selective transfer of Ca2+ to the sarcoplasmic reticulum (SR). Modeling suggested that mitochondrial Ca2+ transfer to the SR via type 2a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is a crucial element of this arrhythmic mechanism. We tested the role of SERCA2a in arrhythmias during ischemic cardiomyopathy. Myocardial infarction (MI) was induced in wild-type (Wt) and SERCA2a heterozygous knockdown (SERCA+/-) mice. Compared with Wt MI mice, SERCA2a heterozygous knockdown (SERCA+/-) MI mice had a substantially lower mortality after 3 weeks of MI without a significant change in MI area. Aside from a significant delay of the cytoplasmic Ca2+ transient decay existed in SERCA+/- compared with Wt, SERCA+/- did not affect cardiac systolic and diastolic function at the whole organ or single cell levels either before or after MI. After MI, SERCA+/- mice had reduced SERCA2a expression in the MI border zone compared with Wt MI mice. SERCA+/- mice had significantly decreased corrected QT intervals and less ventricular tachycardia compared with Wt MI mice. SERCA+/- cardiomyocytes from MI mice showed a reduced action potential duration and reduced triggered activity compared with Wt MI cardiomyocytes. Reduction in arrhythmic risk was accompanied by reduced diastolic SR Ca2+ sparks, reduced SR Ca2+ content, reduced oxidized ryanodine receptor, and increased calsequestrin 2 in SERCA+/- MI mice. SERCA2a knockdown was antiarrhythmic after MI without affecting overall systolic performance. Possible antiarrhythmic mechanisms included reduced SR free Ca2+ and reduced diastolic SR Ca2+ release.

Heterogeneous myocyte remodelling and spatial heterogeneity of repolarization within the myocardial infarction border zone

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fund for Scientific Research-Flanders (FWO) Background Sudden cardiac death due to ventricular arrhythmias is a major cause of mortality after myocardial infarction (MI). The border zone (BZ) surrounding the infarct is the dominant source of arrhythmias. Here a substrate of heterogeneous repolarization is implicated, which could be due to heterogeneous myocyte remodelling. Objective To examine myocyte remodelling within the BZ, in comparison to the remote myocardium, and evaluate the local profile of repolarization of these regions in vivo. Methods MI was induced by 120-minute occlusion of the left anterior descending coronary artery followed by reperfusion in 6 domestic pigs. After 4 weeks, magnetic resonance imaging was performed to assess infarct remodelling and local wall thickness. Within 3 days, electro-anatomical mapping was performed. A non-contact recording of a 64-electrode array was translated to 2048 electrograms distributed over the LV and local activation-recovery-interval (ARI) determined by custom software. After recovery (2-4 days), the pigs were sacrificed, and samples collected from the BZ and remote region for RNA analysis and single cardiomyocyte isolation. Cell dimensions were measured and cellular AP duration (APD) was optically recorded using a fluorescent voltage dye, Di-8-Annepps (stimulation at 1Hz, 37°C). Expression and variability of cardiomyocyte hypertrophy biomarkers were extracted from single nuclear RNA sequencing data (10x Genomics). Results Cardiomyocyte APD in large population samples (&amp;gt; 100 cells per region in each pig) revealed higher heterogeneity in the BZ than the remote region, quantified as the standard deviation (SD) (BZ: 105.9 ± 17.0ms vs remote: 73.9 ± 8.6ms, P = 0.001). Cellular APD heterogeneity correlated strongly with in vivo local ARI heterogeneity, which demonstrated increased heterogeneity in the BZ (R2 = 0.67, P = 0.002). BZ myocytes were hypertrophied with greater increase in cell width than length, and cellular hypertrophy was more heterogeneous by SD in the BZ (BZ: 12.9 ± 2.4μm vs remote: 8.3 ± 1.1μm, P &amp;lt; 0.001). NPPB transcripts reporting on hypertrophic remodelling were higher in BZ than remote (mean lognorm gene expression, BZ: 0.431 ± 0.014 vs remote: 0.107 ± 0.004, P &amp;lt; 0.001), and showed greater heterogeneity in expression between cells by proportion of hypertrophic (NPPB +ve) cells (BZ: 30.86% vs remote: 8.37%, P &amp;lt; 0.001). Wall thickness variance was higher in the BZ compared to the remote region (anterior BZ: 0.15 ± 0.02mm, septal BZ: 0.16 ± 0.04mm vs remote: 0.04 ± 0.02mm, P &amp;lt; 0.001), contributing to increased heterogeneity of local wall stress in BZ. Conclusion Cardiomyocyte remodelling in the BZ is heterogeneous, possibly related to differences in local wall stress, which may contribute to heterogeneous repolarization in vivo and underlie arrhythmia vulnerability within the BZ.

Abstract 12861: Adjoining Myofibroblasts Disrupt Impulse Propagation of Myocardial Tissue via Connexin 43-Mediated Heterocellular Gap Junctional Coupling

Introduction: The composite population of myofibroblasts (MFBs) and cardiomyocytes (CMCs) is known to alter impulse conduction in the heart. A previous study reported that structural disruption between cardiomyocytes by fibrosis or myofibroblast proliferation causes conduction delay due to insulating or reducing the intercellular current. However, it is unknown whether and how the conduction is altered when the myocardial tissue faces adjoining with MFB-rich granulation tissue, like, e.g. , the border zone of myocardial infarcts during infarct healing. Objectives: To clarify the influence of heterocellular gap-junctional coupling between CMCs and MFBs by calcium imaging in an injured myocardial tissue-mimicking model in which CMCs connect with MFBs via microporous membranes. Methods: Fluo-8 fluorescence patterns of impulse propagation were spatiotemporally imaged (27 x 18.9 mm, 333 frames/s, 32 °C) in neonatal rat CMC monolayers cultured on the upper sides of the Boyden chamber (pore diameter, 8 μm) of which MFB monolayer was co-cultured on the reverse sides (CMC-MFB group). For comparison, CMC monolayers were cultured on both membrane sides (CMC-CMC group). Results: During consecutive pacing at 1 - 4 Hz, CMC monolayers showed concentric propagation from the pacing site with slower conduction velocity (CV) and more irregular wavefronts in a frequency-dependent manner. The conduction slowing and its non-uniformity were more remarkable in the CMC-MFB group than in the CMC-CMC group. We confirmed the gap junction coupling between the upper CMC and lower MFB layers using co-immunostaining and calcein staining. Heptanol, gap junction inhibiter, inhibited calcein dye transfer from the upper CMC layer to the lower MFB layer. The knockdown of Cx43 in MFBs on the reverse layer improved the delay of CV and non-uniformity in the CMC-MFB group. Conclusions: In this study, we clarified the electric association between adjoining MFBs and CMCs. Our observations suggest that adjoining MFBs slow down conduction velocity and disrupt conduction uniformity on CMCs via Cx43-mediated heterocellular gap-junctional coupling between CMCs and MFBs.

Discrete sites of frequent premature ventricular complexes cluster within the infarct border zone and coincide with high frequency of delayed afterdepolarizations under adrenergic stimulation.

Sympathetic activation in ischemic heart disease can cause lethal arrhythmias. These often are preceded by premature ventricular complexes (PVCs), which at the cellular level could result from delayed afterdepolarizations. The purpose of this study was to identify and map vulnerable areas for arrhythmia initiation after myocardial infarction (MI) and to explore the link between PVCs and cellular events. Anterior-septal wall MI was induced by 120 minutes of coronary occlusion followed by reperfusion (27 MI and 16 sham pigs). After 4 weeks, EnSite™ electroanatomic mapping combined with imaging was performed to precisely locate PVC sites of origin and subsequently record monophasic action potentials. Cardiomyocytes were isolated from different regions to study regional cellular remodeling. Isoproterenol was used as a surrogate for adrenergic stimulation both invivo and in cardiomyocytes. PVCs originated from the MI border zone (BZ) and occurred at discrete areas with clusters of PVCs within the BZ. At these sites, frequent delayed afterdepolarizations and occasional associated spontaneous action potentials translating to a PVC were present. Cardiomyocytes isolated from the MI BZ exhibited more spontaneous action potentials than cardiomyocytes from remote regions. Sensitivity to adrenergic stimulation was increased in MI, invivo and in cardiomyocytes. In awake, freely moving MI animals, frequent PVCs, ventricular arrhythmia, and sudden cardiac death occurred spontaneously at moderately elevated heart rates. Post-MI, arrhythmias initiate from discrete vulnerable areas within the BZ, where delayed afterdepolarizations, related to increased adrenergic response of BZ cardiomyocytes, can generate PVCs.

Epigenetic State Changes Underlie Metabolic Switch in Mouse Post-Infarction Border Zone Cardiomyocytes.

Myocardial infarction causes ventricular muscle loss and formation of scar tissue. The surviving myocardium in the border zone, located adjacent to the infarct, undergoes profound changes in function, structure and composition. How and to what extent these changes of border zone cardiomyocytes are regulated epigenetically is not fully understood. Here, we obtained transcriptomes of PCM-1-sorted mouse cardiomyocyte nuclei of healthy left ventricle and 7 days post myocardial infarction border zone tissue. We validated previously observed downregulation of genes involved in fatty acid metabolism, oxidative phosphorylation and mitochondrial function in border zone-derived cardiomyocytes, and observed a modest induction of genes involved in glycolysis, including Slc2a1 (Glut1) and Pfkp. To gain insight into the underlying epigenetic regulatory mechanisms, we performed H3K27ac profiling of healthy and border zone cardiomyocyte nuclei. We confirmed the switch from Mef2- to AP-1 chromatin association in border zone cardiomyocytes, and observed, in addition, an enrichment of PPAR/RXR binding motifs in the sites with reduced H3K27ac signal. We detected downregulation and accompanying epigenetic state changes at several key PPAR target genes including Ppargc1a (PGC-1α), Cpt2, Ech1, Fabpc3 and Vldrl in border zone cardiomyocytes. These data indicate that changes in epigenetic state and gene regulation underlie the maintained metabolic switch in border zone cardiomyocytes.

Repolarization heterogeneity within the myocardial infarction border zone correlates with variability of myocyte remodeling

Abstract Background Myocardial infarction (MI) results in a regional scar, with a border zone (BZ) of surviving myocytes interspersed with fibrosis providing an anatomical substrate for re-entry. Heterogeneous repolarization within the BZ may add a functional component aggravating re-entrant arrhythmias. Purpose We studied BZ heterogeneity and developed novel methodology for high resolution mapping of local in vivo activation-repolarization intervals (ARI) within the BZ and for studying the relation to cellular action potential (AP) profiles of cells isolated from the BZ. Methods Anterior-septal myocardial infarction was induced in 5 domestic pigs by 120-minute occlusion of the left anterior descending artery followed by reperfusion (18.9±4.7% of the left ventricle). After 1-month, electro-anatomical mapping was performed. Contact mapping was used to define the BZ (bipolar voltage 0.5–1.5mV). A non-contact recording of a 64-electrode array was translated to 2048 non-contact electrograms distributed over the LV. The non-contact electrograms were processed to determine the ARIs using a custom-made algorithm, validated against monophasic action potential recordings. After 2–4 days recovery, single cardiomyocytes were enzymatically isolated from the anterior-septal BZs and remote regions. Cardiomyocytes were field stimulated at 1Hz at 37°C and cellular AP duration (APD) was optically recorded (fluorescent voltage-sensitive dye Di-8-Annepps). Results In vivo, regional ARIs tended to be longer in the BZs than remote. ARI heterogeneity, quantified as the standard deviation of ARIs in a neighborhood of 1cm radius, was increased in the BZ (anterior BZ: 3.4±1.0 ms, P=0.052, septal BZ: 3.6±1.7 ms, P=0.027 vs remote: 2.0±0.5 ms). Cellular APD was measured in large population samples (&amp;gt;100 cells per region in each pig) and was longer in BZ myocytes compared to the remote region. Cellular APD heterogeneity, measured as the standard deviation within cell population samples pooled by region per animal, was increased in the BZ (anterior BZ: 105.9±17.0 ms, P=0.0010; septal BZ: 98.1±20.8 ms, P=0.0127 vs remote: 73.9±8.6 ms). Cell APD correlated to in vivo ARI (R2=0.34, P=0.021) and cellular heterogeneity correlated strongly with in vivo heterogeneity (R2=0.67, P=0.002). Conclusion In the BZ of MI, in vivo regional heterogeneity adds a functional substrate for re-entry that may result from heterogeneous cellular remodeling and increased cell-cell APD variability. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): KU Leuven BOF-C1 “Blood pressure induced premature ventricular beats as triggers for ventricular arrhythmia in ischemic cardiomyopathy”

Therapeutic potential of clinical-grade human induced pluripotent stem cell-derived cardiac tissues

ObjectivesTo establish a protocol to prepare and transplant clinical-grade human induced pluripotent stem cell (hiPSC)-derived cardiac tissues (HiCTs) and to evaluate the therapeutic potential in an animal myocardial infarction (MI) model. MethodsWe simultaneously differentiated clinical-grade hiPSCs into cardiovascular cell lineages with or without the administration of canonical Wnt inhibitors, generated 5- layer cell sheets with insertion of gelatin hydrogel microspheres (GHMs) (HiCTs), and transplanted them onto an athymic rat MI model. Cardiac function was evaluated by echocardiography and cardiac magnetic resonance imaging and compared with that in animals with sham and transplantation of 5-layer cell sheets without GHMs. Graft survival, ventricular remodeling, and neovascularization were evaluated histopathologically. ResultsThe administration of Wnt inhibitors significantly promoted cardiomyocyte (CM) (P < .0001) and vascular endothelial cell (EC) (P = .006) induction, which resulted in cellular components of 52.0 ± 6.1% CMs and 9.9 ± 3.0% ECs. Functional analyses revealed the significantly lowest left ventricular end-diastolic volume and highest ejection fraction in the HiCT group. Histopathologic evaluation revealed that the HiCT group had a significantly larger median engrafted area (4 weeks, GHM(-) vs HiCT: 0.4 [range, 0.2-0.7] mm2 vs 2.2 [range, 1.8-3.1] mm2; P = .005; 12 weeks, 0 [range, 0-0.2] mm2 vs 1.9 [range, 0.1-3.2] mm2; P = .026), accompanied by the smallest scar area and highest vascular density at the MI border zone. ConclusionsTransplantation of HiCTs generated from clinical-grade hiPSCs exhibited a prominent therapeutic potential in a rat MI model and may provide a promising therapeutic strategy in cardiac regenerative medicine.

Heart Rate Reduction with Ivabradine Prevents Cardiac Rupture after Myocardial Infarction in Mice.

Cardiac rupture is a fatal complication following myocardial infarction (MI). An increase in heart rate (HR) is reportedly an independent risk factor for cardiac rupture during acute MI. However, the role of HR reduction in cardiac rupture after MI remains to be fully elucidated. We aimed to evaluate the therapeutic efficacy of HR reduction with ivabradine (IVA) on post-MI cardiac rupture in mice. We induced MI in mice by ligating the left anterior descending coronary artery. Subsequently, we subcutaneously implanted osmotic pumps filled with IVA solution or vehicle (Veh) in the surviving MI mice at 24h postoperatively. We biochemically analyzed the myocardium on day 5, additionally observed the mice for 10days, and analyzed the rates of cardiac rupture and non-cardiac rupture death, and survival after MI. HR was significantly lower in the IVA-treated mice, whereas blood pressure was comparable between the two groups. Compared to the Veh-treated mice, apoptosis was significantly reduced in the MI border zone in the IVA-treated mice. Although there were no differences in the infarct size of the surviving MI mice between the two groups, HR reduction with IVA significantly reduced cardiac rupture (rupture rate 26 and 8% in the Veh-treated and IVA-treated groups, respectively) and improved survival after MI. Our findings suggest that HR reduction with IVA prevents cardiac rupture after MI. This may be particularly effective in MI patients with a high HR who are either unable to adequately tolerate β-blockers or whose HR remains high despite receiving β-blockers.

Electric Field-Based Dense Spatial Analysis of Unipolar Electrograms to Map Regional Activation-Repolarization Intervals

Objective: We wanted to develop a new method for processing dense spatially sampled unipolar electrograms, that yields physiological spatial distributions in activation-repolarization intervals.Background: Spatial heterogeneity in repolarization plays an important role in genesis and sustainment of cardiac arrhythmias. Reliable determination of repolarization times at high resolution on electrograms remains a difficult task. Methods: The new method describes the electrical activity on the heart's surface in terms of a physical electric field (E-field). Activation and repolarization times are calculated using an amplitude weighted average on QRS and T-waves. Activation-recovery intervals (ARI) obtained with this E-field method were validated against action potential durations (APD90) from monophasic action potential measurements. Relevance of the method was shown by calculating dense spatial distributions of beat-to-beat variability of repolarization (BVR) in myocardial infarction (MI).Results: E-field waveforms were more spatially homogeneous than the unipolar electrograms. The ARI strongly correlated with the APD90 (r2=0.94; p<0.001). Continuous activation and repolarization patterns were obtained, and the method removes non-physiological sharp spatial gradients obtained by currently adopted methods. Spatial BVR analysis revealed an increase in maximum BVR, spatial variance of BVR and spatial gradient of BVR in the border zone versus remote region of MI (all p<0.01). All BVR values were in pathophysiological range as described in other studies.Conclusion: The E-field method allows for a reliable determination of activation and repolarization times over the whole left ventricle. The new methodology confirmed that the border zone of myocardial infarction is a region with patches of increased variability of repolarization.

Abstract 14554: Angiogenic Agent Ono-1301 Enhanced Cardiac Contractile Proteins in Hipsc Derived Cardiac Tissue-like Construct With Functional Angiogenesis

Introduction: Transplantation of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) into myocardial infarcted areas has shown potential in healing myocardial infarction (MI). However, the survival rate of transplanted cells is yet considered limited, restricting the therapeutic effects of the transplantation. We assumed that adding angiogenic agent ONO-1301 could promote early stage angiogenesis between host and graft, and benefit the therapeutic effects of hiPSC-CM transplantation. Methods: This study is based on our former study of Cardiac Tissue-Like Construct (CTLC), where 5 x10e6 of hiPSC-CM was seeded on a 1x1cm poly(lactic-co-glycolic acid) (PLGA) nano fiber (Figure A). In addition, angiogenic agent ONO-1301 was mixed into the fibers. After 24h culture in vitro, 3 layers of CTLCs were transplanted into Nude rat MI models with or without ONO-1301. The rats were raised for another 4 or 8 weeks before sacrifice. Results: In vitro experiments have shown that supernatant of iPS-CM with ONO-1301 underwent a rise in VEGF during a 3 to 6 day culture (Figure B). The VEGF concentration in ONO-1301 positive group is much higer than ONO-1301 negative group (P=0.022) on day3. This difference narrows down over time. In vivo experiments on nude rat have shown that capillary density on MI border zone of ONO fiber+iPS-CM group is 29.95% higher than that of PLGA fiber+iPS-CM group (P=0.004) (figure C). HE and immunofluorescence staining shows remaining transplanted hiPSC-CMs in ONO fiber+iPS-CM group shows more significant TNT2 and human nuclear antigen(HNA) expression than that of PLGA fiber+iPS-CM group (Figure D and E). Also ONO fiber+iPS-CM group showed functional recovery compared to the sham.

Abstract 16640: Neutrophil Expansion is Essential for Adverse Left Ventricular Remodeling and Dysfunction in Chronic Ischemic Heart Failure

Chronic inflammation contributes significantly to disease progression in heart failure (HF), and is characterized by expansion of activated macrophages, dendritic cells, and T cells in the failing heart. Neutrophils play a well-known role as the initial drivers of inflammatory response to myocardial infarction (MI), thereby providing the platform for the subsequent recruitment of macrophages and T cells. However, whether and how neutrophils contribute to inflammation and adverse LV remodeling in chronic HF are unknown. We tested the hypothesis that activated neutrophils are required for tissue remodeling and disease progression in HF. Male C57Bl/6 mice were studied 4 and 8 weeks after left coronary artery ligation during established ischemic HF (sham-operated controls). As assessed by flow cytometry, CD11b + Ly6G + neutrophils were expanded in the peripheral blood (4 w post-MI), and in the bone marrow, spleen, and heart at 8 w post-MI. Immunostaining revealed that in the failing heart, neutrophils were primarily localized to the MI border zone, but also augmented in remote zone myocardium. CXCL1 and CXCL5 chemokine expression was increased in the failing heart, consistent with chemotactic signals for myocardial neutrophil recruitment. Isolated naïve neutrophils also exhibited increased chemotaxis in response to plasma from HF mice (versus plasma from naïve mice), as assessed by time-lapse video microscopy. Moreover, blood neutrophils isolated from HF mice exhibited significantly increased neutrophil extracellular trap (NET) production as compared to control neutrophils, whereas plasma NETs (histone-DNA complexes) were increased in HF mice. Finally, the in vivo role of neutrophils in chronic HF (8 w post-MI) was determined via depletion studies using genetic (Ly6G-diptheria toxin receptor mice) and antibody (1A8) based approaches. In both models, 2 and 4 w of neutrophil depletion, respectively, significantly improved LV systolic function and reduced end systolic volume. 1A8 treated HF mice also exhibited reduced remote zone fibrosis. We conclude: neutrophil expansion and activation are important components of the chronic inflammatory response in HF, and play an obligatory role in the progression of LV remodeling in ischemic cardiomyopathy.

CaMKII inhibition reduces electrical activation heterogeneities caused by mechanical stretch in the myocardium

Abstract Introduction Ca2+/calmodulin-dependant protein kinase II (CaMKII) activity in cardiomyocytes plays a crucial role in their contractility. Increased CaMKII signalling has been associated with mechanical stretch, often caused in the border zone of myocardial infarction. CaMKII upregulation causes a mishandling of intracellular calcium, a precursor of multiple pro-arrhythmic mechanisms, such as early afterdepolarisations. Purpose In this study, we aim to quantify the effects of KN-93 -a CaMKII inhibitor- on wave dynamics, in order to investigate its effectiveness as an anti-arrhythmic agent. Methods An isolated Langendorff model was constructed based on rabbit hearts (n=18) and posteriorly induced to fibrillation. An epicardial multielectrode array (121 electrodes) was used for recording the electrical activity. Mechanical stretch was induced by pushing the anterior myocardial wall from the left-ventricular cavity. Then, a frequency analysis was conducted for the following conditions: before drug infusion, during infusion, during infusion plus stretch, and during infusion post-stretch. Nine hearts represented the untreated group, and the other nine were infused with KN-93 at a concentration of 10 nM (less than 3% of the IC50 value). Results Prior to stretch induction, KN-93 caused no effects in the spectral concentration (SC) and average dominant frequency (ADF) calculated on the infused rabbit hearts. Nevertheless, intrasubject measurements revealed statistically significant differences (p&amp;lt;0.05) between hearts infused with KN-93 and the untreated ones when stretch was induced. Changes in SC were milder in the treated than the untreated group (−6% vs −33%). Also, the stretch-induced increase in ADF was more limited in the treated group (+17% vs +40%). Hearts infused with KN-93 shown a higher resistance to stretch-induced electrical abnormalities, potentially due to better regulated intracellular calcium dynamics. Conclusion CaMKII inhibitors show cardioprotective potential, even at very low concentrations. Further research is required to investigate the therapeutic use of these compounds in conditions of intracellular calcium mishandling and its concomitant life-threatening consequences, such as heart failure or Torsade de Pointes. KN-93 reduces stretch-induced changes Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Universitat de València; Generalitat Valenciana - Prometeo; Carlos III, CIBERCV

Repair of Adult Mammalian Heart After Damages by Oral Intake of Gu Ben Pei Yuan San.

Adult mammalian heart repair after myocardial damage is highly inefficient due to the post-mitotic nature of cardiomyocytes. Interestingly, in traditional Chinese medicine (TCM), there are reported effective treatments of myocardial infarction (MI) and heart failure in adult humans by oral intake of a TCM concoction named Gu Ben Pei Yuan San (GBPYS), which is composed of Panax ginseng, velvet antler, Gekko gecko Linnaeus tail, human placenta, Trogopterus dung, Panax notoginseng, and amber. We fed mice with GBPYS after myocardial damages through everyday self-feeding. We then examined the effect of everyday oral intake of GBPYS on improving cardiac function and myocardial repair in adult mice after apical resection or MI. We found that long-term oral intake of GBPYS significantly improved cardiac function after myocardial damages in adult mice. BrdU, phospho-histone 3, and AuroraB staining indicated increased cell proliferation at the border zone of MI after TCM feeding. GBPYS feeding reduced organ inflammation, induced angiogenesis, and is non-toxic to mice after long-term oral intake. Further, serum derived from TCM-fed MI rats promoted division of both neonatal rat cardiomyocytes and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes in vitro. Oral intake of GBPYS improved heart repair after myocardial damages in adult mice. Our results suggest that there are substances present in GBPYS that help improve adult mammalian heart repair after MI. Also, it could be a good choice of non-invasive alternative therapy for myocardial damages and heart failure after rigorous clinical study in the future.

Regional and temporal changes in left ventricular strain and stiffness in a porcine model of myocardial infarction.

The aim of the present study was to serially track how myocardial infarction (MI) impacts regional myocardial strain and mechanical properties of the left ventricle (LV) in a large animal model. Post-MI remodeling has distinct regional effects throughout the LV myocardium. Regional quantification of LV biomechanical behavior could help explain changes in global function and thus advance clinical assessment of post-MI remodeling. The present study is based on a porcine MI model to characterize LV biomechanics over 28 days post-MI via speckle-tracking echocardiography (STE). Regional myocardial strain and strain rate were recorded in the circumferential, radial, and longitudinal directions at baseline and at 3, 14, and 28 days post-MI. Regional myocardial wall stress was calculated using standard echocardiographic metrics of geometry and Doppler-derived hemodynamic measurements. Regional diastolic myocardial stiffness was calculated from the resultant stress-strain relations. Peak strain and phasic strain rates were nonuniformly reduced throughout the myocardium post-MI, whereas time to peak strain was increased to a similar degree in the MI region and border zone by 28 days post-MI. Elevations in diastolic myocardial stiffness in the MI region plateaued at 14 days post-MI, after which a significant reduction in MI regional stiffness in the longitudinal direction occurred between 14 and 28 days post-MI. Post-MI biomechanical changes in the LV myocardium were initially limited to the MI region but nonuniformly extended into the neighboring border zone and remote myocardium over 28 days post-MI. STE enabled quantification of regional and temporal differences in myocardial strain and diastolic stiffness, underscoring the potential of this technique for clinical assessment of post-MI remodeling. NEW & NOTEWORTHY For the first time, speckle-tracking echocardiography was used to serially track regional biomechanical behavior and mechanical properties postmyocardial infarction (post-MI). We found that changes initially confined to the MI region extended throughout the myocardium in a nonuniform fashion over 28 days post-MI. Speckle-tracking echocardiography-based evaluation of regional changes in left ventricular biomechanics could advance both clinical assessment of left ventricular remodeling and therapeutic strategies that target aberrant biomechanical behavior post-MI.