Myofibroblast Senescence Promotes Arrhythmogenic Remodeling in the Aged Infarcted Rabbit Heart

Progressive tissue remodeling after myocardial infarction (MI) promotes cardiac arrhythmias. This process is well studied in young animals, but little is known about pro-arrhythmic changes in aged animals. Senescent cells accumulate with age and accelerate age-associated diseases. Senescent cells interfere with cardiac function and outcome post-MI with age, but studies have not been performed in large animals, and the mechanisms are unknown. Here, we investigated the role of senescence in regulating inflammation, fibrosis, and arrhythmogenesis in young and aged infarcted rabbits. Aged rabbits exhibited increased peri-procedural mortality and arrhythmogenic electrophysiological remodeling at the infarct border zone (IBZ) compared to young rabbits. Studies of the aged infarct zone revealed persistent myofibroblast senescence and increased inflammatory signaling over a twelve-week timecourse. Senescent IBZ myofibroblasts in aged rabbits appear to be coupled to myocytes, and our computational modeling showed that senescent myofibroblast-cardiomyocyte coupling prolongs action potential duration (APD) and facilitates conduction block permissive of arrhythmias. Aged infarcted human ventricles show levels of senescence consistent with aged rabbits, and senescent myofibroblasts also couple to IBZ myocytes. Our findings suggest that senolytic drugs may mitigate arrhythmias post-MI.

Change in the central control of the bladder function of rats with focal cerebral infarction induced by photochemically-induced thrombosis

The photochemically-induced thrombosis (photothrombosis) method can create focal cerebral infarcts anywhere in the relatively superficial layers of the cerebrum; it is easy to implement and minimally invasive. Taking advantage of this versatility, we aimed to establish a new rat model of urinary frequency with focal cerebral infarction, which was characterized by its simplicity, nonlethal nature, and high reproducibility. The prefrontal cortex and the anterior cingulate cortex, which are involved in lower urinary tract control, were targeted for focal cerebral infarction, and urinary parameters were measured by cystometrogram. Cystometric analysis indicated that micturition intervals significantly shortened in photothrombosis-treated rats compared with those in the sham operative group on Days 1 and 7 (P < 0.01), but prolonged after 14 days, with no difference between the two groups. Immunopathological evaluation showed an accumulation of activated microglia, followed by an increase in reactive astrocytes at the peri-infarct zone after photothrombotic stroke. Throughout this study, all postphotothrombosis rats showed cerebral infarction in the prefrontal cortex and anterior cingulate cortex; there were no cases of rats with fatal cerebral infarction. This model corresponded to the clinical presentation, in that the micturition status changed after stroke. In conclusion, this novel model combining nonlethality and high reproducibility may be a suitable model of urinary frequency after focal cerebral infarction.

Relationship between left ventricular global longitudinal strain, infarct size and left ventricular function in patients with acute myocardial infarction in a stem cell therapy study

Introduction It is critically important to determine the accuracy, and relationships between, non-invasive imaging modalities, such as two-dimensional echocardiography (TTE), gated single-photon emission computed tomography (SPECT) and cardiac magnetic resonance imaging (cMRI) in patients with recent acute myocardial infarction (AMI) because these are used as clinical trial endpoints. Modest improvements in the left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV) and infarct zone size (IS) have been reported in AMI stem cells therapy trials (SCT). Purpose The aim of the study was to evaluate left-ventricular global longitudinal strain (GLS) in patients with AMI enrolled to SCT and assess its relation with infarct zone, LVEF and LVEDV using multimodality imaging including TTE, cMRI and SPECT. Methods Twenty-eight patients (21 male, 7 female, mean age 60.0±8.7 years) with first AMI, 2–5 days after left anterior descending percutaneous coronary intervention (PCI) and IS ≥10% were enrolled. GLS was evaluated with two-dimensional speckle tracking echocardiography (aCMQ, Philips Epiq 7). Infarct zone was measured using SPECT (E.CAM, Siemens) and gadolinium-enhanced cMRI (Siemens Magnetom Sonata 1.5T). LVEF and LVEDV were assessed with TTE (Auto-ROI, Philips), SPECT (GSQUAN, Siemens) and cMRI (MASS Medis). Measurements were obtained independently by blinded analysts. Results Mean GLS was −11.0±2.5% and showed a positive correlation with infarct zone by SPECT and MRI, negative with TTE-LVEF and cMRI-LVEF (Figure 1) and borderline with SPECT-LVEF (r=−0.35, p=0.08). There was no correlation between GLS and TTE-LVEDV (r=−0.25, p=0.25); SPECT-LVEDV (r=−0.38, p=0.077) and MRI-LVEDV (r=−0.20, p=0.365). Patients in the third and fourth GLS quartile had a smaller IS measured by MRI and a trend toward a smaller infarct zone by SPECT (table 1). Patients in the GLS fourth quartile had higher TTE-LVEF and a trend toward higher cMRI-LVEF compared with other quartiles. LVEF measured with TTE and cMRI was higher compared with SPECT-LVEF (+2.6±6.8%, p=0.006 and +4.2±7.8%, p=0.030, respectively) with no difference between TTE-LVEF and MRI-LVEF (p=0.823) (Table 1). LVEDV evaluated by SPECT and MRI was higher compared with TTE-LVEDV (+48.3±24.9 ml, +47.7±29.5 ml, both p&lt;0.001) with no difference between SPECT-LVEDV and MRI-LVEDV (p=0.984) Conclusions In patients with anterior wall AMI, 2–5 days after PCI, GLS showed a good correlation with infarct zone quantified by SPECT and MRI and with LVEF measured with TTE and cMRI. GLS might thus be a valuable tool in the evaluation of myocardial injury in SCT. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): STRATEGMED 265761 “CIRCULATE” National Centre for Research and Development/Poland/ZDS/00564 Jagiellonian University Medical College Table 1 Figure 1

The Influence of the Duration of Acute Coronary Syndrome on the Outcomes of Endovascular Treatment

Multicenter studies have proven the high effectiveness of percutaneous coronary intervention (PCI) in terms of restoring patency of the infarct-related artery (IRA) and improving the prognosis in acute myocardial infarction (AMI). The mechanism of improvement of clinical result after PCI procedure appears to be multifactorial. The aim. To investigate the effect of IRA stenting on the clinical course, prognosis and contractility of the heart in patients with different duration of acute myocardial infarction and its influence on the short-term and long-term effects after intervention. The main determinant for the favorable clinical course and improvement of the prognosis is early (within the first hours of the disease) restoration of antegrade blood flow by IRA stenting. Thus, it is possible to signifi-cantly improve the blood supply to the peri-infarct zone and limit the area of necrosis and maintain heart rate. Materials and methods. The analyzed group included 684 patients with AMI who were endovascularly treated at the Department of Emergency Endovascular Heart Surgery of the National Amosov Institute of Cardiovascular Surgery of the National Academy of Medical Sciences of Ukraine from January 1, 2017 to January 1, 2021. Coronary artery stenting was performed in all the patients. STEMI occurred in 495 (72.4%) patients, and non-STEMI in 189 (27.6%) patients. The mean age of the subjects was 61.8 ± 12.1 years. There were 289 women (42.3%) and 395 men (57.7%). Distribu-tion of the patients depending on the initial Killip class was as follows: 13 (1.9%) had class I, 199 (29.1%) had class II, and 472 (69.0%) had class III myocardial infarction. Atrial fibrillation occurred in 72 (10.5%) patients. Echocardiographic parameters were as follows: left ventricular (LV) end-systolic index 54.1 ± 12.8 ml/m2, LV ejection fraction 0.53 ± 0.05, left atrial diameter 39.5 ± 3.8 mm, systolic pulmonary artery pressure 44.8 ± 7.8 mmHg. In this study, clinical condition and functional capacity of the heart muscle in patients with AMI depending on the condition of the stent segment and the timing of endovascular procedures after the onset of the disease were first inves-tigated in the long term. Conclusions. High efficiency and safety of PCI have been proven, which makes it possible to recommend this pro-cedure for wide application. It has been proven that PCI using matrix and modular stents, as well as statins can reduce the frequency of in-stent stenosis and improve the clinical course of the disease in the long term. It has been proven that stenting in patients with AMI is most effective in the earliest stages of the disease with preservation of LV contractility with possibly complete myocardial revascularization, which contributes to the preservation of viable myocardium in the peri-infarct zone, improvement of myocardial contractility and prevention of myocardium remodeling.

Angiocrine IGFBP3 Spatially Coordinates IGF Signaling During Neonatal Cardiac Regeneration

To identify non cellautonomous effectors of cardiomyocyte mitosis, we analyzed a transcriptomic screen of regenerating and non regenerating neonatal hearts for differentially expressed secreted proteins, which we hypothesized could include candidate mitogens. We identified and validated IGFBP3, which has a Janus-like stabilizing and sequestering effect on IGF growth factors, as a neonatal injury associated secreted protein. IGFBP3 is expressed by and secreted from vascular cells in the neonatal heart after cardiac injury, notably in the infarct border zone. We found that global deletion of IGFBP3 blunted neonatal regeneration, while gain of function experiments using recombinant IGFBP3 and a transgenic mouse model uncovered a pro mitotic effect of IGFBP3 on cardiomyocytes in vitro and in the adult heart. We show that site specific expression of an IGFBP3 protease (PAPPA2) and its inhibitor (STC2) coordinate the spatial release of IGF2 in the infarct zone to regio selectively activate the INSR/ERK/AKT cell growth pathways in cardiomyocytes. Collectively, our work highlights the spatiotemporal orchestration of endothelial cardiomyocyte interactions that are required for neonatal cardiac regeneration.

Abstract 120: Pericytes Contribute To Myofibroblast Expansion And Vascular Maturation In The Infarcted Heart

Infarct healing is dependent on recruitment of inflammatory leukocytes and subsequent activation of myofibroblasts (MF) and neovessel formation, ultimately resulting in formation of a highly vascularized collagen-enriched scar. Though the heart has an abundant population of periendothelial pericytes, its role in wound healing upon myocardial infarction (MI) has not been studied. We hypothesized that in the infarcted myocardium, pericytes may become activated, contributing to inflammatory, fibrotic and angiogenic responses. We used pericyte/fibroblast reporter mice (NG2 DsRed ;PDGFRα GFP ), lineage tracing studies and in vitro approaches to study the fate and role of pericytes in the infarcted myocardium. In normal hearts, NG2+/PDGFRα- pericytes and PDGFRα+/NG2- fibroblasts had distinct transcriptomic profiles. Pericytes expressed mural genes like Acta2 , Pdgfrb and low amounts of extracellular matrix (ECM) genes, whereas fibroblasts synthesized collagens, Timp2/3 and matricellular genes. 7 days post-MI, expansion of the NG2+ population in the infarct zone was associated with emergence of non-mural NG2+/αSMA+ cells with MF characteristics. FACS-sorted NG2+/PDGFRα- cells from 7-day infarcts expressed higher levels of collagens when compared to NG2+/PDGFRα- cells from normal hearts. Infarct pericytes had high integrin and MMP14 expression, reflecting an activated migratory phenotype. Lineage tracing using NG2CreER TM ;Rosa tdTomato ;PDGFRα GFP mice showed that 5.7%±1.04 of PDGFRα+ fibroblasts and 10.49%±2.73 of infarct MFs were derived from NG2+ lineage. Pericyte-derived fibroblasts exhibited higher ECM gene synthesis, in comparison to fibroblasts from non-pericyte origin, while pericyte-derived mural cells showed accentuated inflammatory cytokine gene expression. Immunostaining showed pericytes actively contribute to vascular maturation, forming a mural cell coat enwrapping infarct neovessels. In vitro, TGFβ induced integrins, collagens and MMPs in human pericytes, similar to the changes observed in infarct pericytes. Taken together, our evidences show that after MI, pericytes become activated and contribute to repair by undergoing conversion to a subset of myofibroblasts and by coating infarct neovessels.

Change in the bladder function of rats with focal cerebral infarction induced by photochemically-induced thrombosis

The photochemically-induced thrombosis (photothrombosis) method can create focal cerebral infarcts anywhere in the relatively superficial layers of the cerebrum; it is easy to implement and minimally invasive. Taking advantage of this versatility, we aimed to establish a new rat model of urinary frequency with focal cerebral infarction, which was characterized by its simplicity, nonlethal nature, and high reproducibility. The prefrontal cortex and the anterior cingulate cortex, which are urinary centers, were targeted for focal cerebral infarction, and urinary parameters were measured by cystometrogram. Cystometric analysis indicated that micturition intervals significantly shortened in photothrombosis-treated rats compared with those in the sham operative group on Days 1 and 7 (P < 0.01), but prolonged after 14 days, with no difference between the two groups. Immunopathological evaluation showed an accumulation of activated microglia, followed by an increase in reactive astrocytes at the peri-infarct zone after photothrombotic stroke. Throughout this study, all postphotothrombosis rats showed cerebral infarction in the prefrontal cortex and anterior cingulate cortex; there were no cases of rats with fatal cerebral infarction. This model corresponded to the clinical presentation, in that the micturition status changed after stroke. In conclusion, this novel model combining nonlethality and high reproducibility may be a suitable model of urinary frequency after focal cerebral infarction.

Comparative Dose of Intracarotid Autologous Bone Marrow Mononuclear Therapy in Chronic Ischemic Stroke in Rats

Research on chronic ischemic stroke is limited. One of the more promising approaches showing positive effects in the acute stage is mononuclear bone marrow cell therapy. This research may be the first which presents data about the optimum dose of bone marrow mononuclear cells (BM-MNCs) for chronic ischemic stroke in rats and discusses factors influencing recovery in the chronic stage. We performed temporary middle cerebral artery occlusion (MCAO) procedures on the rats which were then randomly assigned to one of two experimental groups in which they were given either low or high doses of autologous BM-MNCs (5 million or 10 million cells per kg body weight). Rat brains were fixed for HE, CD31, and doublecortin staining for analysis of the effects. Rat behavior was assessed weekly using the cylinder test and a modified neurological severity score (NSS) test. In the four weeks prior to administration of BM-MNC, cylinder test scores improved to near normal, and NSS test scores improved moderately. The infarct zone decreased significantly (p <0,01), there was an improvement in angiogenesis (p = 0.1590) and a significant improvement in neurogenesis (p <0,01). Reduction of the infarct zone was associated with a higher dose whereas both higher and lower doses were found to have a similar effect on improving angiogenesis, and neurogenesis. Recovery was superior after twelve weeks compared with the recovery assessment at eight weeks. In conclusion, a dose of 10 million cells was more effective than a dose of 5 million cells per kg body weight for reducing the infarct zone and ameliorating neurogenesis. There was an improvement of histopathological parameters associated with the longer infarct period.

Myocardial gene expression in patients with myocardial infarction

Funding Acknowledgements Type of funding sources: None. Introduction. Myocardial infarction (MI) and following heart failure (HF) have various clinical scenarios. However, despite the clinical heterogeneity, the management of MI lacks effective personalized approaches. The response to ischemic injury in the infarct zone and remote myocardium has a definite spatio-temporal sequence and underlies the mechanism of adverse cardiac remodeling. Understanding its myocardial biology remains unclear due to insufficiency of heart tissue molecular and genetic analysis. Although animal models play an important role in data accumulation, they failure to reflect all the compounds of response to ischemic injury and following HF syndrome. Purpose. The purpose of the study was to investigate myocardial expression profile in the infarct zone in comparison to remote myocardium in patients with MI. Methods. The study included 4 patients with fatal MI type 1. All patients died within 48 hours of MI. The post-mortem examination was performed according to country policy. In each case, we obtained myocardial samples from the infarct zone and remote myocardium. Genome-wide gene-level expression was assessed by microarray analysis. We used transcriptome analysis software to analyze and visualize global expression patterns of genes. The analysis included normalization process. Differentially expressed genes (DEGs) were defined as gene-level fold change &lt;-2 or &gt;2 and adjusted P-value of &lt;0.05. Results. We studied expression of 21448 genes. A total of 1785 DEGs, including 1692 up-regulated genes and 93 down-regulated genes in the infarct zone, were identified (Figure 1). According to the fold change, the first 10 up-regulated genes were TNFSF10 (TRAIL), SUCLA2, NAE1, PDCL3, OSBPL5, FCGR2C, SELE, CEP63, ST3GAL3, C4orf3. Fold changes for TNFSF10 (TRAIL), SUCLA2, and NAE1 were 19.83 (p = 0.049), 10.68 (p = 0.048), and 10.24 (p = 0.008), respectively. These 3 up-regulated genes were enriched in apoptotic signaling pathways, immune response, angiogenesis, inflammation, tricarboxylic acid cycle, and protein binding. Conclusions. We performed myocardial transcriptomic analysis in patients with MI. DEG analysis clearly distinguishes the infarct zone and remote myocardium. Genes overexpressed in the infarct zone included TNFSF10 (TRAIL), SUCLA2, NAE1. TNFSF10 (TRAIL) regulates vasodilation, angiogenesis, and inflammation. TNFSF10 (TRAIL) regulated effects could be both protective and harmful in ischemic heart disease. The enzyme encoded by SUCLA2 is active in tissues that require a large amount of energy, such as those of the heart, brain, and muscle. Overexpression of NAE1 causes apoptosis through deregulation of NEDD8 conjugation. It is known that the protein encoded by NAE1 binds to the beta-amyloid precursor protein, and it is thought to play a role in the pathogenesis of Alzheimer"s disease. Thus, our pilot study emphasized the potential of translational approach and identified targets to consider for precision diagnostics and therapeutics. Abstract Figure. С1: infarct zone; C2: remote myocardium