Infarct Border Research Articles

GPX3 Overexpression Ameliorates Cardiac Injury Post Myocardial Infarction Through Activating LSD1/Hif1α Axis.

Myocardial infarction (MI) often results in significant loss of cardiomyocytes (CMs), contributing to adverse ventricular remodelling and heart failure. Therefore, promoting CM survival during the acute stage of MI is crucial. This study aimed to investigate the potential role of GPX3 in cardiac repair following MI. First, plasma GPX3 levels were measured in patients with acute MI (AMI), and myocardial GPX3 expression was assessed in a mouse MI model. Furthermore, the effects of GPX3 on MI were investigated through CM-specific overexpression or knockdown invitro and invivo models. RNA sequencing and subsequent experiments were performed to uncover the molecular mechanisms underlying GPX3-related effects. Multi-omics database analysis and experimental verification revealed a significant upregulation of GPX3 expression in ischemic myocardium following MI and in CMs exposed to oxygen-glucose deprivation (OGD). Immunofluorescence results further confirmed elevated cytoplasmic GPX3 expression in CMs under hypoxic conditions. Invitro, GPX3 overexpression mitigated reactive oxygen species (ROS) production and enhanced CM survival during hypoxia, while GPX3 knockdown inhibited these processes. Invivo, CM-specific GPX3 overexpression in the infarct border zone significantly attenuated CM apoptosis and alleviated myocardial injury, promoting cardiac repair and long-term functional recovery. Mechanistically, GPX3 overexpression upregulated LSD1 and Hif1α protein expression, and rescue experiments confirmed the involvement of the LSD1/Hif1α pathway in mediating the protective effects of GPX3. Overall, our findings suggest that GPX3 exerts a protective role in ischemic myocardium post-MI, at least partially through the LSD1/Hif1α axis, highlighting its potential as a therapeutic target for MI treatment.

Restoration effect of chemically modified microRNA-143-3p on acute myocardial infarction in animal models

We investigated whether miR143#12, a synthesized chemically modified miR-143-3p derivative, exerts therapeutic effects on acute myocardial infarction (AMI). Sprague–Dawley rats and Japanese white rabbits underwent 30 min of coronary occlusion followed by 2 weeks of reperfusion. The rat AMI model was intravenously administered with control miRNA (9 μg/kg), 3 μg/kg or 9 μg/kg of miR143#12 1 h after reperfusion, while the rabbit AMI model was intravenously administered with control miRNA (9 μg/kg) or 9 μg/kg of miR143#12. In the rat and rabbit AMI models, 9 μg/kg of miR143#12 significantly reduced infarct sizes and significantly improved cardiac function including LVEF and LVFS at 2 weeks. The tissue miR143 levels in infarct areas significantly decreased after AMI in both models. Electron microscopic study and immunohistochemistry suggested that miR143#12 suppressed autophagic cell death caused by AMI and induced neoangiogenesis in the infarct border. In cultured rat H9c2 cells, miR143#12 significantly inhibited H2O2-induced autophagic cell death by decreasing ROS levels and increased viable cell numbers more than the control by silencing COX-1, -2, and ATG7. Replacement treatment with miR143#12 in the infarct areas, where the expression levels of miR143 were significantly decreased, has a beneficial effect on AMI by silencing COX-1 and -2.

Exploring the molecular mechanisms of comorbidity of myocardial infarction and anxiety disorders by combining multiple data sets with in vivo experimental validation.

The incidence of comorbidity between myocardial infarction (MI) and anxiety disorders is increasing. However, the biological association between them has not been fully understood. This study aims to investigate the molecular mechanisms of comorbidity between MI and anxiety disorders and to predict their key genes and potential therapeutic drugs. We searched Gene Expression Omnibus databases and performed differential analyses using the limma package to identify the functional enrichment of differential genes. Next, we constructed regulatory networks to investigate the relationship between hub genes and autophagy, ferroptosis, and immunity. Furthermore, we predicted transcription factors by R package, constructed a miRNA network, performed the single-cell analysis of key gene expression, and predicted drug targeting of differential genes using the Connectivity Map database. The datasets for MI and anxiety disorders were analyzed for up and down-regulated differential genes, resulting in 35 intersecting differential genes. The top 10 feature genes from each dataset were intersected using Random Forest, resulting in the identification of three intersecting genes: STK17B, AKIRIN2, and WDR77. Validation of the above key genes was carried out by in vitro experiments. We examined the gene expression of STK17B, WDR77 and AKIRIN2 in the hippocampus and myocardial infarction border zone respectively by qPCR and WB, and the results confirmed that the above are the key genes for myocardial infarction and anxiety. There is a significant correlation between the comorbidity mechanism of myocardial infarction and anxiety disorders with ferroptosis and immunity. The construction of the miRNA network revealed that miR-205 and let-7 had higher average connectivity among the three hub genes. The single-cell analysis revealed significant expression of key genes in Endothelial cells, Cardiomyocytes, Macrophages, and Fibroblasts datasets. Cd274 showed a higher correlation with key genes in myocardial infarction and anxiety disorders. Validation by multiple datasets and in vitro experiments showed that STK17B, AKIRIN2, and WDR77 are the key genes in the comorbidity of myocardial infarction and anxiety disorders, and ferroptosis and immunity are the key links in the comorbidity mechanism of myocardial infarction and anxiety disorders.

Abstract 4138380: Cardiac protection of hiPSC-CMs loaded chitosan cardiac patch in myocardial infarcted swine

Background: The low grafting rate of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) following direct injection into the myocardium limits the efficacy of cardiac repair after myocardial infarction (MI). Chitosan-based scaffolds have emerged as functional biomaterials for designing delivery systems in cardiac therapies. This study aimed to enhance the potency of cardiac repair using a hiPSC-CMs loaded chitosan cardiac muscle patch (hCCMP). Methods: The patches were fabricated using chitosan. Characterization of the chitosan patches involved assessing their swelling rate and porosity. The biocompatibility of the hCCMP was analyzed through scanning electron microscopy (SEM) and the CCK8 assay. 16 Yorkshire swine (15-20 kg, 45-50 days of age) were randomly divided into 4 groups (n=4 per group): Sham, MI without further intervention, MI with an empty patch (without hiPSC-CMs), and MI with hCCMP implantation. One and four weeks post-MI, heart function, Infarction size, myocardial perfusion and metabolism were evaluated via cardiac PET-MRI. Engraftment was assessed through human cardiac troponin T staining. Histological analysis including left ventricular anterior wall thickness, cardiac remodeling, angiogenesis, arterialization, and apoptosis in the infarct border area were analyzed using immunofluorescence. Results: SEM showed that the chitosan patch had regular pore size and good 3D structure; porosity was about (96.7±0.37)%. On day3, the patch swelling rate was about (715±64.55)%. SEM, CCK8 assay showed a normal cells growth in the chitosan patch. The hiPSC-CMs grafted well with chitosan patch delivery systems and was associated with significantly improvement in LV function, infarction size, angiogenesis and cardiomyocyte apoptosis in pigs 4 weeks after MI. Conclusions: The 3D chitosan cardiac patch with hiPSC-CMs loaded can enhance the potency of cardiac repair in pigs after MI and present a novel and promising therapic strategy.

Locally increased dysferlin expression improves left-ventricular function by stabilizing membrane nanodomains of cardiomyocytes in the myocardial infarction border zone

Abstract Introduction Dysferlin, a transmembrane protein with multiple Ca2+-binding C2-domains, is expressed in the sarcolemma and vesicle membranes of striated muscle cells. Upon Ca2+ entry, Dysferlin is thought to mediate vesicle fusion and membrane repair events in order to rapidly seal sarcolemmal lesions. Dysferlin mutations are associated with progressive muscular dystrophies and dilated cardiomyopathy. However, the precise role of Dysferlin in protection of membrane nanodomains in ventricular myocytes (VMs) after myocardial infarction (MI) remains elusive. Purpose We hypothesized that Dysferlin is crucial to prevent VMs from loss-of-function and cell death by stabilizing membrane nanodomains like the transverse-axial tubule (TAT) system and the intercalated disc (ICD) cell-cell contact sites post-MI. Methods and Results 1 week post-MI induced by LAD artery ligation, investigator-blinded echocardiography revealed increased infarct sizes and decreased left-ventricular (LV) ejection fraction of Dysferlin-knockout (KO) vs. WT mice (mean±SEM: 21±2 vs. 28±2%, n=20/30 mice, P<0.05). Immunoblotting of WT LV tissue lysates showed an upregulated Dysferlin expression to 148% compared to sham-operated controls. Importantly, immunohistology identified a maximal Dysferlin expression of 230% in the MI border zone that faces a high ischemic and mechanical stress. Hence, we used data-independent mass spectrometry (DIA-MS) to analyse the proteomic profile of the infarction zone, border zone and remote zone from WT vs. KO hearts 1 week post-MI (each n = 5 mice). Overall, we reproducibly quantified 4360 proteins across all LV samples, identifying local genotype-specific proteotypes post-MI. In WT vs. KO samples, DIA-MS revealed 725 differentially abundant proteins in the zone pairwise comparison, highlighting a vital role of Dysferlin for LV remodelling post-MI. In addition, superresolution stimulated emission depletion (STED) microscopy was applied to study the subcellular localization and function of Dysferlin in the MI border zone. Here, we observed VMs with disrupted TAT endomembrane networks and significantly extended ICD regions. Interestingly, STED imaging revealed Dysferlin clusters that highly decorated residual TAT structures and interdigitated ICD membrane folds in the MI border zone. DIA-MS analysis of anti-Dysferlin co-imunoprecipitation experiments from WT vs. KO LV tissues characterized the cardiac interactome of Dysferlin, confirming interactions with proteins of the TAT system and the gap junction hemichannel Connexin-43. Conclusions Our data demonstrate Dysferlin as an endogenous membrane repair protein necessary to stabilize sarcolemmal nanodomains in the MI border zone. Dysferlin not only prevents from loss of TAT structures, but also compensates for increased mechanical stress at the ICDs. Hence, Dysferlin emerges as a novel therapeutic target to control membrane integrity in VMs to prevent from loss-of-function and cell death post-MI.

A new free radical scavenger, resorcimoline, prevents ischaemia/reperfusion injury in the heart

Abstract Introduction Ischaemic heart disease remains the leading cause of death worldwide. Early coronary revascularisation is the most important treatment. Recent findings have revealed that reactive oxygen species (ROS) can lead to tissue damage following coronary artery revascularisation, known as ischaemia-reperfusion injury. In this study, we focused on resorcimoline, a new free radical scavenger that was generated during the synthesis of dimethylresveratrol and dimethylpiceatannol. Last year, we reported the scavenging activity in vitro against multiple ROS, confirmed by the electron spin resonance spectrometry (Ref. 1). Purpose This study aimed to assess the potential of resorcimoline as a novel therapeutic treatment for reducing the degree of infarction caused by myocardial ischaemia, by eliminating ROS generated during myocardial ischaemia-reperfusion. Methods Nine-to-eleven-week-old male Wistar rats were subjected to acute myocardial ischaemia induced by ligation of the left anterior descending coronary artery for 30 minutes, followed by reperfusion for 24 hours. Rats received intravenous injections of resorcimoline at 6 mg/kg before coronary ligation or saline as a control (n = 9 vs. n = 8). The heart sections were double-stained with Evans blue 1% and triphenyltetrazolium chloride 1% to assess infarct area and area-at-risk. For immunohistochemical study, the other ischaemia/reperfusion models were treated with resorcimoline and saline (n = 7 vs. n = 7) as above, and the middle parts of the hearts were embedded in optimal cutting temperature compound and quickly frozen. In the H＆E-stained section, the infarct border zone was defined as a 2-mm band surrounding the necrosis area. Troponin I levels in serum were measured using ELISA. Primary cultured cardiomyocytes were treated with 10 μM angiotensin II for 3.5 hours and then exposed to resorcimoline for 30 minutes at an increasing dosage. A cellular ROS assay kit was used to assess the levels of ROS in the cardiomyocytes and the number of cells was counted with DAPI stain using an image processing software. Results Infarct size in resorcimoline-treated animals was significantly smaller than in control animals (41.8±19.4% vs. 60.8±12.9%, p=0.03, Fig. 1A) and area-at-risk was not significantly different (58.0±27.8% vs. 51.9±18.5%, p=0.61). Apoptotic cells in the border zone were significantly reduced in resorcimoline-treated animals (32.0±11.7% vs. 55.1±16.9%, p=0.01, Fig. 1B). Troponin I levels in resorcimoline-treated animals were significantly lower than in control animals (4524±3596 vs. 7856±4653, p=0.04, Fig. 1C). resorcimoline significantly reduced angiotensin II-induced ROS in cardiomyocytes in a concentration-dependent manner (Fig. 2A,B). Conclusion This novel compound, resorcimoline, can reduce infarct size induced by coronary ischaemia-reperfusion injury by reducing the ROS damage to myocardiocytes in the border zone and cellular ROS levels in cardiomyocytes.

Early CSWT relieves myocardial fibrosis by inhibiting TGF-Smad2/3-MMP-9

Abstract Objective To investigate the correlation between the improvement of cardiac function and myocardial fibrosis and the TGF-β1-Smad2/3-MMP-9 signaling pathway in acute myocardial infarction (AMI) rats after early treatment with extracorporeal cardiac shock wave therapy (CSWT). Methods Thirty SD rats were randomly divided into five groups: sham group (6 rats), AMI group (6 rats), AMI+CSWT group (6 rats), AMI+SB431542 group (6 rats), and AMI+SB431542+CSWT group (6 rats). AMI was induced in all groups except the sham group. The AMI+CSWT group and AMI+SB431542+CSWT group received CSWT treatment. Echocardiography was performed at 4 days and 62 days postoperatively to assess left ventricular ejection fraction (LVEF) in all groups. Additionally, cardiac tissues were sampled and evaluated for pathological changes and degree of fibrosis by HE staining and Masson staining. The TGF-β1-Smad2/3-MMP-9 signaling pathway and protein expression of COL-I and COL-III were detected in the infarcted border area. Results (1) At 4 days postoperatively, EF and FS were significantly lower, and LVESV was enlarged in the AMI group, AMI+CSWT group, AMI+SB431542+CSWT group, and AMI+SB431542 group compared to the sham group (p < 0.05). (2) At 62 days postoperatively, EF and FS were significantly lower, and LVEDV and LVESV were significantly higher in the AMI group compared to the sham group (p < 0.05). EF and FS were significantly higher, and LVEDV and LVESV were significantly lower in the AMI+CSWT group compared to the AMI group (p < 0.05). There was no significant difference in EF, FS, LVEDV, and LVESV between the AMI+CSWT group and the AMI+SB431542+CSWT group (p > 0.05). (3) Histological analysis showed that the AMI group had more blue-stained areas compared to the sham group, while the AMI+CSWT group had fewer blue-stained areas and more neatly arranged red areas compared to the AMI group (p < 0.05). (4) Collagen volume fraction (CVF) was significantly higher in the AMI group compared to the sham group (p < 0.05). CVF was significantly lower in the AMI+CSWT group compared to the AMI group (p < 0.05). There was no significant difference in CVF between the AMI+CSWT group and the AMI+SB431542+CSWT group (p > 0.05). (5) The expression levels of TGF-β1, Smad3, MMP-9, COL-I, and COL-III were significantly higher in the AMI group compared to the sham group (p < 0.05). The expression levels of these proteins were significantly reduced in the AMI+CSWT group compared to the AMI group (p < 0.05). Conclusions Early CSWT improves cardiac function and inhibits myocardial fibrosis associated with the TGF-β1-Smad2/3-MMP-9 signaling pathway.pathological changesdegree of fibrosis

Single cell multi-omics analysis unveils a distinct mechanism of exercise-mediated cardioprotection in ischemic cardiomyopathy

Abstract Background Exercise is pivotal in heart disease prevention, with proven cardioprotective effects in clinical trials. However, its precise molecular mechanisms remain elusive. Recent advancements in single-cell omics have revealed cellular heterogeneity within heart tissues, offering a deeper understanding of the pathophysiology of cardiac diseases. Purpose To investigate the exercise-induced physiological changes in heart tissue at the single-cell level and elucidate the molecular mechanisms underlying its cardioprotective effect on ischemic cardiomyopathy. Methods Murine myocardial infarction (MI) model was established by ligating the left ascending coronary artery, and a six-week voluntary wheel running exercise regimen was implemented to investigate the effect of aerobic exercise on ischemic cardiomyopathy. Integrated multi-omics analyses of single-cell DNA methylation profiling, single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) were conducted to clarify the molecular mechanism of exercise-mediated cardioprotection at single-cell resolution. Overexpression and short hairpin RNA-mediated knockdown of gene X were conducted for murine MI model using cardiomyocyte-specific Myo-AAV2a vector. snRNA-seq of heart tissues from heart failure patients was performed to validate the clinical significance of the expression of gene X in cardiomyocytes. Results Exercise attenuates pathological cardiac remodeling, inducing physiological hypertrophy in non-infarcted areas while suppressing pathological remodeling of cardiomyocytes in infarct border zones. Single-cell epigenetic analysis unveiled intricate molecular mechanisms underlying exercise-induced transcriptome changes, providing insights into the gene regulatory networks modulated by exercise in the heart. In addition, integrated with snRNA-seq, we identified exercise-specific cardiomyocyte clusters, highlighting the substantial role of gene X in the molecular mechanism of exercise. Functional studies confirmed the essential role of gene X in exercise-induced cardioprotection, as its overexpression suppressed the adverse remodeling after MI and knockdown abolished the beneficial effects of exercise. Furthermore, snRNA-seq of heart failure patients revealed that higher expression of gene X was associated with ventricular reverse remodeling and favorable clinical outcomes, underscoring its significance in the pathophysiology of patients with heart failure. Conclusion Our study elucidates exercise-specific gene expression regulation networks in ischemic cardiomyopathy by unraveling the cellular heterogeneity and molecular changes associated with exercise. We identified gene X as a central player in exercise-mediated cardioprotection and further confirmed its significance in clinical settings, providing a potential therapeutic target for heart failure.

CSWT improves ventricular function by promoting angiogenesis through Rho/ROCK/ERK1/2 signal pathway in infarcted myocardium in rats

Abstract Objective This study aimed to investigate the effects of extracorporeal cardiac shock wave therapy (CSWT) on myocardial ischemia and cardiac function in rats with myocardial infarction, and to explore its impact on the expression of myocardial vascular endothelial growth factor and the extent of myocardial fibrosis, thereby elucidating the mechanism of angiogenesis. Methods A rat model of myocardial infarction was induced by ligating the left anterior descending branch of the coronary artery, confirmed by echocardiography. Animals were randomly assigned to five groups: sham operation group, myocardial infarction group, myocardial infarction + CSWT group, myocardial infarction + Y-27632 group, and myocardial infarction + CSWT + Y-27632 group. Cardiac function was evaluated by echocardiography on the 16th and 72nd day post-surgery. Myocardial morphology was assessed by HE staining, and the extent of myocardial fibrosis was determined by Masson's trichrome staining. Immunohistochemical staining for factor VIII was conducted to observe angiogenesis in each infarcted area. Western blotting was employed to detect the protein expression of extracellular regulated kinase (ERK1/2) and pro-angiogenic cytokines in the infarct border zone. Results CSWT treatment significantly reduced myocardial fibrosis and improved cardiac function compared to the myocardial infarction group. Additionally, CSWT upregulated ERK expression and angiogenesis-related factors, promoting angiogenesis. The beneficial effects of CSWT were abolished by inhibiting the Rho/ROCK/ERK1/2 pathway. Furthermore, the area of myocardial fibrosis was significantly reduced in the MI+Y-27632 group compared to the myocardial infarction group (p < 0.01), and left ventricular systolic function index was significantly higher in the myocardial infarction group. Conclusions CSWT may promote angiogenesis post-myocardial infarction by activating the Rho/ROCK/ERK1/2 pathway. Additionally, Y-27632 may improve cardiac function in rats with myocardial infarction, potentially through mitigating myocardial fibrosis.echo images of ratsprotein expression by WB

Specific mode electroacupuncture stimulation opens the blood-brain barrier of the infarcted border zone in rats during MCAO/R recovery via modulation of tight junction protein expression by VEGFA and NF-κB.

The blood-brain barrier (BBB) strictly limits the entry of most exogenous therapeutic drugs into the brain, which brings great challenges to the drug treatment of refractory central diseases, including the treatment of ischemic stroke. Our previous studies have shown that specific mode electroacupuncture stimulation (SMES) can temporarily open the BBB, but with the mechanisms largely unknown. This study explored whether SMES opens the BBB in the infarcted border zone of rats during middle cerebral artery occlusion/reperfusion recovery, and whether this is related to p65 or vascular endothelial growth factor A (VEGFA) modulation of tight junction protein expression through in vivo and in vitro studies. Evans blue, FITC-dextran, mouse-derived nerve growth factor (NGF), and transendothelial electrical resistance values were used to evaluate the permeability of the BBB. Additionally, microvascular endothelial cells and astrocytes were utilized for in vitro study. Immunofluorescence, immunohistochemistry, western blot, and ELISA were employed to assess related protein expression. SMES significantly increased vascular permeability for Evans blue and NGF in the infarcted border zone, and increased the expression of VEGFA by activating p-p65, thereby reducing the expression of tight junction proteins Occludin and ZO-1. Correspondingly, oxygen glucose deprivation/reoxygenation activated p-p65 in and induced VEGFA secretion from astrocytes in vitro. Their conditioned medium reduced the expression of Occludin in bEnd.3 cells and increased the permeability of FITC-dextran. The mechanism of SMES opening infarcted border zone BBB is partly related to its actions on p65, VEGFA, and tight junction proteins.

Novel Insights into Post-Myocardial Infarction Cardiac Remodeling through Algorithmic Detection of Cell-Type Composition Shifts.

Recent advances in single cell sequencing have led to an increased focus on the role of cell-type composition in phenotypic presentation and disease progression. Cell-type composition research in the heart is challenging due to large, frequently multinucleated cardiomyocytes that preclude most single cell approaches from obtaining accurate measurements of cell composition. Our in silico studies reveal that ignoring cell type composition when calculating differentially expressed genes (DEGs) can have significant consequences. For example, a relatively small change in cell abundance of only 10% can result in over 25% of DEGs being false positives. We have implemented an algorithmic approach that uses snRNAseq datasets as a reference to accurately calculate cell type compositions from bulk RNAseq datasets through robust data cleaning, gene selection, and multi-sample cross-subject and cross-cell-type deconvolution. We applied our approach to cardiomyocyte-specific α1A adrenergic receptor (CM-α1A-AR) knockout mice. 8-12 week-old mice (either WT or CM-α1A-KO) were subjected to permanent left coronary artery (LCA) ligation or sham surgery (n=4 per group). Transcriptomes from the infarct border zones were collected 3 days later and analyzed using our algorithm to determine cell-type abundances, corrected differential expression calculations using DESeq2, and validated these findings using RNAscope. Uncorrected DEGs for the CM-α1A-KO X LCA interaction term featured many cell-type specific genes such as Timp4 (fibroblasts) and Aplnr (cardiomyocytes) and overall GO enrichment for terms pertaining to cardiomyocyte differentiation (P=3.1E-4). Using our algorithm, we observe a striking loss of cardiomyocytes and gain in fibroblasts in the α1A-KO + LCA mice that was not recapitulated in WT + LCA animals, although we did observe a similar increase in macrophage abundance in both conditions. This recapitulates prior results that showed a much more severe heart failure phenotype in CM-α1A-KO + LCA mice. Following correction for cell-type, our DEGs now highlight a novel set of genes enriched for GO terms such as cardiac contraction (P=3.7E-5) and actin filament organization (P=6.3E-5). Our algorithm identifies and corrects for cell-type abundance in bulk RNAseq datasets opening new avenues for research on novel genes and pathways as well as an improved understanding of the role of cardiac cell types in cardiovascular disease.

Effect of Intramyocardial Administration of Baculovirus Encoding the Transcription Factor Tbx20 in Sheep With Experimental Acute Myocardial Infarction.

Gene therapy has been proposed as a strategy to induce cardiac regeneration following acute myocardial infarction (AMI). Given that Tbx20, a transcription factor of the T-box subfamily, stimulates cell proliferation and angiogenesis, we designed a baculovirus overexpressing Tbx20 (Bv-Tbx20) and evaluated its effects in cultured cardiomyocytes and in an ovine model of AMI. Cell proliferation and angiogenesis were measured in cardiomyocytes transduced with Bv-Tbx20 or Bv-Null (control). Subsequently, in sheep with AMI, Bv-Tbx20 or Bv-Null was injected in the infarct border. Cardiomyocyte cell cycle activity, angioarteriogenesis, left ventricular function, and infarct size were assessed. Cardiomyocytes transduced with BvTbx20 increased cell proliferation, cell cycle regulatory and angiogenic gene expression, and tubulogenesis. At 7 days posttreatment, sheep treated with Bv-Tbx20 showed increased Tbx20, promitotic and angiogenic gene expression, decreased levels of P21, increased Ki67- (17.09±5.73 versus 7.77±7.24 cardiomyocytes/mm2, P<0.05) and PHH3 (phospho-histone H3)-labeled cardiomyocytes (10.10±3.51 versus 5.23±2.87 cardiomyocytes/mm2, P<0.05), and increased capillary (2302.68±353.58 versus 1694.52±211.36 capillaries/mm2, P<0.001) and arteriolar (146.95±53.14 versus 84.06±16.84 arterioles/mm2, P<0.05) densities. At 30 days, Bv-Tbx20 decreased infarct size (9.89±1.92% versus 12.62±1.33%, P<0.05) and slightly improved left ventricular function. Baculoviral gene transfer-mediated Tbx20 overexpression exerted angiogenic and cardiomyogenic effects invitro. In sheep with AMI, Bv-Tbx20 induced angioarteriogenesis, cardiomyocyte cell cycle activity, infarct size limitation, and a slight recovery of left ventricular function, suggesting that Bv-Tbx20 gene therapy may contribute to cardiac regeneration following AMI.

Acetylcytidine modification of Amotl1 by N-acetyltransferase 10 contributes to cardiac fibrotic expansion in mice after myocardial infarction.

Cardiac fibrosis is a pathological scarring process that impairs cardiac function. N-acetyltransferase 10 (Nat10) is recently identified as the key enzyme for the N4-acetylcytidine (ac4C) modification of mRNAs. In this study, we investigated the role of Nat10 in cardiac fibrosis following myocardial infarction (MI) and the related mechanisms. MI was induced in mice by ligation of the left anterior descending coronary artery; cardiac function was assessed with echocardiography. We showed that both the mRNA and protein expression levels of Nat10 were significantly increased in the infarct zone and border zone 4 weeks post-MI, and the expression of Nat10 in cardiac fibroblasts was significantly higher compared with that in cardiomyocytes after MI. Fibroblast-specific overexpression of Nat10 promoted collagen deposition and induced cardiac systolic dysfunction post-MI in mice. Conversely, fibroblast-specific knockout of Nat10 markedly relieved cardiac function impairment and extracellular matrix remodeling following MI. We then conducted ac4C-RNA binding protein immunoprecipitation-sequencing (RIP-seq) in cardiac fibroblasts transfected with Nat10 siRNA, and revealed that angiomotin-like 1 (Amotl1), an upstream regulator of the Hippo signaling pathway, was the target gene of Nat10. We demonstrated that Nat10-mediated ac4C modification of Amotl1 increased its mRNA stability and translation in neonatal cardiac fibroblasts, thereby increasing the interaction of Amotl1 with yes-associated protein 1 (Yap) and facilitating Yap translocation into the nucleus. Intriguingly, silencing of Amotl1 or Yap, as well as treatment with verteporfin, a selective and potent Yap inhibitor, attenuated the Nat10 overexpression-induced proliferation of cardiac fibroblasts and prevented their differentiation into myofibroblasts in vitro. In conclusion, this study highlights Nat10 as a crucial regulator of myocardial fibrosis following MI injury through ac4C modification of upstream activators within the Hippo/Yap signaling pathway.

Wavefront curvature analysis derived from preprocedural imaging can identify the critical isthmus in patients with postinfarcted ventricular tachycardia

BackgroundWhere activation wavefront curvature is convexly shaped, functional conduction block can occur. ObjectiveThe purpose of this study was to determine whether left ventricular (LV) wall thickness determined from contrast-enhanced computed tomography (CT) is useful in localizing such areas in clinical postinfarction reentrant ventricular tachycardia (VT). MethodsWe evaluated data from 6 patients who underwent catheter ablation for postinfarction VT. CT imaging with inHEART processing was conducted 1–3 days before electrophysiological (EP) study to determine LV wall thickness (T). Activation wavefront curvature was approximated as ΔT/T, where ΔT represents wall thickness change. During EP study, bipolar LV VT electrograms were acquired using a high-density mapping catheter, and activation times were determined. Maps of T, ΔT/T, and VT activation were subsequently compared using statistical analyses. ResultsTwo of 6 cases exhibited dual circuit morphologies, resulting in a total of 8 VT morphologies analyzed. The LV wall near the VT isthmus location tended to be thin, on the order of a few hundred micrometers. Regions of largest ΔT/T partially coincided with the lateral isthmus boundaries where electrical conduction block occurred during VT. ΔT/T at the boundaries, measured from imaging, was significantly larger compared to values at the isthmus midline and to the global LV mean value (P <.001). ConclusionWavefront curvature measured by ΔT/T and caused by source–sink mismatch is dependent on ventricular wall thickness. Areas of high wavefront curvature partly coincide with and may be helpful in locating the VT isthmus in infarct border zones using preprocedural imaging analysis.

Murine matrix metalloproteinase 13 and human matrix metalloproteinase 1 are involved in remodelling processes after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): German Centre for Cardiovascular Research (DZHK) Background Cardiovascular diseases such as myocardial infarction (MI) remain a leading cause of death in the world. Matrix metalloproteinases (MMPs) are not only essential for the cleavage of collagen, but are also modifying inflammatory cytokines. Thus, MMPs are playing a substantial role in the context of fibrotic as well as inflammatory processes in tissue remodelling after MI. Purpose Mmp13 is the highest expressed collagenase in the murine left ventricle (LV). After ligation of the left anterior descending artery (LAD) in mice, Mmp13 is highly upregulated in the LV. Therefore, the aim of this study was to characterize the role of MMP13 in MI. In the human LV, the highest expressed collagenase is MMP1. Single nucleotide polymorphisms (SNPs) in the promotor region of MMP1 can lead to alterations in gene expression level. Therefore, we analysed the genotype of 3 SNPs in about 2000 patients admitted to the emergency department with suspected MI to reveal associations with the development of MI and outcome after MI. Methods A MMP13-knockout (KO) mouse model was examined after induction of MI and gene expression analysis, histological staining, in-situ hybridisation and hemodynamic measurements were conducted. Mmp13 expression in different cardiac cell types was investigated at quiescent stage and under ischaemic conditions, to determine the cellular origin of Mmp13 expression. Out of the human cohort, 2 patient groups (non-MI and MI) were restricted. Hazard ratios adjusted to age and male sex were assessed, to evaluate risk for MI and risk for death after MI in dependency of the SNPs. Results After activation with ischaemic secretome of cardiomyocytes, the Mmp13 expression of macrophages (6.6-fold; p=0.0286) and fibroblasts (4.9-fold; p=0.0079) was significantly increased. Under stimulation with ischaemic secretome of fibroblasts, Mmp13 expression of macrophages (4.3-fold; p=0.0286) and leukocytes (2.3-fold; p=0.0260) was significantly elevated. Additionally, in situ hybridisation revealed Mmp13 expression in immune cells in the border zone of the infarct region. While the survival of WT mice was about 50%, only very few KO mice died after MI (p=0.0107). Moreover, KO mice showed benefits in cardiac function compared to WT littermates 28 days post-MI. In the human cohort, risk for death was significantly altered between the examined genotypes in 2 of 3 investigated SNPs, indicating a role of MMP1 in the remodelling process after MI. Conclusion High levels of Mmp13 after MI originate mainly from activated macrophages and from activated fibroblasts in the infarct border zone. MMP13-deficiency is beneficial for mice after MI, leading to an improved cardiac function 28 days post-MI compared to WT controls. Analysis of SNPs of the human functional homologue of Mmp13 – MMP1 – revealed an association of MMP1 with remodelling processes after MI.

Multipolar Electrograms: A New Configuration That Increases the Measurement Accuracy of Intracardiac Signals

BackgroundAccurate measurements of intracardiac electrograms (EGMs) remain a clinical challenge because of the suboptimal attenuation of far-field potentials. Multielectrode mapping catheters provide an opportunity to construct multipolar instead of bipolar EGMs for rejecting common far-field potentials recorded from a multivectorial space. ObjectivesThe purpose of this study was to develop a multipolar EGM and compare its characteristics to those of bipolar EGMs MethodsUsing a 36-electrode array catheter (Optrell-36, Biosense Webster), a far-field component was mathematically constructed from clusters of electrodes surrounding each inspected electrode. This component was subtracted from the unipolar waveform to produce a local unipolar, referred to as a “multipolar EGM.” The performance of multipolar EGMs was evaluated in 7 swine with healed anteroseptal infarction. ResultsMultipolar EGMs proved superior in attenuating far-field potentials in infarct border zones, increasing the near-field to far-field ratio from 0.92 ± 0.2 to 2.25 ± 0.3 (P < 0.001). Removal of far-field components reduced the voltage amplitude (P < 0.001) and enlarged the infarct surface area (P = 0.02), aligning more closely with histological findings. Of 379 EGMs with ≥20 ms activation time difference between bipolar and multipolar EGMs, 95.3% (361 of 379) were accurately annotated using multipolar EGMs, while annotation based on bipolar EGM was predominantly made on far-field components. ConclusionsMultielectrode array catheters provide a unique platform for constructing multipolar EGMs. This new EGM may be beneficial for "purifying" local potentials within a complex electrical field, resulting in more accurate voltage and activation maps.

Platelets Induce Cell Apoptosis of Cardiac Cells via FasL after Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is one of the leading causes of death worldwide. Cell apoptosis in the myocardium plays an important role in ischemia and reperfusion (I/R) injury, leading to cardiac damage and dysfunction. Platelets are major players in hemostasis and play a crucial role in vessel occlusion, inflammation, and cardiac remodeling after I/R. Here, we studied the impact of platelets on cell apoptosis in the myocardium using a close-chest mouse model of AMI. We found caspase-3-positive resident cardiac cells, while leukocytes were negative for caspase-3. Using two different mouse models of thrombocytopenia, we detected a significant reduction in caspase-3 positive cells in the infarct border zone after I/R injury. Further, we identified platelet FasL to induce cell apoptosis via the extrinsic pathway of Fas receptor activation of target cells. Mechanistically, hypoxia triggers platelet adhesion to FasR, suggesting that platelet-induced apoptosis is elevated after I/R. Platelet-specific FasL knock-out mice showed reduced Bax and Bcl2 expression, suggesting that platelets modulate the intrinsic and extrinsic pathways of apoptosis, leading to reduced infarct size after myocardial I/R injury. Thus, a new mechanism for how platelets contribute to tissue homeostasis after AMI was identified that should be validated in patients soon.

Biomarker Periodic Repolarization Dynamics Indicates Enhanced Risk for Arrhythmias and Sudden Cardiac Death in Myocardial Infarction in Pigs.

Periodic repolarization dynamics (PRD) is an electrocardiographic biomarker that captures repolarization instability in the low frequency spectrum and is believed to estimate the sympathetic effect on the ventricular myocardium. High PRD indicates an increased risk for postischemic sudden cardiac death (SCD). However, a direct link between PRD and proarrhythmogenic autonomic remodeling has not yet been shown. We investigated autonomic remodeling in pigs with myocardial infarction (MI)-related ischemic heart failure induced by balloon occlusion of the left anterior descending artery (n=17) compared with pigs without MI (n=11). Thirty days after MI, pigs demonstrated enhanced sympathetic innervation in the infarct area, border zone, and remote left ventricle paralleled by altered expression of autonomic marker genes/proteins. PRD was enhanced 30 days after MI compared with baseline (pre-MI versus post-MI: 1.75±0.30 deg2 versus 3.29±0.79 deg2, P<0.05) reflecting pronounced autonomic alterations on the level of the ventricular myocardium. Pigs with MI-related ventricular fibrillation and SCD had significantly higher pre-MI PRD than pigs without tachyarrhythmias, suggesting a potential role for PRD as a predictive biomarker for ischemia-related arrhythmias (no ventricular fibrillation versus ventricular fibrillation: 1.50±0.39 deg2 versus 3.18±0.53 deg2 [P<0.05]; no SCD versus SCD: 1.67±0.32 deg2 versus 3.91±0.63 deg2 [P<0.01]). We demonstrate that ischemic heart failure leads to significant proarrhythmogenic autonomic remodeling. The concomitant elevation of PRD levels in pigs with ischemic heart failure and pigs with MI-related ventricular fibrillation/SCD suggests PRD as a biomarker for autonomic remodeling and as a potential predictive biomarker for ventricular arrhythmias/survival in the context of MI.

Sinus rhythm activation signature indicates reentrant ventricular tachycardia inducibility and approximate isthmus location

BackgroundSinus rhythm activation time is useful to assess infarct border zone substrate. ObjectiveWe sought to further investigate sinus activation in ventricular tachycardia (VT). MethodsCanine postinfarction data were analyzed retrospectively. In each experiment, an infarct was created in the left ventricular wall by left anterior descending coronary artery ligation. At 3 to 5 days after ligation, 196–312 bipolar electrograms were recorded from the anterior left ventricular epicardium overlapping the infarct border zone. Sustained monomorphic VT was induced by premature electrical stimulation in 50 experiments and was noninducible in 43 experiments. Acquired sinus rhythm and VT electrograms were marked for electrical activation time, and activation maps of representative sinus rhythm and VT cycles were constructed. The sinus rhythm activation signature was defined as the cumulative number of multielectrode recording sites that had activated per time epoch, and its derivative was used to predict VT inducibility and to define the sinus rhythm slow/late activation sequence. ResultsPlotting mean activation signature derivative, a best cutoff value was useful to separate experiments with reentrant VT inducibility (sensitivity, 42/50) vs noninducibility (specificity, 39/43), with an accuracy of 81 of 93. For the 50 experiments with inducible VT, recording sites overlying a segment of isochrone encompassing the sinus rhythm slow/late activation sequence spanned the VT isthmus location in 32 cases (64%), partially spanned it in 15 cases (30%), but did not span it in 3 cases (6%). ConclusionThe sinus rhythm activation signature derivative is assistive to differentiate substrate supporting reentrant VT inducibility vs noninducibility and to identify slow/late activation for targeting isthmus location.

A Dynamic Gradient Stiffness Material Platform to Manipulate Cardiac Fibroblasts' Spatio‐Temporal Behavior

AbstractAfter myocardial infarction, there exists a spatiotemporal variation of cardiac tissue stiffness across the infarcted border region outward to remote regions, influencing adverse remodeling and cardiac fibrosis, and this stiffness gradient changes over time. Here, a platform with dynamic, tunable, and reversible gradient stiffness can recapitulate in vitro the time‐dependent stiffness range across the infarction border that occurs as part of the remodeling process is presented. This platform enables the observation of time‐dependent interaction between cardiac fibroblasts and their mechanical microenvironment in a spatiotemporal manner. Specifically, the competition and cooperation of a chemical cue (antifibrotic drug) and mechanical cue (gradient softening) in tandem to attenuate the fibrotic responses of cardiac fibroblasts is examined. Applying a combined intervention showed either additive or antagonistic effects on fibrosis‐related gene regulation compared to separate interventions of drug or softening. This work reveals the spatiotemporal variation of fibrotic response in cardiac fibroblasts as well as the complexity of antifibrotic drug dosing with stiffness changes and their combinatory effect on cardiac fibroblasts. This platform provides a unique in vitro tool to study disease progression mechanisms in a more clinically relevant microenvironment and also serves as a cost‐effective model for potential therapeutic screening.