Nonreperfused Myocardial Infarction Research Articles

Correlation between ventricular local impedance and tissue composition by MRI in chronic infarction

Abstract Background Local impedance (LI) mapping is feasible and provides additional tissue characterization of the ventricular tachycardia substrate. Data on tissue composition underlying different LI values are lacking. Purpose To correlate LI with tissue composition evaluated by magnetic resonance imaging (MRI) in a chronic myocardial infarction (MI) swine model. Methods One-month after a non-reperfused anterior MI, eighteen Landrace X Large White pigs underwent delayed-enhancement MRI (deMRI) and endocardial LI mapping. deMRI images were post-processed off-line with commercially available dedicated software; scar subtypes (with thresholds set at 40-60% of maximal pixel signal intensity [PSI]), border-zone corridors and left ventricular wall thickness were measured. LI thresholds were set using the blood-pool LI value to define low, intermediate, and high tissue resistance subtypes. LI maps were co-registered with deMRI for analyses. Results Low LI zones correlated with deMRI dense and transmural scar (exhibiting endo and epicardial PSI >60% in 91% and 80% of the cases, respectively). Intermediate LI tissue exhibited predominantly subendocardial scar with more heterogeneous composition (8%, 47% and 45% of the points had <40%, 40-60% and >60% endocardial PSI, respectively), and with less epicardial involvement (50% of points had epicardial PSI <40%). Border-zone corridors co-localized with intermediate LI tissue in most of the cases (77%). Low LI zones exhibited more pronounced wall thinning compared to intermediate LI tissue (p<0.001) (Figure 1). Conclusions LI correlated with scar density and transmurality on deMRI. Areas of low LI had higher proportion of dense, transmural scar and wall thinning compared to intermediate LI areas. MRI-detected border-zone corridors colocalized with intermediate LI in most cases.LI and deMRI data (endo and epi scar)LI and deMRI data (tissue thickness)

Tissue composition underlying local impedance in chronic infarction: an histological study

Abstract Background Local impedance (LI) mapping is feasible and provides additional tissue characterization of the ventricular tachycardia (VT) substrate. VT isthmuses are usually located in areas of intermediate LI while low LI regions did not actively participate in the VT circuit. Histological studies evaluating the tissue composition underlying different LI values are lacking. Purpose To describe the tissue composition underling different LI subtypes in a chronic myocardial infarction (MI) swine model. Methods One-month after a non-reperfused anterior MI, five Landrace X Large White pigs underwent endocardial LI mapping (figure 1). LI thresholds were set using the blood-pool LI value to define low, intermediate, and high tissue resistance subtypes. A navigated needle-catheter was used to inject methylene-blue at sites with intermediate and low LI to guide the ex vivo tissue localization. Tissue was obtained for further histological analysis. Low and intermediate LI zones were analysed. Results A total of six and eight samples were analysed for low and intermediate LI, respectively. Compared to low LI zones, intermediate LI tissue displayed less Collagen I (1.03±0.26 vs. 0.34±0.35%, p=0.0082), Collagen III (9.92±2.98 vs. 6.47±1.80%, p=0.0380) and Collagen Volume Fraction (10.92±2.77 vs. 6.81±1.92, p=0.0163), without differences in the overall fibrotic and adipocytic deposition (figure 2). No significant differences in the Cx43, CTnI and SERCA2 expression were observed. Conclusion LI subtypes showed distinctive histological composition. Intermediate LI sites displayed less collagen content (I, III and collagen volume fraction) compared to those with low LI.Endocardial LI mappingHystological analysis: Collagen Content

SPTLC3 Is Essential for Complex I Activity and Contributes to Ischemic Cardiomyopathy.

Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3). The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3KO mice subjected to permanent nonreperfused myocardial infarction. Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity. Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.

BAK contributes critically to necrosis and infarct generation during reperfused myocardial infarction

At least seven cell death programs are activated during myocardial infarction (MI), but which are most important in causing heart damage is not understood. Two of these programs are mitochondrial-dependent necrosis and apoptosis. The canonical function of the pro-cell death BCL-2 family proteins BAX and BAK is to mediate permeabilization of the outer mitochondrial membrane during apoptosis allowing apoptogen release. BAX has also been shown to sensitize cells to mitochondrial-dependent necrosis, although the underlying mechanisms remain ill-defined. Genetic deletion of Bax or both Bax and Bak in mice reduces infarct size following reperfused myocardial infarction (MI/R), but the contribution of BAK itself to cardiomyocyte apoptosis and necrosis and infarction has not been investigated. In this study, we use Bak-deficient mice and isolated adult cardiomyocytes to delineate the role of BAK in the pathogenesis of infarct generation and post-infarct remodeling during MI/R and non-reperfused MI. Generalized homozygous deletion of Bak reduced infarct size ∼50% in MI/R in vivo, which was attributable primarily to decreases in necrosis. Protection from necrosis was also observed in BAK-deficient isolated cardiomyocytes suggesting that the cardioprotection from BAK loss in vivo is at least partially cardiomyocyte-autonomous. Interestingly, heterozygous Bak deletion, in which the heart still retains ∼28% of wild type BAK levels, reduced infarct size to a similar extent as complete BAK absence. In contrast to MI/R, homozygous Bak deletion did not attenuate acute infarct size or long-term scar size, post-infarct remodeling, cardiac dysfunction, or mortality in non-reperfused MI. We conclude that BAK contributes significantly to cardiomyocyte necrosis and infarct generation during MI/R, while its absence does not appear to impact the pathogenesis of non-reperfused MI. These observations suggest BAK may be a therapeutic target for MI/R and that even partial pharmacological antagonism may provide benefit.

Abstract P2110: Fibroblast-specific Smad6 Restrains Adverse Remodeling And Dysfunction Following Myocardial Infarction

Signaling induced by the Transforming Growth Factor (TGF)-β superfamily members in fibroblasts plays a central role in repair and remodeling after myocardial infarction (MI). However, TGF-b signals need to be tightly regulated, as excessive or sustained fibroblast activation after MI can accentuate fibrosis, precipitating heart failure. The inhibitory Smads, Smad6 and Smad7, function as endogenous negative regulators that restrain TGF-β and BMP signaling. We have recently demonstrated that fibroblast-specific Smad7 restrains fibrosis after MI acting through TGF-dependent and -independent pathways. However, the role of Smad6 in tissue repair and fibrosis following injury is not known. We hypothesized that Smad6 expression in infarct fibroblasts may play an important role in repair and remodeling of the heart, regulating TGF-b and BMP-mediated responses. In a mouse model of non-reperfused MI, Smad6 expression was increased in infarct fibroblasts and myofibroblasts at 7d post-infarction. To investigate the role of Smad6 in injury and repair, we used CRISPR/Cas9 to generate Smad6 loxp mice. These animals were bred with Col1a2CreER mice to generate fibroblast-specific conditional Smad6 KOs (FS6KO). 7d after MI, FS6KO mice had accentuated systolic and diastolic dysfunction (p<0.01, N=10) associated with scar expansion, accentuated fibrosis, and higher myofibroblast density in the infarct area and border zone. Fibroblast-specific Smad6 loss increased collagen denaturation in the infarct and border zone. In vitro, siRNA knockdown experiments showed that Smad6 suppresses collagen and fibronectin expression, and modulates protease and matricellular gene synthesis. Reactome analysis of RNA-Seq data suggested effects of Smad6 on pathways involved in assembly and crosslinking of collagen fibrils, collagen biosynthesis and degradation, and VEGF-dependent angiogenesis. Furthermore, Ingenuity Pathway Analysis identified signaling by Integrins, Ephrin receptors and Receptor Tyrosine Kinases as key regulators of the effects of Smad6 loss on the fibroblast transcriptome. In conclusion, fibroblast-specific Smad6 restrains adverse remodeling after MI. Ongoing studies explore the molecular mechanisms for the Smad6 effects.

Abstract GS.102: Cd206 + Il-4rα + Macrophages Are Drivers Of Adverse Cardiac Remodeling In Ischemic Cardiomyopathy

Background: Cardiac macrophages are significantly expanded in chronic heart failure (HF); however, their role is unclear. We tested the hypothesis that CD206 + macrophages expressing interleukin (IL)-4Rα in the failing heart are key drivers of adverse left ventricular (LV) remodeling and ischemic cardiomyopathy. Methods and Results: Adult male C57BL/6 (WT) mice underwent non-reperfused myocardial infarction (MI) to induce HF, or sham operation, and cardiac macrophages were profiled using flow cytometry. As compared to sham hearts, both the frequency and absolute number of CD206 + macrophages steadily increased in post-MI hearts during the progression of LV remodeling and HF, such that at 8 w post-MI they comprised ~85% of macrophages. These macrophages were exclusively proliferative and predominantly C-C motif chemokine receptor (CCR2) - with nearly half expressing IL-4Rα, and correlated with LV dysfunction and fibrosis. IL-4-polarized bone marrow derived CD206 + macrophages exhibited marked upregulation of found in inflammatory zone (FIZZ)1 and induced FIZZ1-dependent myofibroblast differentiation of cardiac mesenchymal stem cells (cMSCs), in part related to DLL-4/Jagged1-Notch1 signaling. Intramyocardial adoptive transfer of M[IL-4], but not M[IL-10], macrophages induced progressive LV dilation and dysfunction and augmented cardiac fibrosis over 4 weeks in naïve mice. In vivo IL-4Rα gene silencing in mice with established HF effectively depleted cardiac CD206 + IL-4Rα + macrophages and induced reverse LV remodeling, improving fibrosis, neovascularization, and dysfunction, and suppressing local and systemic inflammation. Lastly, alternatively activated CD206 + and CD163 + macrophages were significantly expanded in human failing hearts and correlated with cardiac fibrosis, with the majority of CD163 + macrophages expressing IL-4Rα and FIZZ3, the human homolog of FIZZ1. Conclusion: CD206 + IL-4Rα + macrophages proliferate and expand in the failing heart and are key mediators of pathological remodeling and fibrosis, in part through the secretion of FIZZ1. Inhibition of CD206 + macrophage IL-4Rα signaling may be a fruitful therapeutic approach for alleviating LV remodeling in HF.

Brown Adipose Tissue and BMP3b Decrease Injury in Cardiac Ischemia-Reperfusion.

Despite advances in treatment, myocardial infarction (MI) is a leading cause of heart failure and death worldwide, with both ischemia and reperfusion (I/R) causing cardiac injury. A previous study using a mouse model of nonreperfused MI showed activation of brown adipose tissue (BAT). Recent studies showed that molecules secreted by BAT target the heart. We investigated whether BAT attenuates cardiac injury in I/R and sought to identify potential cardioprotective proteins secreted by BAT. Myocardial I/R surgery with or without BAT transplantation was performed in wild-type (WT) mice and in mice with impaired BAT function (uncoupling protein 1 [Ucp1]-deficient mice). To identify potential cardioprotective factors produced by BAT, RNA-seq (RNA sequencing) was performed in BAT from WT and Ucp1-/- mice. Subsequently, myocardial I/R surgery with or without BAT transplantation was performed in Bmp3b (bone morphogenetic protein 3b)-deficient mice, and WT mice subjected to myocardial I/R were treated using BMP3b. Dysfunction of BAT in mice was associated with larger MI size after I/R; conversely, augmenting BAT by transplantation decreased MI size. We identified Bmp3b as a protein secreted by BAT after I/R. Compared with WT mice, Bmp3b-deficient mice developed larger MIs. Increasing functional BAT by transplanting BAT from WT mice to Bmp3b-deficient mice reduced I/R injury whereas transplanting BAT from Bmp3b-deficient mice did not. Treatment of WT mice with BMP3b before reperfusion decreased MI size. The cardioprotective effect of BMP3b was mediated through SMAD1/5/8. In humans, the plasma level of BMP3b increased after MI and was positively correlated with the extent of cardiac injury. The results of this study suggest a cardioprotective role of BAT and BMP3b, a protein secreted by BAT, in a model of I/R injury. Interventions increasing BMP3b levels or targeting Smad 1/5 may represent novel therapeutic approaches to decrease myocardial damage in I/R injury.

Targeting myeloid cell coagulation signaling blocks MAP kinase/TGF-β1-driven fibrotic remodeling in ischemic heart failure.

Despite major advances in acute interventions for myocardial infarction (MI), adverse cardiac remodeling and excess fibrosis after MI causing ischemic heart failure (IHF) remain a leading cause of death worldwide. Here we identify a profibrotic coagulation signaling pathway that can be targeted for improved cardiac function following MI with persistent ischemia. Quantitative phosphoproteomics of cardiac tissue revealed an upregulated mitogen-activated protein kinase (MAPK) pathway in human IHF. Intervention in this pathway with trametinib improves myocardial function and prevents fibrotic remodeling in a murine model of non-reperfused MI. MAPK activation in MI requires myeloid cell signaling of protease-activated receptor 2 linked to the cytoplasmic domain of the coagulation initiator tissue factor (TF). They act upstream of pro-oxidant NOX2 NADPH oxidase, ERK1/2 phosphorylation, and activation of profibrotic TGF-β1. Specific targeting with the TF inhibitor nematode anticoagulant protein c2 (NAPc2) starting 1 day after established experimental MI averts IHF. Increased TF cytoplasmic domain phosphorylation in circulating monocytes from patients with subacute MI identifies a potential thromboinflammatory biomarker reflective of increased risk for IHF and suitable for patient selection to receive targeted TF inhibition therapy.

Biophysical Tissue Characterization of Ventricular Tachycardia Substrate With Local Impedance Mapping to Predict Critical Sites

BackgroundNew tools are needed to improve ventricular tachycardia (VT) substrate characterization and optimize outcomes. LI provides biophysical tissue characterization. ObjectivesThe purpose of this study was to test local impedance (LI)-based mapping to predict critical ventricular tachycardia components after myocardial infarction (MI). MethodsOne month after a nonreperfused anterior MI, endo-epicardial high-density electroanatomic mapping and endocardial LI mapping were performed in 23 Landrace Large X White pigs. LI thresholds were set using the blood pool value to define a 10 Ω range: low (<blood pool −1Ω), intermediate (≥blood pool −1Ω and ≤blood pool +9Ω), and high (normal) tissue resistance (>blood pool +9Ω). ResultsLow LI was detected in low-voltage areas in 100% of cases, but intermediate LI was found in both core (87%) and border zone (12.5%) voltage areas. A total of 17 VTs were induced (VT isthmus identified in 9 animals). VT inducibility was associated with the size of intermediate LI area (OR: 1.19 [95% CI: 1.0-1.4]; P = 0.039) and the presence of specific LI patterns: LI corridor (OR: 15.0 [95% CI: 1.3-169.9]; P = 0.029); LI gradient (OR: 30.0 [95% CI: 2.1-421.1]; P = 0.012), high LI heterogeneity (OR: 21.7 [95% CI: 1.8-260.6]; P = 0.015), and presence of ≥2 low LI regions (OR: 11.3 [95% CI: 1.0-130.2]; P = 0.053). Potential VT isthmuses were in areas of intermediate LI and colocalized to LI patterns associated with VT inducibility in all cases (LI corridors or LI gradient). Low LI regions did not actively participate in the VT circuit (0%). ConclusionsLI mapping is feasible and may add useful characterization of the VT substrate. Specific LI patterns (ie, corridors, gradients) were associated with VT inducibility and colocalized with the VT isthmus, thus representing a potential new target for ablation in substrate-based procedures.

Reduction in mitochondrial ROS improves oxidative phosphorylation and provides resilience to coronary endothelium in non-reperfused myocardial infarction.

Recent studies demonstrated that mitochondrial antioxidant MnSOD that reduces mitochondrial (mito) reactive oxygen species (ROS) helps maintain an optimal balance between sub-cellular ROS levels in coronary vascular endothelial cells (ECs). However, it is not known whether EC-specific mito-ROS modulation provides resilience to coronary ECs after a non-reperfused acute myocardial infarction (MI). This study examined whether a reduction in endothelium-specific mito-ROS improves the survival and proliferation of coronary ECs in vivo. We generated a novel conditional binary transgenic animal model that overexpresses (OE) mitochondrial antioxidant MnSOD in an EC-specific manner (MnSOD-OE). EC-specific MnSOD-OE was validated in heart sections and mouse heart ECs (MHECs). Mitosox and mito-roGFP assays demonstrated that MnSOD-OE resulted in a 50% reduction in mito-ROS in MHEC. Control and MnSOD-OE mice were subject to non-reperfusion MI surgery, echocardiography, and heart harvest. In post-MI hearts, MnSOD-OE promoted EC proliferation (by 2.4 ± 0.9 fold) and coronary angiogenesis (by 3.4 ± 0.9 fold), reduced myocardial infarct size (by 27%), and improved left ventricle ejection fraction (by 16%) and fractional shortening (by 20%). Interestingly, proteomic and Western blot analyses demonstrated upregulation in mitochondrial complex I and oxidative phosphorylation (OXPHOS) proteins in MnSOD-OE MHECs. These MHECs also showed increased mitochondrial oxygen consumption rate (OCR) and membrane potential. These findings suggest that mito-ROS reduction in EC improves coronary angiogenesis and cardiac function in non-reperfused MI, which are associated with increased activation of OXPHOS in EC-mitochondria. Activation of an energy-efficient mechanism in EC may be a novel mechanism to confer resilience to coronary EC during MI.

Abstract 15503: Myocardial Infarction Increases Diversity of Neutrophil Immunophenotype

Background: Following myocardial infarction (MI), immune cells are recruited to clear apoptotic cells and facilitate replacement fibrosis. Neutrophils are the first immune cells recruited, and neutrophilia predicts death and adverse outcomes post-MI. Increased demand for neutrophils is met through emergency granulopoiesis. Methods and Results: Our prior investigations stimulating emergency granulopoiesis with injections of exogenous granulocyte-colony stimulating factor (G-CSF; 100 μg/kg/d) indicated neutrophil immunophenotypic diversity with the emergence of CD101 Neg neutrophils in peripheral blood and spleen. In this study, we tested our hypothesis that MI induced emergency granulopoiesis increases neutrophil phenotypic diversity. We used a non-reperfused MI model in C57BL/6J mice (females, 14-20 wk). At 1, 2, 3, and 7 d post-MI, we harvested bone marrow, peripheral blood, spleens, and hearts. Neutrophil immunophenotypes were quantified in dissociated tissues via flow cytometry. Agreeing with previous findings, neutrophil infiltration of the heart was observed at 1-3 d, reaching a climax at 2 d. Our data demonstrates novel CD101 Neg and CD117 Pos neutrophils present in the heart 1-3 d post-MI (n=3-6, p&lt; 0.05). Additionally, elevated numbers of neutrophils and CD101 Neg neutrophil subtypes were observed in bone marrow, peripheral blood, and spleen relative to naïve mice (n=3-10, p&lt;0.05). Conclusion: We conclude that post-MI production of neutrophils results in the recruitment of diverse neutrophil subtypes to the heart. Future studies will seek to understand how post-MI activation of emergency granulopoiesis instigates neutrophil diversity and how CD101 Neg and CD117 Pos neutrophils are involved in replacement fibrosis. Figure 1. Presence of CD101 Neg and CD117 Pos Neutrophils in the Heart in post-MI. Gating (A-C) and quantification (E-G) of neutrophil immunophenotypes. (n=6-9, Mixed Affects Two-way ANOVA with Multiple Comparisons).

PO-676-06 THE ROLE OF REPERFUSION IN MODULATING CYCLIC AMP SIGNALING FOLLOWING MYOCARDIAL INFARCTION

Reperfusion therapy is the standard of care for myocardial infarction (MI) patients, but it is not universally performed and is sometimes unsuccessful. Reperfusion impacts sympathetic innervation of the heart, with reperfused MIs remaining chronically hypo-innervated, whereas hyper-innervation is more common in non-reperfused MIs. We hypothesize that reperfusion status and resulting sympathetic innervation will differentially impact myocardial β-adrenergic signaling and electrophysiological responses.

Metabolic transformation of fat in obesity determines the inflammation resolving capacity of splenocardiac and cardiorenal networks in heart failure.

All fats are not created equal, and despite the extensive literature, the effect of fat intake is the most debated question in obesity, cardiovascular, and cardiorenal research. Cellular and molecular mechanisms underlying cardiac dysfunction and consequent heart failure in the setting of obesity are not well understood. Our understanding of how fats are metabolically transformed after nonreperfused myocardial infarction (MI), in particular, is incomplete. Here, using male C57BL/6J mice (2 mo old), we determined the role of omega-6 fatty acids, provided as safflower oil (SO) for 12 wk, followed by supplementation with docosahexaenoic acid (DHA; n-3 fatty acids) for 8 wk before MI. With SO feeding, inflammation resolution was impaired. Specialized proresolving mediators (SPMs) increased in DHA-fed mice to reverse the effects of SO, whereas prostaglandins and thromboxane B2 were reduced in the spleen and amplified multiple resolving mechanisms in heart and kidney post-MI. DHA amplified the number of resolving macrophages and cardiac reparative pathways of the splenocardiac and cardiorenal networks in acute heart failure, with higher Treg cells in chronic heart failure and marked expression of Foxp3+ in the myocardium. Our findings indicate that surplus ingestion of SO intensified systemic, baseline, nonresolving inflammation, and DHA intake dominates splenocardiac resolving phase with the biosynthesis of SPMs and controlled cardiorenal inflammation in heart failure survivor mice.NEW & NOTEWORTHY Chronic and surplus dietary intake of safflower oil (SO) increased plasma creatinine dysregulated post-MI splenocardiac inflammation coincides with the dysfunctional cardiorenal network. In contrast, docosahexaenoic acid (DHA) increases post-MI survival in chronic heart failure. DHA transforms into specialized proresolving mediators (SPMs) and limited proinflammatory prostaglandins and thromboxanes following myocardial infarction (MI). DHA promotes Ly6Clow resolving macrophages and T regulatory cells (Foxp3+) in a splenocardiac manner post-MI.

Transient cardioprotective effects of remote ischemic postconditioning on non-reperfused myocardial infarction: longitudinal evaluation study in pigs

BackgroundRemote ischemic postconditioning (RIPostC) has recently emerged as a potential novel therapeutic strategy to achieve protection against acute myocardial infarction (AMI) injury. We aimed to evaluate the longitudinal cardioprotective effects of RIPostC on AMI using 99mTc-MIBI SPECT myocardial perfusion imaging (MPI) and gated 18F-FDG PET (GPET) in pigs. MethodsMI was induced in 12 Chinese pigs. RIPostC was conducted by four 5 min cycles of blood pressure cuff inflation applied to the lower limb immediately after AMI. MPI and GPET were performed longitudinally at baseline, 3rd, 14th, 28th, and 56th days after AMI. Total perfusion defect (TPD), hibernating myocardium (HM), scar, left ventricular (LV) remodeling (End diastolic volume, EDV), and bone marrow (BM) metabolic activity were analyzed, and inflammation biomarkers were measured. ResultsIn outcome evaluation, there was a significant attenuation in TPD (Δ value, at 14th, 28th and 56th days), HM (Δ value, at 14th, 56th days) and Scar (Δ value, at 14th, 28th days) in the RIPostC group compared with the control group (P < 0.05). Additionally, RIPostC attenuated LV enlargement (ΔEDV, at 14th day) (P < 0.05) in comparison to controls. BM 18F-FDG uptake activity in the RIPostC group was lower than the control group (P < 0.05) at the 3rd day after AMI. There was a non-statistically significant trend of decreased MMP-2 levels in the RIPostC group post-AMI (P > 0.05). ConclusionsRIPostC presented the longitudinal cardioprotective effects by preserving myocardial viability, reducing infarct size, attenuating LV remodeling at early stage post-AMI, and may also have an anti-inflammatory effect at the acute phase.

Abstract 14035: IL-4Ra-Mediated CD206 + Macrophage Activation Promotes Adverse Remodeling in the Failing Heart

Background: In heart failure (HF), cardiac macrophages are largely comprised of CD206 + cells that locally self-renew independent of the blood monocyte pool. We tested the hypothesis that cardiac CD206 + macrophages expressing interleukin (IL)-4Rα are key mediators of adverse left ventricular (LV) remodeling in HF. Methods and Results: Adult male C57BL/6 mice underwent non-reperfused myocardial infarction (MI) to induce HF. HF mice (vs. sham mice) exhibited progressive expansion of cardiac CD206 + macrophages up to 8 w post-MI (accounting for ~80% total macrophages), whereas cardiac CD206 - macrophages peaked at 48 h and then decreased by 90% at 1 w post-MI and beyond. Cardiac CD206 + macrophage levels negatively correlated with LV ejection fraction, and positively correlated with remote zone fibrosis. These cells were predominantly CCR2 - and nearly half expressed IL-4Rα, whereas CD206 - macrophages, monocytes, neutrophils, and T-cells had markedly lower IL-4Rα expression. Bone marrow-derived macrophages (BMDMs) polarized with IL-4 (M[IL-4]) induced myofibroblast activation in vitro . Intramyocardial adoptive transfer of M[IL-4] macrophages (1 x 10 6 cells) into naïve mice induced progressive LV chamber dilation and dysfunction, and augmented cardiac fibrosis over 4 w. Next, sham and HF mice with similar LV remodeling at 4 w post-MI were given IL-4Rα-specific or control antisense oligonucleotides (ASOs) 40 mg/kg i.p. every 3 d for 4 w. HF mice receiving IL-4Rα ASOs exhibited profoundly diminished cardiac CD206 + IL-4Rα + (and total CD206 + ) macrophage levels, substantially less macrophage proliferation and phagocytosis, and improvement of LV dysfunction, hypertrophy, and fibrosis. Moreover, both M[IL-4] CD206 + BMDMs and CD206 + IL-4Rα + macrophages in failing hearts expressed high levels of Fizz1 (found in inflammatory zone 1). Blocking FIZZ1 expression and/or secretion blunted the profibrotic capacity of M[IL-4] CD206 + BMDMs in vitro . Conclusion: CD206 + IL-4Rα + macrophages proliferate and expand in the failing heart and are key drivers of pathological remodeling and fibrosis, in part through the secretion of FIZZ1. Inhibition of CD206 + macrophage IL-4Rα signaling may be a fruitful therapeutic approach for alleviating LV remodeling in HF.

Preservation of Contractile Reserve and Diastolic Function by Inhibiting the NLRP3 Inflammasome with OLT1177® (Dapansutrile) in a Mouse Model of Severe Ischemic Cardiomyopathy Due to Non-Reperfused Anterior Wall Myocardial Infarction.

Interleukin-1β (IL-1β), a product of the NLRP3 inflammasome, modulates cardiac contractility and diastolic function. We proposed that OLT1177® (dapansutrile), a novel NLRP3 inhibitor, could preserve contractile reserve and diastolic function after myocardial infarction (MI). We used an experimental murine model of severe ischemic cardiomyopathy through the ligation of the left coronary artery without reperfusion, and after 7 days randomly assigned mice showing large anterior MI (>4 akinetic segments), increased left ventricular (LV) dimensions ([LVEDD] > 4.4 mm), and reduced function (LV ejection fraction < 40%) to a diet that was enriched with OLT1177® admixed with the chow in the diet at 3.75 g/kg (Group 1 [n = 10]) or 7.5 g/kg (Group 2 [n = 9]), or a standard diet as the no-treatment control group (Group 3 [n = 10]) for 9 weeks. We measured the cardiac function and contractile reserve with an isoproterenol challenge, and the diastolic function with cardiac catheterization at 10 weeks following the MI surgery. When compared with the control (Group 3), the mice treated with OLT1177 (Group 1 and 2) showed significantly greater preservation of their contractile reserve (the percent increase in the left ventricular ejection fraction [LVEF] after the isoproterenol challenge was +33 ± 11% and +40 ± 6% vs. +9 ± 7% in the standard diet; p < 0.05 and p < 0.005 for Group 1 and 2, respectively) and of diastolic function measured as the lower left ventricular end-diastolic pressure (3.2 ± 0.5 mmHg or 4.5 ± 0.5 mmHg vs. 10.0 ± 1.6 mmHg; p < 0.005 and p < 0.009 respectively). No differences were noted between the resting LVEF of the MI groups. These effects were independent of the effects on the ventricular remodeling after MI. NLRP3 inflammasome inhibition with OLT1177® can preserve β-adrenergic responsiveness and prevent left ventricular diastolic dysfunction in a large non-reperfused anterior MI mouse model. OLT1177® could therefore be used to prevent the development of heart failure in patients with ischemic cardiomyopathy.

Collagen denaturation in the infarcted myocardium involves temporally distinct effects of MT1-MMP-dependent proteolysis and mechanical tension

Tissue injury results in profound alterations in the collagen network, associated with unfolding of the collagen triple helix, proteolytic degradation and generation of fragments. In the infarcted myocardium, changes in the collagen network are critically involved in the pathogenesis of left ventricular rupture, adverse remodeling and chronic dysfunction. We hypothesized that myocardial infarction is associated with temporally and spatially restricted patterns of collagen denaturation that may reflect distinct molecular mechanisms of collagen unfolding. We used a mouse model of non-reperfused myocardial infarction, and in vitro assays in fibroblast-populated collagen lattices. In healing infarcts, labeling with collagen hybridizing peptide (CHP) revealed two distinct patterns of collagen denaturation. During the inflammatory and proliferative phases of infarct healing, collagen denaturation was pericellular, localized in close proximity to macrophages and myofibroblasts. qPCR array analysis of genes associated with matrix remodeling showed that Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) is markedly upregulated in infarct macrophages and fibroblasts, suggesting its involvement in pericellular collagen denaturation. In vitro, MT1-MMP-mediated pericellular collagen denaturation is involved in cardiac fibroblast migration. The effects of MT1-MMP on collagen denaturation and fibroblast migration involve the catalytic site, and require hemopexin domain-mediated actions. In contrast, during the maturation phase of infarct healing, extensive collagen denaturation was noted in the hypocellular infarct, in the infarct border zone and in the mitral valve annulus, in the absence of MT1-MMP. In vitro, mechanical tension in attached collagen lattices was sufficient to induce peripheral collagen denaturation. Our study suggests that in healing infarcts, early pericellular collagen denaturation may be important for migration of macrophages and reparative myofibroblasts in the infarct. Extensive denaturation of collagen fibers is noted in mature scars, likely reflecting mechanical tension. Chronic collagen denaturation may increase susceptibility of the matrix to proteolysis, thus contributing to progressive cardiac dilation and post-infarction heart failure.

Implication of anti-angiogenic VEGF-A165b in angiogenesis and systolic function after reperfused myocardial infarction

Abstract Background Angiogenesis participates in re-establishing microcirculation after myocardial infarction (MI). Purpose In this study, we aim to further understand the role of the anti-angiogenic isoform vascular endothelial growth factor (VEGF)-A165b after MI and explore its potential as a co-adjuvant therapy to coronary reperfusion. Methods Two mice MI models were formed: 1) permanent coronary ligation (non-reperfused MI), 2) transient 45-min coronary occlusion followed by reperfusion (reperfused MI); in both models, animals underwent echocardiography before euthanasia at day 21 after MI induction. Serum and myocardial VEGF-A165b levels were determined. In both experimental MI models, functional and structural implication of VEGF-A165b blockade was assessed. In a cohort of 104 ST-segment elevation MI patients, circulating VEGF-A165b levels were correlated with cardiovascular magnetic resonance-derived left ventricular ejection fraction at 6-months and with the occurrence of adverse events (death, heart failure and/or re-infarction). Results In both models, circulating and myocardial VEGF-A165b presence was increased 21 days after MI induction. Serum VEGF-A165b levels inversely correlated with systolic function evaluated by echocardiography. VEGF-A165b blockage increased capillary density, reduced infarct size, and enhanced left ventricular function in reperfused, but not in non-reperfused MI experiments. In patients, higher VEGF-A165b levels correlated with depressed ejection fraction and worse outcomes. Conclusions In experimental and clinical studies, higher serum VEGF-A165b levels associates with a worse systolic function. Its blockage enhances neoangiogenesis, reduces infarct size, and increases ejection fraction in reperfused, but not in non-reperfused MI experiments. Therefore, VEGF-A165b neutralization represents a potential co-adjuvant therapy to coronary reperfusion. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): This study was funded by “Instituto de Salud Carlos III” and “Fondos Europeos de Desarrollo Regional FEDER” (Exp. PIE15/00013, PI17/01836, PI18/00209 and CIBERCV16/11/00486).

Ivabradine is as effective as metoprolol in the prevention of ventricular arrhythmias in acute non-reperfused myocardial infarction in the rat

Ventricular arrhythmias are a major source of early mortality in acute myocardial infarction (MI) and remain a major therapeutic challenge. Thus we investigated effects of ivabradine, a presumably specific bradycardic agent versus metoprolol, a β-blocker, at doses offering the same heart rate (HR) reduction, on ventricular arrhythmias in the acute non-reperfused MI in the rat. Immediately after MI induction a single dose of ivabradine/ metoprolol was given. ECG was continuously recorded and ventricular arrhythmias were analyzed. After 6 h epicardial monophasic action potentials (MAPs) were recorded and cardiomyocyte Ca2+ handling was assessed. Both ivabradine and metoprolol reduced HR by 17% and arrhythmic mortality (14% and 19%, respectively, versus 33% in MI, p < 0.05) and ventricular arrhythmias in post-MI rats. Both drugs reduced QTc prolongation and decreased sensitivity of ryanodine receptors in isolated cardiomyocytes, but otherwise had no effect on Ca2+ handling, velocity of conduction or repolarization. We did not find any effects of potential IKr inhibition by ivabradine in this setting. Thus Ivabradine is an equally effective antiarrhythmic agent as metoprolol in early MI in the rat. It could be potentially tested as an alternative antiarrhythmic agent in acute MI when β-blockers are contraindicated.

Validation of diagnostic criteria and histopathological characterization of cardiac rupture in the mouse model of nonreperfused myocardial infarction.

In patients with myocardial infarction (MI), cardiac rupture is an uncommon but catastrophic complication. In the mouse model of nonreperfused MI, reported rupture rates are highly variable and depend not only on the genetic background and sex of animals but also on the method used for documentation of rupture. In most studies, diagnosis of cardiac rupture is based on visual inspection during autopsy; however, criteria are poorly defined. We performed systematic histopathological analysis of whole hearts from C57BL/6J mice dying after nonreperfused MI and evaluated the reliability of autopsy-based criteria in identification of rupture. Moreover, we compared the cell biological environment of the infarct between rupture-related and rupture-independent deaths. Histopathological analysis documented rupture in 50% of mice dying during the first week post-MI. Identification of a gross rupture site was highly specific but had low sensitivity; in contrast, hemothorax had high sensitivity but low specificity. Mice with rupture had lower myofibroblast infiltration, accentuated macrophage influx, and a trend toward reduced collagen content in the infarct. Male mice had increased mortality and higher incidence of rupture. However, infarct myeloid cells harvested from male and female mice at the peak of the incidence of rupture had comparable inflammatory gene expression. In conclusion, the reliability of autopsy in documentation of rupture in infarcted mice is dependent on the specific criteria used. Macrophage-driven inflammation and reduced activation of collagen-secreting reparative myofibroblasts may be involved in the pathogenesis of post-MI cardiac rupture.NEW & NOTEWORTHY We show that cardiac rupture accounts for 50% of deaths in C57BL/6J mice undergoing nonreperfused myocardial infarction protocols. Overestimation of rupture events in published studies likely reflects the low specificity of hemothorax as a criterion for documentation of rupture. In contrast, identification of a gross rupture site has high specificity and low sensitivity. We also show that mice dying of rupture have increased macrophage influx and attenuated myofibroblast infiltration in the infarct. These findings are consistent with a role for perturbations in the balance between inflammatory and reparative responses in the pathogenesis of postinfarction cardiac rupture. We also report that the male predilection for rupture in infarcted mice is not associated with increased inflammatory activation of myeloid cells.