Increased Infarct Size Research Articles

Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia.

Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion (I/R) injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodeling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodeling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodeling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction.

Investigating the temporal link between PM2.5 exposure and acceleration of myocardial ischemia reperfusion injury: Emphasizing the hazardous presence of metals in inhaled air

Exposure to PM2.5 is widely acknowledged to induce cardiotoxic effects, leading to decreased myocardial tolerance to revascularization procedures and subsequent ischemia reperfusion injury (IR). However, the temporal relationship between PM2.5 exposure and vulnerability to IR, along with the underlying mechanisms, remains unclear and is the focus of this study. Female Wistar rats were exposed to PM2.5 at a concentration of 250 μg/m³ for 3 h daily over varying durations (7, 14, and 21 days), followed by IR induction. Our results demonstrated a significant increase in cardiac injury, as evidenced by increased infarct size and elevated cardiac injury markers, starting from day 14 of PM2.5 exposure, accompanied by declined cardiac function. These adverse effects were associated with apoptosis and impaired mitochondrial function, including reduced bioenergetics, mitochondrial DNA copy number and quality control mechanisms, along with inactivation of the PI3K/AKT/AMPK signalling pathways. Furthermore, analysis of myocardial tissue revealed elevated metal accumulation, particularly within mitochondria. Chelation of PM2.5 -associated metals using EDTA significantly mitigated the toxic effects on cardiac IR pathology, as confirmed in both rat myocardium and H9c2 cells. These findings suggest that metals in PM2.5 play a crucial role in inducing cardiotoxicity, impairing myocardial resilience to stress through mitochondrial accumulation and dysfunction.

Examining the Impact of Environmental and Genetic Factors that Influence Sarcospan Expression in the Heart

Objective: Susceptibility to cardiac injury can be hereditary and the most common causes include genetic variations in proteins regulating lipid metabolism, arterial hypertension, diabetes, body-mass index as well as environmental causes such as smoking. Sarcospan (SSPN) is an integral member of the dystrophin-glycoprotein complex (DGC) and has an important role in stabilizing the muscle membrane of striated muscle. Genetic variants in the SSPN gene have been linked with increased cardiovascular disease risk and studies are ongoing to identify underlying mechanisms. Our studies using perfused mouse hearts subjected to acute ischemia/reperfusion injury indicate that SSPN deletion increases infarct size and arrhythmia risk (Hwang et al, IJMS, 2023). In addition, adult SSPN−/− cardiomyocytes were found to have decreased antioxidative capacity and heightened susceptibility to oxidative stress. Therefore, we hypothesize that genetic factors that decrease cardiac SSPN expression will increase cardiac susceptibility of mice exposed to conditions of prolonged oxidative stress. In this study we examine the role of SSPN to heart injury caused by chronic oxidative stress mediated by cigarette smoke (CS). Methods: To assess the impact of chronic oxidative stress conditions on SSPN expression in the heart 2 month-old WT C57BL/6J mice were exposed to 4 months CS. We found that SSPN expression was significantly reduced the CS exposed hearts. Since CS increases oxidative damage and deteriorates antioxidant defenses of the heart and vascular system, we examined the combined impact of SSPN deficiency and chronic oxidative stress. To address this, 7-month-old WT and SSPN−/− male and female mice were exposed to CS for 4 months. The mice were assessed after CS for body composition (EchoMRI), glucose tolerance testing and echocardiography. Tissue histology, immunoblotting of oxidative and inflammatory stress markers, serum inflammatory markers were assessed. Lung fibroblasts were isolated from the smoked and non-smoked groups to evaluate inflammatory, fibrotic, and oxidative stress pathway activation to respective stimuli. Results: Preliminary findings indicate that after CS SSPN−/− hearts exhibit larger areas of myocardial damage than WT mice. Surprisingly, after CS the SSPN−/− mice showed better glucose tolerance than WT mice and no significant changes in body mass. Minor decrements were found in the cardiac function of the CSE mice. Exposure of lung fibroblasts obtained from non-smoked SSPN−/− mice to innate immune ligands revealed activation of specific pro-inflammatory pathways.Conclusions: SSPN-deficiency increases susceptibility to cardiac injury in mice subjected to CS exposure, likely through crosstalk mechanisms that exist between oxidative and inflammatory pathways that SSPN appears to influence. Funding Sources: Florida Department of Health, James and Esther King #21K12. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dim light at night shifts microglia to a pro-inflammatory state after cerebral ischemia, altering stroke outcome in mice

Circadian rhythms are endogenous biological cycles that regulate physiology and behavior and are set to precisely 24-h by light exposure. Light at night (LAN) dysregulates physiology and function including immune response; a critical component that contributes to stroke pathophysiological progression of neuronal injury and may impair recovery from injury. The goal of this study is to explore the effects of dim LAN (dLAN) in a murine model of ischemic stroke to assess how nighttime lighting from hospital settings can affect stroke outcome. Further, this study sought to identify mechanisms underlying pathophysiological changes to immune response after circadian disruption. Male and female adult Swiss Webster (CFW) mice were subjected to transient or permanent focal cerebral ischemia, then were subsequently placed into either dark night conditions (LD) or one night of dLAN (5 lx). 24 h post-stroke, sensorimotor impairments and infarct sizes were quantified. A single night of dLAN following MCAO increased infarct size and sensorimotor deficits across both sexes and reduced survival in males after 24 h. Flow cytometry was performed to assess microglial phenotypes after MCAO, and revealed that dLAN altered the percentage of microglia that express pro-inflammatory markers (MHC II+ and IL-6) and microglia that express CD206 and IL-10 that likely contributed to poor ischemic outcomes. Following these results, microglia were reduced in the brain using Plexxikon 5622 (PLX 5622) a CSFR1 inhibitor, then the mice received an MCAO and were exposed to LD or dLAN conditions for 24 h. Microglial depletion by PLX5622 resulted in infarct sizes that were comparable between lighting conditions. This study provides supporting evidence that environmental lighting exacerbates ischemic injury and post-stroke mortality by a biological mechanism that exposure to dLAN causes a fundamental shift of activated microglial phenotypes from beneficial to detrimental at an early time point after stroke, resulting in irreversible neuronal death.

Abstract WP324: MCA/FeCl 3 Thromboembolic Model, an Easily Reproducible Model for COVID-19-Induced Embolic Cerebrovascular Complications

COVID-19 neurological complications such as strokes and cognitive impairment have been reported. Yet the molecular mechanisms are under investigation. A mild and reproducible thromboembolic model will be a helpful tool for investigating COVID-thromboembolic complications. The aim of this study is to investigate the middle cerebral artery/ferric chloride (MCA/FeCl 3 ) thromboembolic model as an easily and reproducible model for COVID-19-induced cerebrovascular embolic complications. Methods: SARS-CoV-2 spike-protein of the alpha variant were injected intravenously (4 ug/animal) in K18 (transgenic knock-in humanized ACE2 in epithelial cells). Seven days later, MCA/FeCl 3 thromboembolic model was induced in K18 mice by exposing the MCA and placing filter paper wet with FeCl 3 . Laser speckle imaging was used for the assessment of blood flow and recanalization after 1,2,6, and 24 hrs. TTC was used to assess the infarct volume. Cognitive function was assessed using a novel object recognition test. Cerebral vascular density and blood-brain barrier (BBB) were assessed by immunohistochemistry staining using Iso-B4 lectin and Occludin-5 antibody. Results: SARS-CoV-2 spike-protein caused a significant increase in the time required for vascular recanalization as detected by laser speckle imaging compared to control K18 mice. (P<0.05). In parallel, spike protein caused a significant increase in infarct size compared to control K18 mice (P<0.05). Our immunohistochemistry showed that spike protein decreased vascular density and increased vascular rarefaction compared to control K18 mice (P<0.05). A novel object recognition test showed that spike protein increased cognitive dysfunction compared to control (P<0.05). Western blot analysis showed that spike protein increased inflammatory markers and decreased BBB tight junction protein compared to control K18 mice (P<0.05). In conclusion , the MCA/FeCl 3 thromboembolic model is an easily reproducible mouse model to examine COVID-19-induced stroke and cerebrovascular complications.

Abstract WP301: STAT3 Inhibition Reduces Infarct and Neuroinflammation in Experimental Stroke

Signal Transducer and Activator of Transcription 3 (STAT3) activation is increased in the brain following cerebral ischemia. STAT3, a transcription factor, is a positive regulator of cell survival and is implicated in cytoprotection following ischemia in multiple cell types, including neurons and endothelial cells. We hypothesized that pharmacological inhibition of STAT3 activation in mice during the reperfusion phase after middle cerebral artery occlusion (MCAO) will lead to increased infarct size. We subjected mice to 1-hour MCAO, following which they received STAT3 inhibitor Stattic (20 mg/kg), or vehicle, for 24 hours delivered subcutaneously via osmotic pump. Twenty-four hours later, infarct was measured by TTC and inflammatory cells were measured by flow cytometry. For experiments involving splenectomy, spleens were surgically removed 14 days prior to MCAO surgery. Surprisingly, we found that STAT3 inhibition reduces infarct after MCAO, with infarct in the hemisphere reducing from 49.90 ± 2.44 % in vehicle to 32.86 ± 1.55 % in Stattic-treated mice. Since STAT3 is involved in inflammatory processes, as well as cytoprotection, this led us to hypothesize that the reduction in infarct we observe may be due to decreased inflammation. We carried out FACS analysis and observed that Stattic inhibits MCAO-induced infiltration of B-, T- and dendritic cells into the brain. Neutrophil and monocyte infiltration into the brain were unaffected by Stattic. Since MCAO is known to induce splenic atrophy, with spleen size being inversely proportional to infarct volume, we measured spleen weights. Interestingly Stattic-treated mice had larger spleens (66.31 ± 5.40 g) compared to vehicle-treated mice (46.43 ± 4.60 g), indicating a dampened peripheral immune response upon inhibition of STAT3. Furthermore, Stattic was no longer effective in reducing infarct size in splenectomized mice. We conclude that pharmacological inhibition of STAT3 reduces infarct following MCAO, with the mechanism of protection involving differential regulation of the peripheral immune response.

Rhein alleviates myocardial ischemic injury by inhibiting mitochondrial division, activating mitochondrial autophagy and suppressing myocardial cell apoptosis through the Drp1/Pink1/Parkin pathway.

Rhein, which has antioxidant and anti-inflammatory response properties, is a beneficial treatment for different pathologies. However, the mechanism by which rhein protects against myocardial ischemic injury is poorly understood. To establish an acute myocardial infarction (AMI) rat model, we performed left anterior descending (LAD) ligation. Sprague‒Dawley rats were randomly divided into four groups: sham, AMI, AMI + rhein (AMI + R), and AMI + mitochondrial fission inhibitor (AMI + M). The extent of myocardial injury was evaluated by TTC staining, serum myocardial injury markers, and HE and Masson staining. Cardiac mitochondria ultrastructure was visualized by transmission electron microscopy. TUNEL assay and flow cytometry analysis were used to estimate cell apoptosis. Protein expression levels were measured by Western blotting. In vitro, the efficacy of rhein was assessed in H9c2 cells under hypoxic condition. Our results revealed that rats with AMI exhibited increased infarct size and indicators of myocardial damage, along with activation of Drp1-dependent mitochondrial fission, decreased mitophagy and increased apoptosis rates. However, pretreatment with rhein significantly reversed these effects and demonstrated similar efficacy to Mdivi-1. Furthermore, rhein pretreatment protected against myocardial ischemic injury by inhibiting mitochondrial fission, as evidenced by decreased Drp1 expression. It also enhanced mitophagy, as indicated by increased expression of Beclin1, Pink1 and Parkin, an increased LC3-II/LC3-I ratio and increased formation of autolysosomes. Additionally, rhein pretreatment mitigated apoptosis in AMI. These results were also confirmed in vitro in H9c2 cells. Our results demonstrate that rhein pretreatment exerts cardioprotective effects against myocardial ischemic injury via the Drp1/Pink1/Parkin pathway.

Novel Role for Cardiolipin as a Target of Therapy to Mitigate Myocardial Injury Caused by Venoarterial Extracorporeal Membrane Oxygenation.

Cardiolipin is a mitochondrial-specific phospholipid that maintains integrity of the electron transport chain (ETC) and plays a central role in myocardial ischemia/reperfusion injury. Tafazzin is an enzyme that is required for cardiolipin maturation. Venoarterial extracorporeal membrane oxygenation (VA-ECMO) use to provide hemodynamic support for acute myocardial infarction has grown exponentially, is associated with poor outcomes, and is under active clinical investigation, yet the mechanistic effect of VA-ECMO on myocardial damage in acute myocardial infarction remains poorly understood. We hypothesized that VA-ECMO acutely depletes myocardial cardiolipin and exacerbates myocardial injury in acute myocardial infarction. We examined cardiolipin and tafazzin levels in human subjects with heart failure and healthy swine exposed to VA-ECMO and used a swine model of closed-chest myocardial ischemia/reperfusion injury to evaluate the effect of VA-ECMO on cardiolipin expression, myocardial injury, and mitochondrial function. Cardiolipin and tafazzin levels are significantly reduced in the left ventricles of individuals requiring VA-ECMO compared with individuals without VA-ECMO before heart transplantation. Six hours of exposure to VA-ECMO also decreased left ventricular levels of cardiolipin and tafazzin in healthy swine compared with sham controls. To explore whether cardiolipin depletion by VA-ECMO increases infarct size, we performed left anterior descending artery occlusion for a total of 120 minutes followed by 180 minutes of reperfusion in adult swine in the presence and absence of MTP-131, an amphipathic molecule that interacts with cardiolipin to stabilize the inner mitochondrial membrane. Compared with reperfusion alone, VA-ECMO activation beginning after 90 minutes of left anterior descending artery occlusion increased infarct size (36±8% versus 48±7%; P<0.001). VA-ECMO also decreased cardiolipin and tafazzin levels, disrupted mitochondrial integrity, reduced electron transport chain function, and promoted oxidative stress. Compared with reperfusion alone or VA-ECMO before reperfusion, delivery of MTP-131 before VA-ECMO activation reduced infarct size (22±8%; P=0.03 versus reperfusion alone and P<0.001 versus VA-ECMO alone). MTP-131 restored cardiolipin and tafazzin levels, stabilized mitochondrial function, and reduced oxidative stress in the left ventricle. We identified a novel mechanism by which VA-ECMO promotes myocardial injury and further identify cardiolipin as an important target of therapy to reduce infarct size and to preserve mitochondrial function in the setting of VA-ECMO for acute myocardial infarction.

Phenylacetyl glutamine (PAGln) enhances cardiomyocyte death after myocardial infarction through β1 adrenergic receptor.

This study aims to explore the roles of phenylacetyl glutamine (PAGln) on myocardial infarction (MI) pathogenesis. Here, using targeted metabolomics analysis, it was found that the plasma metabolite PAGln was upregulated in coronary artery disease (CAD) patients and MI mice and could be an independent risk factor for CAD. In vivo and in vitro functional experiments revealed that PAGln pretreatment enhanced MI-induced myocardial injury and cardiac fibrosis, as evident by the increased infarct size, cardiomyocyte death, and the upregulated expression of cardiac fibrosis markers (Col1a1 and α-SMA). Combined with RNA-sequencing analysis and G protein-coupled receptor (GPCR) inhibitor, we found that the GPCR signaling activation is essential for PAGln-mediated effects on cardiomyocyte death. Furthermore, drug affinity responsive target stability and cellular thermal shift assay demonstrated that PAGln could interact with β1-adrenergic receptor (AR). Moreover, β1-AR blocker treatment indeed extended the cardiac remodeling after PAGln-enhanced MI. These results suggest that PAGln might be a potential therapeutic target for extending the cardiac remodeling window in MI patients that signals via β1-AR.

YTHDF2 Promotes Cardiac Ferroptosis via Degradation of SLC7A11 in Cardiac Ischemia-Reperfusion Injury.

Myocardial ischemia-reperfusion (I/R) injury facilitates cardiomyocyte death and endangers human health. N6-methyladenosine (m6A) methylation plays a critical role in cardiovascular diseases. The m6A reader YTHDF2 identifies m6A-modified RNA and promotes target RNA degradation. Hence, we hypothesized that YTHDF2 affects I/R injury by regulating RNA stability. Both mRNA and protein levels of YTHDF2 were upregulated in I/R mice and hypoxia-reoxygenation (H/R)-induced cardiomyocytes. Silencing of endogenous YTHDF2 abrogated cardiac dysfunction and lowered the infarct size in I/R mice, and forced expression of YTHDF2 aggravated these adverse pathological processes. Consistently, the protective effect of silencing YTHDF2 occurred in cardiomyocytes exposed to H/R and erastin. Furthermore, RNA-seq and RIP revealed that YTHDF2 recognized the m6A modification sites of the ferroptosis-related gene SLC7A11 mRNA to promote its degradation both in vivo and in vitro. Inhibition of SLC7A11 impaired cardiac function, increased infarct size, and the release of LDH in I/R mice after silencing YTHDF2. The beneficial effects of si-YTHDF2 on H/R injury were reversed by co-transfection with si-SLC7A11, which substantially exacerbated ferroptosis and the production of ROS. The cardioprotective effects of silencing YTHDF2 are accomplished by increasing SLC7A11 stability and expression and reducing ferroptosis, providing novel potential therapeutic targets for treating ischemic cardiac diseases.

Platelet-derived microRNAs play a pivotal role in cardiac remodeling after myocardial ischemia and reperfusion

Abstract Background Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of miRNAs, which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate, that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. Purpose The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. Methods and Results Combining an in vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (LSFM, PET&amp;MRI, speckle-tracking echocardiography), in vitro immune cell migration assays and Next-generation deep sequencing analysis of platelet miRNA expression pattern in mice with a MK/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating LV remodeling after myocardial I/R following transient LCA ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial ischemia and reperfusion. Worsened cardiac remodeling after myocardial infarction (MI) in mice with a platelet-specific Dicer deletion (Dicer Pf4Δ/PfΔ4) resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at d28 post-MI. Altogether, these observations culminated in an impaired LV function and hampered long-term cardiac recovery after experimental MI and reperfusion therapy. Treatment with the P2Y12 antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in Dicer Pf4Δ/Pf4Δ mice. Conclusions The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial ischemia and reperfusion.

Circulating growth differentiation factor 11 exacerbates myocardial injury in mice and associates with increased infarct size in humans

Abstract Background The heart rejuvenating effects of circulating growth differentiation factor 11 (GDF11), a TGF-β superfamily member that shares 90% homology with myostatin (MSTN), remains controversial. Here, we aimed to probe the role of GDF11 in acute myocardial infarction (MI), a frequent cause of heart failure and premature death during ageing. Methods Young (3-4 months) and old (22-24 months) C57Bl/6 mice were subjected to either systemic recombinant GDF11 (rGDF11; 0.1 mg/kg BW daily) or control treatment over 30 days before myocardial ischaemia-reperfusion (I/R) injury was induced. Dissected hearts underwent in-depth profiling that ranged from histological workup and the assessment of inflammatory pathways to cardiomyocyte viability testing and IPA-guided -omics. Key regulatory mechanisms were studied in vitro using human adult stromal cardiac-specific mesenchymal cells (cMSCs) and HL-1 cardiomyocytes. By harnessing liquid chromatography–tandem mass spectrometry (LC-MS/MS), plasma levels of GDF11/MSTN were determined in prospectively recruited patients with MI and the independent association with myocardial infarct size was estimated using hierarchical regression models. Results In contrast to endogenous Mstn, myocardial Gdf11 mRNA expression levels declined upon ageing, and were further reduced following I/R injury, suggesting a therapeutic potential of GDF11 signalling in MI. Unexpectedly, boosting systemic Gdf11 by rGDF11 delivery prior to myocardial I/R augmented rather than reduced infarct size in mice irrespective of their age, predominantly by accelerating pro-apoptotic signalling. By applying a targeted transcriptomics approach on dissected hearts subjected to I/R, we identified the mesenchymal marker Nkx2-5 to be blunted upon rGDF11 supplementation, pointing toward a cMSC-mediated mechanism. Accordingly, human cMSCs exposed to high rGDF11 in vitro showed similar reductions in NKX2-5 expression levels, coinciding with diminished anti-apoptotic effects conferred by resident cMSCs on HL-1 cardiomyocytes. Finally, by harnessing a highly-specific LC–MS/MS based assay, we show that in patients with MI receiving contemporary management circulating GDF11 but not MSTN levels inclined with age. Moreover, circulating GDF11 emerged as an independent predictor of myocardial infarct size, as estimated by standardized peak creatine kinase-MB levels. Notably, GDF11 levels were particularly elevated in those at high risk for adverse outcomes following the acute event. Conclusions Our data challenge the initially reported rejuvenating effects of circulating GDF11 and suggest that high levels of systemic GDF11 exacerbate myocardial injury in mice by impinging on the cardioprotective effects of resident cMSCs. Persistently high GDF11 levels during ageing may contribute to the age-dependent loss of endogenous cardioprotective mechanisms and thus poor outcomes post-MI.

Revealing concealed cardioprotection by platelet Mfsd2b-released S1P in human and murine myocardial infarction

Abstract Background/Introduction Platelet activation on ruptured or eroded atherosclerotic lesions leads to thrombus formation and ischemic events. Hence, platelet inhibition is the cornerstone of primary and secondary prevention in patients with cardiovascular disease. Recently, non-canonical effects of platelets in multiple disease processes were revealed. However, others have demonstrated the opposite: a protective and regenerative role of platelets during myocardial healing. Sphingosine-1-phosphate (S1P) is an important bioactive lipid released from platelets upon activation and has potent cardioprotective properties in AMI when acutely administered or kept constitutively high due to pharmacological or genetic inhibition of its degradation. In humans, S1P levels in plasma are dynamically regulated during AMI. Purpose We hypothesized that platelets may be an important source of the S1P burst during AMI and that platelet-derived S1P is relevant for the extent of myocardial damage due to AMI. Methods We have addressed these questions in experimental AMI in mice lacking components of the S1P synthesis, release and signaling pathways. In additon, we conducted a hypothesis generating, prospective, monocentric, time-series, translational analysis in 127 patients with ST-elevation myocardial infarction (STEMI) to analyze the association between S1P plasma concentration and infarct size. Results Injection of cell-free supernatant of activated platelets (SNT+) prior AMI in mice led to decreased infarct size after 24 h of reperfusion as compared to supernatant of non-activated platelets (SNT-). This reduction of infarct size by SNT+ was associated with reduced neutrophil accumulation and reduced apoptosis after AMI. S1P-concentrations in SNT+ was 25% higher as compared to SNT-. S1P concentrations in SNT- of mice lacking Sphingosinkinase-1 (SphK1) were 70% lower and did not increase in SNT+. Accordingly, SNT+ of SphK1- deficient platelets failed to reduce infarct size. Deficiency of platelet S1P exporter Mfsd2b shows increased infarct size during in vivo AMI compared to littermate controls. SNT+ of WT mice led to improved outcome after ischemia in a model of Langendorff-perfused hearts. This was blunted by inhibition of S1P receptor 1 and genetic cardiomyocyte deficiency of S1PR1. Platelet S1P release during activation was reduced by P2Y12 inhibition but preserved during GPIIb/IIIa (tirofiban) inhibition. This translated to blunted cardioprotection by SNT+ of cangrelor treated platelets but sustained cardioprotection during tirofiban treatment. Plasma S1P concentrations was associated with cardiovascular death and infarct size in patients with ST-elevation myocardial infarction. Conclusion We have identified the mechanisms underlying platelet-associated cardioprotection and finally, revealed the importance of platelet-released S1P on admission in patients with ST-elevation myocardial infarction (STEMI) regarding to infarct size and long-term prognosis.

Stimulation of soluble guanylyl cyclase in erythrocytes induces export of cGMP and cardioprotection in type 2 diabetes

Abstract Background Red blood cells (RBCs) are known to export cardioprotective nitric oxide (NO) bioactivity. Although, RBCs contain a functional soluble guanylyl cyclase (sGC), the signalling behind the export of NO bioactivity remains unclear. Impairment of NO signalling in RBCs has been suggested to contribute to augmented cardiac ischemia-reperfusion injury in type 2 diabetes (T2D). We hypothesized that increased NO signaling by stimulation of sGC in RBCs protects from myocardial ischemia-reperfusion injury by release of a cardioprotective signal. Purpose To investigate whether stimulation of sGC, the intracellular NO receptor, in RBCs from T2D patients induces export of a cardioprotective factor and to determine its nature. Methods RBCs were collected from patients with T2D and from gender- and age-matched healthy subjects and incubated with different pharmacological compounds affecting the NO-signalling pathway. The RBCs or their supernatant were then administered to isolated Langendorff-perfused rat hearts subjected to 25 min global ischemia and 60 min reperfusion. Left ventricular developed pressure and infarct size were measured. ELISA and immunofluorescence were used to determine levels of cyclic guanosine monophosphate (cGMP) and substrate of protein kinase G activation. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85-23, revised 1996). The present study was performed following The Code of Ethics of the World Medical Association outlined in the Declaration of Helsinki of 1975 and revised in 1983 for experiments that involve human subjects. Results RBCs from T2D patients impaired post-ischemic cardiac function and increased infarct size compared to RBCs from healthy subjects, and these effects were prevented by pre-incubation of the RBCs with the sGC stimulator CYR715. Administration of the supernatant from RBCs incubated with the sGC stimulator resulted in similar cardioprotection, indicating release of a cardioprotective factor from the RBCs (Figure 1A and B). The cardioprotection was abolished by inhibition of transport of cGMP with MK571, an inhibitor of cyclic nucleotide transport. Extracellular cGMP levels in the supernatant from RBCs were increased following sGC stimulation, and administration of exogenous cGMP induced cardioprotection (Figure 1C and D). Expression of protein kinase G-dependent phosphorylated vasodilator-specific phosphoprotein in cardiomyocytes was increased following administration of supernatant from RBCs incubated with the sGC stimulator (Figure 1E). Conclusions This study shows that stimulation of sGC in RBCs leads to release of a cardioprotective mediator, identified as cGMP, that activates cardiac protein kinase G and protects from ischemia-reperfusion injury. It further identifies RBC sGC as a novel therapeutic target to prevent cardiovascular injury in T2D.Figure 1

Abstract 18418: Osteoprotegerin Mediates T-Cell Response in Myocardial Wound Healing Post-Ischemic Injury

Introduction: Myocardial Infarction (MI) triggers an inflammatory response associated with cardiac wound healing. T-regulatory cells (Tregs) play a crucial role in the MI heart during this inflammatory response via their regulation of cardiac repair and function. Stimulating and sustaining the pro-reparative T cell response after MI would aid in preserving cardiac structure and function. Cortical Bone derived Stem Cells (CBSCs) possess a diverse array of inflammatory signaling molecules that can influence the inflammatory microenvironment of the heart after MI. A paracrine factor, Osteoprotegerin (OPG), is abundantly present in CBSCs, but it is unknown if OPG can mediate Tregs in myocardial injury. Hypothesis: CBSCs-derived Osteoprotegerin can regulate cardiac wound healing post-MI by modulating T cell response. Methods and Results: T cells exposed to CBSC secretome underwent an upregulation of a Treg population (p≤0.0001) characterized by the expression of Tumor Necrosis Factor Receptor II (TNFRII). Further investigation into the CBSC secretome highlighted OPG as a potent signaling molecule responsible for inducing TNFRII+ Tregs (p≤0.0001) measured by FACS analysis. siRNA-mediated depletion of OPG studies showed compromised TNFRII+ Treg induction (p≤0.0001). In vivo studies, adult FoxP3-GFP (12-16 week) mice underwent left anterior descending artery ligation followed by intramyocardial injection of CBSCs, Mesenchymal Stem Cells (MSCs), or vehicle (saline) along the infarct border zone. Resident T-lymphocyte populations were assessed at 1- and 8-weeks post-MI via flow cytometry. CBSC-treated hearts yielded a greater prevalence of TNFRII+ Tregs at 1- and 8-weeks following MI compared to MSCs (p≤0.01; p≤0.0001) and vehicle (p≤0.0001; p≤0.05). CBSC induction following MI reduced infarct size compared to the vehicle (p≤0.05). Ablation of the Treg population in the ischemic zone following MI increased infarct size and solicited adverse cardiac remodeling (p≤0.05). Conclusion: Osteoprotegerin can promote cardiac wound healing by facilitating the expansion and preservation of pro-reparative TNFRII+ Tregs.

A New Target of Electroacupuncture Pretreatment Mediated Sympathetic Nervous to Improve MIRI: Glutamatergic Neurons in Fastigial Nucleus of the Cerebellum

Ischemic heart disease is a fatal cardiovascular disease that irreversibly impairs the function of the heart, followed by reperfusion leading to a further increase in infarct size. Clinically, we call it myocardial ischemia–reperfusion injury (MIRI). A growing number of clinical observations and experimental studies have found electroacupuncture (EA) to be effective in alleviating MIRI. This study attempts to investigate whether glutamatergic neurons in fastigial nucleus (FN) of the cerebellum are involved in EA pretreatment to alleviate MIRI via sympathetic nerves, and the potential mechanisms of EA pretreatment process. A MIRI model was established by ligating the coronary artery of the left anterior descending branch of the heart for 30 minutes, followed by 2 hours of reperfusion. Multichannel physiological recordings, electrocardiogram, cardiac ultrasound, chemical genetics, enzyme-linked immunosorbent assay and immunofluorescence staining methods were combined to demonstrate that EA pretreatment inhibited neuronal firing and c-Fos expression in FN of the cerebellum and reduced cardiac sympathetic firing. Meanwhile, EA pretreatment significantly reduced cardiac ejection fraction (EF), shortening fraction (SF), percentage infarct area, decreased myocardial norepinephrine (NE), creatine kinase isoenzyme MB (CK-MB) concentrations, and improved MIRI-induced myocardial tissue morphology. The results were similar to the inhibition of glutamatergic neurons in FN. However, the activation of glutamatergic neurons in FN diminished the aforementioned effects of EA pretreatment. This study revealed that glutamatergic neurons in FN of the cerebellum is involved in EA pretreatment mediated sympathetic nervous and may be a potential mediator for improving MIRI.

Inflammatory Bowel Disease Increases the Severity of Myocardial Infarction after Acute Ischemia-Reperfusion Injury in Mice.

(1) Background: Increased risk of myocardial infarction (MI) has been linked to several inflammatory conditions, including inflammatory bowel disease (IBD). However, the relationship between IBD and MI remains unclear. Here, we implemented an original mouse model combining IBD and MI to determine IBD's impact on MI severity and the link between the two diseases. (2) Methods: An IBD model was established by dextran sulfate sodium (DSS) administration in drinking water, alone or with oral C. albicans (Ca) gavage. IBD severity was assessed by clinical/histological scores and intestinal/systemic inflammatory biomarker measurement. Mice were subjected to myocardial ischemia-reperfusion (IR), and MI severity was assessed by quantifying infarct size (IS) and serum cardiac troponin I (cTnI) levels. (3) Results: IBD mice exhibited elevated fecal lipocalin 2 (Lcn2) and IL-6 levels. DSS mice exhibited almost two-fold increase in IS compared to controls, with serum cTnI levels strongly correlated with IS. Ca inoculation tended to worsen DSS-induced systemic inflammation and IR injury, an observation which is not statistically significant. (4) Conclusions: This is the first proof-of-concept study demonstrating the impact of IBD on MI severity and suggesting mechanistic aspects involved in the IBD-MI connection. Our findings could pave the way for MI therapeutic approaches based on identified IBD-induced inflammatory mediators.

Macrophage neogenin deficiency exacerbates myocardial remodeling and inflammation after acute myocardial infarction through JAK1-STAT1 signaling.

Immune response plays a crucial role in post-myocardial infarction (MI) myocardial remodeling. Neogenin (Neo1), a multifunctional transmembrane receptor, plays a critical role in the immune response; however, whether Neo1 participates in pathological myocardial remodeling after MI is unclear. Our study found that Neo1 expression changed significantly after MI in vivo and after LPS + IFN-γ stimulation in bone marrow-derived macrophages (BMDMs) in vitro. Neo1 functional deficiency (using a neutralizing antibody) and macrophage-specific Neo1 deficiency (induced by Neo1flox/flox;Cx3cr1cre mice) increased infarction size, enhanced cardiac fibrosis and cardiomyocyte apoptosis, and exacerbated left ventricular dysfunction post-MI in mice. Mechanistically, Neo1 deficiency promoted macrophage infiltration into the ischemic myocardium and transformation to a proinflammatory phenotype, subsequently exacerbating the inflammatory response and impairing inflammation resolution post-MI. Neo1 deficiency regulated macrophage phenotype and function, possibly through the JAK1-STAT1 pathway, as confirmed in BMDMs in vitro. Blocking the JAK1-STAT1 pathway with fludarabine phosphate abolished the impact of Neo1 on macrophage phenotype and function, inflammatory response, inflammation resolution, cardiomyocyte apoptosis, cardiac fibrosis, infarction size and cardiac function. In conclusion, Neo1 deficiency aggravates inflammation and left ventricular remodeling post-MI by modulating macrophage phenotypes and functions via the JAK1-STAT1 signaling pathway. These findings highlight the anti-inflammatory potential of Neo1, offering new perspectives for therapeutic targets in MI treatment. Neo1 deficiency aggravated inflammation and left ventricular remodeling after MI by modulating macrophage phenotypes and functions via the JAK1-STAT1 signaling pathway.

Mitigating the risk of flow deterioration by deferring stent optimization in STEMI patients with large thrombus burden: Insights from a prospective cohort study

ObjectivesIt is uncertain, if omitting post-dilatation and stent oversizing (stent optimization) is safe and may decrease the risk for distal thrombus embolization (DTE) in STEMI patients with large thrombus burden (LTB).BackgroundIn patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (pPCI) with stenting, (DTE) and flow deterioration are common and increase infarct size leading to worse outcomes.MethodsFrom a prospective registry, 74 consecutive STEMI patients with LTB undergoing pPCI with stenting and intentionally deferred stent optimization were analyzed. Imaging data and outcomes up to 2 years follow-up were analyzed.ResultsOverall, 74 patients (18% females) underwent deferred stent optimization. Direct stenting was performed in 13 (18%) patients. No major complications occurred during pPCI. Staged stent optimization was performed after a median of 4 (interquartile range (IQR) 3; 7) days. On optical coherence tomography, under-expansion and residual thrombus were present in 59 (80%) and 27 (36%) cases, respectively. During deferred stent optimization, we encountered no case of flow deterioration (slow or no-reflow) or side branch occlusion. Minimal lumen area (mm2) and stent expansion (%) were corrected from 4.87±1.86mm to 6.82±2.36mm (p<0.05) and from 69±18% to 91±12% (p<0.001), respectively. During follow-up, 1 patient (1.4%) required target lesion revascularization and 1 (1.4%) patient succumbed from cardiovascular death.ConclusionsAmong STEMI patients with LTB, deferring stent optimization in the setting of pPCI appears safe and potentially mitigates the risk of DTE. The impact of this approach on infarct size and clinical outcomes warrants further investigation in a dedicated trial.

Circulating GDF11 exacerbates myocardial injury in mice and associates with increased infarct size in humans.

The heart rejuvenating effects of circulating growth differentiation factor 11 (GDF11), a transforming growth factor-β superfamily member that shares 90% homology with myostatin (MSTN), remains controversial. Here, we aimed to probe the role of GDF11 in acute myocardial infarction (MI), a frequent cause of heart failure and premature death during ageing. In contrast to endogenous Mstn, myocardial Gdf11 declined during the course of ageing and was particularly reduced following ischaemia/reperfusion (I/R) injury, suggesting a therapeutic potential of GDF11 signalling in MI. Unexpectedly, boosting systemic Gdf11 by recombinant GDF11 delivery (0.1 mg/kg body weight over 30 days) prior to myocardial I/R augmented myocardial infarct size in C57BL/6 mice irrespective of their age, predominantly by accelerating pro-apoptotic signalling. While intrinsic cardioprotective signalling pathways remained unaffected by high circulating GDF11, targeted transcriptomics and immunomapping studies focusing on GDF11-associated downstream targets revealed attenuated Nkx2-5 expression confined to CD105-expressing cells, with pro-apoptotic activity, as assessed by caspase-3 levels, being particularly pronounced in adjacent cells, suggesting an indirect effect. By harnessing a highly specific and validated liquid chromatography-tandem mass spectrometry-based assay, we show that in prospectively recruited patients with MI circulating GDF11 but not MSTN levels incline with age. Moreover, GDF11 levels were particularly elevated in those at high risk for adverse outcomes following the acute event, with circulating GDF11 emerging as an independent predictor of myocardial infarct size, as estimated by standardized peak creatine kinase-MB levels. Our data challenge the initially reported heart rejuvenating effects of circulating GDF11 and suggest that high levels of systemic GDF11 exacerbate myocardial injury in mice and humans alike. Persistently high GDF11 levels during ageing may contribute to the age-dependent loss of cardioprotective mechanisms and thus poor outcomes of elderly patients following acute MI.