Determination Of 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.

Aloperine Alleviates Myocardial Injury Induced by Myocardial Ischemia and Reperfusion by Activating the ERK1/2/β-catenin Signaling Pathway.

Myocardial ischemia/reperfusion (I/R) injury can cause severe cardiac damage. Aloperine is a quinolizidine alkaloid found in the leaves and seeds of Sophora alopecuroides L. It has been recognized that aloperine has organ-protective properties; however, its role in cardioprotection is poorly characterized. This study aimed to evaluate the cardioprotective effects of aloperine against myocardial I/R injury in vivo. Adult male Sprague‒Dawley rats were randomly divided into sham-operated, control, and aloperine groups. All rats except for the sham-operated rats were subjected to 45min of myocardial ischemia (by left anterior descending ligation) followed by 3h of reperfusion. Aloperine (10mg/kg) was given intravenously at the onset of reperfusion. The cardioprotective effects of aloperine were evaluated by determining infarct size, hemodynamics, histological changes, cardiac biomarkers, and cardiac apoptosis. Aloperine limited infarct size; improved hemodynamics; attenuated myocardial I/R-induced histological deterioration; decreased serum LDH, CK-MB, and α-HBDH levels; and inhibited apoptosis after myocardial I/R injury. Moreover, aloperine stimulated the phosphorylation of ventricular ERK1/2, which is a major module of MAPK signaling pathways. Furthermore, aloperine increased the ventricular expression levels of β-catenin. Pharmacological inhibition of ERK1/2 diminished aloperine-induced cardioprotection and blocked ERK1/2/β-catenin signaling. These data support the cardioprotective effect of aloperine against myocardial I/R injury, which is mediated, at least in part, by the ERK1/2/β-catenin signaling pathway.

Hyperbaric Oxygen Upregulates Mst1 to Activate Keap1/Nrf2/HO-1 Pathway Resisting Oxidative Stress in a Rat Model of Acute Myocardial Infarction.

This study aimed to investigate the protective effects and mechanisms of hyperbaric oxygen (HBO) preconditioning in a rat model of acute myocardial infarction (MI) established by ligation of the left anterior descending (LAD) coronary artery. Microarray, real-time PCR, and western blotting (WB) results demonstrated that the Mst1 gene was downregulated in the heart tissue of the MI rat model. HBO preconditioning significantly increased Mst1 expression in cardiac tissues of rats after MI modeling. Lentiviral infection was used to silence the Mst1 gene in rats treated with HBO to probe the effect of Mst1 on HBO cardioprotection. HBO preconditioning decreased heart infarct size and ameliorated cardiac function in MI rats, whereas Mst1 silencing reversed the effect of HBO administration, as indicated after heat infarct size determination via TTC staining, histological examination via HE staining, and measurements of cardiac function. HBO preconditioning reduced oxidative stress and inflammation in cardiac tissue of MI rat model, evidenced by alteration of malondialdehyde (MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), and protein carbonyl contents, as well as production of inflammation-associated myeloperoxidase (MPO), IL-1β, and TNF-α. These findings provide a new signaling mechanism through which HBO preconditioning can protect against acute MI injury through the Mst1-mediating Keap1/Nrf2/HO-1-dependent antioxidant defense system.

Malonate protection against acute infarction in isolated mouse hearts is lost in the presence of insulin

Abstract Background & Objectives Malonate, an inhibitor of mitochondrial succinate dehydrogenase (SDH), is a promising novel protectant against acute ischaemia-reperfusion injury (IRI) through reduction of ischaemic succinate accumulation and ROS production during early reperfusion. Malonate is still protective when given only at reperfusion, making it an ideal agent for treating AMI patients. However, numerous studies demonstrated that the metabolic milieu could affect the potency of cardioprotective treatments. Therefore, in this study, we explored whether and how the severity of cardiac I/R injury and cardioprotection by malonate is affected by the metabolic milieu. We hypothesized that protection by malonate is affected by metabolic milie. Methods Isolated Langendorff-perfused hearts (n=8 to 19 per group) of C57Bl/6N mice were perfused with perfusate to which substrates and hormones were added in a step-wise manner: 1) glucose (G), 2) glucose + glutamine (GG), 3) glucose + glutamine + palmitate (GGP), or 4) glucose + glutamine + palmitate + insulin (GGPI), and subjected to 30 min ischaemia (I) and 90 min reperfusion (R). Heart were treated with malonate (5mM) or vehicle (saline) during the first 5 min of reperfusion only. Cardiac function of the left ventricle was monitored by a balloon in the left ventricle. Coronary effluent was collected at 7 time points of reperfusion to detect the activity of lactate dehydrogenase (LDH) as an indicator of cell death. At the end reperfusion, hearts were used for infarct size determination through TTC staining. Data are presented as mean ± SD. Results Going from glucose-only perfusate to a perfusate containing all examined substrates and hormones, the severity of cardiac I/R injury increased: infarct size (%) went from 31.9 ± 10.9% to 37.7 ± 10.6% to 52.3 ± 14.2%, and finally to 68 ± 9.9 %. Similar trends for increased IRI were observed for LDH, End-Diastolic Pressure (EDP) and recovery of the Rate-Pressure Product (RPP). When compared with vehicle control, malonate significantly decreased IS% (control vs malonate: 31.9 ± 10.9% vs. 23.3 ± 4.8%, 37.7 ± 10.6% vs. 20.9 ± 8.5%, 68 ± 9.9% vs. 40.2 ± 9.2 %), and increased the RRP recovery (64.1 ± 16.6 %vs. 83.3 ± 17.9 %, 55.6 ± 11% vs. 72.3 ± 4%, 39.3 ± 15.6% vs. 55.9 ± 8.3%) in the G, GG and GGP group. The EDP was significantly decreased by malonate in the G and GG group (32.8 ± 14.3 mmHg vs. 20.9 ± 9.8 mmHg, 35.1 ± 8.7 mmHg vs. 22.2 ± 8.6 mmHg ). LDH was only significantly decreased by malonate in the GG group (35.2 ± 12.9 vs. 17.1 ± 7.7 U / GWW). Most importantly, adding insulin to the perfusate nullified protection by malonate for all four outcomes. Conclusions The severity of cardiac I/R injury is lowest with glucose-only perfusate, and increased with increased complexity of the metabolic milieu. Although malonate is a strong protectant against cardiac IRI for most metabolic milieus, in the isolated heart, protection is lost in the presence of insulin.

Abstract 18459: A Cell Permeable Protein Kinase C Beta II Peptide Inhibitor Mitigates the Generation of Reactive Oxygen Species in Rat ex-vivo and Porcine in-vivo Ischemia-Reperfusion Injury

Introduction: Restoration of blood flow following myocardial infarction is necessary to salvage ischemic tissue, but reperfusion is known to cause myocardial ischemia/reperfusion injury (MIR). Cytokine induced translocation of protein kinase C beta II (PKCβII) during reperfusion (R) stimulates the production of reactive oxygen species (ROS). Dual conjugation of PKCβII peptide inhibitor (PKCβII-) with myristic acid (myr) and trans-activator of transcription (Tat) (myr-Tat-CC-SLNPEWNET [myr-Tat-PKCβII-]) enhances intracellular delivery and reduces phorbol ester-induced neutrophil ROS. We hypothesize that 20ng/kg of myr-Tat-PKCβII- would exert cardioprotective effects in porcine MIR in-vivo by reducing stimulation of ROS. Methods: Ex vivo: We tested 100nM - 10fmol myr-Tat-PKCβII- in ex-vivo rat MIR. Isolated hearts from anesthetized male SD rats underwent global I (30-min)/R(50-min). Myr-Tat-PKCβII- was given during first 5min of R. DP/dt max was measured via pressure transducer in the left ventricle. 1% TTC staining was used to determine infarct size. Data was analyzed via Fisher’s PLSD. In vivo: Regional I (1 hr)/R (3 hrs) was induced in male Yorkshire pigs. At R, myr-Tat-PKCβII- or scrambled control peptide (20ng/kg; ~100 pM blood volume [vol.] concentration [conc.]) was given by intra-arterial bolus. Ejection fraction (EF) was calculated. Infarct size was determined with Evans Blue dye and 1% TTC staining. Data was analyzed using Student’s t-test. Results: Ex-vivo: Myr-Tat-PKCβII- (100nM - 100fmol; 10-14±3%; n=3-6) significantly reduced infarct size compared to controls (21±3%; n=21; p<0.05). DP/dt max was significantly improved (100pM-1pM; 961±274; n=5-6) compared to 100nM (163±77, n=3; p<0.05), 100fM (481±86, n=5; p<0.05), and 100pMscrambled myr-Tat-PKCβII- (386±86, n=4; p<0.05). In-vivo: Myr-Tat-PKCβII- 20ng/kg (~100pM blood vol. conc.) significantly restored EF to within 1.4±0.7% of baseline and reduced infarct size to (10.0±2.8%; n=4) compared to control (6.4±2.1%; 28.5±8.3%; n=6; p<0.05). Conclusions: Results suggest 20ng/kg (~100pM blood vol. conc.)) should continue to be used to test in porcine MIR in-vivo. Future studies include a 12-week porcine survival study to evaluate infarct size and cardiac function.

FTO represses NLRP3-mediated pyroptosis and alleviates myocardial ischemia-reperfusion injury via inhibiting CBL-mediated ubiquitination and degradation of β-catenin.

Cardiac ischemia/reperfusion (I/R) injury is a complicated pathological event, which has close association with pyroptosis. This study uncovered the regulatory mechanisms of fat mass and obesity-associated protein (FTO) in NLRP3-mediated pyroptosis during cardiac I/R injury. H9c2 cells were stimulated with oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability and pyroptosis were detected by CCK-8 and flow cytometry. Western blotting or RT-qPCR was performed to analyze target molecule expression. NLRP3 and Caspase-1 expression was observed by immunofluorescence staining. IL-18 and IL-1β production was detected by ELISA. The total m6A and m6A level of CBL was determined by dot blot assay and methylated RNA immunoprecipitation-qPCR, respectively. The interaction between IGF2BP3 and CBL mRNA was confirmed by RNA pull-down and RIP assays. The protein interaction between CBL and β-catenin and β-catenin ubiquitination were evaluated by Co-IP. Myocardial I/R model was established in rats. We determined infarct size by TTC staining and pathological changes by H&E staining. LDH, CK-MB, LVFS, and LVEF were also assessed. FTO and β-catenin were down-regulated, while CBL was up-regulated by OGD/R stimulation. FTO/β-catenin overexpression or CBL silencing restrained OGD/R-induced NLRP3 inflammasome-mediated pyroptosis. CBL repressed β-catenin expression via ubiquitination and degradation. FTO reduced the mRNA stability of CBL by inhibiting m6A modification. CBL-mediated ubiquitination and degradation of β-catenin were involved in FTO-induced pyroptosis inhibition during myocardial I/R injury. FTO inhibits NLRP3-mediated pyroptosis to attenuate myocardial I/R injury via repressing CBL-induced ubiquitination degradation of β-catenin.

Naltrindole Pretreatment Elicits Potent Dose Dependent Effects on Reducing Infarct Size During Ischemia Reperfusion In-Vivo

Over one million individuals experience a myocardial infarction (MI) every year. Currently, no therapeutic is available to salvage necrotic cardiac tissue following an MI. The use of a prophylactic agent to reduce infarct size in patients at high risk for an MI would attenuate the development of heart failure. Interventional cardiologists state that a 25% reduction in infarct size is clinically meaningful to reduce heart failure. Previously, a 5 min pretreatment with high dose (HD) naltrindole (NTI) (7.5 mg/kg x 5 min = 37.5 mg/kg, n=3) resulted in a robust 74% (15±6%) reduction in infarct size compared to control in an in-vivo model of ischemia reperfusion injury (IRI). This effect was accompanied by a reduction in irreversible hypercontracture known as rigor. However, it caused a transient reduction in cardiac function during the infusion period. In this study, we aim to determine the effect of intermediate (ID; 3.75 mg/kg x 5 min = 18.75 mg/kg) and low dose (LD; 0.2-1.0 mg/kg x 5 min = 1.0-5.0 mg/kg) NTI pretreatment on infarct size and cardiac function in a rat in vivo model of IRI.Female Sprague-Dawley rats (~250g) were divided into four cohorts: non-treated control (n=15), LD (n=18), ID (n=5), and HD (n=5) NTI groups. All cohorts were anesthetized with Ketamine-xylazine (90 mg/kg, 9 mg/kg). Control rats received normal saline (0.9% NaCl), while treatment groups received either HD, ID, or LD NTI infused through the jugular vein (0.4mL/min) over 5 mins before inducing regional ischemia by occluding the left descending coronary artery for 30 min followed by 3 hr reperfusion. Cardiac function was measured through a pressure transducer catheter placed into the left ventricle (LV) in NTI treated groups. Planimetered photographs of heart slices were used to determine infarct size. Data were analyzed via ANOVA, Bonferroni-Dunn, and post-hoc analysis. Probability values <0.05 were considered statistically significant.HD, ID, and LD NTI reduced infarct size by 81% (11±4%) p<0.05, 55% (26±7%) p<0.05, and 4% (55±4%) respectively compared to control (57±2%). Cardiac function parameters such as dP/dt min, the maximal rate of LV pressure relaxation, was not significantly affected by ID NTI compared to LD NTI in contrast to the cardiac depression elicited by HD NTI. These results indicate that the cardiac depressive effects of NTI are dose dependent and the infarct reducing effects of NTI can be achieved through doses that spare cardiac function. Therefore, ID NTI prophylaxis may be an effective strategy to limit cardiac tissue death in populations at high risk of an MI. Future studies will be conducted on porcine cardiac IRI models. Funding was supported by Young Therapeutics LLC and internal grants from Philadelphia College Of Osteopathic Medicine This is the full abstract presented at the American Physiology Summit 2023 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.

Abstract 10387: Macrophage Migration Inhibitory Factor Maintains Mitochondrial Integrity in Aging During Ischemic Insults

Introduction: The expression and secretion of cardiac MIF (macrophage migration inhibitory factor) are downregulated in aging. A small molecule MIF agonist, MIF20 can increase the binding affinity of MIF with CD74 receptor to modulate AMPK signaling. However, the pharmacological mechanisms by which MIF20 triggers MIF-AMPK signaling cascade to benefit heart in response to ischemia and reperfusion (I/R) stress remain unknown. Hypothesis: MIF20 rescues an impaired MIF-AMPK signaling in aging through augmenting the interaction between MIF and CD74 receptor to maintain mitochondrial integrity of aged heart under ischemic stress. Methods: Young (3-5 months) and aged (21-24 months) C57BL/6J mice were subjected to 45 min of ligation of LAD (left anterior descending coronary artery) and injection of MIF20 (0.15 μg/kg, i.v.) 5 min before 24 hr of reperfusion with release of LAD. Echocardiography measures cardiac functions, and TTC/Evans blue staining for determination of myocardial infarction size. Seahorse XF Analyzer for mitochondrial respiration functions of isolated cardiomyocytes. Results: The echocardiography data showed that I/R caused cardiac systolic dysfunctions but not effect on cardiac diastolic functions of both young and aged C57BL/6J mice. There were no differences between male and female groups. Administration of MIF20 significantly ameliorated I/R-induced cardiac dysfunctions in both young and aged mice, while more improvement observed in aged versus young mice. MIF20 treatment reduced myocardial infarct size caused by I/R in both young and aged mice, but the reduction extent of aged hearts was higher than that of young hearts (p<0.05). Mitochondrial respiration rates of isolated cardiomyocytes as indicted by ATP production were impaired by I/R versus sham operations in both young and aged hearts. Intriguingly, MIF20 treatment significantly rescued the impaired mitochondrial functions caused by I/R in both young and aged cardiomyocytes. Moreover, the mitochondrial functions of young and aged cardiomyocytes were improved to a similar level under I/R with MIF20. Conclusions: MIF agonist MIF20 rescues the impaired energy metabolism in aged heart. The mitochondrial integrity under I/R by MIF20 could limit ischemic insults in aging.

Dexmedetomidine attenuates myocardial ischemia/reperfusion-induced ferroptosis via AMPK/GSK-3β/Nrf2 axis

The present study aimed to investigate whether dexmedetomidine (Dex) exerts cardioprotection effect through inhibiting ferroptosis. Myocardial ischemia/reperfusion injury (MIRI) was induced in Sprague-Dawley rats in Langendorff preparation. The hemodynamic parameters were recorded. Triphenyltetrazolium chloride (TTC) staining was used to determine infarct size. In the in vitro study, the model of hypoxia/reoxygenation (HR) was established in H9c2 cells. Cell viability and apoptosis were detected using cell counting kit 8 (CCK-8), and AV/PI dual staining respectively. Lipid peroxidation as measured by the fluorescence of the fatty acid analog C11-BODIPY581/591 probe and intracellular ferrous iron levels were measured by fluorescence of Phen Green SK (PGSK) probe, whereas immunofluorescence and transmission electron microscopy were also used to examine ferroptosis. Protein levels were investigated by Western blot. The interactions of AMPK/GSK-3β signaling with Nrf2 were also assessed through AMPK inhibition and GSK-3β overexpression. Our findings indicated that Dex significantly alleviated myocardial infarction, improved heart function, and decreased HR-induced accumulation of Fe2+ and lipid peroxidation in cardiomyocytes. Dex significantly increased the expression levels of Nrf2, SLC7A11, and GPX4. However, inhibition of Nrf2 by ML385 blunted the protective effect of Dex in HR-treated H9c2 cells. Inhibition of AMPK with a specific inhibitor or siRNA decreased the expression levels of phosphorylation of GSK-3β and Nrf2 induced by Dex. Overexpression of GSK-3β resulted in lower levels of nuclear Nrf2, whereas depression of GSK-3β enhanced expressions of nuclear Nrf2. In conclusion, Dex protects hearts against MIRI-induced ferroptosis via activation of Nrf2 through AMPK/GSK-3β signaling pathway.

Abstract P1048: Dual Myristic Acid And Trans Activator Of Transcription Conjugated Peptide Facilitates Delivery Of Protein Kinase C Beta II Peptide Inhibitor To Attenuate Myocardial Ischemia/Reperfusion Injury

Peptides conjugated to myristic acid (myr) or trans activator of transcription (tat) have long been used to increase cell permeability via simple diffusion and endocytosis, respectively. Previously myr-PKCβII inhibitor (myr-PKCβII-) exerted cardioprotective effects in myocardial ischemia/reperfusion (I/R) and inhibited superoxide (SO) release from rat leukocytes by 40% (20 μM). By contrast, dual conjugated myr-tat-PKCβII inhibitor (myr-tat-PKCβII-) inhibited SO release by 90% at the same concentration. The purpose of this study was to determine the potency of myr-tat-PKCβII- compared to myr-PKCβII- in mitigating infarct size and restoring cardiac function in an ex-vivo rat model of myocardial I/R injury. Isolated hearts from male Sprague-Dawley rats (~300g) were subjected to global I(30-min)/R(50-min) . Left ventricular cardiac function was recorded using a pressure transducer. Treatments were infused during the first 5 min of R. After R, the hearts were sectioned (2 mm) from base to apex and stained with 1% triphenyltetrazolium chloride. Infarcted tissue was excised to determine infarct size (i.e. infarct tissue weight/ total tissue weight). Data were analyzed using ANOVA Fisher’s LSD analysis. Compared to untreated controls (23.2±3.1%, n=17), myr-tat-PKCβII- exerted a significant and similar decrease in infarct size that was observed from 100 nM (9.7±0.59%, n=3, p<0.05), 1 nM (10.0±2.3%, n=5, p<0.05), to 100 pM (12.6±2.3%, n=5, p<0.05); but not at 10 nM (13.8±3.6%, n=5). Cardiac function for all treatment groups did not significantly improve from control. However, at 100pM (1070±170 mmHg/s), +dP/dt max was significantly improved compared to all other treatment groups (p<0.05). The addition of myr to tat-conjugated peptides improved the cardioprotective effects ~200-fold compared to myr-PKCβII- and ~2000-fold compared to native peptide presumably by enhanced intracellular delivery of cargo. The ~50% reduction in infarct size suggests that myr-tat-PKCβII- is cardioprotective in I/R. Future studies will test our most effective dose determined in ex vivo hearts to restore post-reperfusion cardiac function combined with significant cardiac salvaging effect (reduced infarct size) in a porcine myocardial I/R model.

Modulation of Mitochondrial Respiration During Early Reperfusion Reduces Cardiac Injury in Donation After Circulatory Death Hearts.

Donation after circulatory death (DCD) donors are a potential source for heart transplantation. The DCD process has unavoidable ischemia and reperfusion (I/R) injury, primarily mediated through mitochondria, which limits routine utilization of hearts for transplantation. Amobarbital (AMO), a transient inhibitor of the electron transport chain, is known to decrease cardiac injury following ex vivo I/R. We studied whether AMO treatment during reperfusion can decrease injury in DCD hearts. Sprague Dawley rat hearts subjected to 25 minutes of in vivo ischemia (DCD hearts), or control beating donor hearts, were treated with AMO or vehicle for the first 5 minutes of reperfusion, followed by Krebs-Henseleit buffer reperfusion for 55 minutes (for mitochondrial isolation) or 85 minutes (for infarct size determination). Compared with vehicle, AMO treatment led to decreased infarct size (25.2% ± 1.5% vs. 31.5% ± 1.5%; P ≤ 0.05) and troponin I release (4.5 ± 0.05 ng/mL vs. 9.3 ± 0.24 ng/mL, P ≤ 0.05). AMO treatment decreased H 2 O 2 generation with glutamate as complex I substrate in both subsarcolemmal mitochondria (SSM) (37 ± 3.7 pmol·mg -1 ·min -1 vs. 56.9 ± 4.1 pmol·mg -1 ·min -1 ; P ≤ 0.05), and interfibrillar mitochondria (IFM) (31.8 ± 2.8 pmol·mg -1 ·min -1 vs. 46 ± 4.8 pmol·mg -1 ·min -1 ; P ≤ 0.05) and improved calcium retention capacity in SSM (360 ±17.2 nmol/mg vs. 277 ± 13 nmol/mg; P ≤ 0.05), and IFM (483 ± 20 nmol/mg vs. 377± 19 nmol/mg; P ≤ 0.05) compared with vehicle treatment. SSM and IFM retained more cytochrome c with AMO treatment compared with vehicle. In conclusion, brief inhibition of mitochondrial respiration during reperfusion using amobarbital is a promising approach to decrease injury in DCD hearts.

The temporal impact of erythropoietin administration on mitochondrial function and dynamics in cardiac ischemia/reperfusion injury

Erythropoietin (EPO) has been shown to provide protection against ischemia/reperfusion (I/R) injury in various experimental models. However, clinical trials revealed unsatisfactory results when EPO was given to patients with myocardial infarction following reperfusion. The timing of EPO administration and its relation to mitochondrial function may largely involve in this controversy. We hypothesized that EPO given at different time points exert varying cardioprotective effects in terms of myocardial infarct size, left ventricular (LV) function, arrhythmia, apoptosis, mitochondrial function, and mitochondrial dynamics in rats with cardiac I/R injury. Male Wistar rats were subjected to either sham (n = 6) or cardiac I/R operation (n = 48). Rats undergoing cardiac I/R operation (30-min ischemia, followed by 120-min reperfusion) were allotted into 4 subgroups (n = 12/group): vehicle, EPO pretreatment, EPO given during ischemia, and EPO given at reperfusion. EPO was administered intravenously at 5000 unit/kg. Arrhythmia and LV function were monitored throughout the protocol. Next, the hearts were collected to determine infarct size, mitochondrial function, mitochondrial dynamics, gap junction protein, and apoptosis. Cardiac I/R promoted arrhythmias, LV dysfunction, infarct size expansion, apoptosis, mitochondrial dysfunction and increased mitochondrial fission. EPO given either before or during ischemia, but not at reperfusion, attenuated arrhythmia scores, LV dysfunction, infarct size, and apoptosis. Only EPO given either before or during ischemia alleviated mitochondrial swelling, mitochondrial depolarization, and reduced the levels of p-Drp1ser616/Drp1. Data from in vitro study also confirmed that EPO directly attenuated mitochondrial dysfunction in H9c2 cells subjected to hypoxia/reoxygenation. In conclusion, the EPO administration, either before or during ischemia, exerted cardioprotection against I/R injury by attenuating mitochondrial dynamic imbalance, mitochondrial dysfunction, and apoptosis, leading to reduced infarct size and improved LV function.

Clinical risk factors associated with ventricular fibrillation during first ST-elevation myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union’s Horizon 2020 research and innovation programme under acronym ESCAPE-NET, registered under grant agreement No 733381 Introduction Sudden cardiac death (SCD) remains a major public health issue. Most cases in the general population are caused by ischemic heart disease, and often occur in patients without known ischemic heart disease. The assessment of risk factors may point to novel causal pathways or new targets for intervention and risk prediction of SCD. Objective The aim of this study was to evaluate the effect of family history of sudden death, prior history of atrial fibrillation (AF), and anterior infarct location on ECG on the development of ventricular fibrillation (VF) during first ST-elevation myocardial infarction (STEMI). Methods We performed individual participant data meta-analyses of three European case-control studies including first STEMI patients (aged 18-80 years) with VF (cases) and without VF (controls) before revascularization (GEVAMI, AGNES, and PREDESTINATION). Analyses were done using fixed-effect, inverse variance weighted meta-analysis and multivariable logistic regression. Potential confounding variables were identified using causal diagrams and missing data were handled with multiple imputation for each cohort separately. Results We included 1664 cases and 2497 controls (median age (IQR) = 59 (51-67) years, 20% females) in the analyses. After adjusting for potential confounding, we found an independent and additive association between the three exposures and VF (see picture): for family history of sudden death odds ratio (OR) 1.59 (95% confidence interval: 1.37-1.85), for AF OR 2.41 (1.49-3.89), and for anterior myocardial infarction OR 1.50 (1.32-1.71). Further investigation indicated increased effect of family history with multiple sudden deaths in the family, a stronger effect of AF on VF developing within the first minutes of symptoms, and the effect of anterior infarctions being modified by enzymatically determined infarct size. The three risk factors showed an additive effect: with one factor present OR 1.59 (1.38-1.84), two factors OR 2.41 (1.95-2.99), and all three factors OR 5.49 (1.43-21.1). Complete case analysis gave similar results for all analyses. Conclusions Family history of sudden death, history of AF, and anterior infarct location with significant interaction with enzymatic infarct size were all independently and additively associated with an increased risk of VF in patients with a first STEMI.

Cardioprotective effects of redox regulator MnTnBuOE‐2‐PyP 5+ in myocardial ischemia/reperfusion injury

BackgroundMyocardial infarction is a leading cause of death worldwide and occurs due to decrease or complete blockage of coronary blood flow. Re‐establishment of blood flow after a brief period of ischemia is accompanied by exacerbation of the cardiomyocytes and its death, a phenomenon known as myocardial ischemia‐reperfusion (I/R) injury. Much evidence suggests that oxidative stress is involved in the pathogenesis of myocardial ischemia‐reperfusion injury and is potential target for therapeutic interventions. The aim of this study is to evaluate cardioprotective role of redox active Mn(III) N‐n‐butoxyethylpyridinium‐2‐yl)porphyrin, MnTnBuOE‐2‐PyP5+ (BMX‐001) in myocardial I/R injury. This drug is now in 4 Phase II clinical trials where the radioprotection of normal issue and anticancer efficacy have been evaluated.MethodsMyocardial I/R injury in mice C57BL/6 was induced by ligating left anterior descending artery for 1 hour followed by 24 hours of reperfusion. At the end of reperfusion, hearts were sectioned (1mm) and stained with 1% 2, 3, 5‐Triphenyltetrazolium chloride (TTC) to determine infarct size. Cardiac function was evaluated by M‐mode echocardiography at the end of the reperfusion. For in vitro model, H9c2 cells were subjected to hypoxia for 3 hours followed by 24 hours of reperfusion. Cell apoptosis was measured by TUNEL assay. Mitochondrial superoxide and Cardiolipin peroxidation were measured by using MitoSOX and 10‐N‐nonyl‐Acridin Orange, respectively.ResultsIn the current study, we demonstrated that treatment with MnTnBuOE‐2‐PyP5+ (2mg/kg) 4 hours before I/R injury in mice ameliorate cardiac injury as evident by limited infarct size and improved cardiac function (P<0.05). Consistently, MnTnBuOE‐2‐PyP5+ improved mitochondrial SOD2 expression (P<0.05) and reduced cardiomyocytes apoptosis in mice heart (P<0.05). In our in vitro model of hypoxia/reoxygenation injury in H9c2 cells, treatment with MnTnBuOE‐2‐PyP5+ (10µM) 24 hours before hypoxia/ reoxygenation injury decreased cardiolipin peroxidation, mitochondrial superoxide level and 4‐HNE adducted proteins (P<0.01). Additionally we explored role of MnTnBuOE‐2‐PyP5+ on cell apoptosis following ischemia/reperfusion injury in H9c2 cells. Redox regulator significantly reduced TUNEL positive cells following I/R injury indicating reduced cardiomyocytes apoptosis (p<0.05). In‐vitro, redox‐active Mn porphyrin improved cell viability and lactate dehydrogenase release following hypoxia/reoxygenation in H9c2 cells (p<0.05).ConclusionThis result provided evidence that MnTnBuOE‐2‐PyP5+ elicited cytoprotective effects through reduction of mitochondrial oxidative stress and cardiomyocyte apoptosis suggesting prospective therapeutics in myocardial I/R Injury.

Role of Calcium Homeostasis in Ischemic Stroke: A Review.

Stroke is the second most common cause of death worldwide. It occurs due to the insufficient supply of oxygen-rich blood to the brain. It is a complex disease with multiple associated risk factors, including smoking, alcoholism, age, sex, ethnicity, etc. Calcium ions are known to play a vital role in cell death pathways, which is a ubiquitous intracellular messenger during and immediately after an ischemic period. Disruption in normal calcium homeostasis is known to be a major initiator and activator of the ischemic cell death pathway. Under ischemic stroke conditions, glutamate is released from the neurons and glia, which further activates the N-methyl-D-aspartate (NMDA) receptor and triggers the rapid translocation of Ca2+ from extracellular to intracellular spaces in cerebral tissues and vice versa. Various studies indicated that Ca2+ could have harmful effects on neurons under acute ischemic conditions. Mitochondrial dysfunction also contributes to delayed neuronal death, and it was established decades ago that massive calcium accumulation triggers mitochondrial damage. Elevated Ca2+ levels cause mitochondria to swell and release their contents. As a result, oxidative stress and mitochondrial calcium accumulation activate mitochondrial permeability transition and lead to depolarization-coupled production of reactive oxygen species. This association between calcium levels and mitochondrial death suggests that elevated calcium levels might have a role in the neurological outcome in ischemic stroke. Previous studies have also reported that elevated Ca2+ levels play a role in the determination of infarct size, outcome, and recurrence of ischemic stroke. The current review has been compiled to understand the multidimensional role of altered Ca2+ levels in the initiation and alteration of neuronal death after an ischemic attack. The underlying mechanisms understood to date have also been discussed.

Mediation of the Cardioprotective Effects of Mannitol Discovered, with Refutation of Common Protein Kinases.

The osmodiuretic agent Mannitol exerts cardioprotection against ischemia and reperfusion (I/R) injury when applied as a pre- and/or postconditioning stimulus. Previously, we demonstrated that these properties are mediated via the activation of mitochondrial ATP-sensitive potassium (mKATP) channels. However, considering Mannitol remains in the extracellular compartment, the question arises as to which receptor and intracellular signaling cascades are involved in myocardial protection by the osmodiuretic substance. Protein kinase B (Akt) and G (PKG), as part of the reperfusion injury salvage kinase (RISK) and/or endothelial nitric oxide (eNOS)/PKG pathway, are two well-investigated intracellular targets conferring myocardial protection upstream of mitochondrial potassium channels. Adenosine receptor subtypes have been shown to trigger different cardioprotective pathways, for example, the reperfusion injury. Further, Mannitol induces an increased activation of the adenosine 1 receptor (A1R) in renal cells conferring its nephroprotective properties. Therefore, we investigated whether (1) Akt and PKG are possible signaling targets involved in Mannitol-induced conditioning upstream of the mKATP channel and/or whether (2) cardioprotection by Mannitol is mediated via activation of the A1R. All experiments were performed on male Wistar rats in vitro employing the Langendorff isolated heart perfusion technique with infarct size determination as the primary endpoint. To unravel possible protein kinase activation, Mannitol was applied in combination with the Akt (MK2206) or PKG (KT5823) inhibitor. In further groups, an A1R blocker (DPCPX) was given with or without Mannitol. Preconditioning with Mannitol (Man) significantly reduced the infarct size compared to the control group. Co-administration of the A1R blocker DPXPC fully abolished myocardial protection of Mannitol. Interestingly and in contrast to the initial hypothesis, neither administration of the Akt nor the PKG blocker had any impact on the cardioprotective properties of Mannitol-induced preconditioning. These results are quite unexpected and show that the protein kinases Akt and PKG—as possible targets of known protective signaling cascades—are not involved in Mannitol-induced preconditioning. However, the cardioprotective effects of Mannitol are mediated via the A1R.

Innate Lymphoid Cells Promote Recovery of Ventricular Function After Myocardial Infarction

BackgroundInnate lymphoid cells type 2 (ILC2s) play critical homeostatic functions in peripheral tissues. ILC2s reside in perivascular niches and limit atherosclerosis development. ObjectivesILC2s also reside in the pericardium but their role in postischemic injury is unknown. MethodsWe examined the role of ILC2 in a mouse model of myocardial infarction (MI), and compared mice with or without genetic deletion of ILC2. We determined infarct size using histology and heart function using echocardiography. We assessed cardiac ILC2 using flow cytometry and RNA sequencing. Based on these data, we devised a therapeutic strategy to activate ILC2 in mice with acute MI, using exogenous interleukin (IL)-2. We also assessed the ability of low-dose IL-2 to activate ILC2 in a double-blind randomized clinical trial of patients with acute coronary syndromes (ACS). ResultsWe found that ILC2 levels were increased in pericardial adipose tissue after experimental MI, and genetic ablation of ILC2 impeded the recovery of heart function. RNA sequencing revealed distinct transcript signatures in ILC2, and pointed to IL-2 axis as a major upstream regulator. Treatment of T-cell–deficient mice with IL-2 (to activate ILC2) significantly improved the recovery of heart function post-MI. Administration of low-dose IL-2 to patients with ACS led to activation of circulating ILC2, with significant increase in circulating IL-5, a prototypic ILC2-derived cytokine. ConclusionsILC2s promote cardiac healing and improve the recovery of heart function after MI in mice. Activation of ILC2 using low-dose IL-2 could be a novel therapeutic strategy to promote a reparative response after MI.

Inhibition of serum and glucocorticoid regulated kinases by GSK650394 reduced infarct size in early cerebral ischemia-reperfusion with decreased BBB disruption

Blood-brain barrier (BBB) disruption is one of the most important pathological changes following cerebral ischemia-reperfusion. We tested whether inhibition of the serum and glucocorticoid regulated kinase 1 (SGK1) would decrease BBB disruption and contribute to decreasing infarct size in the first few hours of cerebral ischemia-reperfusion within the thrombolysis therapy time window. After transient middle cerebral artery occlusion (MCAO), an SGK1 inhibitor GSK650394, or vehicle was administered into the lateral ventricle of rats. After one hour of MCAO and two hours of reperfusion, we determined BBB disruption using the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid, and also determined infarct size, phosphorylation of NDRG1, and MMP2 protein level. Ischemia-reperfusion increased (+34%, p < 0.05) and GSK650394 decreased (−25%, p < 0.05) the Ki in the ischemic-reperfused cortex. GSK650394 decreased the percentage of cortical infarct (−31%, p < 0.001). At the same time GSK650394 reduced NDRG1 phosphorylation and MMP2 protein level in the ischemic-reperfused cortex suggesting that SGK1 was inhibited by GSK650394 and that lower MMP2 could be one of the mechanisms of decreased BBB disruption. Collectively our data suggest that GSK650394 could be neuroprotective and one of the mechanisms of the neuroprotection could be decreased BBB disruption. SGK1 inhibition within the thrombolysis therapy time window might reduce cerebral ischemia-reperfusion injury.

Left ventricular function, strain, and infarct characteristics in patients with transient ST-segment elevation myocardial infarction compared to ST-segment and non-ST-segment elevation myocardial infarctions.

AimsThis study aims to explore cardiovascular magnetic resonance (CMR)-derived left ventricular (LV) function, strain, and infarct size characteristics in patients with transient ST-segment elevation myocardial infarction (TSTEMI) compared to patients with ST-segment and non-ST-segment elevation myocardial infarctions (STEMI and NSTEMI, respectively).Methods and resultsIn total, 407 patients were enrolled in this multicentre observational prospective cohort study. All patients underwent CMR examination 2–8 days after the index event. CMR cine imaging was performed for functional assessment and late gadolinium enhancement to determine infarct size and identify microvascular obstruction (MVO). TSTEMI patients demonstrated the highest LV ejection fraction and the most preserved global LV strain (longitudinal, circumferential, and radial) across the three groups (overall P ≤ 0.001). The CMR-defined infarction was less frequently observed in TSTEMI than in STEMI patients [77 (65%) vs. 124 (98%), P < 0.001] but was comparable with NSTEMI patients [77 (65%) vs. 66 (70%), P = 0.44]. A remarkably smaller infarct size was seen in TSTEMI compared to STEMI patients [1.4 g (0.0–3.9) vs. 13.5 g (5.3–26.8), P < 0.001], whereas infarct size was not significantly different from that in NSTEMI patients [1.4 g (0.0–3.9) vs. 2.1 g (0.0–8.6), P = 0.06]. Whilst the presence of MVO was less frequent in TSTEMI compared to STEMI patients [5 (4%) vs. 53 (31%), P < 0.001], no significant difference was seen compared to NSTEMI patients [5 (4%) vs. 5 (5%), P = 0.72].ConclusionTSTEMI yielded favourable cardiac LV function, strain, and infarct-related scar mass compared to STEMI and NSTEMI. LV function and infarct characteristics of TSTEMI tend to be more similar to NSTEMI than STEMI.

Effects of three types of bariatric interventions on myocardial infarct size and vascular function in rats with type 2 diabetes mellitus

AimsThe effects of three types of bariatric interventions on myocardial infarct size were tested in the rat model of type 2 diabetes mellitus (T2DM). We also evaluated the effects of bariatric surgery on no-reflow phenomenon and vascular dysfunction caused by T2DM. Main methodsRats with T2DM were assigned into groups: without surgery, sham-operated, ileal transposition, Roux-en-Y gastric bypass, and sleeve gastrectomy. Oral glucose tolerance, glucagon-like peptide-1, and insulin levels were measured. Six weeks after surgery, the animals were subjected to myocardial ischemia-reperfusion followed by histochemical determination of infarct size (IS), no-reflow zone, and blood stasis area size. Vascular dysfunction was characterized using wire myography. Key findingsAll bariatric surgery types caused significant reductions in animal body weight and resulted in T2DM compensation. All bariatric interventions partially normalized glucagon-like peptide-1 responses attenuated by T2DM. IS was significantly smaller in animals with T2DM. Bariatric surgery provided no additional IS limitation compared with T2DM alone. Bariatric surgeries reversed T2DM-induced enhanced contractile responses of the mesenteric artery to 5-hydroxytryptamine. Sleeve gastrectomy normalized decreased nitric oxide synthase contribution to the endothelium-dependent vasodilatation in T2DM. SignificanceT2DM resulted in a reduction of infarct size and no-reflow zone size. Bariatric surgery provided no additional infarct-limiting effect, but it normalized T2DM-induced augmented vascular contractility and reversed decreased contribution of nitric oxide to endothelium-dependent vasodilatation typical of T2DM. All taken together, we suggest that this type of surgery may have a beneficial effect on T2DM-induced cardiovascular diseases.