Measurement Of Infarct Size Research Articles

L-Citrulline Supplementation Reduces Blood Pressure and Myocardial Infarct Size under Chronic Intermittent Hypoxia, a Major Feature of Sleep Apnea Syndrome.

Intermittent hypoxia (IH) is a landmark of obstructive sleep apnea (OSA) at the core of the cardiovascular consequences of OSA. IH triggers oxidative stress, a major underlying mechanism for elevated blood pressure (BP) and increased infarct size. L-citrulline is an amino acid that has been demonstrated to be protective of the cardiovascular system and exert pleiotropic effects. Therefore, we tested the impact of citrulline supplementation on IH-induced increase in BP and infarct size. Four groups of rats exposed to normoxia (N) or IH [14 days (d), 8 h/day, 30 s-O2 21%/30 s-O2 5%] and were supplemented or not with citrulline (1 g·kg-1·d-1). After 14 d, BP was measured, and hearts were submitted to global ischemia-reperfusion to measure infarct size. Histological and biochemical analyses were conducted on hearts and aorta to assess oxidative stress. Citrulline significantly reduced BP (-9.92%) and infarct size (-18.22%) under IH only. In the aorta, citrulline supplementation significantly decreased superoxide anion and nitrotyrosine levels under IH and abolished the IH-induced decrease in nitrite. Citrulline supplementation significantly decreased myocardial superoxide anion levels and xanthine oxidase enzyme activity under IH. Citrulline shows a cardioprotective capacity by limiting IH-induced pro-oxidant activity. Our results suggest that citrulline might represent a new pharmacological strategy in OSA patients with high cardiovascular risk.

Abstract 11206: Adipose-Derived Mesenchymal Stem Cells Overexpressing PD-L1 and Akt Confer Myocardial Protection Through Upregulation of CD25 + T Cells in a Rat Model of Acute Myocardial Infarction

Background: Translational studies showed positive results of stem cell therapy against myocardial infarction (MI)-induced cardiac dysfunction. Programmed death ligand 1 (PD-L1) is a key immune receptor, which modulates immune system and maintain the stability of coronary plaques. Akt is one of the signaling that exert its cardioprotective effect through regulating PD-L1. In the present study, we overexpressed PD-L1 and Akt in adipose-derived mesenchymal stem cells (AdMSC) and determined the protection against MI. Methods and Results: Adult Wistar rats were randomly separated into four groups: sham, MI, treatment of AdMSC or AdMSC overexpressed with PD-L1 and Akt (AdMSC-PDL1-Akt) immediately after MI. MI was induced by LAD ligation and ADMSC were injected into the heart around the area at risk. After 4 weeks, rats were examined by echocardiography, pressure-volume analysis, infarct size measurement, and immunohistochemistry to evaluate the efficacy of the ADMSCs on myocardium. Results demonstrated AdMSC-PDL1-Akt was more resistant to ROS in vitro , and could restore MI-induced contractile dysfunction in vivo by increasing ESPVR (P=0.048) and PRSW (P=0.015). AdMSC-PDL1-Akt could also protect hearts from MI-increased infarct size (P=0.002). Immunohistochemistry staining with caspase 3 and NFκB were upregulated in MI hearts and significantly reversed in AdMSC-PDL1-Akt group (P<0.001 and P=0.036). To clarify mechanisms underlying the effect of AdMSC-PDL1-Akt, we found it could induce normal T cells into CD25 + regulatory T cells in vitro , which is in parallel with the data shown in our in vivo immunohistochemistry study that CD25 was upregulated in AdMSC-PDL1-Akt tissues (P=0.03). Conclusions: Overexpression of PD-L1 and Akt in AdMSC could increase its resistance to ROS-related apoptosis in vitro and enhance the protective effects against MI-induced cardiac dysfunction through modulation of T cells in vivo . Keywords: AdMSC; PD-L1; myocardial infarction; regulatory T cells

Abstract 10786: Cardiac Muscle Patches Containing Four Types of Cardiac Cells Derived From Human Pluripotent Stem Cells Improve Recovery From Cardiac Injury

Introduction: We have shown that human cardiac muscle patches (hCMPs) containing three different types of cardiac cells—cardiomyocytes (CMs), smooth-muscle cells (SMCs), and endothelial cells (ECs), all of which were differentiated from human pluripotent stem cells (hPSCs)—significantly improved cardiac function, infarct size, and hypertrophy in a pig model of myocardial infarction (MI). However, hPSC-CMs are phenotypically immature, which may lead to arrythymogenic concerns; thus, since hPSC-derived cardiac fibroblasts (hPSC-CFs) appear to enhance the maturity of hPSC-CMs, we compared hCMPs containing hPSC-CMs, -SMCs, -ECs, and -CFs (4TCC-hCMPs) with a second hCMP construct that lacked hPSC-CFs but was otherwise identical (3TCC-hCMPs). Methods: hCMPs were generated in a fibrin scaffold. MI was induced in SCID mice through permanent coronary artery (LAD) ligation, followed by treatment with cardiac muscle patches. Animal groups included: MI heart treated with 3TCC-hCMP; with 4TCC-hCMP; MI heart treated with no patch (MI group) and sham group. Cardiac function was evaluated via echocardiography, and cell engraftment rate while infarct size was evaluated histologically at four weeks after patch transplantation. Results: The results from experiments in cultured hCMPs demonstrate: 1) that structural, metabolic, and ion-channel markers for CM maturation were significantly more abundant in 4TCC-hCMPs; and 2) that conduction velocities and action-potential durations were significantly greater in hPSC-CMs from 4TCC-hCMPs (18 cm/s ± 0.72 vs. 31 cm/s ± 3.23, p<0.005, 3TCC vs. 4TCC). Furthermore, when 3TCC- and 4TCC-hCMPs were compared in a mouse MI model, measures of cardiac function ( EF: 41.55% ± 0.92 vs. 49.18% ± 0.86, p<0.05, 3TCC vs. 4TCC), infarct size (39.23% ± 4.28 vs. 22.72% ± 0.98, p<0.05, 3TCC vs. 4TCC), hypertrophy, and CM engraftment were significantly better in animals treated with 4TCC-hCMPs than in the 3TCC-hCMP-treatment group (8.56% ± 1.79 vs. 15.99% ± 1.56, p<0.05, 3TCC vs. 4TCC). Conclusions: These results demonstrate that the inclusion of hPSC-CFs in hCMP manufacture promotes hPSC-CM maturation and increases the efficacy of hCMPs for myocardial repair.

Cardioprotection by Hypothyroidism Is Not Mediated by Favorable Hemodynamics-Role of Canonical Thyroid Hormone Receptor Alpha Signaling.

Hypothyroidism has been shown to reduce infarct size in rats, but the underlying mechanisms are unclear. We used isolated pressure-constant perfused hearts of control, hypothyroid and hyperthyroid mice and measured infarct size, functional parameters and phosphorylation of key molecules in cardioprotective signaling with matched heart rate. Compared with controls, hypothyroidism was cardioprotective, while hyperthyroidism was detrimental with enlarged infarct size. Next, we asked how thyroid hormone receptor α (TRα) affects ischemia/reperfusion (IR) injury. Thus, canonical and noncanonical TRα signaling was investigated in the hearts of (i) mice lacking TRα (TRα0), (ii) with a mutation in TRα DNA-binding domain (TRαGS) and (iii) in hyperthyroid TRα0 (TRα0hyper) and TRαGS mice (TRαGShyper). TRα0 mouse hearts were protected against IR injury. Furthermore, infarct size was reduced in the hearts of TRαGS mice that lack canonical TRα signaling but maintain noncanonical TRα action. Hyperthyroidism did not increase infarct size in TRα0 and TRαGS mouse hearts. These cardioprotective effects were not associated with increased phosphorylation of key proteins of RISK, SAFE and eNOS pathways. In summary, chronic hypothyroidism and the lack of canonical TRα signaling are cardioprotective in IR injury and protection is not due to favorable changes in hemodynamics.

GPR39 Knockout Worsens Microcirculatory Response to Experimental Stroke in a Sex-Dependent Manner.

No current treatments target microvascular reperfusion after stroke, which can contribute to poor outcomes even after successful clot retrieval. The G protein-coupled receptor GPR39 is expressed in brain peri-capillary pericytes, and has been implicated in microvascular regulation, but its role in stroke is unknown. We tested the hypothesis that GPR39 plays a protective role after stroke, in part due to preservation of microvascular perfusion. We generated GPR39 knockout (KO) mice and tested whether GPR39 gene deletion worsens capillary blood flow and exacerbates brain injury and functional deficit after focal cerebral ischemia. Stroke was induced in male and female GPR39 KO and WT littermates by 60-min middle cerebral artery occlusion (MCAO). Microvascular perfusion was assessed via capillary red blood cell (RBC) flux in deep cortical layers in vivo using optical microangiography (OMAG). Brain injury was assessed by measuring infarct size by 2,3,5-triphenyltetrazolium chloride staining at 24h or brain atrophy at 3weeks after ischemia. Pole and cylinder behavior tests were conducted to assess neurological function deficit at 1 and 3weeks post-stroke. Male but not female GPR39 KO mice exhibited larger infarcts and lower capillary RBC flux than WT controls after stroke. Male GPR39 KO mice also exhibited worse neurologic deficit at 1week post-stroke, though functional deficit disappeared in both groups by 3weeks. GPR39 deletion worsens brain injury, microvascular perfusion, and neurological function after experimental stroke. Results indicate that GPR39 plays a sex-dependent role in re-establishing microvascular flow and limiting ischemic brain damage after stroke.

Prognostic significance of myocardial salvage assessed by cardiac magnetic resonance in reperfused ST-segment elevation myocardial infarction.

AimsMyocardial salvage index (MSI) is attracting increasing attention for predicting prognosis in acute myocardial infarction (AMI); however, the evaluation of MSI is mainly based on contrast agent-dependent cardiac magnetic resonance (CMR) scanning sequences. This study aims to investigate the prognostic value of MSI in reperfused ST-segment elevation myocardial infarction (STEMI) through the contrast agent-free CMR technique.Methods and resultsNighty-two patients with acute STEMI, who underwent CMR after primary percutaneous coronary intervention (PPCI), were finally enrolled. Patients were subcategorized into two groups according to median MSI. T1 and T2 mapping were conducted for measuring infarct size (IS) and area at risk (AAR). IS was significantly larger in < median MSI group than ≥ median MSI group (P < 0.001). AAR between the two groups showed no obvious differences (P = 0.108). Left ventricular ejection fraction (LVEF) was lower in < median MSI group than ≥ median MSI group (P = 0.014). There was an obvious inverse correlation between MSI and reperfusion time (R = –0.440, P < 0.001) and a strong inverse correlation between MSI and IS (R = –0.716, P = 0.011). As for the relationship LVEF, MSI showed positive but weak correlation (R = 0.2265, P < 0.001). Over a median follow-up period of 263 (227–238) days, prevalence of MACEs was significantly higher in the < median MSI group [HR: 0.15 (0.04–0.62); Log-rank P = 0.008]. The univariate Cox regression analysis revealed that LVEF, IS, and MSI were significant predictors for major adverse cardiovascular events (MACEs) (all P < 0.05). In the stepwise multivariate Cox regression analysis, LVEF and MSI were identified as independent parameters for predicting MACEs (both P < 0.05). In the receiver-operating characteristic analysis, LVEF, IS, and MSI showed prognostic value in predicting MACEs with AUCs of 0.809, 0.779, and 0.896, respectively, all (P < 0.05). A combination of MSI with LVEF showed the strongest prognostic value of MACEs (AUC: 0.901, sensitivity: 77.78%, specificity: 98.80%, P < 0.001). Delong’s test showed that the combination of LVEF with MSI had an incremental value than LVEF itself in predicting MACEs (P = 0.026).ConclusionContrast agent-free CMR technique provides a reliable evaluation of MSI, which contributes to assessing the efficacy of reperfusion therapy and predicting the occurrence of MACEs.

Abstract P2076: β-hydroxybutyrate Administered At Reperfusion Alleviates Myocardial Ischemia-reperfusion Injury By Enhancing Autophagy And Mitochondrial Homeostasis

Introduction: ST-elevation myocardial infarction (STEMI) is the leading cause of heart failure. Ischemia/reperfusion (I/R) injury after revascularization contributes ~50% of infarct size, which we don’t have a clinical therapy. β-hydroxybutyrate (β-OHB) is a metabolite that increases during stress. In addition to providing energy, it serves as a signaling molecule (such as histone deacetylase inhibitor) to regulate many protective processes. Increasing the β-OHB level before I/R is cardiac protective. However, it has not been tested when administered at reperfusion. Hypothesis: The physiological concentration of β-OHB administered at the time of reperfusion reduces I/R injury by enhancing autophagy and mitochondrial homeostasis. Methods: C57BL6 wild-type mice were randomized into 2 groups and subjected to I/R surgery (I: 45 mins; R: 24 hrs). At reperfusion, normal saline or 10 mmol/kg of β-OHB (4-6 mM blood level) was given intraperitoneally. Echo was used to evaluate cardiac functions. We measured Infarct size, autophagic flux, and mitochondrial DNA levels in the heart. CAG-RFP-GFP-LC3 (RFLC3) mice were used to detect autophagic flux. Neonatal rat ventricular myocytes (NRVMs) were subjected to simulated I/R to test the effect of β-OHB in vitro . Small interfering RNA of ATG7 gene was used to assess the role of autophagy. Seahorse assay was performed to evaluate mitochondrial function after I/R. Results: β-OHB gave at reperfusion reduced infarct size by 50% (N=15 in control and N=11 in β-OHB group), and preserved systolic function in mice. In the border zone, the LC3II and the mtDNA level increased, and β-OHB increased autophagic flux in RFLC3 mice (N=5). In NRVMs subjected to I/R, β-OHB increased histone acetylation, cell viability, and maintained mitochondrial homeostasis (mitochondrial mass, membrane potential), and reduced ROS generation (N=4-6). Seahorse assay showed β-OHB enhanced ATP production after I/R. ATG7 knockdown blocked the protective effect of β-OHB in NRVMs. Conclusions: β-OHB administered at reperfusion reduces infarct size by increasing autophagic flux and maintaining mitochondrial homeostasis. Since β-OHB has been used in heart failure patients safely, it is a viable therapeutics to reduce infarct size in STEMI patients.

Left Ventricular Remodeling After Anterior-STEMI PCI: Imaging Observations in the Door-to-Unload (DTU) Pilot Trial.

To determine the predictive value of cardiac magnetic resonance (CMR) and echocardiographic parameters on left ventricular (LV) remodeling in ST-segment elevation myocardial infarction (STEMI) patients without cardiogenic shock and treated with mechanical LV unloading followed by immediate or delayed percutaneous coronary intervention (PCI)-mediated reperfusion. In STEMI, infarct size (IS) directly correlates with major cardiovascular outcomes. Preclinical models demonstrate mechanical LV unloading before reperfusion reduces IS. The door-to-unload (DTU)-STEMI pilot trial evaluated the safety and feasibility of LV unloading and delayed reperfusion in patients with STEMI. This multicenter, prospective, randomized, safety and feasibility trial evaluated patients with anterior STEMI randomized 1:1 to LV unloading with the Impella CP (Abiomed) followed by immediate reperfusion vs delayed reperfusion after 30 minutes of unloading. Patients were assessed by CMR at 3-5 days and 30 days post PCI. Echocardiographic evaluations were performed at 3-5 and 90 days post PCI. At 3-5 days post PCI, patients were compared based on IS as percentage of LV mass (group 1 ≤25%, group 2 >25%). Selection of IS threshold was performed post hoc. Fifty patients were enrolled from April 2017 to May 2018. At 90 days, group 1 (IS ≤25%) exhibited improved LV ejection fraction (from 53.1% to 58.9%; P=.001) and group 2 (IS >25%) demonstrated no improvement (from 37.6% to 39.1%; P=.55). LV end-diastolic volume and end-systolic volume were unchanged in group 1 and worsened in group 2. There was correlation between 3-5 day and 30-day CMR measurements of IS and 90-day echocardiography-derived LV ejection fraction. Immediate 3-5 day post-therapy IS by CMR correlates with 90-day echocardiographic LVEF and indices of remodeling. Patients with post-therapy IS >25% demonstrated evidence of adverse remodeling. Larger studies are needed to corroborate these findings with implications on patient management and prognosis.

Curcumin attenuates myocardial ischemia and reperfusion-induced proinflammatory response through activation of the Nrf-2/HO-1 signaling pathway

Acute myocardial infarction (MI) is a global issue with high incidence and mortality. Rapid recovery of the blocked coronary flow represents the most effective strategy to reduce the size of myocardial infarction and improve the cardiac function. The aim of the study is to investigate the effects of curcumin in attenuates myocardial ischemia and reperfusion-induced proinflammatory response through activation of the Nrf-2/HO-1 signaling pathway. Wild‐type C57BL/6J mice (male, 8–10 weeks old) were used and murine myocardial ischemia and reperfusion injury (IRI) model was conducted, cardiac function was evaluated by echocardiography. Infarct size measurement, cardiac troponin-I assays, oxidative stress analysis and histopathological analyzed. The resulted data showed that curcumin alleviates myocardial inflammatory responses and oxidative stress during myocardial IRI. Also, curcumin reduces cell apoptosis induced by myocardial IRI, curcumin reduces cell apoptosis induced by myocardial IRI and activates the Nrf-2/HO-1 signaling pathway during myocardial. Inconclusion, this study demonstrates that curcumin attenuates myocardial IRI by inhibiting proinflammatory cytokines through a mechanism that may be related to activation of the Nrf-2/HO-1 signaling pathway.

L-citrulline supplementation reduces systolic arterial pressure and myocardial infarct size in a rat model of sleep apnea syndrome

Sleep apnea syndrome is characterized by intermittent hypoxia (IH), leading to an increase in blood pressure (BP) and infarct size. Independently, IH has been demonstrated to alter arginine (Arg) metabolism. As Arg availability directly impacts nitric oxide production, we speculate that plasmatic Arg deficiency during IH may contribute to cardiovascular alterations induced by IH. Knowing that citrulline (Cit) is a better precursor of Arg than Arg itself, we propose to assess the impact of chronic Cit supplementation on IH-induced an increase in BP and infarct size. Four groups of rats ( n = 11–14), were submitted to normoxia (N) or IH [14 days (d), 8 h per d, 30 s-O2 21%/30 s-O2 5%] and supplemented or not with Cit (1 g.kg −1 .d −1 ). After 14 d, BP was measured by carotid catheterization; hearts were perfused by the Langendorff method and submitted to global and total ischemia (30 min)-reperfusion (120 min) before measuring infarct size related to left ventricle area (I/V) by colorimetry and planimetry. Another set of rats was added ( n = 5–6) in order to collect the heart and aorta for histological and biochemical analyses. IH increases systolic BP (144.5 ± 3.5 vs. 165.1 ± 4.2 mmHg in N and IH respectively, n = 11–14, P < 0.01) and I/V (33.7 ± 4.4 vs. 46 ± 2.5% in N and IH, respectively, n = 10–12, P < 0.05). Cit abolishes IH-induced increase in both systolic BP (139.8 ± 4. vs. 155.6 ± 6.6 mmHg NCit and IHCit, respectively) and I/V (35.7 ± 3.3 vs. 38.2 ± 2.1% in NCit and IHCit). Preliminary western blot results show that IH decreases T495P-eNOS in heart whereas it did not affect S1177P-eNOS. Interestingly, Cit supplementation seems to decrease even more the T495P-eNOS in the IHCit group compared to IH and NCit groups. Cit supplementation abolishes the deleterious effects of IH in terms of BP and infarct size. This suggests that citrulline could be a complementary or alternative treatment to the current sleep apnea standard treatment that is not fully efficient. However, biochemical and histological studies (NOx, oxidative stress, S-nitrosylation…) are ongoing to enlighten the specific mechanisms involved in cardiovascular protection induced by citrulline chronic supplementation during IH.

Pre-transplantation of Bone Marrow Mesenchymal Stem Cells Amplifies the Therapeutic Effect of Ultrasound-Targeted Microbubble Destruction-Mediated Localized Combined Gene Therapy in Post-Myocardial Infarction Heart Failure Rats.

Although stem cell transplantation and single-gene therapy have been intensively discussed separately as treatments for myocardial infarction (MI) hearts and have exhibited ideal therapeutic efficiency in animal models, clinical trials turned out to be disappointing. Here, we deliver sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) and connexin 43 (Cx43) genes simultaneously via an ultrasound-targeted microbubble destruction (UTMD) approach to chronic MI hearts that have been pre-treated with bone marrow mesenchymal stem cells (BMSCs) to amplify cardiac repair. First, biotinylated microbubbles (BMBs) were fabricated, and biotinylated recombinant adenoviruses carrying the SERCA2a or Cx43 gene were conjugated to the surface of self-assembled BMBs to form SERCA2a-BMBs, Cx43-BMBs or dual gene-loaded BMBs. Then, the general characteristics of these bubbles, including particle size, concentration, contrast signal and gene loading capacity, were examined. Second, a rat myocardial infarction model was created by ligating the left anterior descending coronary artery and injecting BMSCs into the infarct and border zones. Four weeks later, co-delivery of SERCA2a and Cx43 genes to the infarcted heart were delivered together to the infarcted heart using the UTMD approach. Cardiac mechano-electrical function was determined 4 wk after gene transfection, and the infarcted hearts were collected for myocardial infarct size measurement and detection of expression of SERCA2a, Cx43 and cardiac-specific markers. Finally, to validate the role of BMSC transplantation, MI rats transplanted or not with BMSCs were transfected with SERCA2a and Cx43, and the cardiac mechano-electrical function of these two groups of rats was recorded and compared. General characteristics of the self-assembled gene-loaded BMBs were qualified, and the gene loading rate was satisfactory. The self-assembled gene-loaded BMBs were in microscale and exhibit satisfactory dual-gene loading capacity. High transfection efficiency was achieved under ultrasound irradiation in vitro. In addition, rats in which SERCA2a and Cx43 were overexpressed simultaneously had the best contractile function and electrical stability among all experimental groups. Immunofluorescence assay revealed that the levels of SERCA2a and/or Cx43 proteins were significantly elevated, especially in the border zone. Moreover, compared with rats that did not receive BMSCs, rats pre-treated with BMSCs have better mechano-electrical function after transfection with SERCA2a and Cx43. Collectively, we report a promising cardiac repair strategy for post-MI hearts that exploits the providential advantages of stem cell therapy and UTMD-mediated localized co-delivery of specific genes.

Integrated chemical profiling, network pharmacology and pharmacological evaluation to explore the potential mechanism of Xinbao pill against myocardial ischaemia–reperfusion injury

Context Xinbao pill (XBW), a traditional Chinese herbal formula, is widely used in clinical treatment for cardiovascular diseases; however, the therapeutic effect of XBW on myocardial ischaemia–reperfusion injury (MI/RI) is unclear. Objective This study evaluates the cardioprotective effect and molecular mechanism of XBW against MI/RI. Materials and methods A phytochemistry-based network pharmacology analysis was used to uncover the mechanism of XBW against MI/RI. Ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry method was used to identify chemicals. MI/RI-related targets of XBW were predicted using TargetNet database, OMIC database, etc. Sprague-Dawley (SD) rats under anterior descending artery ligation model were divided into Sham, MI/RI and XBW (180 mg/kg, intragastric administration). After 30 min ischaemia and 24 h reperfusion, heart tissues were collected for measurement of myocardial infarct size. After oxygen glucose deprivation for 6 h, H9c2 cells were treated with XBW (60, 240 and 720 μg/mL) and diazoxide (100 μM) for 18 h of reperfusion. Results Thirty-seven chemicals were identified in XBW; 50 MI/RI-related targets of XBW were predicted using indicated databases. XBW significantly reduced infarct size and creatine kinase MB (CK-MB) level after MI/RI; XBW protected H9c2 cells against OGD/R injury. Gene ontology (GO) and KEGG pathway enrichment analyses by String database showed that the cardioprotective effect of XBW was associated with autophagy and apoptosis signalling pathways. Experimental investigation also verified that XBW suppressed apoptosis, autophagy and endoplasmic reticulum (ER) stress. Conclusions XBW showed therapeutic effects against MI/RI mainly via attenuating apoptosis though suppressing excessive autophagy and ER stress.

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Layer-By-Layer Fabrication of Thicker and Larger Human Cardiac Muscle Patches for Cardiac Repair in Mice.

The engineered myocardial tissues produced via most manufacturing techniques are typically just a few dozen micrometers thick, which is too thin for therapeutic applications in patients. Here, we used a modified layer-by-layer (LBL) fabrication protocol to generate thick human cardiac muscle patches (hCMPs) with thicknesses of ~3.75 mm. The LBL-hCMPs were composed of a layer of endothelial cells (ECs) sandwiched between two layers of cardiomyocytes (CMs): both cell populations were differentiated from the same human induced pluripotent stem cell line (hiPSCs) and suspended in a fibrin matrix, and the individual layers were sutured together, leaving channels that allowed the culture medium to access the internal cell layer. The LBL-hCMPs were cultured on a dynamic culture platform with electrical stimulation, and when compared to Control-hCMPs consisting of the same total number of hiPSC-ECs and -CMs suspended in a single layer of fibrin, hiPSC-CMs in the LBL-hCMPs were qualitatively more mature with significantly longer sarcomeres and expressed significantly higher levels of mRNA transcripts for proteins that participate in cardiomyocyte contractile activity and calcium handing. Apoptotic cells were also less common in LBL- than in Control-hCMPs. The thickness of fabricated LBL-hCMP gradually decreased to 0.8 mm by day 28 in dynamic culture. When the hCMP constructs were compared in a mouse model of myocardial infarction, the LBL-hCMPs were associated with significantly better measurements of engraftment, cardiac function, infarct size, hypertrophy, and vascularity. Collectively these observations indicate that our modified LBL fabrication protocol produced thicker hCMPs with no decline in cell viability, and that LBL-hCMPs were more potent than Control-hCMPs for promoting myocardial repair in mice.

Usual echo parameters versus global longitudinal strain for measuring infarct size and left ventricular functional remodeling after primary percutaneous coronary intervention

Usual echo parameters versus global longitudinal strain for measuring infarct size and left ventricular functional remodeling after primary percutaneous coronary intervention

Prognostic value of divergent pattern detection by 99mTc-sestamibi gated SPECT in patients with anterior acute myocardial infarction

PurposeIn gated myocardial perfusion SPECT, apical remodeling may be identified by the presence of a divergent pattern (DP) of the left ventricle (LV). Methods and ResultsWe examined 150 anterior ST-elevation myocardial infarction (STEMI) patients, all successfully treated with primary percutaneous coronary interventions (PCI). Perfusion gated-SPECT to measure infarct size, LV end-diastolic (ED) and end-systolic (ES) volumes and ejection fraction (EF) was acquired before hospital discharge and repeated at 6-month follow-up. DP was observed in 26 patients, who had larger infarct size (28 ± 19% vs. 15.7 ± 17%, P < 0.02), and lower EF (33 ± 7% vs. 41 ± 10%, P < 0.001) than patients without DP. At follow-up, DP patients had significantly larger EDV (156 ± 54 vs. 107 ± 44 mL, P < 0.0001), ESV (104 ± 47 vs. 59 ± 36 mL, P < 0.0001) and lower EF (35 ± 12% vs. 48 ± 13%, P < 0.0001). 54% of DP patients developed remodeling at follow-up vs. 12% of those without DP (P < 0.001). During follow up, 7 events in the DP group (27%) and 11 events in patients without DP (9%; P < 0.02) occurred. Kaplan–Meier survival curves showed a worse prognosis for DP patients. ConclusionIn patients with anterior AMI, early DP detection is related to subsequent LV dysfunction, larger infarct size, and worse severity. It is helpful for predicting LV remodeling at short-term follow-up and has prognostic implications.

Abstract 13089: Reperfusion Injury is Reduced by Antagonism of NLRP3's Caspase-1

VX-765, inhibitor of caspases-1, 3 and 4, at reperfusion reduces infarction in isolated rat hearts by 50% after ischemia/reperfusion (I/R). It was assumed cardioprotection by this pharmaceutical-grade drug was the result of inhibition of caspase-1 (cas-1) activation by the NLRP3 inflammasome which would trigger lethal pyroptosis. However, there has been no direct proof of that hypothesis. To test which, if any, caspase was responsible for protection, we measured infarct size after 50-min global ischemia/2-h reperfusion in isolated wild type (WT) mouse hearts and hearts from mice in which cas-1 had been genetically deleted (offspring of breeders from Jackson Labs genotyped to confirm mutation). Infarct size was measured objectively by Image J analysis of tetrazolium-stained heart slices using the same threshold settings for all hearts. No gender difference in infarct size was found in WT hearts (see figure) so genders were mixed, especially in the in the knockout (KO) groups. Because Krebs perfusate lacks blood-borne esterases required to convert pro-drug VX-765 to its active metabolite, VRT-043198 (VRT), we employed VRT in our model. Infarct size in hearts reperfused with buffer containing 180 nM VRT was halved to 43.7±4.8% of ventricular mass (p&lt;0.001 vs either cohort of WT hearts). Infarct size in cas-1 KO mice averaged 42.2±5.1% (pNS vs VRT). To eliminate possibility of a protected phenotype unrelated to cas-1, cas-1 KO mice were treated with 180 nM VRT. There was no further change in infarct size (44.0±4.3%) confirming cas-1 is VRT’s only target. NLRC4 also activates cas-1, but NLRC4 KO mice exposed to I/R were not protected (89.2±1.2% infarction), whereas inhibition of NLRP3 with MCC950 resulted in modest protection (65.5±5.3% infarction). These results strongly support NLRP3 as the source of the lethal cas-1. VX-765 at reperfusion is a promising adjunct intervention to decrease infarct size in patients treated for ST-segment elevation myocardial infarction.

Cardiac MRI to Visualize Myocardial Damage after ST-Segment Elevation Myocardial Infarction: A Review of Its Histologic Validation.

Cardiac MRI is a noninvasive diagnostic tool using nonionizing radiation that is widely used in patients with ST-segment elevation myocardial infarction (STEMI). Cardiac MRI depicts different prognosticating components of myocardial damage such as edema, intramyocardial hemorrhage (IMH), microvascular obstruction (MVO), and fibrosis. But how do cardiac MRI findings correlate to histologic findings? Shortly after STEMI, T2-weighted imaging and T2* mapping cardiac MRI depict, respectively, edema and IMH. The acute infarct size can be determined with late gadolinium enhancement (LGE) cardiac MRI. T2-weighted MRI should not be used for area-at-risk delineation because T2 values change dynamically over the first few days after STEMI and the severity of T2 abnormalities can be modulated with treatment. Furthermore, LGE cardiac MRI is the most accurate method to visualize MVO, which is characterized by hemorrhage, microvascular injury, and necrosis in histologic samples. In the chronic setting post-STEMI, LGE cardiac MRI is best used to detect replacement fibrosis (ie, final infarct size after injury healing). Finally, native T1 mapping has recently emerged as a contrast material-free method to measure infarct size that, however, remains inferior to LGE cardiac MRI. Especially LGE cardiac MRI-defined infarct size and the presence and extent of MVO may be used to monitor the effect of new therapeutic interventions in the treatment of reperfusion injury and infarct size reduction. © RSNA, 2021 Online supplemental material is available for this article.

Preconditioning the rat heart with 5-azacytidine attenuates myocardial ischemia/reperfusion injury via PI3K/GSK3β and mitochondrial KATP signaling axis.

5-Azacytidine is well known for its clinical usage in cancer treatments. The present study investigates the role of 5-azacytidine as a cardioprotective agent to ameliorate ischemia/reperfusion (I/R) injury. The cardioprotective effect of 5-azacytidine was evaluated in three experimental models: in vitro, ex vivo, and in vivo. The cardioprotective effect was evaluated via cell viability, hemodynamic indices, infarct size measurement, and assessment of histopathology, oxidative stress, and mitochondrial function. The experiments were repeated in the presence of PI3K/GSK3β and mitochondrial KATP (mtKATP ) cardioprotective signaling pathway inhibitors to understand the underlying mechanism. 5-Azacytidine improved the cell viability by 29% in I/R-challenged H9C2 cells. Both isolated rat heart and LAD ligation model confirmed the infarct sparing effect of 5-azacytidine against I/R. It also provided a beneficial effect by normalizing the altered hemodynamics, reducing the infarct size and cardiac injury markers, reversing the perturbation of mitochondria, reduced oxidative stress, and improved the pPI3K and pAKT protein expression from I/R. In addition, it also augmented the activation of PI3K/AKT and mtKATP signaling pathway, confirmed by using wortmannin (PI3K inhibitor), SB216763 (GSK3β inhibitor), and glibenclamide (mtKATP channel closer). The effectiveness of 5-azacytidine as a cardioprotective agent is attributed to its activation of the PI3K/GSK3β and mtKATP channel signaling axis, thereby preserving mitochondrial function and reducing oxidative stress.

Morphine Prevents Ischemia/Reperfusion-Induced Myocardial Mitochondrial Damage by Activating δ-opioid Receptor/EGFR/ROS Pathway.

The purpose of this study was to determine whether the epidermal growth factor receptor (EGFR), which is a classical receptor tyrosine kinase, is involved in the protective effect of morphine against ischemia/reperfusion (I/R)-induced myocardial mitochondrial damage. Isolated rats hearts were subjected to global ischemia followed by reperfusion. Cardiac H9c2 cells were exposed to a simulated ischemia solution followed by Tyrode's solution to induce hypoxia/reoxygenation (H/R) injury. Triphenyltetrazolium chloride (TTC) was used to measure infarct size. The mitochondrial morphological and functional changes were determined using transmission election microscopy (TEM), mitochondrial stress assay, and mitochondrial swelling, respectively. Mitochondrial fluorescence indicator JC-1, DCFH-DA, and Mitosox Red were used to determine mitochondrial membrane potential (△Ψm), intracellular reactive oxygen species (ROS) and mitochondrial superoxide. A TUNUL assay kit was used to detect the level of apoptosis. Western blotting analysis was used to measure the expression of proteins. Treatment of isolated rat hearts with morphine prevented I/R-induced myocardial mitochondrial injury, which was inhibited by the selective EGFR inhibitor AG1478, suggesting that EGFR is involved in the mitochondrial protective effect of morphine under I/R conditions. In support of this hypothesis, the selective EGFR agonist epidermal growth factor (EGF) reduced mitochondrial morphological and functional damage similarly to morphine. Further study demonstrated that morphine may alleviate I/R-induced cardiac damage by inhibiting autophagy but not apoptosis. Morphine increased protein kinase B (Akt), extracellular regulated protein kinases (ERK) and signal transducer and activator of transcription-3 (STAT-3) phosphorylation, which was inhibited by AG1478, and EGF had similar effects, indicating that morphine may activate Akt, ERK, and STAT-3 via EGFR. Morphine and EGF increased intracellular reactive oxygen species (ROS) generation. This effect of morphine was inhibited by AG1478, indicating that morphine promotes intracellular ROS generation by activating EGFR. However, morphine did not increase ROS generation when cells were transfected with siRNA against EGFR. In addition, EGFR activity was markedly increased by morphine, but the effect of morphine was reversed by naltrindole. These results suggest that morphine may activate EGFR via δ-opioid receptor activation. Morphine may prevent I/R-induced myocardial mitochondrial damage by activating EGFR through δ-opioid receptors, in turn increasing RISK and SAFE pathway activity via intracellular ROS. Moreover, morphine may reduce myocardial injury by regulating autophagy but not apoptosis.