Infective endocarditis with systemic bacterial embolism caused by Staphylococcus aureus in a free-ranging Amami rabbit (Pentalagus furnessi).

The Amami rabbit (Pentalagus furnessi) is found only on the two islands of Amami-Oshima and Tokunoshima in southwest Japan. It has a primitive appearance and ecology, is an evolutionarily valuable animal and has been assigned to the International Union for Conservation of Nature Red List of Threatened Species. We describe a case with mild purulent wounds on the distal digital skin of both forelimbs and multiple nodular lesions in various organs, including the heart and kidney. Microscopically, the heart lesions were characterized by disruption of the mitral valve and multifocal myocardial necrosis and abscesses due to infection with gram-positive cocci. Similar bacterial infarctions were also found in other organs, including the kidneys. The bacteria were identified as Staphylococcus aureus by immunohistochemical and molecular biological examinations. This first report of infective endocarditis and systemic infarctions caused by S. aureus in an Amami rabbit indicates the importance of monitoring purulent injuries, even if mild, to prevent secondary infections in this species.

Remote ischemic preconditioning-induced late cardioprotection: possible role of melatonin-mitoKATP-H2S signaling pathway

Purpose: Remote ischemic preconditioning (RIPC) confers cardioprotection against ischemia reperfusion (IR) injury. However, the precise mechanisms involved in RIPC-induced cardioprotection are not fully explored. The present study was aimed to identify the role of melatonin in RIPC-induced late cardioprotective effects in rats and to explore the role of H2 S, TNF-α and mitoKATP in melatoninmediated effects in RIPC. Methods: Wistar rats were subjected to RIPC in which hind limb was subjected to four alternate cycles of ischemia and reperfusion of 5 min duration by using a neonatal blood pressure cuff. After 24 h of RIPC or ramelteon-induced pharmacological preconditioning, hearts were isolated and subjected to IR injury on the Langendorff apparatus. Results: RIPC and ramelteon preconditioning protected the hearts from IR injury and it was assessed by a decrease in LDH-1, cTnT and increase in left ventricular developed pressure (LVDP). RIPC increased the melatonin levels (in plasma), H2 S (in heart) and decreased TNF-α levels. The effects of RIPC were abolished in the presence of melatonin receptor blocker (luzindole), ganglionic blocker (hexamethonium) and mitochondrial KATP blocker (5-hydroxydecanoic acid). Conclusion: RIPC produce delayed cardioprotection against IR injury through the activation of neuronal pathway, which may increase the plasma melatonin levels to activate the cardioprotective signaling pathway involving the opening of mitochondrial KATP channels, decrease in TNF-α production and increase in H2 S levels. Ramelteon-induced pharmacological preconditioning may also activate the cardioprotective signaling pathway involving the opening of mitochondrial KATP channels, decrease in TNF-α production and increase in H2 S levels.

Subepicardial burn injuries in bullfrog heart induce electrocardiogram changes mimicking inferior wall myocardial infarction.

Using bullfrog hearts, we previously reproduced a ST segment elevation in electrocardiogram (ECG), mimicking human ischemic heart disease. In the present study, by inducing subepicardial burn injuries on the inferior part of the frog heart ventricle, we could reproduce typical ECG changes observed in human inferior wall myocardial infarction, such as the marked elevation of the ST segments in inferior limb leads (II, III, aVF) and their reciprocal depression in the opposite limb leads (I, aVL). Due to the decrease in Na+/K+-ATPase protein expression, the resting membrane potential of injured cardiomyocytes shifted toward depolarization. Such induced electrical difference between the injured and intact cardiomyocytes was thought to be responsible for the creation of "currents of injury" and the subsequent ST segment changes.

It takes time to heal a broken heart: ventricular plasticity improves heart performance after myocardial infarction in rainbow trout, Oncorhynchus mykiss.

Coronary arteriosclerosis is a common feature of both wild and farmed salmonid fishes and may be linked to stress-induced cardiac pathologies. Yet, the plasticity and capacity for long-term myocardial restructuring and recovery following a restriction in coronary blood supply are unknown. Here, we analyzed the consequences of acute (3 days) and chronic (from 33 to 62 days) coronary occlusion (i.e. coronary artery ligation) on cardiac morphological characteristics and in vivo function in juvenile rainbow trout, Oncorhynchus mykiss. Acute coronary artery occlusion resulted in elevated resting heart rate and decreased inter-beat variability, which are both markers of autonomic dysfunction following acute myocardial ischemia, along with severely reduced heart rate scope (maximum-resting heart rate) relative to sham-operated trout. We also observed a loss of myocardial interstitial collagen and compact myocardium. Following long-term coronary artery ligation, resting heart rate and heart rate scope normalized relative to sham-operated trout. Moreover, a distinct fibrous collagen layer separating the compact myocardium into two layers had formed. This may contribute to maintain ventricular integrity across the cardiac cycle or, alternatively, demark a region of the compact myocardium that continues to receive oxygen from the luminal venous blood. Taken together, we demonstrate that rainbow trout may cope with the aversive effects caused by coronary artery obstruction through plastic ventricular remodeling, which, at least in part, restores cardiac performance and myocardium oxygenation.

Transient deep and giant negative T waves in dogs with myocardial injury.

INTRODUCTION: Although transient deep and giant negative T waves (NTWs) may develop during myocardial injury (MI) in humans, no data exist on this repolarization abnormality in canine MI. Therefore, this study aimed to describe the occurrence of transient deep/giant NTWs in dogs with MI. ANIMALS, MATERIALS AND METHODS: Medical records were retrospectively searched to identify dogs with MI and transient deep/giant NTWs. Signalment, history, and selected diagnostic test results were reviewed. Data analysis was descriptive. RESULTS: Six cases were diagnosed with MI associated with deep (n = 1) and giant (n = 5) transient NTWs. Myocardial injury was classified as acute in all cases and was due to snake envenomation (n = 3), sepsis (n = 2), and systemic inflammatory response syndrome (n = 1). At the time of deep/giant NTWs identification, all dogs had elevated cardiac troponin I and ≥1 echocardiographic abnormality of the left ventricular structure and/or function. Moreover, all dogs with giant NTWs had prolonged QT intervals. After the MI resolution, T-wave polarity and QT-interval duration became normalized in all dogs. Moreover, left ventricular morphological and functional parameters were completely normalized in four dogs. In contrast, ventricular echogenicity remained heterogeneous in two dogs, despite otherwise normalized ventricular parameters. Five dogs were still alive at the conclusion of the study. CONCLUSIONS: Transient deep/giant NTWs may develop in dogs with acute MI and T-wave polarity changes seem to occur synchronously with the evolution of myocardial damage. Moreover, transient deep/giant NTWs seem associated with a favorable prognosis in canine MI.

Inflammatory Responses with Left Ventricular Compromise after Induction of Myocardial Infarcts in Sheep (Ovis aries).

Ischemic myocardial disease is a major cause of death among humans worldwide; it results in scarring and pallor of the myocardium and triggers an inflammatory response that contributes to impaired left ventricular function. This response includes and is evidenced by the production of several inflammatory cytokines including TNFα, IL1ß, IL4, IFNγ, IL10 and IL6. In the current study, myocardial infarcts were induced in 6 mo old male castrated sheep by ligation of the left circumflex obtuse marginal arteries (OM 1 and 2). MRI was used to measure parameters of left ventricular function that include EDV, ESV, EF, SVI, dp/dt max and dp/dt min at baseline and at 4 wk and 3 mo after infarct induction. We also measured serum concentrations of an array of cytokines. Postmortem histologic findings corroborate the existence of left ventricular myocardial injury and deterioration. Our data show a correlation between serum cytokine concentrations and the development of myocardial damage and left ventricular functional compromise.

Hydromorphone-induced Neurostimulation in a Yorkshire Swine (Sus scrofa) after Myocardial Infarction Surgery.

Opiates play an important role in the control of pain associated with thoracotomy in both people and animals. However, key side effects, including sedation and respiratory depression, could limit the use of opiates in animals that are lethargic due to cardiac disease. In addition, a rare side effect-neuroexcitation resulting in pathologic behavioral changes (seizures, mania, muscle fasciculation)-after the administration of morphine or hydromorphone is well-documented in many species. In pigs, however, these drugs have been shown to stimulate an increase in normal activity. In the case presented, we describe a Yorkshire-cross pig which, after myocardial infarction surgery, went from nonresponsive to alert, responsive, and eating within 30 min of an injection of hydromorphone. This pig was not demonstrating any signs associated with pain at this time, suggesting that the positive response was due to neural stimulation. This case report is the first to describe the use of hydromorphone-a potent, pure µ opiate agonist-for its neurostimulatory effect in pigs with experimentally-induced cardiac disease.

Cardiomyocytes capture stem cell-derived, anti-apoptotic microRNA-214 via clathrin-mediated endocytosis in acute myocardial infarction.

Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication that have the potential to improve cardiac function when used in cell-based therapy. However, the means by which cardiomyocytes respond to EVs remains unclear. Here, we sought to clarify the role of exosomes in improving cardiac function by investigating the effect of cardiomyocyte endocytosis of exosomes from mesenchymal stem cells on acute myocardial infarction (MI). Exposing cardiomyocytes to the culture supernatant of adipose-derived regenerative cells (ADRCs) prevented cardiomyocyte cell damage under hypoxia in vitro. In vivo, the injection of ADRCs into the heart simultaneous with coronary artery ligation decreased overall cardiac infarct area and prevented cardiac rupture after acute MI. Quantitative RT-PCR-based analysis of the expression of 35 known anti-apoptotic and secreted microRNAs (miRNAs) in ADRCs revealed that ADRCs express several of these miRNAs, among which miR-214 was the most abundant. Of note, miR-214 silencing in ADRCs significantly impaired the anti-apoptotic effects of the ADRC treatment on cardiomyocytes in vitro and in vivo To examine cardiomyocyte endocytosis of exosomes, we cultured the cardiomyocytes with ADRC-derived exosomes labeled with the fluorescent dye PKH67 and found that hypoxic culture conditions increased the levels of the labeled exosomes in cardiomyocytes. Chlorpromazine, an inhibitor of clathrin-mediated endocytosis, significantly suppressed the ADRC-induced decrease of hypoxia-damaged cardiomyocytes and also decreased hypoxia-induced cardiomyocyte capture of both labeled EVs and extracellular miR-214 secreted from ADRCs. Our results indicate that clathrin-mediated endocytosis in cardiomyocytes plays a critical role in their uptake of circulating, exosome-associated miRNAs that inhibit apoptosis.

Identification of very early inflammatory markers in a porcine myocardial infarction model.

BACKGROUND: Acute myocardial infarction (AMI) is one of the most deleterious conditions leading to cardiovascular diseases and mortality. The importance of an early and accurate diagnosis assures immediate medical treatments, which are fundamental to reduce mortality and improve prognoses. AMI is associated to an inflammatory response which includes the increase of circulating inflammatory cytokines, chemokines and immune cell activation. This study aimed to identify which are the very early immune-related biomarkers that may be used as predictors of myocardial infarction severity. In order to mimic the pathophysiological events involved in human myocardial infarction, a temporary occlusion (90 min) of the mid-left anterior descending coronary artery was performed in a swine animal model. RESULTS: Lymphocyte subsets analysis in peripheral blood revealed significant alterations in CD4+/CD8+ ratio and naïve and effector/memory T cell percentages at 1 h post-myocardial infarction. Changes in TH1/TH2-related cytokine, monocyte and neutrophil markers gene expression were observed in peripheral blood lymphocytes, as well. Additionally, significant correlations between cardiac parameters (cardiac enzymes, left ventricular ejection fraction and % infarct) and blood-derived parameters (cytokine expression and lymphocyte subset distribution) were found. CONCLUSIONS: Peripheral blood lymphocyte alterations are easily and swiftly detectable, so they may be good biomarkers for a very early prognosis and to predict myocardial infarction severity.

Systematic Review and Meta-Analysis of the Impact of Hypoxia on Infarcted Myocardium: Better or Worse?

BACKGROUND/AIMS: Patients with myocardial infarction and hypoxemia require supplemental oxygen. However, the current therapeutic paradigm is contradicted by several recent studies in which the post-infarcted heart appears to benefit from systemic hypoxia. With this systematic review and meta-analysis, we aimed to discover whether systemic hypoxia is beneficial or detrimental to the infarcted myocardium. METHODS: We conducted an electronic search of the PubMed, EMBASE, and Web of Science databases and extracted the outcomes of cardiac function, geometry, and hemodynamics. A random-effect model was applied when the I2 value of greater than 50%. The sensitivity analysis was performed by omitting one study at a time, and publication bias was assessed using Egger's test. In addition, the quality of studies was evaluated using the risk of bias tool devised by the Systematic Review Centre for Laboratory Animal Experimentation. RESULTS: Six reports comprising 14 experiments were ultimately screened from among 10,323 initially identified preclinical studies. Few studies reported the method of randomization and none described allocation concealment, random outcome assessment or blinding. Overall, chronic hypoxia was found to have a beneficial effect on the ejection fraction (standard mean difference [SMD] = 5.39; 95% confidence interval [CI], 3.83 to 6.95; P < 0.001) of the infarcted heart, whereas acute hypoxia significantly improved hemodynamics, as indicated by an increase in the maximal rate of rise of left ventricular pressure (SMD = 1.27; 95% CI, 0.27 to 2.28; P = 0.013) and cardiac output (SMD = 1.26; 95% CI, 0.34 to 2.18; P = 0.007) and a decrease in total systematic vascular resistance (SMD = -0.89; 95% CI, -1.24 to -0.53; P < 0.001). Furthermore, a reduced oxygen content increased the stroke volume (P = 0.010). However, hypoxia reduced the end-systolic (SMD = -2.67; 95% CI, -4.09 to -1.26; P < 0.001) and end-diastolic (SMD = -3.61; 95% CI, -4.65 to -2.57; P < 0.001) left ventricular diameters and increased the total pulmonary resistance (SMD = 0.76; 95% CI, 0.20 to 1.33; P = 0.008), pulmonary arterial mean pressure (SMD = 2.02; 95% CI, 0.23 to 3.81; P = 0.027), and left atrial pressure (SMD = 1.20; 95% CI, 0.57 to 1.82; P < 0.001). CONCLUSION: Hypoxia significantly improved heart function after infarction, with particular beneficial effects on systolic function and hemodynamics. However, it had slightly adverse effects on pulmonary circulation and left ventricular geometry. A lower inspired oxygen concentration may improve cardiac function, although further research is needed to determine the optimum level of hypoxia. Finally, more studies of hypoxia and myocardial infarction in larger species are required before these findings can be incorporated into therapeutic guidelines.

Effects of Triple-Mutated Hypoxia-Inducible Factor-1α on Angiogenesis and Cardiac Function Improvement in Rats with Myocardial Infarction.

BACKGROUND/AIMS: The effects of hypoxia-inducible factor-1α (HIF-1α) on angiogenesis and cardiac function improvement in rats with myocardial infarction (MI) is unknown and our current study was to evaluate whether HIF-1α would be beneficial for angiogenesis and cardiac function improvement in MI rats. METHODS: A mutant of adenovirus HIF-1α (Ad-HIF-1α-Trip) was constructed by three sites mutation (Pro402, Pro564 and Asn803) in HIF-1α. The rat MI model was produced by permanent ligation of left anterior descending artery and 1×109 PFU adenovirus (Ad) vector particles of Ad-Null, Ad- HIF-1a-564/402, Ad- HIF-1a-Trip, 250ng vascular endothelial growth factor (VEGF) in 0.5ml saline or only 0.5 ml saline were injected intramuscularly around the infarct border zone. Real-time PCR, ELISA and western blotting were used to evaluate angiogenesis factors expression. Capillary density and necrotic areas were detected by immunohistochemistry staining and TTC staining, respectively. Cardiac function assessment was done by echocardiography before operation and on day 7, 14 and 28 after MI. Blood samples were drawn for the measurement of cardiac biomarkers, liver function and kidney function. RESULTS: On day 7, compared to the other groups, the expressions of HIF-1α and angiogenesis factors, and the capillary density were all significantly higher in the Ad-HIF-1α-Trip group. However, on day 28, no significant between-group differences were observed. After 72 hours of MI, serum level of cardiac biomarkers and the necrotic areas were significantly lower in the Ad-HIF-1a-Trip group compared to the other groups. Echocardiography showed that on day 7, cardiac functions were significantly reduced in all groups compared to the baseline. Cardiac function in the Ad-HIF-1α-Trip group was decreased less profoundly through day 7 to day 28 compared to the other groups. Importantly, no significant differences in liver and renal function were observed. CONCLUSION: Mutation of Pro402, Pro564 and Asn803 are beneficial for enhancement of the efficacy of HIF-1α. Ad-HIF-1α-Trip is able to improve angiogenesis and cardiac function, which may be a promising avenue for treatment of ischemic heart disease in the future.

Inhibition of MicroRNA-124 Reduces Cardiomyocyte Apoptosis Following Myocardial Infarction via Targeting STAT3.

BACKGROUND/AIMS: MicroRNAs play an important role in regulating myocardial infarction (MI)-induced cardiac injury. MicroRNA-124 (miR-124) plays a vital role in regulating cellular proliferation, differentiation and apoptosis. Although the alteration of miR-124 was confirmed in peripheral blood of MI patients, little is known regarding the biological functions of miR-124 in cardiomyocytes. This study was designed to explore the role of miR-124 in MI and its underlying mechanisms. METHODS: Real-time PCR was used to quantify the microRNAs levels. TUNEL and Flow cytometry were performed to measure cell apoptosis. Western blot analysis was employed to detect expression of Bcl-2, Bax, Caspase-3 and STAT3 proteins. RESULTS: We revealed that miR-124 was significantly up-regulated in a mice model of MI and in neonatal rat ventricular myocytes (NRVMs) with H2O2 treatment. H2O2 treatment induced cardiomyocyte injury with reduced cell viability and enhanced apoptotic cell death, whereas silencing expression of miR-124 by AMO-124 (antisense inhibitor oligodeoxyribonucleotides) alleviated these deleterious changes. AMO-124 decreased the expression of Bax and cleaved-caspase-3 and upregulated the expression of Bcl-2 in H2O2-treated NRVMs. Besides, AMO-124 improved mitochondrial dysfunction of NRVMs induced by H2O2 treatment. Moreover, antagomir-124 markedly decreased the infarct area and apoptotic cardiomyocytes and improved cardiac function in MI mice. Furthermore, we identified STAT3 as a direct target of miR-124, and downregulation of miR-124 ameliorated the diminished levels of STAT3 and p-STAT3 (Tyr705) in response to H2O2 or MI. STAT3 inhibitor, stattic, was shown to attenuate the elevation of p-STAT3 in NRVMs with AMO-124 transfection. Inhibiting of STAT3 activity by stattic abrogated protective effects of AMO-124 on H2O2-induced cardiomyocytes apoptosis. CONCLUSION: Taken together, our data demonstrate that downregulation of miR-124 inhibits MI-induced apoptosis through upregulating STAT3, which suggests the therapeutic potential of miR-124 for myocardial infarction.

Divergent Effects of Cyclophilin-D Inhibition on the Female Rat Heart: Acute Versus Chronic Post-Myocardial Infarction.

BACKGROUND/AIMS: The mitochondrial permeability transition pore opening plays a critical role in the pathogenesis of myocardial infarction. Inhibition of cyclophilin-D (CyP-D), a key regulator of the mitochondrial permeability transition pore, has been shown to exert cardioprotective effects against ischemia-reperfusion injury on various animal models, mostly in males. However, failure of recent clinical trials requires a detailed elucidation of the cardioprotective efficacy of CyP-D inhibition. The aim of this study was to examine whether cardioprotective effects of sanglifehrin A, a potent inhibitor of CyP-D, on post-infarcted hearts depends on reperfusion. METHODS: Acute or chronic myocardial infarction was induced by coronary artery ligation with/without subsequent reperfusion for 2 and 28 days in female Sprague-Dawley rats. Cardiac function was estimated by echocardiography. Oxygen consumption rates, ROS production, permeability transition pore opening, protein carbonylation and respiratory supercomplexes were analyzed in isolated cardiac mitochondria. RESULTS: Sanglifehrin A significantly improved cardiac function of reperfused hearts at 2 days but failed to protect after 28 days. No protection was observed in non-reperfused post-infarcted hearts. The respiratory control index of mitochondria was significantly reduced in reperfused infarcted hearts at 2-days with no effect at 28-days post-infarction on reperfused and non-reperfused hearts. Likewise, only a minor increase in reactive oxygen species production was observed at 2-days in non-reperfused post-infarcted hearts. CONCLUSION: This study demonstrates that CyP-D inhibition exerts cardioprotective effects in reperfused but not in non-reperfused infarcted hearts of female rats, and the effects are observed only during acute post-infarction injury.

Zinc Supplementation Attenuates Cardiac Remodeling After Experimental Myocardial Infarction.

BACKGROUND/AIMS: The objective of our study was to evaluate the effects of zinc supplementation on cardiac remodeling following acute myocardial infarction in rats. METHODS: Animals were subdivided into 4 groups and observed for 3 months: 1) Sham Control; 2) Sham Zinc: Sham animals receiving zinc supplementation; 3) Infarction Control; 4) Infarction Zinc. After the followup period, we studied hypertrophy and ventricular geometry, functional alterations in vivo and in vitro, changes related to collagen, oxidative stress, and inflammation, assessed by echocardiogram, isolated heart study, western blot, flow cytometer, morphometry, and spectrophotometry. RESULTS: Infarction induced a significant worsening of the functional variables. On the other hand, zinc attenuated both systolic and diastolic cardiac dysfunction induced by infarction. Considering the infarct size, there was no difference between the groups. Catalase and superoxide dismutase decreased in infarcted animals, and zinc increased its activity. We found higher expression of collagens I and III in infarcted animals, but there was no effect of zinc supplementation. Likewise, infarcted animals had higher levels of IL-10, but without zinc interference. Nrf-2 values were not different among the groups. Infarction increased the amount of Treg cells in the spleen as well as the amount of total lymphocytes. Zinc increased the amount of CD4+ in infarcted animals, but we did not observe effects in relation to Treg cells. CONCLUSION: zinc attenuates cardiac remodeling after infarction in rats; this effect is associated with modulation of antioxidant enzymes, but without the involvement of collagens I and III, Nrf-2, IL-10, and Treg cells.

Astragaloside IV Protects Rat Cardiomyocytes from Hypoxia-Induced Injury by Down-Regulation of miR-23a and miR-92a.

BACKGROUND/AIMS: Astragaloside IV (AS-IV), a traditional Chinese medicine isolated from Astragalus membranaceus, has been shown to exert cardioprotective effect previously. This study aimed to reveal the effects of AS-IV on hypoxia-injured cardiomyocyte. METHODS: H9c2 cells were treated with various doses of AS-IV for 24 h upon hypoxia. CCK-8 assay, flow cytometry/Western blot, and qRT-PCR were respectively conducted to measure the changes in cell viability, apoptosis, and the expression of miR-23a and miR-92a. Sprague-Dawley rats were received coronary ligation, and were administrated by various doses of AS-IV for 14 days. The infarct volume and outcome of rats followed by ligation were tested by ultrasound, arteriopuncture and nitrotetrazolium blue chloride (NBT) staining. RESULTS: We found that 10 µg/ml of AS-IV exerted myocardioprotective effects against hypoxia-induced cell damage, as AS-IV significantly increased H9c2 cells viability and decreased apoptosis. Interestingly, the myocardioprotective effects of AS-IV were alleviated by miR-23a and/or miR-92a overexpression. Knockdown of miR-23a and miR-92a activated PI3K/AKT and MAPK/ ERK signaling pathways. Bcl-2 was a target gene for miR-23a, and BCL2L2 was a target gene for miR-92a. In the animal model of myocardial infarction (MI), AS-IV significantly reduced the infarct volume, ejection fraction (EF), shortening fraction (FS) and LV systolic pressure (LVSP), and significantly increased left ventricular end-diastolic internal diameter (LVEDd). And also, the elevated expression of miR-23a and miR-92a in MI rat was reduced by AS-IV. CONCLUSION: AS-IV protected cardiomyocytes against hypoxia-induced injury possibly via down-regulation of miR-23a and miR-92a, and via activation of PI3K/AKT and MAPK/ERK signaling pathways.

IL (Interleukin)-10-STAT3-Galectin-3 Axis Is Essential for Osteopontin-Producing Reparative Macrophage Polarization After Myocardial Infarction.

BACKGROUND: Both osteopontin (OPN) and galectin-3 have been implicated in phagocytic clearance of dead cells and reparative fibrosis during wound healing. CD206+ macrophages are involved in tissue repair through phagocytosis and fibrosis after myocardial infarction (MI). However, the relationship among OPN, galectin-3, and macrophage polarization in the context of MI remains unclear. METHODS: The time course of Spp1 (encoding OPN) expression in the heart after MI showed a strong activation of Spp1 on day 3 after MI. To identify where in the body and in which cells the transcriptional activity of Spp1 increased after MI, we analyzed EGFP (enhanced green fluorescent protein)- Spp1 knockin reporter mice on day 3 after MI. RESULTS: The transcriptional activity of Spp1 increased only in CD206+ macrophages in the infarct myocardium, and most of CD206+ macrophages have strong transcriptional activation of Spp1 after MI. The temporal expression pattern of Lgal3 (encoding galectin-3) in cardiac macrophages after MI was similar to that of Spp1, and OPN is almost exclusively produced by galectin-3hiCD206+ macrophages. Although both interleukin (IL)-4 and IL-10 were reported to promote CD206+ macrophage-mediated cardiac repair after MI, IL-10- but not IL-4-stimulated CD11b+Ly6G- cells could differentiate into OPN-producing galectin-3hiCD206+ macrophages and showed enhanced phagocytic ability. Inhibition of STAT3 tyrosine phosphorylation suppressed IL-10-induced expression of intracellular galectin-3 and transcriptional activation of Spp1. Knockdown of galectin-3 suppressed their ability to differentiate into OPN-producing cells, but not STAT3 activation. The tyrosine phosphorylation of STAT3 and the appearance rate of galectin-3hiCD206+ cells on cardiac CD11b+Ly6G- cells in Spp1 knockout mice were the same as those in wild-type mice. Spp1 knockout mice showed vulnerability to developing post-MI left ventricular chamber dilatation and the terminal deoxynucleo-tidyltransferase 2'-Deoxyuridine-5'-triphosphate nick-end labeling (TUNEL)-positive cells in the infarcted myocardium after MI remained higher in number in Spp1 knockout mice than in wild-type mice. CONCLUSIONS: OPN is almost exclusively produced by galectin-3hiCD206+ macrophages, which specifically appear in the infarct myocardium after MI. The IL-10-STAT3-galectin-3 axis is essential for OPN-producing reparative macrophage polarization after myocardial infarction, and these macrophages contribute to tissue repair by promoting fibrosis and clearance of apoptotic cells. These results suggest that galectin-3 may contribute to reparative fibrosis in the infarct myocardium by controlling OPN levels.

MicroRNA-223 Regulates Cardiac Fibrosis After Myocardial Infarction by Targeting RASA1.

BACKGROUND/AIMS: Percutaneous coronary intervention reduces acute myocardial infarction (MI)-induced mortality to a great extent, but effective treatments for MI-induced cardiac fibrosis and heart failure are still lacking. MicroRNAs (miRNAs) play a variety of roles in cells and have thus been investigated extensively. MicroRNA-223 (miR-223) expression has been reported to be altered in post-MI heart failure in humans; however, the roles of miR-223 in MI remain unknown. Our study aimed to elucidate the roles of miR-223 in cardiac fibrosis. METHODS: Cultured cardiac fibroblasts (CFs) were activated by TGF-ß1 stimulation. Gain and loss of miR-223 and RAS p21 protein activator 1 (RASA1) knockdown in CFs were achieved by transfecting the cells with miR-223 mimics and inhibitors, as well as small interfering RNA-RASA1 (siRASA1), respectively. Quantitative real-time reverse transcriptase-polymerase chain reactions (qRT-PCR) was used to determine miR-223-3p and RASA1 expression levels, and Cell Counting Kit-8 (CCK-8), transwell migration and scratch assays were performed to assess CFs viability and migration, respectively. Western blotting was used to detect collagen I, collagen III, alpha-smooth muscle actin (a-SMA), RASA1, p-Akt/t-Akt, p-MEK1/2/t-MEK1/2, and p-ERK1/2/t-ERK1/2 protein expressions, and immunofluorescence assays were used to detect the expression of α-actin, vimentin and α-SMA. Luciferase assays were carried out to determine whether miR-223 binds to RASA1. Rat models of MI were established by the ligation of the left anterior descending (LAD) coronary artery. MiR-223 inhibition in vivo was achieved via intramyocardial injections of the miR-223 sponge carried by adeno-associated virus 9 (AAV9). The cardiac function was detected by echocardiography, and cardiac fibrosis was shown by Masson's trichrome staining. RESULTS: miR-223 was increased in CFs compared to cardiomypcytes, and TGF-ß1 treatment increased miR-223 expression in CFs. The miR-223 mimics enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs, while the miR-223 inhibitors had contrasting effects and partially prevented the promoting effects of TGF-ß1. qRT-PCR and western blotting revealed that miR-223 negatively regulated RASA1 expression, and the luciferase assays showed that miR-223 suppressed the luciferase activity of the RASA1 3' untranslated region (3'UTR), indicating that miR-223 binds directly to RASA1. Similar to transfection with the miR-223 mimics, RASA1 knockdown enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs. Moreover, RASA1 knockdown partially reversed the inhibitory effects of the miR-223 inhibitor on cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression, indicating that the effects of miR-223 in CFs are partially mediated by the regulation of RASA1 expression. Further exploration showed that miR-223 mimics and siRASA1 promoted MEK1/2, ERK1/2 and AKT phosphorylation, while the miR-223 inhibitors had contrasting effects. The in vivo experiments confirmed the results of the in vitro experiments and showed that miR-223 inhibition prevented cardiac functional deterioration and cardiac fibrosis. CONCLUSIONS: miR-223 enhanced cell proliferation, migration, and differentiation in CFs, thus mediated cardiac fibrosis after MI partially via the involvement of RASA1.

Self-Gated Late Gadolinium Enhancement at 7T to Image Rats with Reperfused Acute Myocardial Infarction.

Objective: A failed electrocardiography (ECG)-trigger often leads to a long acquisition time (TA) and deterioration in image quality. The purpose of this study was to evaluate and optimize the technique of self-gated (SG) cardiovascular magnetic resonance (CMR) for cardiac late gadolinium enhancement (LGE) imaging of rats with myocardial infarction/reperfusion. Materials and Methods: Cardiovascular magnetic resonance images of 10 rats were obtained using SG-LGE or ECG with respiration double-gating (ECG-RESP-gating) method at 7T to compare differences in image interference and TA between the two methods. A variety of flip angles (FA: 10°-80°) and the number of repetitions (NR: 40, 80, 150, and 300) were investigated to determine optimal scan parameters of SG-LGE technique based on image quality score and contrast-to-noise ratio (CNR). Results: Self-gated late gadolinium enhancement allowed successful scan in 10 (100%) rats. However, only 4 (40%) rats were successfully scanned with the ECG-RESP-gating method. TAs with SG-LGE varied depending on NR used (TA: 41, 82, 154, and 307 seconds, corresponding to NR of 40, 80, 150, and 300, respectively). For the ECG-RESP-gating method, the average TA was 220 seconds. For SG-LGE images, CNR (42.5 ± 5.5, 43.5 ± 7.5, 54 ± 9, 59.5 ± 8.5, 56 ± 13, 54 ± 8, and 41 ± 9) and image quality score (1.85 ± 0.75, 2.20 ± 0.83, 2.85 ± 0.37, 3.85 ± 0.52, 2.8 ± 0.51, 2.45 ± 0.76, and 1.95 ± 0.60) were achieved with different FAs (10°, 15°, 20°, 25°, 30°, 35°, and 40°, respectively). Optimal FAs of 20°-30° and NR of 80 were recommended. Conclusion: Self-gated technique can improve image quality of LGE without irregular ECG or respiration gating. Therefore, SG-LGE can be used an alternative method of ECG-RESP-gating.

Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration.

Human diseases are often caused by loss of somatic cells that are incapable of re-entering the cell cycle for regenerative repair. Here, we report a combination of cell-cycle regulators that induce stable cytokinesis in adult post-mitotic cells. We screened cell-cycle regulators expressed in proliferating fetal cardiomyocytes and found that overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 efficiently induced cell division in post-mitotic mouse, rat, and human cardiomyocytes. Overexpression of the cell-cycle regulators was self-limiting through proteasome-mediated degradation of the protein products. In vivo lineage tracing revealed that 15%-20% of adult cardiomyocytes expressing the four factors underwent stable cell division, with significant improvement in cardiac function after acute or subacute myocardial infarction. Chemical inhibition of Tgf-ß and Wee1 made CDK1 and cyclin B dispensable. These findings reveal a discrete combination of genes that can efficiently unlock the proliferative potential in cells that have terminally exited the cell cycle.

Arctigenin exerts protective effects against myocardial infarction via regulation of iNOS, COX­2, ERK1/2 and HO­1 in rats.

The present study aimed to determine the protective effects of arctigenin against myocardial infarction (MI), and its effects on oxidative stress and inflammation in rats. Left anterior coronary arteries of Sprague­Dawley rats were ligated, in order to generate an acute MI (AMI) model. Arctigenin was administered to AMI rats at 0, 50, 100 or 200 µmol/kg. Western blotting and ELISAs were performed to analyze protein expression and enzyme activity. Arctigenin was demonstrated to effectively inhibit the levels of alanine transaminase, creatine kinase­MB and lactate dehydrogenase, and to reduce infarct size in AMI rats. In addition, the activity levels of malondialdehyde, interleukin (IL)­1ß and IL­6 were significantly suppressed, and the levels of glutathione peroxidase, catalase and superoxide dismutase were significantly increased by arctigenin treatment. Arctigenin treatment also suppressed the protein expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX­2) and heme oxygenase 1 (HO­1), and increased the protein expression levels of phosphorylated­extracellular signal­regulated kinase 1/2 (p­ERK1/2) in AMI rats. Overall, the results of the present study suggest that arctigenin may inhibit MI, and exhibits antioxidative and anti­inflammatory effects through regulation of the iNOS, COX­2, ERK1/2 and HO­1 pathways in a rat model of AMI.