Murine Model Of Myocardial Infarction Research Articles

Effects on cardiac function, remodeling and inflammation following myocardial ischemia\u2013reperfusion injury or unreperfused myocardial infarction in hypercholesterolemic APOE*3-Leiden mice

Many novel therapies to treat myocardial infarction (MI), yielding promising results in animal models, nowadays failed in clinical trials for several reasons. The most used animal MI model is based on permanent ligation of the left anterior descending (LAD) coronary artery in healthy mice resulting in transmural MI, while in clinical practice reperfusion is usually accomplished by primary percutaneous coronary interventions (PCI) limiting myocardial damage and inducing myocardial ischemia–reperfusion (MI-R) injury. To evaluate a more similar murine MI model we compared MI-R injury to unreperfused MI in hypercholesterolemic apolipoprotein (APO)E*3-Leiden mice regarding effects on cardiac function, left ventricular (LV) remodeling and inflammation. Both MI-R and MI resulted in significant LV dilation and impaired cardiac function after 3 weeks. Although LV dilation, displayed by end-diastolic (EDV) and end-systolic volumes (ESV), and infarct size (IS) were restricted following MI-R compared to MI (respectively by 27.6% for EDV, 39.5% ESV, 36.0% IS), cardiac function was not preserved. LV-wall thinning was limited with non-transmural LV fibrosis in the MI-R group (66.7%). Two days after inducing myocardial ischemia, local leucocyte infiltration in the infarct area was decreased following MI-R compared to MI (36.6%), whereas systemic circulating monocytes were increased in both groups compared to sham (130.0% following MI-R and 120.0% after MI). Both MI-R and MI models against the background of a hypercholesterolemic phenotype appear validated experimental models, however reduced infarct size, restricted LV remodeling as well as a different distributed inflammatory response following MI-R resemble the contemporary clinical outcome regarding primary PCI more accurately which potentially provides better predictive value of experimental therapies in successive clinical trials.

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Interleukin-1alpha deficiency reduces cardiac remodeling in a murine model of myocardial infarction without reperfusion

Early development of myocardial inflammation following myocardial infarction (MI) is essential for healing of the injury. During this phase, necrotic cardiomyocytes (CMs) release pro-inflammatory “damage associated molecular patterns”, or DAMPs. DAMPs trigger innate immune signaling pathways in the myocardium leading to a primary immune response orchestrated by inflammatory phagocytes. It is followed by a resolution and proliferation phase characterized by the activation of fibroblasts and reparative scar formation. Dysregulation in these processes promote alterations in the myocardium and lead to adverse cardiac remodeling. We previously demonstrated that Interleukin-1alpha (IL-1a) is the primary DAMP released by necrotic CMs in vitro and is essential for a full magnitude early inflammatory response in the myocardium in vivo. Present study aims at defining the role of IL-1a in the process of post-MI inflammation, cardiac remodeling and heart failure by comparing the response to MI of IL-1a-competent (WT), systemic IL-1a knockout (KO) mice and CMs conditional KO (cKO) mice. Mice underwent permanent ligation of the LAD coronary artery. Inflammatory and fibrotic responses of the myocardium were analyzed by western blotting, ELISA, qPCR and FACS for the phagocytic immune cells content within the myocardium during the first week post-MI. Echographic measurements of cardiac dimensions and function were made 4 to 6 weeks post-MI. KO mice showed a slight decrease in inflammatory parameters along with decreased ratio of pro-inflammatory macrophages in the first week after MI as compared to WT. We observed a strong decrease in fibrotic markers and TGF-beta responsive genes 7 days post-MI and reduced dimensions of left ventricle 4 to 6 weeks post-MI in KO compared to WT mice, which was not the case in cKO. IL-1a, from an origin other than CMs, exacerbates post-MI remodeling, making it a presumed therapeutic target.

The molecular mechanism involved in cardioprotection by the dietary flavonoid fisetin as an agonist of PPAR-γ in a murine model of myocardial infarction

The methodology exploring the cardioprotective potential of the flavonoid Fisetin through its ability to modulate PPAR-γ was unraveled in the present study. Computational modelling through molecular docking based binding study of interactions between Fiestin and PPAR-γ revealed the potential role of Fisetin as an agonist of PPAR-γ. A murine model of cardiac ischemia-reperfusion injury was used to explore this further. Male Wistar Rats were randomly assigned to five groups. Fisetin (20 mg/kg; p. o) was administered for 28 days. Ischemia was induced for 45 min on the 29th day followed by 60 min of reperfusion. Fisetin pretreatment upregulated the expression of PPAR-γ in heart tissue significantly Cardioprotection was assessed by measurement of hemodynamic parameters, infarct size, ELISA for oxidative stress, immunohistochemistry and TUNEL assay for apoptosis, and western blot analysis for MAPK proteins and inflammation. PPAR-γ activation by fisetin led to significantly reduced infarct size, suppression of oxidative stress, reduction of cardiac injury markers, alleviation of inflammation, and inhibition of apoptosis The MAPK-based molecular mechanism showed a rise in a key prosurvival kinase, ERK1/ERK2 and suppression of JNK and p38 proteins. The aforementioned beneficial findings of fisetin were reversed on the administration of a specific antagonist of PPAR-γ. In conclusion, through our experiments, we have proved that fisetin protects the heart against ischemia-reperfusion injury and the evident cardioprotection is PPAR-γ dependant. In conclusion, our study has revealed a prime mechanism involved in the cardioprotective effects of fisetin. Hence, Fisetin may be evaluated in further clinical studies as a cardioprotective agent in patients undergoing reperfusion interventions.

Cardiac repair in a murine model of myocardial infarction with human induced pluripotent stem cell-derived cardiomyocytes

BackgroundCellular replacement strategies using human induced pluripotent stem cells (iPSCs) and their cardiac derivatives are emerging as novel treatments for post-myocardial infarction (MI) heart failure (HF); however, the mechanism of recovery of heart function is not very clear. The purpose of this study was to investigate the efficiency of using highly purified human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) for myocardial repair in a mouse model of MI and to clarify the mechanism of recovery of heart function.MethodsAnimals modelling MI were randomly assigned to receive direct intramyocardial injection of culture medium (MI group) or 4 × 105 iPS-CMs (cell group) at the infarct border zone. Left ventricle (LV) performance was assessed with serial cardiac electrophysiology and was measured 1, 2 and 4 weeks post-MI. Invasive LV pressure measurement was measured at 4 weeks and was followed by sacrifice for histological examination.ResultsCompared to the MI group, the left ventricle ejection fraction (LVEF), left ventricular internal diameter in end-diastole (LVIDd) and end-systole (LVIDs) and maximal positive and negative pressure derivative (±dP/dt) were significantly improved in the iPS-CM group at 4 weeks post-MI. Histological examination revealed a very limited number of iPS-CMs 4 weeks after transplantation. Nonetheless, there was a significant enhancement of angiogenesis and a reduction in apoptosis of native cardiomyocyte after iPS-CM transplantation.ConclusionsOur results demonstrate that transplantation of human iPS-CMs can improve heart function via paracrine action in a mouse model of myocardial infarction.

Loss of miR-132/212 Has No Long-Term Beneficial Effect on Cardiac Function After Permanent Coronary Occlusion in Mice.

Background: Myocardial infarction (MI) is caused by occlusion of the coronary artery and induces ischemia in the myocardium and eventually a massive loss in cardiomyocytes. Studies have shown many factors or treatments that can affect the healing and remodeling of the heart upon infarction, leading to better cardiac performance and clinical outcome. Previously, miR-132/212 has been shown to play an important role in arteriogenesis in a mouse model of hindlimb ischemia and in the regulation of cardiac contractility in hypertrophic cardiomyopathy in mice. In this study, we explored the role of miR-132/212 during ischemia in a murine MI model.Methods and Results: miR-132/212 knockout mice show enhanced cardiac contractile function at baseline compared to wild-type controls, as assessed by echocardiography. One day after induction of MI by permanent occlusion, miR-132/212 knockout mice display similar levels of cardiac damage as wild-type controls, as demonstrated by infarction size quantification and LDH release, although a trend toward more cardiomyocyte cell death was observed in the knockout mice as shown by TUNEL staining. Four weeks after MI, miR-132/212 knockout mice show no differences in terms of cardiac function, expression of cardiac stress markers, and fibrotic remodeling, although vascularization was reduced. In line with these in vivo observation, overexpression of miR-132 or miR-212 in neonatal rat cardiomyocyte suppress hypoxia induced cardiomyocyte cell death.Conclusion: Although we previously observed a role in collateral formation and myocardial contractility, the absence of miR-132/212 did not affect the overall myocardial performance upon a permanent occlusion of the coronary artery. This suggests an interplay of different roles of this miR-132/212 before and during MI, including an inhibitory effect on cell death and angiogenesis, and a positive effect on cardiac contractility and autophagic response. Thus, spatial or tissue specific manipulation of this microRNA family may be essential to fully understand the roles and to develop interventions to reduce infarct size.

Collagen-Based Microcapsules As Therapeutic Materials for Stem Cell Therapies in Infarcted Myocardium.

As cell therapies emerged, it was quickly realized that pro-regenerative cells directly injected into injured tissue struggled within the inflammatory microenvironment. By using microencapsulation, i.e., encapsulating cells within polymeric biomaterials, they are henceforth protected from the harmful extracellular cues, while still being able to receive oxygen and nutrients and release secreted factors. Previous work showed that stem cells encapsulated within a biologically inert material (agarose) were able to significantly improve the function of the infarcted mouse heart. With the aim of using more bioresponsive microcapsules, we sought to develop an enzymatically degradable, type I collagen-based microcapsule for the intramyocardial delivery of bone marrow-derived mesenchymal stromal cells in a murine model of myocardial infarction.

Volumetric Optoacoustic Tomography Differentiates Myocardial Remodelling.

Myocardial healing following myocardial infarction (MI) is a complex process that is yet to be fully understood. Clinical attempts in regeneration of the injured myocardium using cardiac stem cells faced major challenges, calling for a better understanding of the processes involved at a more basic level in order to foster translation. We examined the feasibility of volumetric optoacoustic tomography (VOT) in studying healing of the myocardium in different models of MI, including permanent occlusion (PO) of the left coronary artery, temporary occlusion (ischemia-reperfusion-I/R) and infarcted c-kit mutants, a genetic mouse model with impaired cardiac healing. Murine hearts were imaged at 100Hz frame rate using 800nm excitation wavelength, corresponding to the peak absorption of indocyanine green (ICG) in plasma and the isosbestic point of haemoglobin. The non-invasive real-time volumetric imaging capabilities of VOT have allowed the detection of significant variations in the pulmonary transit time (PTT), a parameter affected by MI, across different murine models. Upon intravenous injection of ICG, we were able to track alterations in cardiac perfusion in I/R models, which were absent in wild-type (wt) PO or kitW/kitW-v PO mice. The wt-PO and I/R models further exhibited irregularities in their cardiac cycles. Clear differences in the PTT, ICG perfusion and cardiac cycle patterns were identified between the different models and days post MI. Overall, the results highlight the unique capacity of VOT for multi-parametric characterization of morphological and functional changes in murine models of MI.

Hexarelin targets neuroinflammatory pathways to preserve cardiac morphology and function in a mouse model of myocardial ischemia-reperfusion

Acute myocardial ischemia and reperfusion injury (IRI) underly the detrimental effects of coronary heart disease on the myocardium. Despite the ongoing advances in reperfusion therapies, there remains a lack of effective therapeutic strategies for preventing IRI. Growth hormone secretagogues (GHS) have been demonstrated to improve cardiac function, attenuate inflammation and modulate the autonomic nervous system (ANS) in models of cardiovascular disease. Recently, we demonstrated a reduction in infarct size after administration of hexarelin (HEX), in a murine model of myocardial infarction. In the present study we employed a reperfused ischemic (IR) model, to determine whether HEX would continue to have a cardioprotective influence in a model of higher clinical relevance.Myocardial ischemia was induced by transient ligation of the left descending coronary artery (tLAD) in C57BL/6 J mice followed by HEX (0.3 mg/kg/day; n = 20) or vehicle (VEH) (n = 18) administration for 21 days, first administered immediately prior-to reperfusion. IR-injured and sham mice were subjected to high-field magnetic resonance imaging to assess left ventricular (LV) function, with HEX-treated mice demonstrating a significant improvement in LV function compared with VEH-treated mice. A significant decrease in interstitial collagen, TGF-β1 expression and myofibroblast differentiation was also seen in the HEX-treated mice after 21 days. HEX treatment shifted the ANS balance towards a parasympathetic predominance; combined with a significant decrease in cardiac troponin-I and TNF-α levels, these findings were suggestive of an anti-inflammatory action on the myocardium mediated via HEX.In this model of IR, HEX appeared to rebalance the deregulated ANS and activate vagal anti-inflammatory pathways to prevent adverse remodelling and LV dysfunction. There are limited interventions focusing on IRI that have been successful in improving clinical outcome in acute myocardial infarction (AMI) patients, this study provides compelling evidence towards the translational potential of HEX where all others have largely failed.

MiRNA-181a is a novel regulator of aldosterone-mineralocorticoid receptor-mediated cardiac remodelling.

The aldosterone-mineralocorticoid receptor (Aldo-MR) pathway is activated during cardiac stress, such as hypertension, myocardial infarction (MI), and heart failure. The importance of Aldo and MR in the pathogenesis of cardiac diseases is well established; however, the regulatory mechanisms behind Aldo/MR-induced cardiac remodelling remain uncertain. We here investigated potential miRNA-mediated regulation of the Aldo-MR pathway to improve mechanistic understanding. High-throughput screening of 2,555 miRNAs using an MR responsive stable cardiomyocyte cell line (MMTV-GFP-HL-1) identified miR-181a as a potential regulator of Aldo-MR pathway. MiR-181a was found to downregulate the expression of Ngal (lipocalin-2), a well-established downstream effector molecule of Aldo-MR. In addition, Aldo-induced cellular hypertrophy decreased significantly upon miR-181a overexpression. Genetic miR-181 knockout in murine MI model led to deteriorated cardiac function, cardiac remodelling, and activation of Aldo-MR pathway while AAV9-mediated miR-181a overexpression improved cardiac function and deactivated Aldo-MR pathway proving a cardio-protective role of miR-181a. Global RNA sequencing of cells under Aldo treatment with/without miR-181a overexpression identified potential miR-181a targets functionally contributing to Aldo-MR pathway. Adamts1, a direct target of miR-181a, was found to be downregulated with miR-181a overexpression and upregulated with inhibition. Similar to miR-181a overexpression, siRNA-mediated inhibition of Adamts1 inhibited Aldo-MR pathway. We here show that miR-181a is a novel regulator of the Aldo-MR pathway regulating the levels of Ngal via direct targeting of Adamts1. This new insight establishes miR-181a as a factor of immense value participating in downstream networks of Aldo-MR pathway. Our in vivo studies further confirmed miR-181a as cardio-protective under MI stress. Thus, miR-181a's involvement in Aldo-MR-mediated cardiac remodelling confers it with tremendous potential to be developed further as a new therapeutic target.

Substrate for the Myocardial Inflammation–Heart Failure Hypothesis Identified Using Novel USPIO Methodology

ObjectivesThe purpose of this study was to identify where ultrasmall superparamagnetic particles of iron oxide (USPIO) locate to in myocardium, develop a methodology that differentiates active macrophage uptake of USPIO from passive tissue distribution; and investigate myocardial inflammation in cardiovascular diseases. BackgroundMyocardial inflammation is hypothesized to be a key pathophysiological mechanism of heart failure (HF), but human evidence is limited, partly because evaluation is challenging. USPIO-magnetic resonance imaging (MRI) potentially allows specific identification of myocardial inflammation but it remains unclear what the USPIO-MRI signal represents. MethodsHistological validation was performed using a murine acute myocardial infarction (MI) model. A multiparametric, multi-time-point MRI methodology was developed, which was applied in patients with acute MI (n = 12), chronic ischemic cardiomyopathy (n = 7), myocarditis (n = 6), dilated cardiomyopathy (n = 5), and chronic sarcoidosis (n = 5). ResultsUSPIO were identified in myocardial macrophages and myocardial interstitium. R1 time-course reflected passive interstitial distribution whereas multi-time-point R2* was also sensitive to active macrophage uptake. R2*/R1 ratio provided a quantitative measurement of myocardial macrophage infiltration. R2* behavior and R2*/R1 ratio were higher in infarcted (p = 0.001) and remote (p = 0.033) myocardium in acute MI and in chronic ischemic cardiomyopathy (infarct: p = 0.008; remote p = 0.010), and were borderline higher in DCM (p = 0.096), in comparison to healthy controls, but were no different in myocarditis or sarcoidosis. An R2*/R1 threshold of 25 had a sensitivity and specificity of 90% and 83%, respectively, for detecting active USPIO uptake. ConclusionsUSPIO are phagocytized by cardiac macrophages but are also passively present in myocardial interstitium. A multiparametric multi-time-point MRI methodology specifically identifies active myocardial macrophage infiltration. Persistent active macrophage infiltration is present in infarcted and remote myocardium in chronic ischemic cardiomyopathy, providing a substrate for HF.

Cardiac‐derived Erythropoietin: A Novel Therapeutic Strategy to Treat Myocardial Infarction?

BackgroundIn response to hypoxia, the kidney is considered the major source for erythropoietin (EPO) – a protein responsible for stimulating hematopoiesis. Interestingly, recombinant human EPO (rhEPO) also has known anti‐apoptotic, cardioprotective, and inotropic effects. Preclinically, supraphysiological concentrations of rhEPO, given at the time of permanent coronary artery occlusion, is effective at reducing apoptosis in the area‐at‐risk, infarct size, and left ventricular remodeling and functional deficits. Clinically, researchers have encountered significant translational difficulties using EPO post‐myocardial infarction, as the hematopoietic effect of chronic rhEPO dosing limits its therapeutic use in patients.Emerging findings demonstrate that EPO mRNA expression occurs in non‐renal tissues, including the liver, bone, and reproductive organs, yet the evidence is divided with regards to the heart. Our preliminary data shows that cardiac EPO expression is upregulated during embryonic development, suggesting it has a paracrine role in cardiac development. Therefore, whether the adult heart produces EPO under a stress (e.g., myocardial infarction) and has physiological relevance remains unknown. Notably, in humans, serum EPO levels are elevated at 3 days post‐myocardial infarction, which indicates that the injured/hypoxic heart may produce EPO in vivo. Accordingly, our objective was to improve our understanding of the regulation and physiological significance of cardiac‐derived EPO using a murine model of myocardial infarction. It was hypothesized that a myocardial infarction would increase cardiac EPO mRNA expression, which may serve as a paracrine factor to preserve cardiac structure and function following an ischemic injury.Methods and ResultsMale CD1 mice were subjected to permanent ligation of the left anterior descending coronary artery to induce a myocardial infarction. At 12 h post‐surgery, hearts were harvested for qPCR analyses, which showed a significant upregulation in EPO mRNA expression. At 2, 4, and 9 weeks post‐myocardial infarction (when hearts were anoxic), hematocrit was significantly elevated, compared to age‐matched shams, indicating that serum EPO levels were still increased at these timepoints. To investigate whether cardiac EPO is driven solely by hypoxia, we subjected mice to severe hypoxia (9% O2) for 24 h and evaluated EPO mRNA expression in the heart and kidney. Indeed, EPO expression was significantly increased in the kidney, while we observed a very modest increase in the heart.ConclusionsHere we show that the heart is a significant non‐renal source of EPO post‐myocardial infarction. Further, profound hypoxia does not significantly drive cardiac‐derived EPO expression, suggesting it is regulated by a hypoxia‐independent mechanism post‐injury. Taken together, endogenous cardiac EPO production may be elevated to provide paracrine cardioprotective support following a myocardial infarction.Support or Funding InformationCanadian Institutes of Health Research. Natural Sciences and Engineering Research Council of Canada.

Induction of Nrg1 and ErbB4 Is Specific to Virus Induced Injury of the Myocardium and May be Detected during Pathogenesis of Viral Myocarditis as Blood‐Based Biomarkers for Diagnosis

BackgroundMyocarditis, inflammation of the myocardium, globally affects >3 million people annually. Heterogeneous histological findings and clinical presentation ranging from flu‐like illness to acute cardiogenic shock make diagnosis exceedingly difficult. The current gold standard for diagnosis requires histological examination of invasive endomyocardial biopsies, which provides a sensitivity of <30% in independently published studies. Although etiologies are diverse, viruses are the most prominent causes of myocarditis. Our studies demonstrated that the coxsackievirus B3 (CVB3), a common pathogen associated with viral myocarditis, manipulates the expression of Neuregulin 1 (Nrg1) and its ligand, receptor tyrosine‐protein kinase, ErbB4. Moreover, previous studies in other viruses have demonstrated preferential expression of the higher affinity Nrg1β versus the less potent Nrg1α isoform. The goal of this research is to determine the specificity of Nrg1 and ErbB4 induction to viral myocarditis as compared to other forms of acute myocardial injury, particularly myocardial infarction (m.i.). Additionally, we aim to analyze expression, tissue and isoform specificity of Nrg1 and ErbB4 in blood and heart tissue during the pathogenesis of viral myocarditis towards developing a non‐invasive blood‐based diagnostic assay via novel biomarkers.Methods4‐week old male A/J mice were sham or CVB3 infected. Blood and tissue were harvested at time points corresponding to the acute, sub‐acute and chronic phases of disease. Specimens were analyzed for mRNA and protein expression and sub‐cellular localization using RT‐qPCR, Western blotting and confocal microscopy, respectively. Expression of Nrg1 and ErbB4 in viral myocarditis models was compared to murine myocardial infarction models via confocal microscopy.ResultsUpregulation (p<0.05) of Nrg1 and ErbB4 protein fragments were observed in murine heart tissue at different phases of viral pathogenesis as compared to m.i. and non‐infected controls. The observed upregulation was specific to the heart and not observed in the infected pancreas or lung. In addition, ~35 kDa and ~26 kDa fragments of Nrg1 and ErbB4 respectively were detected in the plasma of infected mice, while absent in the non‐infected controls. Confocal microscopy revealed that Nrg1 localized to the nuclear periphery while a diffuse increase in cellular ErbB4 expression was observed during infection. mRNA levels of Nrg1β were significantly upregulated (~3‐fold) at the acute phase (6–7 dpi) of viral myocarditis, while the differences in expression of Nrg1α were not significant.ConclusionTissue specific upregulation and fragments detected in the plasma of both ErbB4 and Nrg1, with preferential expression of Nrg1β, were observed as a result of CVB3 infection. Moreover, upregulation was not observed in an ischemic model of acute myocardial injury, indicating a viral specific process. These composite observations support the concept that these biomarkers may ultimately yield a non‐invasive blood‐based diagnostic assay for viral myocarditis.Support or Funding InformationThis research is supported by the Myocarditis Foundation and the Michael Smith Foundation for Health Research.

Protease-activated receptor 2 deficiency in hematopoietic lineage protects against myocardial infarction through attenuated inflammatory response and fibrosis

Ischaemic heart disease is one of the leading causes of death. Protease-activated receptor 2 (PAR2) is widely expressed within the cardiovascular system and is known to mediate inflammatory processes in various immunocytes, such as macrophages, mastocytes and neutrophils. Here, we investigated whether activating macrophage PAR2 modulates cardiac remodelling in a murine model of myocardial infarction. Myocardial infarction was produced by the permanent ligation of the left anterior descending coronary artery (LAD) in C57BL/6J background wild-type (WT) mice transplanted with bone marrow from WT or PAR2 knockout (PAR2 KO) mice. Hematopoietic deficiency of PAR2 had improvement of left ventricular systolic dysfunction and dilatation and decreased fibrosis deposition in remote zone at 1 week after LAD ligation. Inactivation of PAR2 also led to less recruitment of macrophages in myocardium, which was accompanied by decreased expression of pro-inflammatory cytokines. Furthermore, cultured cardiac fibroblasts (CFs) were activated and showed a fibrotic phenotype after being co-cultured in medium containing PAR2-activating macrophage, which enhances interferon-beta (INF-β) expression. The beneficial effects of macrophages with INF-β neutralisation or PAR2-deletion ameliorates the JAK/STAT3 pathway in CFs, which might be attributed to CF activation. These data suggest that macrophage-derived IFN-β plays a crucial role in adverse cardiac remodelling after myocardial infarction, at least in part, through a PAR2-dependent mechanism.

Cardioprotective Potential of Human Endothelial-Colony Forming Cells from Diabetic and Nondiabetic Donors.

Objective: The potential therapeutic role of endothelial progenitor cells (EPCs) in ischemic heart disease for myocardial repair and regeneration is subject to intense investigation. The aim of the study was to investigate the proregenerative potential of human endothelial colony-forming cells (huECFCs), a very homogenous and highly proliferative endothelial progenitor cell subpopulation, in a myocardial infarction (MI) model of severe combined immunodeficiency (SCID) mice. Methods: CD34+ peripheral blood mononuclear cells were isolated from patient blood samples using immunomagnetic beads. For generating ECFCs, CD34+ cells were plated on fibronectin-coated dishes and were expanded by culture in endothelial-specific cell medium. Either huECFCs (5 × 105) or control medium were injected into the peri-infarct region after surgical MI induction in SCID/beige mice. Hemodynamic function was assessed invasively by conductance micromanometry 30 days post-MI. Hearts of sacrificed animals were analyzed by immunohistochemistry to assess cell fate, infarct size, and neovascularization (huECFCs n = 15 vs. control n = 10). Flow-cytometric analysis of enzymatically digested whole heart tissue was used to analyze different subsets of migrated CD34+/CD45+ peripheral mononuclear cells as well as CD34−/CD45− cardiac-resident stem cells two days post-MI (huECFCs n = 10 vs. control n = 6). Results: Transplantation of human ECFCs after MI improved left ventricular (LV) function at day 30 post-MI (LVEF: 30.43 ± 1.20% vs. 22.61 ± 1.73%, p < 0.001; ΔP/ΔTmax 5202.28 ± 316.68 mmHg/s vs. 3896.24 ± 534.95 mmHg/s, p < 0.05) when compared to controls. In addition, a significantly reduced infarct size (50.3 ± 4.5% vs. 66.1 ± 4.3%, p < 0.05) was seen in huECFC treated animals compared to controls. Immunohistochemistry failed to show integration and survival of transplanted cells. However, anti-CD31 immunohistochemistry demonstrated an increased vascular density within the infarct border zone (8.6 ± 0.4 CD31+ capillaries per HPF vs. 6.2 ± 0.5 CD31+ capillaries per HPF, p < 0.001). Flow cytometry at day two post-MI showed a trend towards increased myocardial homing of CD45+/CD34+ mononuclear cells (1.1 ± 0.3% vs. 0.7 ± 0.1%, p = 0.2). Interestingly, we detected a significant increase in the population of CD34−/CD45−/Sca1+ cardiac resident stem cells (11.7 ± 1.7% vs. 4.7 ± 1.7%, p < 0.01). In a subgroup analysis no significant differences were seen in the cardioprotective effects of huECFCs derived from diabetic or nondiabetic patients. Conclusions: In a murine model of myocardial infarction in SCID mice, transplantation of huECFCs ameliorated myocardial function by attenuation of adverse post-MI remodeling, presumably through paracrine effects. Cardiac repair is enhanced by increasing myocardial neovascularization and the pool of Sca1+ cardiac resident stem cells. The use of huECFCs for treating ischemic heart disease warrants further investigation.

Cardiomyocyte‐derived exosomal microRNA‐92a mediates post‐ischemic myofibroblast activation both in vitro and ex vivo

AimsWe hypothesize that specific microRNAs (miRNAs) within cardiomyocyte‐derived exosomes play a pivotal role in the phenoconversion of cardiac myofibroblasts following myocardial infarction (MI).Methods and resultsWe used an established murine model of MI, obtained in vivo via ligation of the left anterior descending coronary artery. We isolated adult cardiomyocytes and fibroblasts, and we assessed the functional role of cardiomyocyte‐derived exosomes and their molecular cargo in the activation of cardiac fibroblasts. We identified and biologically validated miR‐92a as a transcriptional regulator of mothers against DPP homologues 7 (SMAD7), a known inhibitor of α‐smooth muscle actin (α‐SMA), established marker of myofibroblast activation. We found that miR‐92a was significantly (P < 0.05) upregulated in cardiomyocyte‐derived exosomes and in fibroblasts isolated after MI compared with SHAM conditions (n ≥ 6/group). We tested the activation of myofibroblasts by measuring the expression levels of αSMA, periostin, and collagen. Primary isolated cardiac fibroblasts were activated both when incubated with cardiomyocyte‐derived exosomes isolated from ischemic cardiomyocytes and when cultured in conditioned medium of post‐MI cardiomyocytes, whereas no significant difference was observed following incubation with exosomes or medium from sham cardiomyocytes. These effects were attenuated when an inhibitor of exosome secretion, GW4869 (10 μM for 12 h) was included in the experimental setting. Through means of specific miR‐92a mimic and miR‐92a inhibitor, we also verified the mechanistic contribution of miR‐92a to the activation of cardiac fibroblasts.ConclusionsOur results indicate for the first time that miR‐92a is transferred to fibroblasts in form of exosomal cargo and is critical for cardiac myofibroblast activation.

Cardiosomal microRNAs Are Essential in Post-Infarction Myofibroblast Phenoconversion.

The inclusion of microRNAs (miRNAs) in extracellular microvesicles/exosomes (named cardiosomes when deriving from cardiomyocytes) allows their active transportation and ensures cell-cell communication. We hypothesize that cardiosomal miRNAs play a pivotal role in the activation of myofibroblasts following ischemic injury. Using a murine model of myocardial infarction (MI), we tested our hypothesis by measuring in isolated fibroblasts and cardiosomes the expression levels of a set of miRNAs, which are upregulated in cardiomyocytes post-MI and involved in myofibroblast phenoconversion. We found that miR-195 was significantly upregulated in cardiosomes and in fibroblasts isolated after MI compared with SHAM conditions. Moreover, primary isolated cardiac fibroblasts were activated both when incubated with cardiosomes isolated from ischemic cardiomyocytes and when cultured in conditioned medium of post-MI cardiomyocytes, whereas no significant effect was observed following incubation with cardiosomes or medium from sham cardiomyocytes. Taken together, our findings indicate for the first time that a cardiomyocyte-specific miRNA, transferred to fibroblasts in form of exosomal cargo, is crucial in the activation of myofibroblasts.

Targeting Endothelin Receptors in a Murine Model of Myocardial Infarction Using a Small Molecular Fluorescent Probe.

The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)methyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo.

D-Limonene Ameliorates Myocardial Infarction Injury by Reducing Reactive Oxygen Species and Cell Apoptosis in a Murine Model.

Myocardial infarction (MI) leads to high mortality, and pharmacological or percutaneous primary interventions do not significantly inhibit ischemia/reperfusion injuries, particularly those caused by oxidative stress. Recently, research groups have evaluated several naturally occurring antioxidant compounds for possible use as therapeutic alternatives to traditional treatments. Studies have demonstrated that d-limonene (DL), a monoterpene of citrus fruits, possesses antioxidant and cardiovascular properties. Thus, this work sought to elucidate the mechanisms of protection of DL in an isoproterenol-induced murine MI model. It was observed that DL (10 μmol) attenuated 40% of the ST elevation, reduced the infarct area, prevented histological alterations, abolished completely oxidative stress damage, restored superoxide dismutase activity, and suppressed pro-apoptotic enzymes. In conclusion, the present study demonstrated that DL produces cardioprotective effects from isoproterenol-induced myocardial infarction in Swiss mice through suppression of apoptosis.