Permanent Coronary Artery Occlusion Research Articles

Multidimensional immunoimaging to characterize the inflammatory reaction after permanent and temporary coronary artery occlusion in mice

Abstract Background The immune response following acute myocardial infarction encompasses a delicate balance between inflammatory and reparative programs. Our knowledge of these complex mechanisms is mainly derived from studies using a murine model of permanent coronary artery occlusion. In this study we developed, validated and implemented multiparametric imaging methods to investigate cardiac function and the systemic immune response in transient or permanent coronary artery occlusion mouse models. Methods The myocardial infarction models encompassed either transient (40 min) or permanent LAD occlusion in C57BL/6 mice, and non-infarcted mice were used as controls. Two or seven days later, the animals subjected to systemic immunoimaging of the bone marrow, spleen and myocardium with late gadolinium enhancement cardiac MRI (LGE cMRI), 18F-fluorodeoxyglucose (18F-FDG) PET, 18F-Fluorothymidine (18F-FLT) PET, 64Cu-CCR2 PET targeting inflammatory monocytes, 89Zr-CD11b nanobody PET and 19F-HDL-PERFECTA MRI, both targeting myeloid cells. In addition, the same myocardial infarction models were applied to atherosclerosis-prone Apoe−/− and systemic inflammation and plaque progression were assessed by flow cytometry and immunohistochemistry four weeks after infarction. Results Through LGE cMRI and 18F-FDG PET, we observed that temporary coronary occlusion resulted in a smaller infarct size, better cardiac function and viability compared to permanent occlusion. Multiparametric immunoimaging targeting CD11b+ cells by 89Zr-CD11b nanobody PET and Ly6Chi inflammatory monocytes by 64Cu-CCR2 PET demonstrated that mice subjected to transient coronary occlusion had less immune cell influx to the ischemic myocardium. This finding was confirmed by flow cytometry analysis of the infarct zone. In contrast, both myocardial infarction models cause a similar systemic immune response in the bone marrow and spleen as observed with multimodal imaging with subsequent similar numbers of CD11b+ cells in the blood. Both permanent and temporary coronary artery occlusion aggravate atherosclerosis in Apoe−/− mice with higher macrophage and Ly6Chi monocyte numbers in aortas and larger plaque size compared to Apoe−/− mice without myocardial infarction. Conclusions We developed and employed multimodal, multiparametric imaging protocols to characterize the immune response in the heart, bone marrow and spleen in two models of myocardial infarction. While cardiac function was superior in the ischemia reperfusion model, both types of myocardial infarction accelerated atherosclerosis. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): NIH and DFG

Antagonism of N/OFQ attenuates externalization of β1-adrenergic receptor and ventricular arrhythmias in acute myocardial ischemia rat model

Nociceptin/orphanin FQ (N/OFQ) and adrenergic activations play roles in promoting cardiac arrhythmia in acute myocardial ischemia but whether N/OFQ and β1-adrenergic activities interact and how they interact in the arrhythmogenesis are still unknown. We designed this study to investigate the potential interaction of N/OFQ and β1-adrenergic activities and the underlying mechanism in arrhythmogenesis in acute myocardial ischemia. Ventricular arrhythmia was evaluated in anaesthetized rats following permanent coronary artery occlusion (CAO), in presence and absence of UFP-101 (a selective antagonist of N/OFQ receptor). The changes of β1-adrenergic receptor (β1-AR) in plasma membrane of cardiomyocytes were quantitatively evaluated and the relations with the alterations of phosphorylated Raf kinase inhibitor protein (p-RKIP) and phosphorylated connexin 43 (p-Cx43) were investigated. The ventricular arrhythmia was 59% less in the animals pre-treated with UFP-101 than the placebo-treated control (difference of means = −2.41; 95% confidence interval (CI) −2.84 to −1.99; P < 0.001). Meanwhile, p-RKIP and membrane β1-AR in the myocardium were downregulated by 59% and 24%, respectively (p-RKIP: difference of means = −6.91; 95% CI -8.38 to −5.45; P < 0.001; membrane β1-AR difference of means = −27.06; 95% CI -29.89 to −24.23; P < 0.001). Artificial upregulation of RKIP by didymin significant increased β1-AR in plasma membrane of the cardiomyocytes in the animals prone to ventricular arrhythmia. The findings may suggest that activation of N/OFQ receptor in acute myocardial ischemia induces upregulation of p-RKIP, externalization of β1-adrenergic receptor and downregulation of p-Cx43 in the cardiomyocytes, which promotes ventricular arrhythmia.

Chronic treatment with serelaxin mitigates adverse remodeling in a murine model of ischemic heart failure and modulates bioactive sphingolipid signaling

Relaxin is a pleiotropic hormone demonstrated to confer cardioprotection in animal models of myocardial infarction and ischemic heart failure by modulating inflammation, fibrosis and arrhythmogenesis. Several of these pathways in the ischemic myocardium are intricately tied with the downstream signaling of bioactive sphingolipids, which play an active role during post-infarction remodeling. In this current study, we examined the effects of relaxin on sphingosine 1-phosphate (S1P), and the potential benefits of relaxin treatment on cardiac health in a rodent model of ischemic heart failure. Acute (30 min) and sub-acute (24 h) treatment of primary cardiomyocytes with serelaxin (recombinant human relaxin-2) increased the cardiomyocyte content of S1P. In the rodent model, treatment with relaxin for 28 days following myocardial ischemia by way of permanent left coronary artery occlusion improved survival and cardiac function, reduced fibrosis and apoptosis, and mitigated the expression of several pro-inflammatory and pro-fibrotic markers. The expression of beclin-1 (autophagy marker) was also reduced. The expression of S1P was significantly higher in cardiac tissue and plasma samples extracted from serelaxin-treated mice at day 28. In conclusion, our studies show a significant protection from relaxin in ischemic heart disease, and demonstrate the association between relaxin signaling and S1P generation.

Isosteviol improves cardiac function and promotes angiogenesis after myocardial infarction in rats.

Isosteviol has been indicated as a cardiomyocyte protector. However, the underlying mechanism remains unclear. Thus, we sought to confirm the protective effect of isosteviol after myocardial infarction in a model of permanent coronary artery occlusion and investigate the potential proangiogenic activity in vitro and in vivo. A 4-week permanent coronary artery occlusion rat model was generated, and the protective effect of isosteviol was evaluated by echocardiographic imaging and hemodynamics assays. The coronary capillary density was tested by immunochemistry and micro-computed tomography (μCT) imaging. The effect of isosteviol on endothelial cells was determined in human umbilical vein endothelial cells (HUVECs) in vitro and Tg (kdrl: EGFP) zebrafish in vivo. We also examined the expression of related transcription factors by real-time polymerase chain reaction (RT-qPCR). Isosteviol increased ejection fraction (EF), fractional shortening (FS), cardiac systolic index (CI), maximum rate of increase of left ventricular pressure (Max dp/dt), and left ventricular systolic pressure (LVSP) by 32%, 40%, 25%, 26%, and 10%, respectively, in permanent coronary artery occlusion rats. Interestingly, it also promoted coronary capillary density by 2.5-fold. In addition, isosteviol promoted the proliferation and branching of HUVECs in vitro. It also rescued intersegmental vessel (ISV) development and improved endothelial cell proliferation by approximately fivefold (4-6) in zebrafish embryos in vivo. Isosteviol also upregulated the expression of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor A (VEGFA) in zebrafish by fourfold and 3.5-fold, respectively. Our findings suggest that isosteviol is a proangiogenic agent and that this activity is related to its protective effects against myocardial ischemia. After using the permanent coronary artery occlusion model, we demonstrated that isosteviol promotes angiogenesis directly and increases capillary density in myocardial ischemia rats. Isosteviol promotes angiogenesis in zebrafish in vivo and increases vascular endothelial cell proliferation in HUVECs and zebrafish. The angiogenesis activity of isosteviol may be correlated with VEGFA and HIF-1α signaling.

Macrophage depletion impairs adequate cardiac repair in mouse models of myocardial infarction with variable transmurality - insights from multimodality molecular imaging

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Introduction Balanced myocardial tissue inflammation following acute myocardial infarction (MI) is needed for optimal cardiac repair. Macrophages contribute to wound healing, but may also be deleterious. Purpose We investigated the impact of macrophage depletion on early cardiac inflammation and later functional outcome in two models of MI with variable transmurality. Methods C57BL/6N mice received clodronate-liposomes for macrophage depletion (n = 49) or control PBS-liposomes (n = 23). After 24h, mice underwent permanent occlusion (PO) or transient ischemia-reperfusion (I/R, 60min) of the left coronary artery or sham surgery. Cardiac inflammation was assessed on MI + 1d, 3d, and 7d by CXCR4-targeted PET/CT using 68Ga-pentixafor. 99mTc-sestamibi SPECT/CT and cardiac magnetic resonance (CMR) calculated infarct sizes and left ventricular (LV) function at 1wk and 6wks. 18F-NaF PET/CT measured tissue microcalcification at 4wks. Imaging signals were validated by ex vivo autoradiography and immunohistochemistry. Results Clodronate macrophage depletion did not affect infarct size compared to PBS, but perfusion defects at 6wks were significantly larger after PO compared to I/R (%LV, 32 ± 11 vs 14 ± 10, p = 0.01). In both models, infarct CXCR4 expression was higher after macrophage depletion vs PBS at all timepoints (%injected dose (ID)/g; d3: PO: 1.4 ± 0.2 vs 0.9 ± 0.1; I/R: 1.4 ± 0.2 vs 1.0 ± 0.02; p &amp;lt; 0.05), and confirmed by ex vivo autoradiography. Immunostaining demonstrated fewer macrophages and higher neutrophil content within the myocardium after macrophage depletion vs PBS at 1d, 3d, and 7d post-MI. Acute LV rupture after PO was more frequent in macrophage-depleted than PBS mice (37% vs 17%). Conversely, surviving PO mice showed a similarly impaired ejection fraction (EF) after macrophage depletion vs PBS at 6wks (%, 32 ± 9 vs 32 ± 11, p = 0.84). No acute LV rupture was observed after I/R, but macrophage depletion led to worse EF (%, 42 ± 11 vs 54 ± 3, p = 0.1). Macrophage-depleted mice exhibited a dense intracavity thrombus adherent to the infarct wall after either injury, as visualized on CMR at 1wk. 18F-NaF PET identified active calcification localized to the thrombus region 4wks after MI, which was colocalized to CT opaque regions at 6wks. Conclusion Macrophage depletion impairs cardiac repair via several mechanisms including neutrophil-dominated inflammation, LV thrombus formation and tissue calcification. This observation underscores the requirement of macrophages for effective healing and may explain adverse response to broad anti-inflammatory therapy in myocardial ischemia.

CircRNA 010567 plays a significant role in myocardial infarction via the regulation of the miRNA-141/DAPK1 axis.

BackgroundMyocardial infarction (MI), caused by temporary or permanent coronary artery occlusion, poses a serious threat to patients’ lives. Circular RNAs (circRNAs), a new kind of endogenous noncoding RNAs, have been widely studied recently. This study was designed to illustrate and potential molecular mechanisms of circRNA 010567 in hypoxia-induced cardiomyocyte injury in vitro, so as to provide new strategies for the therapy of MI.MethodsH9c2 cells were cultured in anoxic conditions with 94% N2, 5% CO2, and 1% O2 to establish the in vitro MI model. Cell viability and apoptosis were checked using MTT and flow cytometry assay, respectively, Moreover, the levels of circRNA 010567, miR-141, and DAPK1 was determined using qRT-PCR. The putative targets of circRNA 010567 and miR-141 were confirmed by dual-luciferase reporter system and the RNA immunoprecipitation (RIP) assay. The release of creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), and the viability of mitochondria were detected using assay kits.ResultsThe current study revealed that circRNA 010567 and DAPK1 were over-expressed, and miR-141 was low-expressed in hypoxia-induced MI. circRNA 010567 sponges miR-141 and DAPK1 was a direct target of miR-141. Mechanistic investigations revealed that circRNA 010567-siRNA impaired the release of CK-MB and cTnI, and promoted the viability of mitochondria in hypoxia-induced H9c2 cells, while these findings were reversed by the miR-141 inhibitor. In addition, the miR-141 mimic markedly reduced the release of CK-MB and cTnI, and promoted the viability of mitochondria, and these results were reversed by the DAPK1-plasmid. Subsequently, functional experiments revealed that hypoxia-stimulated decreases in H9c2 cell viability, as well as increases in apoptosis and caspase-3 activity, were induced by the miR-141 mimic and circRNA 010567-siRNA. However, these results were reversed by the miR-141 inhibitor and DAPK1-plasmid.ConclusionsOur results demonstrated that circRNA 010567-siRNA played a protective role in hypoxia-induced cardiomyocyte damage via regulating the miR-141/DAPK1 axis, indicating that circRNA 010567-siRNA may be a promising target for MI therapy.

Hepatocyte growth factor intervention to reduce myocardial injury and improve cardiac function on diabetic myocardial infarction rats.

Acute myocardial infarction (AMI) is recognized to be a severe threat to people’s health conditions and life quality. The accumulation of hepatocyte growth factor (HGF) in ischemic myocardium has been observed in both processes of experimental ischemia and reperfusion (I/R) and permanent coronary artery occlusion. The aim of the study was to investigate the effect of HGF on myocardial cell apoptosis, ventricular remodeling and cardiac function after myocardial infarction (MI) in diabetic rats, and to explore whether the effect is mediated by HGF/c-Met signaling pathway. MI significantly increases LVWI and RVWI and myocardial apoptotic index, and up-regulates the expression of HGF and c-Met at mRNA and protein levels in MI control group. The LVWI and RVWI, and myocardial apoptosis were reduced by treatment with HGF, which also increased the myocardial cell viability and the expression of HGF and c-Met. In summary, HGF significantly attenuates myocardial apoptosis and improves cardiac function after AMI in diabetic rats by further enhancing the activation of HGF/c-Met pathway.

Effects of progranulin on the pathological conditions in experimental myocardial infarction model

Progranulin is a secreted growth factor associated with multiple physiological functions in ischemic pathophysiology. However, it is still not fully understood how progranulin is involved in ischemic lesion and cardiac remodeling after myocardial infarction (MI). In this study, we investigated the effects of progranulin on myocardial ischemia and reperfusion injury. We investigated progranulin expression using Western blotting and immunostaining after permanent left coronary artery (LCA) occlusion in mice. Infarct size and the number of infiltrating neutrophils were measured 24 h after permanent LCA occlusion. Recombinant mouse progranulin was administered before LCA occlusion. In addition, we evaluated cardiac function using cardiac catheterization and echocardiography, and fibrosis size by Masson’s trichrome staining after myocardial ischemia/reperfusion in rabbits. Recombinant human progranulin was administered immediately after induction of reperfusion. Progranulin expression increased in the myocardial ischemic area 1, 3, and 5 days after permanent LCA occlusion in mice. The administration of recombinant mouse progranulin significantly attenuated infarct size and infiltrating neutrophils 24 h after permanent LCA occlusion in mice. We also found that administration of recombinant human progranulin ameliorated the deterioration of cardiac dysfunction and fibrosis after myocardial ischemia/reperfusion in rabbits. These findings suggest that progranulin may protect myocardial ischemia/reperfusion injury.

Secreted frizzled-related protein 2, a novel mechanism to induce myocardial ischemic protection through angiogenesis.

Our hypothesis is that Secreted Frizzled-Related Protein 2 (sFPR2) is an important mechanism mediating ischemic cardioprotection, since it is the most upregulated gene in the third window of ischemic preconditioning. One week after permanent coronary artery occlusion (CAO), sFRP2 TG mice exhibited a 49% higher LV ejection fraction and a 36% reduction in infarct size, p < 0.05, and reduced fibrosis in both adjacent and remote zones, along with an increase in collagen type III and a decrease in the collagen type I/III ratio compared with WTL. The ischemic cardioprotection was associated with increased angiogenesis and arteriogenesis, reflected by increased capillary and arteriolar proliferation in the ischemic zone, thereby preserving blood flow after CAO. The angiogenesis and arteriogenesis were mediated by cross talk between myocytes and endothelial cells. The mechanism for cardioprotection and angiogenesis/arteriogenesis did not involve a traditional vascular growth hormone, e.g., VEGF or FGF, but rather cTGF, and ATF6 through the stress signaling pathway. The ATF6 inhibitor, AEBSF, blocked the upregulation of cTGF and both the angiogenesis and arteriogenesis, resulting in abolition of the reduced infarct size and protection of cardiac function in the sFRP2 TG mouse following permanent CAO. sFRP2 is a novel mechanism to induce angiogenesis/arteriogenesis, mediated through the endoplasmatic reticulum (ER) stress signaling pathway, ATF6 and cTGF, which protects the heart from myocardial ischemia.

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.

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.

Accurate identification of myocardial viability after myocardial infarction with novel manganese chelate-based MR imaging.

We developed a novel manganese (Mn2+ ) chelate for magnetic resonance imaging (MRI) assessment of myocardial viability in acute and chronic myocardial infarct (MI) models, and compared it with Gadolinium-based delay enhancement MRI (Gd3+ -DEMRI) and histology. MI was induced in 14 rabbits by permanent occlusion of the left circumflex coronary artery. Gd3+ -DEMRI and Mn2+ chelate-based delayed enhancement MRI (Mn2+ chelate-DEMRI) were performed at 7days (acute MI, n=8) or 8weeks (chronic MI, n=6) after surgery with sequential injection of 0.15mmol/kg Gd3+ and Mn2+ chelate. The biodistribution of Mn2+ in tissues and blood was measured at 1.5 and 24h. Blood pressure, heart rate (HR), left ventricular (LV) function, and infarct fraction (IF) were analyzed, and IF was compared with the histology. The Mn2+ chelate group maintained a stable hemodynamic status during experiment. For acute and chronic MI, all rabbits survived without significant differences in HR or LV function before and after injection of Mn2+ chelate or Gd3+ (p>0.05). Mn2+ chelate mainly accumulated in the kidney, liver, spleen, and heart at 1.5h, with low tissue uptake and urine residue at 24h after injection. In the acute MI group, there was no significant difference in IF between Mn2+ chelate-DEMRI and histology (22.92±2.21% vs. 21.79±2.25%, respectively, p=0.87), while Gd3+ -DEMRI overestimated IF, as compared with histology (24.54±1.73%, p=0.04). In the chronic MI group, there was no significant difference in IF between the Mn2+ chelate-DEMRI, Gd3+ -DEMRI, and histology (29.50±11.39%, 29.95±9.40%, and 29.00±10.44%, respectively, p>0.05), and all three were well correlated (r=0.92-0.96, p<0.01). We conclude that the use of Mn2+ chelate-DEMRI is reliable for MI visualization and identifies acute MI more accurately than Gd3+ -DEMRI.

DPP-4 inhibition by linagliptin prevents cardiac dysfunction and inflammation by targeting the Nlrp3/ASC inflammasome.

We compared the effects of linagliptin (Lina, a DPP4 inhibitor) and GLP-1 receptor activation by exenatide followed by exendin-4 in an infusion pump (EX) on infarct size (IS), post-infarction activation of the inflammasome and remodeling in wild-type (WT) and db/db diabetic mice. Mice underwent 30min ischemia followed by 24h reperfusion. IS was assessed by TTC. Additional mice underwent permanent coronary artery occlusion. Echocardiography was performed 2w after infarction. Activation of the inflammasome in the border zone of the infarction was assessed by rt-PCR and ELISA 2w after reperfusion. Further in vitro experiments were done using primary human cardiofibroblasts and cardiomyocytes exposed to simulated ischemia-reoxygenation. Lina and EX limited IS in both the WT and the db/db mice. Lina and EX equally improved ejection fraction in both the WT and the db/db mice. mRNA levels of ASC, NALP3, IL-1β, IL-6, Collagen-1, and Collagen-3 were higher in the db/db mice than in the WT mice. Infarction increased these levels in the WT and db/db mice. Lina more than EX attenuated the increase in ASC, NALP3, IL-1β, IL-6, Collagen-1 and Collagen-3, TNFα and IL-1β, and decreased apoptosis, especially in the db/db mice. In vitro experiments showed that Lina, but not EX, attenuated the increase in TLR4 expression, an effect that was dependent on p38 activation with downstream upregulation of Let-7i and miR-146b levels. Lina and EX had similar effects on IS and post-infarction function, but Lina attenuated the activation of the inflammasome and the upregulation of collagen-1 and collagen-3 more than direct GLP-1 receptor activation. This effect depends on p38 activation with downstream upregulation of miR-146b levels that suppresses TLR4 expression.

Abstract 590: EphrinA1-Fc Attenuates Dysfunction and Fibrosis in Nonreperfused Myocardium at 4 weeks Post-MI in WT B6 but Not EphA2-R Mutant Mice

Each year, an estimated 805,000 US citizens have a new or recurrent myocardial infarction (MI), causing over 360,000 deaths. We have previously reported that a single intramyocardial administration of recombinant ephrinA1-Fc, a member of the ephrinA/EphA family of membrane-anchored receptor tyrosine kinases, significantly reduced ischemic injury and remodeling 4 days in nonreperfused WT B6 mice via reduced apoptosis and inflammation and increased autophagic flux. After acute ischemia/reperfusion (30 min I/24hr or 4 days R) in WT B6 mice, ephrinA1-Fc demonstrated anti-inflammatory effects coupled with complete functional preservation due to modulation of cardiomyocyte ultrastructure and metabolism. The goal of this study is to assess the capacity of a single intramyocardial administration of ephrinA1-Fc at the time of permanent coronary artery ligation to maintain the structural and functional salvage effects through the wound healing process to stable scar formation in both B6 and EphA2-R-M (EphA2 receptor null mutant) mice. At 4 weeks post-MI in WT B6 mice, ephrinA1-Fc improved ejection fraction by 11%, attenuated cardiomyocyte hypertrophy by 38%, and decreased interstitial fibrosis by 35%. In contrast, these parameters were not different or worsened in EphA2-R-M mice treated with ephrinA1-Fc, suggesting that this receptor plays an important role in ephrinA1-Fc-mediated mitigation of the deleterious ventricular remodeling caused by permanent coronary artery occlusion. To further investigate the effects of ephrinA1-Fc directly on fibroblast function, isolated primary mouse cardiac fibroblasts were cultured with ephrinA1-Fc both with and without pro-fibrotic TGF-β stimulation to determine the direct effects of ephrinA1-Fc on col I and III production, remodeling mediators MMP-2, MMP-9, and TIMP-1, as well as signaling molecules DDR2 and SMADs2/3 as potential mechanistic pathways involved. The results of these assays are forthcoming. This study demonstrates the potential of a single administration of ephrinA1-Fc to not only attenuate acute ischemic injury but also mitigate pathological accumulation of interstitial fibrosis and remodeling, thereby reducing ventricular stiffening and the resultant progression to heart failure.

Pretreatment of carprofen impaired initiation of inflammatory- and overlapping resolution response and promoted cardiorenal syndrome in heart failure

Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are commonly used to control pain, inflammation, and limit the cardinal signs of injury in humans. However, prolonged use of NSAIDs increases the risk of heart attack (myocardial infarction; MI) and the subsequent risk of heart and renal failure. The molecular and cellular mechanism of action for this adverse effect, particularly along the cardiorenal network, is incomplete. To define the mechanism, carprofen (CAP), an NSAID was administered at the dose of 5 mg/kg to C57BL/6 male mice for two weeks. After last dose of CAP treatment mice were subjected to permanent occlusion of coronary artery that induces irreversible cardiac remodeling while maintaining naive and MI-controls. After MI, cardiac pathology and dysfunction were confirmed, along with additional measurements of kidney function, histology, and injury markers, such as plasma creatinine. CAP treatment increased plasma creatinine levels and subsequently, myocardial structural disorganization increased. Kidney neutrophil gelatinase associated lipocalin (NGAL) and protein expression were increased post-MI. After two weeks CAP treatment, the expression of pyrogenic pro-inflammatory cytokines TNF-α and IL-1β was increased compared to non-CAP treated mice, indicative of amplified inflammatory response. There was also evidence that renal injury of both the post-CAP treatment controls and post-CAP MI were much greater than the non-CAP treated naïve controls, as serum creatinine and NGAL levels were elevated along with obvious structural impairment of the glomerulus. Therefore, CAP treatment tampers with the acute inflammatory response that promotes cardiorenal syndrome and non-resolving inflammation post-MI in acute heart failure.

Abstract 16750: Angiogenesis/Arteriogenesis, the Mechanism for Cardioprotection, in Secreted Frizzled Related Protein 2 Transgenic Mice

Secreted frizzled related protein 2 (sFRP2) has been shown to protect against myocardial ischemia primarily mediated by reducing fibrosis. Our hypothesis is that sFRP2 myocardial ischemic protection induced by permanent coronary artery occlusion (CAO) must involve a primary angiogenesis/arteriogenesis mechanism in order to protect the heart in the chronic absence of blood flow. To test this hypothesis, we induced permanent CAO for 1 week in cardiac specific sFRP2 transgenic (TG) mice (n=12), which exhibited reduced infarct size vs. their wild type littermates (WTL; n=13) (16±1 vs. 25±2%) and improved left ventricular ejection fraction (LVEF) (61±6 vs. 45±6%). These salutary effects were accompanied by angiogenesis (170% increase in capillary proliferation and a 54% increase in capillary density) and arteriogenesis (106% increase in arteriole proliferation). sFRP2 increased in vitro tubule formation in HUVEC cells by 46% (p&lt;0.05), compared to vehicle. As expected, sFRP2 TG mice also exhibited decreased remote fibrosis, compared to WTL (1.6±0.1 vs.± 3.7±0.3%; n=4-6/group). ATF6, an ER stress response transcription factor, was upregulated in sFRP2 TG mice. When ATF6 was blocked with AEBSF, sFRP2 TG mice no longer demonstrated cardioprotection with permanent CAO, as reflected by similar infarct size, LV EF, capillary and arteriolar proliferation observed in WTL. cTGF, a pro-angiogenic factor, was also upregulated in sFRP2 TG mice. Blocking cTGF with a cTGF antibody, eliminated the increased tubule formation with sFRP2 treatment. AEBSF also eliminated the increased cTGF in sFRP2 TG, confirming ATF6 as the upstream regulator of cTGF, responsible for the angiogenesis and arteriogenesis observed in the sFRP2 TG mice. To determine if angiogenesis/arteriogenesis was the initiating mechanism inducing cardioprotection, blood flow, measured ultrasonically at 1 day following CAO, was increased significantly more, p&lt;0.05, in the ischemic zone of sFRP2 TG mice (2.1±0.46 A.U.) compared with blood flow in WTL (0.4 ±0.09 A.U.). Thus, angiogenesis/arteriogenesis appears to be the primary cardioprotective mechanism induced by sFRP2, with reduced fibrosis playing a secondary role.

Abstract 398: GLP-1 Receptor Agonist Administered After the Infarct Suppresses miR-33 Expression in Lean Mice Subjected to Permanent Coronary Artery Occlusion

MicroRNAs (miRs) regulate post-transcriptional gene expression and protein translation. There is now increasing evidence that miRs regulate mitochondrial turnover. miR-33 regulates cholesterol metabolism and mitochondrial function in cardiac fibroblasts and macrophages and has been implicated in promoting fibrosis. Excessive fibrosis contributes to the development of adverse LV remodeling. Given our recent findings that a glucagon-like peptide-1 receptor agonist (GLP1Ra) [Sigma] induces mitophagy and mitigates post-MI LV remodeling, the purpose of this study was to determine its effects on miR-33 expression after MI. We analyzed miR-33 expression in the viable regions of hearts in lean mice after permanent coronary arterial ligation (PCAL) and subsequent treatment with GLP1Ra administered 2h and 48h after the infarction (total of 2 doses). PCAL was associated with a marked increase in miR-33 that peaked at day 3 (data not shown). In the presence of GLP1Ra, miR-33 expression was suppressed 3 days post-PCAL ( A ). Polysome profiling revealed that miR-33 was decreased in the translating fractions of GLP1Ra-treated mice and increased in the non-translating fraction (80S free ribosomes) compared to vehicle ( B and C ). Decreased association of miR-33 with polysomes in GLP1Ra-treated mice would allow for increased target translation. Given that suppression of miR-33 is implicated in promoting fibrosis, a suppression of this miR by acute GLP1Ra treatment could explain the beneficial effects of this drug in limiting adverse cardiac remodeling.

Abstract 350: Brown Adipose Tissue, a Novel Mechanism to Induce both Angiogenesis and Arteriogenesis

Brown adipose tissue (BAT) is known to protect metabolism, but the extent to which it mediates angiogenesis and cardioprotection, the goal of this investigation, is not known. The Regulator of G Protein Signaling 14 (RGS14) knockout (KO) mouse is unique in that it not only extends longevity, but also enhances several aspects of healthful aging, i.e., protects against diabetes, obesity, exercise intolerance and increased brown adipose tissue by 47% compared with wild type littermates (WT). However, less is known about its role in cardioprotection. Accordingly we examined the effects of chronic myocardial ischemia after 1 and 3 weeks of permanent coronary artery occlusion (CAO) in 3-4 month old RGS14 KO and their WT. Scar size after 3 weeks CAO, determined by histology, was decreased by 33±5% in RGS14 KO, which was only possible in the face of permanent CAO if angiogenesis/arteriogenesis developed in the RGS14 KO mice hearts. Indeed, both angiogenesis assessed by Ki 67 endothelial cell staining and arteriogenesis, assessed by Ki 67 arteriole staining, more than doubled compared with WT. This was associated with a 206±25.5% increase in VEGF in the RGS14 KO hearts compared with WT. We then examined the role of BAT by using a simulated BAT KO, i.e., BAT was transplanted from the RGS14 KO to WT. In the WT with BAT transplants VEGF was increased by 90%. Infarct size, assessed as the fraction of area at risk, was lower, p&lt;0.05, in the BAT transplants (27±2.8%), compared with infarct size in RGS14 KO donors (44±1.9%), a value similar to that observed in WT without the BAT transplant (46±1.0%). Thus, brown adipose tissue appears to be a novel mechanism to induce both angiogenesis and arteriogenesis, resulting in vascular protection from ischemia.

Essential role for smooth muscle cell stromal interaction molecule-1 in myocardial infarction.

Stromal interacting molecule-1 (STIM1) plays a role in coordinating calcium signaling in different cell types. The increase or deletion of STIM1 expression in cardiomyocyte causes cardiac complication. Moreover, the deletion of STIM1 in endothelial cell causes vascular endothelial dysfunction. However, the disruption of STIM1 in smooth muscle cells (SMC) has no effect on endothelial function but protects vascular function when mice are infused with angiotensin-II. Nevertheless, the role of SMC-STIM1 in acute and chronic myocardial infarction (MI) induced by acute ischemia-reperfusion injury and permanent coronary artery occlusion is unknown. Stim1 were generated and crossed into the SM22α-Cre backgrounds. SM22α-Cre causes deletion of STIM1 floxed genes in adult SMC (Stim1). Control and Stim1 mice were subjected to acute ischemia-reperfusion injury. Hearts were then harvested and incubated with triphenyltetrazolium chloride to determine the infarct size. In control mice which are subjected to ischemia-reperfusion, the heart developed a significant infarct associated with an increase in STIM1 expression. Interestingly, the infarct size was substantially reduced in Stim1 mice. The protection in Stim1 mice against ischemia-reperfusion injury involves the modulation of endoplasmic reticulum stress, apoptosis, oxidative stress, protein kinase B, and mitogen-activated protein (MAP) kinase (ERK1/2 and p38) signaling, and inflammation. Furthermore, in another model of chronic MI induced by permanent coronary artery occlusion, SMC-STIM1 disruption significantly reduced myocardial infarct size and improved cardiac function. Our results provide new evidence that SMC-STIM1 disruption is a novel mechanism that protects the heart from MI through reduction of endoplasmic reticulum stress, oxidative stress, MAP-Kinase, apoptosis, and inflammation.

Allogeneic pASC transplantation in humanized pigs attenuates cardiac remodeling post-myocardial infarction.

Cell therapy repair strategies using adult mesenchymal stromal cells have shown promising evidence to prevent cardiac deterioration in rodents even in the absence of robust differentiation of the cells into cardiomyocytes. We tested whether increasing doses of porcine adipose-tissue derived mesenchymal stem cells (pASCs) increase cardiac tissue perfusion in pigs post-myocardial infarction (MI) receiving angiotensin-converting-enzyme inhibitor (ACE inhibitors) and Beta-blockers similarly to patients. Female pigs were subjected to MI induction by sponge permanent occlusion of left circumflex coronary artery (LCx) generating approximately 10% of injured LV area with minimum hemodynamic impact. We assessed tissue perfusion by real time myocardial perfusion echocardiography (RTMPE) using commercial microbubbles before and following pASCs treatment. Four weeks after the occlusion of the left circumflex artery, we transplanted placebo or pASCs (1, 2 and 4x106 cells/Kg BW) into the myocardium. The highest dose of pASCs increased myocardial vessel number and blood flow in the border (56% and 3.7-fold, respectively) and in the remote area (54% and 3.9-fold, respectively) while the non-perfused scar area decreased (up to 38%). We also found an increase of immature collagen fibers, although the increase in total tissue collagen and types I and III was similar in all groups. Our results provide evidence that pASCs-induced stimulation of tissue perfusion and accumulation of immature collagen fibers attenuates adverse remodeling post-MI beyond the normal beneficial effects associated with ACE inhibition and beta-blockade.