Left Coronary Ligation Research Articles

Abstract 5470: Cardiomyocyte-specific Overexpression of Nox4 Attenuates Adverse Cardiac Remodeling After Myocardial Infarction

Background. Reactive oxygen species (ROS) production is implicated in the development of cardiac remodeling after myocardial infarction (MI). NADPH oxidases are major sources of cardiovascular ROS, with cardiomyocytes expressing both Nox2 and Nox4 isoforms. We previously showed that Nox2 contributes significantly to the processes underlying adverse cardiac remodeling and contractile dysfunction post-MI, but the effect of Nox4 remains unclear. The aim of this study was to investigate the role of Nox4 in cardiac remodelling after MI. Methods and Results. We generated transgenic mice (TG) with cardiomyocyte-specific overexpression of Nox4, expressing the mouse full-length Nox4 cDNA under control of the α -MHC promoter. TG were backcrossed onto a C57BL6/J background and wild-type littermates (WT) used as controls. Nox4 TG mice were grossly similar to WT in terms of body weight, growth, activity, fertility and basal cardiac function. TG displayed heart-specific expression of the Nox4 transgene, had significantly higher Nox4 protein expression, over 60% higher heart NADPH oxidase activity and 42% increased hydrogen peroxide production (all P<0.05). Compared with WT, p22 phox protein expression was increased by 2.4-fold, which may be related to an increased protein stability. There was no change in protein expression level of Nox2 or eNOS. MI was achieved by permanent left coronary ligation for 4 weeks. Compared with WT/MI, TG/MI groups displayed significantly high survival rate (72 % vs 95%) and less hypertrophy at 4 weeks post MI in terms of heart/body weight ratio (6.95±0.16 vs 6.44±0.14, p<0.01), although the infarct size was similar (38.3±2.0 vs 39.9±2.8%). TG mice also exhibited less apoptosis and interstitial cardiac fibrosis (0.52±0.03 vs 0.65±0.05%, p<0.05), and lower increases in expression of fibronectin and procollagen III mRNA. Both echocardiography and cardiac catheterization demonstrated less left ventricular cavity dilatation and a preservation of cardiac function in TG than WT mice. Conclusions. In contrast to Nox2, which contributes to cardiac dilation, contractile dysfunction and fibrosis, cardiomyocyte Nox4 was found to exert protective effects against adverse remodelling post-MI.

Survival and Cardiac Remodeling After Myocardial Infarction Are Critically Dependent on the Host Innate Immune Interleukin-1 Receptor-Associated Kinase-4 Signaling

The innate immune system greatly contributes to the inflammatory process after myocardial infarction (MI). Interleukin-1 receptor-associated kinase-4 (IRAK-4), downstream of Toll/interleukin-1 receptor signaling, has an essential role in regulating the innate immune response. The present study was designed to determine the mechanism by which IRAK-4 is responsible for the cardiac inflammatory process, which consequently affects left ventricular remodeling after MI. Experimental MI was created in IRAK-4(-/-) and wild-type mice by left coronary ligation. Mice with a targeted deletion of IRAK-4 had an improved survival rate at 4 weeks after MI. IRAK-4(-/-) mice also demonstrated attenuated cardiac dilation and decreased inflammation in the infarcted myocardium, which was associated with less proinflammatory and Th1 cytokine expression mediated by suppression of nuclear factor-kappaB and c-Jun N-terminal kinase activation. IRAK-4(-/-) mice had fewer infiltrations of CD45+ leukocytes and CD11c+ dendritic cells, inhibition of apoptosis, and reduced fibrosis and nitric oxide production. Cardiac dendritic cells in IRAK-4(-/-) mice were relatively immature or functionally naïve after MI in that they demonstrated less cytokine and costimulatory molecule gene expression. Furthermore, IRAK-4(-/-) dendritic cells have less mobilization capacity. Transfer of wild type-derived bone marrow dendritic cells into IRAK-4(-/-) mice for functional dendritic cell reconstitution negated the survival advantage and reduced the cardiac dilation observed with IRAK-4(-/-) mice at 28 days after MI. Deletion of IRAK-4 has favorable effects on survival and left ventricular remodeling after MI through modification of the host inflammatory process by blunting the detrimental bone marrow dendritic cells mobilization after myocardial ischemia.

Naive Rat Amnion-Derived Cell Transplantation Improved Left Ventricular Function and Reduced Myocardial Scar of Postinfarcted Heart

Stem cells contained in the amniotic membrane may be useful for cellular repair of the damaged heart. Previously, we showed that amnion-derived cells (ADCs) express embryonic stem cell surface markers and pluripotent stem cell-specific transcription factor genes. These ADCs also possess the potential for mesoderm (cardiac) lineage differentiation. In the present study we investigated whether untreated naive ADC transplantation into the injured left ventricular (LV) myocardium is beneficial as a cell-based cardiac repair strategy in a rat model. ADCs were isolated from Lewis rat embryonic day 14 amniotic membranes. FACS analysis revealed that freshly isolated ADCs contained stage-specific embryonic antigen-1 (SSEA-1), Oct-4-positive cells, and mesenchymal stromal cells, while hematopoietic stem cell marker positive cells were absent. Reverse transcription-PCR revealed that naive ADCs expressed cardiac and vascular specific genes. We injected freshly isolated ADCs (2 x 10(6) cells suspended in PBS, ADC group) into acutely infarcted LV myocardium produced by proximal left coronary ligation. PBS was injected in postinfarction controls (PBS group). Cardiac function was assessed at 2 and 6 weeks after injection. ADC treatment attenuated LV dilatation and sustained LV contractile function at 2 and 6 weeks in comparison to PBS controls (p < 0.05, ANOVA). LV peak systolic pressure and maximum dP/dt of ADC-treated heart were higher and LV end-diastolic pressure and negative dP/dt were lower than in PBS controls (p < 0.05). Histological assessment revealed that infarcted myocardium of the ADC-treated group had less fibrosis, thicker ventricular walls, and increased capillary density (p < 0.05). The fate of injected ADCs was confirmed using ADCs derived from EGFP(+) transgenic rats. Immunohistochemistry at 6 weeks revealed that EGFP(+) cells colocalized with von Willebrand factor, alpha-smooth muscle actin, or cardiac troponin-I. Our results suggest that naive ADCs are a potential cell source for cellular cardiomyoplasty.

Hypoxic preconditioning protects rat hearts against ischaemia–reperfusion injury: role of erythropoietin on progenitor cell mobilization

Preconditioning, such as by brief hypoxic exposure, has been shown to protect hearts against severe ischaemia. Here we hypothesized that hypoxic preconditioning (HPC) protects injured hearts by mobilizing the circulating progenitor cells. Ischaemia-reperfusion (IR) injury was induced by left coronary ligation and release in rats kept in room air or preconditioned with 10% oxygen for 6 weeks. To study the role of erythropoietin (EPO), another HPC + IR group was given an EPO receptor (EPOR) antibody via a subcutaneous mini-osmotic pump 3 weeks before IR induction. HPC alone gradually increased haematocrit, cardiac and plasma EPO, and cardiac vascular endothelial growth factor (VEGF) only in the first two weeks. HPC improved heart contractility, reduced ischaemic injury, and maintained EPO and EPOR levels in the infarct tissues of IR hearts, but had no significant effect on VEGF. Interestingly, the number of CD34(+)CXCR4(+) cells in the peripheral blood and their expression in HPC-treated hearts was higher than in control. Preconditioning up-regulated cardiac expression of stromal derived factor-1 (SDF-1) and prevented its IR-induced reduction. The EPOR antibody abolished HPC-mediated functional recovery, and reduced SDF-1, CXCR4 and CD34 expression in IR hearts, as well as the number of CD34(+)CXCR4(+) cells in blood. The specificity of neutralizing antibody was confirmed in an H9c2 culture system. In conclusion, exposure of rats to moderate hypoxia leads to an increase in progenitor cells in the heart and circulation. This effect is dependent on EPO, which induces cell homing by increased SDF-1/CXCR4 and reduces the heart susceptibly to IR injury.

Downregulation of ferritin heavy chain increases labile iron pool, oxidative stress and cell death in cardiomyocytes

Ferritin heavy chain (FHC) protein was significantly reduced in murine failing hearts following left coronary ligation or thoracic transverse aortic constriction. The mRNA expression of FHC was not significantly altered in failing hearts, compared to that in control sham-operated hearts. Prussian blue staining revealed spotty iron depositions in myocardial infarct failing hearts. Oxidative stress was enhanced in the myocardial infarct failing hearts, as evidenced by increases in 4-hydroxy-2-nonenal and 8-hydroxy-2′-deoxyguanosine immunoreactivity. To clarify the functional significance of FHC downregulation in hearts, we infected rat neonatal cardiomyocytes with adenoviral vector expressing short hairpin RNA targeted to FHC (Ad-FHC-RNAi). The downregulation of FHC induced a reduction in the viability of cardiomyocytes. The relative number of iron deposition-, 4-hydroxy-2-nonenal- or 8-hydroxy-2′-deoxyguanosine-positive cardiomyocytes was significantly higher in Ad-FHC-RNAi-infected cardiomyocytes than in control vector-infected cardiomyocytes. Treatment of Ad-FHC-RNAi-infected cardiomyocytes with desferrioxamine, an iron chelator, significantly reduced the number of iron, 4-hydroxy-2-nonenal or 8-hydroxy-2′-deoxyguanosine-positive cells, and increased viability. In addition, treatment with N-acetyl cysteine, an antioxidant, significantly reduced the number of 4-hydroxy-2-nonenal- or 8-hydroxy-2′-deoxyguanosine-positive cells. Reduced viability in Ad-FHC-RNAi-infected cardiomyocytes was significantly improved with N-acetyl cysteine treatment. These findings indicate that excessive free iron and the resultant enhanced oxidative stress caused by downregulation of FHC lead to cardiomyocyte death. The decrease in FHC expression in failing hearts may play an important role in the pathogenesis of heart failure.

Proteomic Analysis of Left Ventricular Remodeling in an Experimental Model of Heart Failure

The development of chronic heart failure (CHF) following myocardial infarction is characterized by progressive alterations of left ventricle (LV) structure and function called left ventricular remodeling (LVR), but the mechanism of LVR remains still unclear. Moreover, information concerning the global alteration protein pattern during the LVR will be helpful for a better understanding of the process. We performed differential proteomic analysis of whole LV proteins using an experimental model of CHF in which myocardial infarction was induced in adult male rats by left coronary ligation. Among 1000 protein spots detected in 2D-gels, 49 were differentially expressed in LV of 2-month-old CHF-rats, corresponding to 27 different identified proteins (8 spots remained unidentified), classified in different functional groups as being heat shock proteins, reticulum endoplasmic stress proteins, oxidative stress proteins, glycolytic enzymes, fatty acid metabolism enzymes, tricarboxylic acid cycle proteins and respiratory chain proteins. We validated modulation of selected proteins using Western blot analysis. Our data showed that proteins involved in cardiac metabolism and oxidative stress are modulated during LVR. Interestingly, proteins of stress response showed different adaptation pathways in the early and late phase of LVR. Expression of four proteins, glyceraldehyde-3-phosphate dehydrogenase, alphaB-crystallin, peroxiredoxin 2, and isocitrate dehydrogenase, was linked to echographic parameters according to heart failure severity.

Reduction in Hemoglobin–Oxygen Affinity Results in the Improvement of Exercise Capacity in Mice With Chronic Heart Failure

This study examined whether a reduction in hemoglobin-oxygen affinity improves exercise capacity in mice with heart failure. Exercise intolerance is a major determinant of quality of life in patients with chronic heart failure. One of the major goals of the treatment for chronic heart failure is to improve quality of life. Four weeks after left coronary ligation, we transplanted bone marrow cells isolated from the transgenic mice expressing a hemoglobin variant with low oxygen affinity, Presbyterian, into the lethally irradiated mice with heart failure or administered a synthetic allosteric modifier of hemoglobin. The mice were then exercised on a treadmill. Four weeks after the left coronary artery ligation, mice showed cardiac dysfunction and chamber dilation, which were characteristics of heart failure. The transplantation led to a reduction in hemoglobin-oxygen affinity and an increase in oxygen supply for skeletal muscle without changes in muscle properties. The transplanted mice showed improved running performance on a treadmill despite impaired cardiac contractility. Furthermore, administration of the synthetic allosteric modifier of hemoglobin showed a similar effect. Allosteric modification of hemoglobin represents a therapeutic option for improving exercise capacity in patients with chronic heart failure. One mechanism of improvement in exercise capacity is enhanced oxygen delivery in the skeletal muscle.

Effect of a long‐term treatment with a low‐dose granulocyte colony‐stimulating factor on post‐infarction process in the heart

Although beneficial effects of granulocyte colony-stimulating factor (G-CSF) have been demonstrated on post-myocardia infarction (MI) process, the mechanisms and feasibility are not fully agreed yet. We investigated effects of a long-term treatment with a low-dose G-CSF started 1 day after the onset of MI, on post-infarction process. One day after being made MI by left coronary ligation, mice were given G-CSF (10 μg/kg/day) for 4 weeks. The G-CSF treatment resulted in a significant mitigation of cardiac remodelling and dysfunction. In the G-CSF-treated hearts, the infarcted scar was smaller with less fibrosis and abundant vessels while in the non-infarcted area, hypertrophic cardiomyocytes with attenuated degenerative changes and reduced fibrosis were apparent. These effects were accompanied by activation of signal transducer and activator of transcription 3 (STAT3) and Akt and also by up-regulation of GATA-4, myosin heavy chain and matrix metalloproteinases-2 and -9. Apoptosis of cardiomyocytes appeared insignificant at any stages. Parthenolide, a STAT3 inhibitor, completely abolished the beneficial effects of G-CSF on cardiac function and remodelling with loss of effect on both anti-cardiomyocyte degeneration and anti-fibrosis. In contrast, wortmannin, an Akt inhibitor, did not affect G-CSF-induced benefis despite cancelling vessel increase. In conclusion, treatment with G-CSF at a small dose but for a long duration beneficially affects the post-infarction process possibly through STAT3-mediated anti-cardiomyocyte degeneration and anti-fibrosis, but not through anti-cardiomyocyte apoptosis or Akt-mediated angio-genesis. The findings may also imply a more feasible way of G-CSF administration in the clinical settings.

Time course of echocardiographic and electrocardiographic parameters in myocardial infarct in rats

In animal models the evaluation of myocardial infarct size in vivo and its relation to the actual lesion found post mortem is still a challenge. The purpose of the current study was to address if the conventional electrocardiogram (ECG) and/or echocardiogram (ECHO) could be used to adequately predict the size of the infarct in rats. Wistar rats were infarcted by left coronary ligation and then ECG, ECHO and histopathology were performed at 1, 7 and 28 days after surgery. Correlation between infarct size by histology and Q wave amplitude in lead L1 was only found when ECGs were performed one day post-surgery. Left ventricular diastolic and systolic dimensions correlated with infarct size by ECHO on day 7 post-infarction. On days 7 and 28 post-infarction, ejection indexes estimated by M-mode also correlated with infarct size. In summary we show that conventional ECG and ECHO methods can be used to estimate infarct size in rats. Our data suggest that the 7-day interval is actually the most accurate for estimation of infarct size by ECHO.

Reply to “Letter to the editor: Infarct size measurements are critically important when comparing interventions affecting ventricular remodeling”

Reply to “Letter to the editor: Infarct size measurements are critically important when comparing interventions affecting ventricular remodeling”

BMS-747158-02: A novel PET myocardial perfusion imaging agent

BMS-747158-02 is a fluorine 18-labeled pyridaben derivative designed as a new myocardial perfusion imaging agent for use with positron emission tomography (PET). This study evaluated BMS-747158-02 in animal models of cardiac perfusion and compared it with established single photon emission computed tomography agents. In a rat biodistribution study, BMS-747158-02 (15 microCi) had substantially higher myocardial uptake than technetium 99m sestamibi (100 microCi) at 15 minutes (3.5% +/- 0.3% %ID/g vs 1.9% +/- 0.1% %ID/g) and 120 minutes (3.2% +/- 0.4% of injected dose per gram vs 1.8% +/- 0.0% of injected dose per gram) after intravenous administration. Uptake ratios of heart to lung and liver at 60 minutes were also higher for BMS-747158-02 (12.7 +/- 1.4 and 3.7 +/- 0.2, respectively) than Tc-99m sestamibi (5.9 +/- 0.5 and 2.4 +/- 0.4, respectively). In an isolated rabbit heart model at flow rates of 1.66 to 5.06 mL x min(-1).g(-1) wet left ventricular weight, the net BMS-747158-02 heart uptake increased proportionally (0.93 +/- 0.15 to 2.44 +/- 0.40 mL.min(-1) x g(-1)) and to a greater extent than that of thallium 201 (0.76 +/- 0.02 to 1.11 +/- 0.02 mL x min(-1) x g(-1)) or Tc-99m sestamibi (0.49 +/- 0.03 to 0.77 +/- 0.08 mL x min(-1) x g(-1)). PET imaging with BMS-747158-02 showed a clear and sustained cardiac uptake in rats, rabbits, and nonhuman primates with minimal lung interference and rapid liver clearance. Myocardial perfusion deficit zones created by either permanent left coronary ligation or reperfusion after ligation in rats were both clearly identified on PET cardiac images of BMS-747158-02 and had good agreement with in vitro histology. BMS-747158-02 exhibited high and sustained cardiac uptake that was proportional to blood flow, and it represents a new class of PET myocardial perfusion imaging agent.

Abstract 1395: Role of High Mobility Group Box 1 Protein in Post-Infarction Healing Process and Left Ventricular Remodeling

Background: The appropriate infarct healing process is prerequisite for preventing left ventricular (LV) remodeling after myocardial infarction (MI). High mobility group box1 protein (HMGB1) is derived from necrotic cells and activated macrophages and involved in tissue repair as immunostimulatory signal. However, the role of HMGB1 in post-MI LV remodeling is unknown. Methods and Results: We examined 35 patients with first ST-elevated acute MI. Peak serum HMGB1 level, determined by serial measurements (q. 6h), did not differ in age, sex, coronary risk factors and infarct site. Its peak level was markedly higher in patients with adverse in-hospital clinical outcomes, including pump failure, cardiac rupture, LV aneurysm and cardiac death, compared with those without (all p&lt;0.05). Then we performed experimental study using rat MI model, induced by permanent left coronary ligation. Anti-HMGB1 antibodies (1 mg/day, MI/antiH, n=20) or control antibodies (MI/C, n=20) were injected subcutaneously every 24 hours for 7 days. Sham-operated rats served as controls (n=10). Quantitative RT-PCR and immunoblotting showed that HMGB1 expression was increased in the infarcted area peaking at day 3 in MI/C compared with sham (p&lt;0.05). Its expression was localized in myocytes and inflammatory cells by immunohistochemistry. The mRNA level of TNF-α (−48%, p=0.03) and IL1-β (−44%, p=0.02) and the number of infiltrated macrophages (p=0.04) in the infarcted area were markedly reduced at day 3 in MI/antiH compared with MI/C. Echocardiographic and hemodynamic studies at day 7 showed increased LV end-diastolic dimension (p=0.03), LVEDP (p=0.03), and decreased LV max dP/dt (p=0.01) in MI/antiH compared with MI/C. Histologically, scar thinning in the infarcted area (0.53±0.23 vs 0.68±0.21 mm, p=0.02) and hypertrophy in the non-infarcted myocardium (2.01±0.23 vs 1.63±0.21 mm, p=0.03) were more prominent in MI/antiH compared with MI/C at day 14. Conclusions: Elevation of serum HMGB1 level is associated with adverse clinical outcomes in patients with acute MI. However, blockade of HMGB1 in rat MI model aggravated early LV remodeling through impaired infarct healing and advanced scar thinning, suggesting an essential role of HMGB1 in the appropriate repairing process after MI.

Thyroid Function Disturbance and Type 3 Iodothyronine Deiodinase Induction after Myocardial Infarction in Rats—A Time Course Study

In humans, there is a significant decrease in serum T(3) and increase in rT(3) at different time points after myocardial infarction, whereas serum TSH and T(4) remain unaltered. We report here a time course study of pituitary-thyroid function and thyroid hormone metabolism in rats subjected to myocardial infarction by left coronary ligation (INF). INF- and sham-operated animals were followed by serial deiodination assays and thyroid function tests, just before, and 1, 4, 8, and 12 wk after surgery. At 4 and 12 wk after INF, liver type 1 deiodinase activity was significantly lower, confirming tissue hypothyroidism. Type 3 deiodinase (D3) activity was robustly induced 1 wk after INF only in the infarcted myocardium. Reminiscent of the consumptive hypothyroidism observed in patients with large D3-expressing tumors, this induction of cardiac D3 activity was associated with a decrease in both serum T(4) ( approximately 50% decrease) and T(3) (37% decrease), despite compensatory stimulation of the thyroid. Thyroid stimulation was documented by both hyperthyrotropinemia and radioiodine uptake. Serum TSH increased by 4.3-fold in the first and 3.1-fold in the fourth weeks (P < 0.01), returning to the basal levels thereafter. Thyroid sodium/iodide-symporter function increased 1 wk after INF, accompanying the increased serum TSH. We conclude that the acute decrease in serum T(4) and T(3) after INF is due to increased thyroid hormone catabolism from ectopic D3 expression in the heart.

An Elastic, Biodegradable Cardiac Patch Induces Contractile Smooth Muscle and Improves Cardiac Remodeling and Function in Subacute Myocardial Infarction

Our objective in this study was to apply an elastic, biodegradable polyester urethane urea (PEUU) cardiac patch onto subacute infarcts and to examine the resulting cardiac ventricular remodeling and performance. Myocardial infarction induces loss of contractile mass and scar formation resulting in adverse left ventricular (LV) remodeling and subsequent severe dysfunction. Lewis rats underwent proximal left coronary ligation. Two weeks after coronary ligation, a 6-mm diameter microporous PEUU patch was implanted directly on the infarcted LV wall surface (PEUU patch group, n = 14). Sham surgery was performed as an infarction control (n = 12). The LV contractile function, regional myocardial wall compliance, and tissue histology were assessed 8 weeks after patch implantation. The end-diastolic LV cavity area (EDA) did not change, and the fractional area change (FAC) increased in the PEUU patch group (p < 0.05 vs. week 0), while EDA increased and FAC decreased in the infarction control group (p < 0.05). The PEUU patch was largely resorbed 8 weeks after implantation and the LV wall was thicker than infarction control (p < 0.05 vs. control group). Abundant smooth muscle bundles with mature contractile phenotype were found in the infarcted myocardium of the PEUU group. The myocardial compliance of the PEUU group was distributed between normal myocardium and infarction control (p < 0.001). Implantation of a novel biodegradable PEUU patch onto a subacute myocardial infarction promoted contractile phenotype smooth muscle tissue formation and improved cardiac remodeling and contractile function at the chronic stage. Our findings suggest a new therapeutic option against post-infarct cardiac failure.

Skeletal muscle gene transfer and regulated expression of IGF‐I increases function of the failing heart

Current methods of cardiac gene transfer include intramuscular injection (im) into heart muscle or coronary delivery. When applied to rodents with heart failure, these surgical approaches are associated with high mortality, an impediment that would also complicate clinical studies. To circumvent this, we selected a transgene, Insulin-like Growth Factor-I (IGF-I), which may have favorable effects on heart function when secreted from a remote site. This study examines the feasibility of skeletal muscle injection of adeno-associated virus-5 encoding IGF-I under tet regulation (AAV5.IGFI-tet). Left coronary ligation was performed in rats and MI confirmed 1wk later by echocardiography. Rats with MI then received 2×1012 vp of AAV5.IGFI-tet in the anterior tibialis (im). Four wks later they were randomly assigned to receive doxycycline in the drinking water (IGF-On; n=5) or not (IGF-Off; n=3). Ten wks post MI anesthetized rats underwent a final echo and terminal study in which a 1.8F pressure-volume transducer was placed in the left ventricle (LV). IGF-On rats showed an increased in LV fractional area change (p=0.04), ejection fraction (p=0.04), and velocity of circumferential fiber shortening (p=0.04), and had smaller LV end-diastolic (p=0.002) and end-systolic (p=0.003) dimensions. LV function was improved with dobutamine infusion (Table; IGF-On, n=4; IGF-Off, n=3). Serum IGF-I was increased 5 wk after activation (IGF-On: 218±7 ng/ml; IGF-Off: 158±24 ng/ml; p=0.046). These data indicate that intramuscular injection of AAV5.IGFI-tet increases function of the failing heart.

Circulating Angiotensin II Upregulates Brain Angiotensin Type 1 Receptors in Normal and Heart Failure Rats

In heart failure (HF), angiotensin type 1 receptors (AT1-R) are upregulated in multiple brain regions crucial for the regulation of cardiovascular responses and sympathetic drive. The mechanisms responsible for this phenomenon remain unknown. Blood-borne angiotensin II (ANG II) affects the brain by binding to AT1-R in circumventricular organs that lack a blood-brain barrier. In this study, we examined the hypothesis that the increased circulating ANG II in HF upregulates AT1-R in the brain. Male Sprague-Dawley (n=8) rats received a subcutaneous infusion of ANG II (0.25 mg/hr, 4wks) concomitant with an intracerebroventricular (ICV) infusion of the AT1-R antagonist losartan (LOS, 20 mg/hr, 4wks, n=4) or vehicle (VEH, n=4). Control rats received saline (n=4). ANG II increased AT1-R expression (by Western blot, units/μg) from 24.0 ± 3.3 to 40.6 ± 4.9 (saline vs. ANG II, P<0.05) in a section of brain containing paraventricular nucleus of hypothalamus and subfornical organ; LOS completely blocked that response. A second group of rats underwent left coronary ligation to induce HF and were treated with ICV LOS (20 mg/hr, 4wks, n=4) or VEH (n=4). LOS decreased AT1-R expression from 46.8 ± 3.7 to 30.5 ± 5.2 (VEH vs. LOS, P<0.05). These data indicate that increases in circulating ANG II upregulate brain AT1-R, and that upregulation of brain AT1-R in ANG II infused and HF rats can be inhibited by a centrally administered AT1-R antagonist. These results suggest that increased circulating ANG II contributes to upregulation of brain AT1-R in HF rats.

In Vitro and In Vivo Characterization of CK-1827452, a Selective Cardiac Myosin Activator

Current inotropic therapies improve contractility by activating the adenylyl cyclase pathway, by delaying cAMP degradation through inhibition of phosphodiesterase (PDE), or by inhibiting the Na/K exchanger, all of which converge on a final common pathway of increasing the intracellular calcium transient of the cardiac myocyte. However, these calcium dependent treatments can be detrimental to patients with heart failure (HF). A calcium independent approach, increasing cardiac contractility by directly activating cardiac myosin, may address the liabilities of the current inotropic agents. Biochemical specificity, cellular mechanism of action, and in vivo cardiac function in Sprague Dawley (SD) rats, SD rats with HF, and anesthetized dogs was determined with the myosin activator CK-1827452. Biochemical assays demonstrate that CK-1827452 activates the ATPase of an intact cardiac sarcomere by directly activating cardiac myosin. In isolated left ventricular myocytes, CK-1827452 (0.2 μM) increased myocyte contractility without altering the calcium transient. Combination of CK-1827452 with isoproterenol (β-adrenergic agonist) resulted only in an additive increase in contractility with no further change in the calcium transient unlike cAMP dependent inotropic mechanisms. CK-1827452 does not inhibit PDE Type III isolated from human platelets. In vivo contractile function in anesthetized SD rats and dogs was quantitated using echocardiography (M-mode), and simultaneous pressure measurements. CK-1827452 infusion in SD rats (0.25 - 2.5 mg/kg/hr) or dogs (0.03 - 1.0 mg/kg/hr) significantly increased fractional shortening (FS) in a dose-dependent manner with no significant change in peripheral blood pressure or heart rate. Rats with defined heart failure induced by left coronary ligation, or sham treated animals had similar and significant increases in FS when treated with CK-1827452. In summary, CK-1827452 is a cardiac specific myosin activator which increases myocyte contractility without changing the calcium transient, increases FS in rats and dogs, and is efficacious in a rat model of HF. These data indicate that CK-1827452 may be a useful therapeutic in the treatment of human heart failure.

Transient reduction in myocardial free oxygen radical levels is involved in the improved cardiac function and structure after long-term allopurinol treatment initiated in established chronic heart failure

Oxidative stress, i.e. imbalance between reactive oxygen species (ROS) and antioxidant defences, contributes to the progression of chronic heart failure (CHF). Acute inhibition of xanthine oxidase (XO), which produces ROS, improves mechanical efficiency of the failing heart, but whether long-term XO inhibition exerts beneficial effects in CHF is unknown. In rats with established CHF induced by left coronary ligation, we assessed the effects of a 5-day and a 10-week treatment with the XO inhibitor allopurinol (50 mg kg(-1) day(-1)) on haemodynamics and left ventricular (LV) function and structure. Both acute and chronic allopurinol treatment increase cardiac output without modification of arterial pressure, but only chronic allopurinol treatment reduces LV end-diastolic pressure and LV relaxation constant. Chronic allopurinol treatment decreases both LV systolic and diastolic diameters, but acute allopurinol treatment only decreases LV systolic diameter. Moreover, chronic allopurinol decreases LV weight and collagen density. Despite XO inhibition after acute and chronic allopurinol treatment, as both treatments reduce uric acid plasma levels, only acute allopurinol treatment reduces LV ROS determined using electron spin resonance spectroscopy. However, the CHF-enhanced myocardial thiobarbituric acid reactive substances levels were never modified. In experimental CHF, long-term allopurinol treatment, initiated in a pathological state of overt CHF, improves LV haemodynamics and function and prevents LV remodelling. These long-term effects are, at least partially, caused by a transient reduction of myocardial ROS shortly after initiation of allopurinol treatment, but whether other mechanism(s), independent of myocardial redox 'status', such as reduced inflammation, are implicated remains to be confirmed.

Reciprocal regulation of angiopoietin-1 and angiopoietin-2 following myocardial infarction in the rat.

This study sought to characterize changes in the angiopoietin system in a rat model of myocardial infarction (MI). Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) bind to the endothelial-specific receptor tyrosine kinase, TIE-2. Ang-2 has been suggested to be an antagonist of TIE-2, possibly acting to release endothelial cells from the tonic stabilizing influence of Ang-1. However, on prolonged exposure, Ang-2 has been shown to acquire agonistic activity at TIE-2, raising the possibility that this isoform may play a direct role in neovascularization. Sprague-Dawley rats were subjected to left coronary ligation and myocardial tissues were harvested from the infarct and peri-infarct regions, or from non-infarcted myocardium. Changes in gene expression were determined by RT-PCR and confirmed by Northern analysis. Changes in protein expression were confirmed by Western analysis and immunocytochemistry, and TIE-2 activity was determined by immunoprecipitation with anti-TIE-2 and antiphosphotyrosine immunoblotting. At 24 h, Ang-1 mRNA and protein expression within the infarct and peri-infarct regions were decreased compared to non-infarcted myocardium, whereas Ang-2 mRNA levels were markedly increased and TIE-2 expression was unchanged. Immunohistochemical staining revealed Ang-1 and TIE-2 immunoreactivity localized to vascular endothelium. In the infarct territory, Ang-2 immunostaining was localized primarily to invading leukocytes at 24 h. At 1 week, Ang-1 expression was partially restored, whereas Ang-2 expression remained elevated. At the time of peak elevation in Ang-2, Tie2 phosphorylation was found to be markedly increased, consistent with receptor activation. Thus, myocardial ischemia induced by left coronary artery ligation resulted in a sustained increase in Ang-2 expression and a reciprocal decrease in Ang-1, consistent with a predominant role for Ang-2 in the angiogenic response to MI.

Postinfarction gene therapy against transforming growth factor-β signal modulates infarct tissue dynamics and attenuates left ventricular remodeling and heart failure

We examined effects of transforming growth factor-β (TGF-β) signal inhibition on post-myocardial infarction (MI) ventricular remodeling and dysfunction. MI was made by left coronary ligation in mice, and adenovirus encoding soluble TGF-β type II receptor (sTβRII), a competitive inhibitor of TGF-β, was injected into the hindlimb muscles on the 3rd day of MI (control, LacZ). Post-MI survival was significantly improved in the sTβRII mice (96% vs. control, 71%, p < 0.05). The sTβRII gene treatment resulted in a significant attenuation in ventricular dilatation and dysfunction 4 weeks post-MI, where a reduction in ventricular fibrosis was histologically confirmed. Although the MI size was not different, the MI thickness was greater and the MI circumference was smaller in the sTβRII group; in the infarct area, α-smooth muscle actin-positive cells were abundant, which might have contributed for infarct contraction. Myofibroblast apoptosis in granulation tissue during the subacute stage (10 days old MI) was less frequent in the sTβRII group, and an in vitro experiment revealed an apoptosis-inhibitory effect of sTβRII on cultured myofibroblasts. Finally, treatment of MI bearing mice with sTβRII was ineffective when started at the chronic stage (scar phase) of MI. In conclusion, the postinfarction gene therapy against TGF-β signal improved cardiac remodeling through affecting infarct tissue dynamics (apoptosis inhibition and infarct contraction) in addition to cardiac fibrosis.