James T. Thackeray

Sympathetic nervous dysregulation in the absence of systolic left ventricular dysfunction in a rat model of insulin resistance with hyperglycemia

BackgroundDiabetes mellitus is strongly associated with cardiovascular dysfunction, derived in part from impairment of sympathetic nervous system signaling. Glucose, insulin, and non-esterified fatty acids are potent stimulants of sympathetic activity and norepinephrine (NE) release. We hypothesized that sustained hyperglycemia in the high fat diet-fed streptozotocin (STZ) rat model of sustained hyperglycemia with insulin resistance would exhibit progressive sympathetic nervous dysfunction in parallel with deteriorating myocardial systolic and/or diastolic function.MethodsCardiac sympathetic nervous integrity was investigated in vivo via biodistribution of the positron emission tomography radiotracer and NE analogue [11C]meta-hydroxyephedrine ([11C]HED). Cardiac systolic and diastolic function was evaluated by echocardiography. Plasma and cardiac NE levels and NE reuptake transporter (NET) expression were evaluated as correlative measurements.ResultsThe animal model displays insulin resistance, sustained hyperglycemia, and progressive hypoinsulinemia. After 8 weeks of persistent hyperglycemia, there was a significant 13-25% reduction in [11C]HED retention in myocardium of STZ-treated hyperglycemic but not euglycemic rats as compared to controls. There was a parallel 17% reduction in immunoblot density for NE reuptake transporter, a 1.2 fold and 2.5 fold elevation of cardiac and plasma NE respectively, and no change in sympathetic nerve density. No change in ejection fraction or fractional area change was detected by echocardiography. Reduced heart rate, prolonged mitral valve deceleration time, and elevated transmitral early to atrial flow velocity ratio measured by pulse-wave Doppler in hyperglycemic rats suggest diastolic impairment of the left ventricle.ConclusionsTaken together, these data suggest that sustained hyperglycemia is associated with elevated myocardial NE content and dysregulation of sympathetic nervous system signaling in the absence of systolic impairment.

Presence of Specific 11C-meta-Hydroxyephedrine Retention in Heart, Lung, Pancreas, and Brown Adipose Tissue

11C-meta-Hydroxyephedrine (HED) is used in cardiac PET as an index of norepinephrine (NE) reuptake transporter (NET) density and synaptic NE levels. Whereas cardiac uptake is well documented, tracer retention in other tissues with rich noradrenergic innervation is unclear. Dysfunctional sympathetic nervous system (SNS) function in extracardiac metabolic storage tissues (i.e., adipose tissue and skeletal muscle) and endocrine organs contributes to several disorders. The aim of this study was to determine the potential of HED as an index of NE function in brown adipose tissue, lung, pancreas, skeletal muscle, and kidney by identifying NET-specific retention and determining the presence of radiolabeled metabolites. Methods: Male Sprague–Dawley rats were administered HED and sacrificed at 30 min after tracer injection. Tissues were rapidly excised and counted for radioactivity, and relative tracer retention was quantified. Pretreatment with NET inhibitors established specific HED accumulation. The effect of elevated NE was tested by subcutaneous minipump NE infusion or inhibition of monoamine oxidase. Column-switch high-performance liquid chromatography (HPLC) was used to analyze the presence of radiolabeled metabolites in heart, brown adipose tissue, pancreas, and plasma. Results: NET-specific retention was observed in heart, brown adipose tissue, lung, and pancreas but not in liver, skeletal muscle, or kidney. A dose-dependent response of HED accumulation to treatments elevating NE levels was established in tissues exhibiting specific uptake. At 30 min after tracer administration, HPLC analysis revealed 93%–95% of total radioactivity signal derived from unchanged HED in heart, pancreas, and brown adipose tissue compared with 61% ± 8% unchanged HED in plasma. Conclusion: In addition to the heart, lung, pancreas, and brown adipose tissue exhibit specific and NE-responsive uptake of HED, supporting the potential for novel PET studies of SNS integrity in these tissues.

Test–retest repeatability of quantitative cardiac 11C-meta-hydroxyephedrine measurements in rats by small animal positron emission tomography

INTRODUCTION The norepinephrine analogue (11)C-meta-hydroxyephedrine (HED) has been used to interrogate sympathetic neuronal reuptake in cardiovascular disease. Application for longitudinal studies in small animal models of disease necessitates an understanding of test-retest variability. This study evaluated the repeatability of multiple quantitative cardiac measurements of HED retention and washout and the pharmacological response to reuptake blockade and enhanced norepinephrine levels. METHODS Small animal PET images were acquired over 60 min following HED administration to healthy male Sprague Dawley rats. Paired test and retest scans were undertaken in individual animals over . Additional HED scans were conducted following administration of norepinephrine reuptake inhibitor desipramine or continuous infusion of exogenous norepinephrine. HED retention was quantified by retention index, standardized uptake value (SUV), monoexponential and one-compartment washout. Plasma and cardiac norepinephrine were measured by high performance liquid chromatography. RESULTS Test retest variability was lower for retention index (15% ± 12%) and SUV (19% ± 15%) as compared to monoexponential washout rates (21% ± 13%). Desipramine pretreatment reduced myocardial HED retention index by 69% and SUV by 85%. Chase treatment with desipramine increased monoexponential HED washout by 197% compared to untreated controls. Norepinephrine infusion dose-dependently reduced HED accumulation, reflected by both retention index and SUV, with a corresponding increase in monoexponential washout. Plasma and cardiac norepinephrine levels correlated with HED quantitative measurements. CONCLUSION The repeatability of HED retention index, SUV, and monoexponential washout supports its suitability for longitudinal PET studies in rats. Uptake and washout of HED are sensitive to acute increases in norepinephrine concentration.

Reduced CGP12177 binding to cardiac β-adrenoceptors in hyperglycemic high-fat-diet-fed, streptozotocin-induced diabetic rats

INTRODUCTION Abnormal sympathetic nervous system and β-adrenoceptor (β-AR) signaling is associated with diabetes. [(3)H]CGP12177 is a nonselective β-AR antagonist that can be labeled with carbon-11 for positron emission tomography. The aim of this study was to examine the suitability of this tracer for evaluation of altered β-AR expression in diabetic rat hearts. METHODS Ex vivo biodistribution with [(3)H]CGP12177 was carried out in normal Sprague-Dawley rats for evaluation of specific binding and response to continuous β-AR stimulation by isoproterenol. In a separate group, high-fat-diet feeding imparted insulin resistance and a single intraperitoneal injection of streptozotocin (STZ) or vehicle evoked hyperglycemia (blood glucose >11 mM). [(3)H]CGP12177 biodistribution was assessed at 2 and 8 weeks post-STZ to measure β-AR binding in heart, 30 min following tracer injection. Western blotting of β-AR subtypes was completed in parallel. RESULTS Infusion of isoproterenol over 14 days did not affect cardiac binding of [(3)H]CGP12177. Approximately half of rats treated with STZ exhibited sustained hyperglycemia and progressive hypoinsulinemia. Myocardial [(3)H]CGP12177 specific binding was unchanged at 2 weeks post-STZ but significantly reduced by 30%-40% at 8 weeks in hyperglycemic but not euglycemic STZ-treated rats compared with vehicle-treated controls. Western blots supported a significant decrease in β(1)-AR in hyperglycemic rats. CONCLUSIONS Reduced cardiac [(3)H]CGP12177 specific binding in the presence of sustained hyperglycemia corresponds to a decrease in relative β(1)-AR expression. These data indirectly support the use of [(11)C]CGP12177 for assessment of cardiac dysfunction in diabetes.

Targeting Amino Acid Metabolism for Molecular Imaging of Inflammation Early After Myocardial Infarction

Acute tissue inflammation after myocardial infarction influences healing and remodeling and has been identified as a target for novel therapies. Molecular imaging holds promise for guidance of such therapies. The amino acid 11C-methionine is a clinically approved agent which is thought to accumulate in macrophages, but not in healthy myocytes. We assessed the suitability of positron emission tomography (PET) with 11C-methionine for imaging post-MI inflammation, from cell to mouse to man. Uptake assays demonstrated 7-fold higher 11C-methionine uptake by polarized pro-inflammatory M1 macrophages over anti-inflammatory M2 subtypes (p<0.001). C57Bl/6 mice (n=27) underwent coronary artery ligation or no surgery. Serial 11C-methionine PET was performed 3, 5 and 7d later. MI mice exhibited a perfusion defect in 32-50% of the left ventricle (LV). PET detected increased 11C-methionine accumulation in the infarct territory at 3d (5.9±0.9%ID/g vs 4.7±0.9 in remote myocardium, and 2.6±0.5 in healthy mice; p<0.05 and <0.01 respectively), which declined by d7 post-MI (4.3±0.6 in infarct, 3.4±0.8 in remote; p=0.03 vs 3d, p=0.08 vs healthy). Increased 11C-methionine uptake was associated with macrophage infiltration of damaged myocardium. Treatment with anti-integrin antibodies (anti-CD11a, -CD11b, -CD49d; 100µg) lowered macrophage content by 56% and 11C-methionine uptake by 46% at 3d post-MI. A patient study at 3d after ST-elevation MI and early reperfusion confirmed elevated 11C-methionine uptake in the hypoperfused myocardial region. Targeting of elevated amino acid metabolism in pro-inflammatory M1 macrophages enables PET imaging-derived demarcation of tissue inflammation after MI. 11C-methionine-based molecular imaging may assist in the translation of novel image-guided, inflammation-targeted regenerative therapies.

Gauging cardiac repair and regeneration with new molecular probes

Regeneration of cardiac muscle has been the ultimate objective of cell-based therapy over the last decade. The first generation of clinical stem cell trials in cardiology, however, have borne only modest success, such that the regeneration of mature cardiomyocytes remains elusive (1). Rather, the observed benefits are ascribed to enhanced damage repair via paracrine mechanisms and endogenous cell recruitment. Accordingly, regenerative cardiology has begun to move away from transplantation of exogenous stem cells, rather aiming to support and enhance the natural healing process (2). As such, infarct healing and the cellular mechanisms underlying this process have emerged as a target for novel molecular imaging probes. Infarct healing is a complex and multifaceted process that offers several molecular targets for therapy and imaging. After myocardial infarction, there is an immediate and organized infiltration of inflammatory leukocytes, which enact a series of both protective and adverse consequences to the damaged myocardium. The protective role of inflammatory leukocytes lies in the engulfment and removal of dead and dying cells, and isolation of cellular damage from the healthy myocardium, allowing for the generation of stable, collagen-rich scar tissue. Invading leukocytes also secrete chemotactic factors to recruit a secondary wave of more reparative inflammatory cell types, including M2-like macrophages, some lymphocytes, and a small proportion of bone marrow–derived progenitor cells, which contribute to angiogenesis and cardioprotection (Fig. 1A). The inflammatory cascade can be detrimental, however, as it is believed that the early wave of granulocytes, lymphocyte antigen 6 complexhigh (Ly6Chigh) monocytes, and differentiated M1-like macrophages perpetuates inflammation and contributes to infarct expansion, leading to higher degrees of remodeling (3,4). Striking a balance between leukocyte-mediated healing and rigorous inflammation is optimal but can be complicated in numerous ways (Figs. 1B–1E). Exacerbated proinflammatory cell activity leads to scar instability and left ventricular rupture in mice, which is thought to manifest as infarct expansion in humans. A prolonged proinflammatory response may increase infarct size and contribute to worse remodeling. Shifting proportions of leukocyte subpopulations, including altered granulocyte content or decreased reparative cell recruitment, can have deleterious effects on cardiac function. Complete early suppression of inflammation increases the rate of left ventricular rupture and late remodeling. Novel treatment strategies seeking to augment the protective mechanisms while dampening the maladaptations can be challenging because the substrate is constantly changing and comprised of a diverse collection

Cardiac β-Adrenoceptor Expression Is Reduced in Zucker Diabetic Fatty Rats as Type-2 Diabetes Progresses.

Objectives Reduced cardiac β-adrenoceptor (β-AR) expression and cardiovascular dysfunction occur in models of hyperglycemia and hypoinsulinemia. Cardiac β-AR expression in type-2 diabetes models of hyperglycemia and hyperinsulinemia, remain less clear. This study investigates cardiac β-AR expression in type-2 diabetic Zucker diabetic fatty (ZDF) rats. Methods Ex vivo biodistribution experiments with [3H]CGP12177 were performed in Zucker lean (ZL) and ZDF rats at 10 and 16 weeks of age as diabetes develops. Blood glucose, body mass, and diet consumption were measured. Western blotting of β-AR subtypes was completed in parallel. Echocardiography was performed at 10 and 16 weeks to assess systolic and diastolic function. Fasted plasma insulin, free fatty acids (FFA), leptin and fed-state insulin were also measured. Results At 10 weeks, myocardial [3H]CGP12177 was normal in hyperglycemic ZDF (17±4.1mM) compared to ZL, but reduced 16-25% at 16 weeks of age as diabetes and hyperglycemia (22±2.4mM) progressed. Reduced β-AR expression not apparent at 10 weeks also developed by 16 weeks of age in ZDF brown adipose tissue. In the heart, Western blotting at 10 weeks indicated normal β1-AR (98±9%), reduced β2-AR (76±10%), and elevated β3-AR (108±6). At 16 weeks, β1-AR expression became reduced (69±16%), β2-AR expression decreased further (68±14%), and β3-AR remained elevated, similar to 10 weeks (112±9%). While HR was reduced at 10 and 16 weeks in ZDF rats, no significant changes were observed in diastolic or systolic function. Conclusions Cardiac β-AR are reduced over 6 weeks of sustained hyperglycemia in type-2 diabetic ZDF rats. This indicates cardiac [3H]CGP12177 retention and β1- and β2-AR expression are inversely correlated with the progression of type-2 diabetes.

Insulin therapy normalizes reduced myocardial β-adrenoceptors at both the onset and after sustained hyperglycemia in diabetic rats.

AIMS Reduced cardiac β-adrenoceptors (β-AR) and cardiovascular (CV) dysfunction occur in diabetes mellitus (DM) and can be normalized by insulin. It is unclear how the duration of untreated hyperglycemia prior to intervention impacts insulins effects. This study assesses insulins effect on reduced myocardial β-AR and CV function, comparing insulin therapy at the onset of hyperglycemia and after a sustained period of hyperglycemia in streptozotocin (STZ) rats. MAIN METHODS Ex vivo biodistribution experiments with [(3)H]CGP12177 were performed in high-fat fed STZ rats after 8 weeks of hyperglycemia evaluating cardiac β-AR expression. Western blotting of β-AR subtypes was completed in parallel. Serial echocardiography at 0, 6, and 8 weeks post-STZ investigated CV function. Sub-groups of hyperglycemic rats were treated with insulin early, at 1 week post-STZ (InsE) for 7 weeks, or late at 6 weeks post-STZ (InsL) for 2 weeks to observe how the duration of hyperglycemia prior to insulin impacts its effects. KEY FINDINGS Reduced myocardial [(3)H]CGP12177 binding occurred 8 weeks post-STZ in hyperglycemics, but was normal in both insulin treatments. Western blotting supported reduced β1-AR in hyperglycemics, but not in either treatment. InsE and InsL treatments improved prolonged mitral valve deceleration (MVD) observed in hyperglycemic animals, but hyperglycemic and InsL still displayed reduced heart rates (HR). SIGNIFICANCE This work supports that glycemic control with insulin normalizes cardiac β-AR effectively regardless of prior hyperglycemia but HR may not recover as readily, indirectly supporting the utility of [(11)C]CGP12177 positron emission tomography (PET) in assessing cardiac β-AR and their modulation with glycemic therapy.

Introducing Fellowship Programs: Cardiovascular Nuclear Imaging at Hannover Medical School, Germany

The Department of Nuclear Medicine at Hannover Medical School(MHH), Germany, accepts international research fellows in Cardiovascular Nuclear Imaging at any time, depending on pre-existing experience, scientific track record and availability of external funding. The program, which is introduced in this article, covers both preclinical as well as clinical projects, with a focus on translational molecular imaging. Work may be conducted in the areas of radiotracer development (radiochemistry), imaging technology development, imaging of basic systems biology and/or mechanistic clinical studies. Research projects typically relate to the major cross-sectional research areas of MHH in the fields of immunology/inflammation, biomedical device technology, regeneration and transplantation.

Insulin supplementation attenuates cancer-induced cardiomyopathy and slows tumor disease progression

Advanced cancer induces fundamental changes in metabolism and promotes cardiac atrophy and heart failure. We discovered systemic insulin deficiency in cachectic cancer patients. Similarly, mice with advanced B16F10 melanoma (B16F10-TM) or colon 26 carcinoma (C26-TM) displayed decreased systemic insulin associated with marked cardiac atrophy, metabolic impairment, and function. B16F10 and C26 tumors decrease systemic insulin via high glucose consumption, lowering pancreatic insulin production and producing insulin-degrading enzyme. As tumor cells consume glucose in an insulin-independent manner, they shift glucose away from cardiomyocytes. Since cardiomyocytes in both tumor models remained insulin responsive, low-dose insulin supplementation by subcutaneous implantation of insulin-releasing pellets improved cardiac glucose uptake, atrophy, and function, with no adverse side effects. In addition, by redirecting glucose to the heart in addition to other organs, the systemic insulin treatment lowered glucose usage by the tumor and thereby decreased tumor growth and volume. Insulin corrected the cancer-induced reduction in cardiac Akt activation and the subsequent overactivation of the proteasome and autophagy. Thus, cancer-induced systemic insulin depletion contributes to cardiac wasting and failure and may promote tumor growth. Low-dose insulin supplementation attenuates these processes and may be supportive in cardio-oncologic treatment concepts.