K Clarke

Iron particles for noninvasive monitoring of bone marrow stromal cell engraftment into, and isolation of viable engrafted donor cells from, the heart.

Stem cells offer a promising approach to the treatment of myocardial infarction and prevention of heart failure. We have used iron labeling of bone marrow stromal cells (BMSCs) to noninvasively track cell location in the infarcted rat heart over 16 weeks using cine‐magnetic resonance imaging (cine‐MRI) and to isolate the BMSCs from the grafted hearts using the magnetic properties of the donor cells. BMSCs were isolated from rat bone marrow, characterized by flow cytometry, transduced with lentiviral vectors expressing green fluorescent protein (GFP), and labeled with iron particles. BMSCs were injected into the infarct periphery immediately following coronary artery ligation, and rat hearts were imaged at 1, 4, 10, and 16 weeks postinfarction. Signal voids caused by the iron particles in the BMSCs were detected in all rats at all time points. In mildly infarcted hearts, the volume of the signal void decreased over the 16 weeks, whereas the signal void volume did not decrease significantly in severely infarcted hearts. High‐resolution three‐dimensional magnetic resonance (MR) microscopy identified hypointense regions at the same position as in vivo. Donor cells containing iron particles and expressing GFP were identified in MR‐targeted heart sections after magnetic cell separation from digested hearts. In conclusion, MRI can be used to track cells labeled with iron particles in damaged tissue for at least 16 weeks after injection and to guide tissue sectioning by accurately identifying regions of cell engraftment. The magnetic properties of the iron‐labeled donor cells can be used for their isolation from host tissue to enable further characterization.

REAL-TIME ASSESSMENT OF KREBS CYCLE METABOLISM WITH HYPERPOLARISED [2-13C]PYRUVATE

The Krebs cycle is fundamental to cardiac energy production, and is often implicated in energetic imbalances characteristic of heart disease. To date, Krebs cycle flux has been measured using 13C-magnetic resonance spectroscopy with isotopomer analysis; however, this approach is limited to the study of steady-state metabolism …

T2 Factor inhibiting HIF (FIH1) modulates cardiac function and metabolism

Hypoxia-inducible factor (HIF) plays a pivotal role in the cellular response to reduced oxygen availability. HIF activity is regulated by two families of oxygen sensitive enzymes; the prolyl hydroxylase domain (PHD) family, and factor-inhibiting HIF (FIH1). FIH1 is thought to be an essential regulator of metabolism but its role in the heart is unknown. Mice with a null mutation in the FIH1 gene (FIH1-/-, n≥5) had 18% lower body weight (p<0.02) than wild type littermate controls (WT, n≥6), but normal total cardiac mass. Right ventricular mass determined via MRI was 25% greater in FIH1-/- hearts (p<0.01). Cine MRI revealed a 15% reduction in stroke volume in FIH1-/- hearts, from 27.4±2.3 µl in WT to 23.4±1.5 µl in FIH1-/- (p<0.05). Impaired contractility was also observed in individual myocytes (sarcomere shortening was 3.01%±0.20% in FIH1-/- compared to 3.92%±0.17% in WT, p<0.05) and was associated with reduced Ca2+ transient amplitude (fura-2 ratio 0.21±0.02 and 0.29±0.02 for FIH1-/- and WT respectively, p<0.05). Glycolytic flux (µmol/min/g) was significantly higher in Langendorff perfused FIH1-/- hearts (1.17±0.04) than WT (0.79±0.12, p<0.05) although no changes in lactate efflux were detected. There were no differences in pyruvate dehydrogenase kinase 1 and 4 protein expression and citrate synthase activity (µmol/min/mg) was similar for both WT (1.01±0.02) and FIH1-/- (0.96±0.03) hearts. Our data suggest a novel role for FIH1 in modulating cardiac contractility and metabolism, with FIH1 ablation producing cardiac effects comparable to those associated with activation of the hypoxic signalling pathway.

216 IMPAIRED IN VIVO MITOCHONDRIAL KREBS CYCLE ACTIVITY FOLLOWING MYOCARDIAL INFARCTION ASSESSED USING HYPERPOLARIZED MAGNETIC RESONANCE SPECTROSCOPY

An increasing body of evidence links alterations in cardiac metabolism with the progression of heart disease. Using the recently developed technique of hyperpolarized 13C magnetic resonance spectroscopy, in vivo alterations in mitochondrial metabolism were assessed following myocardial infarction (MI). Hyperpolarization of 13C containing compounds can increase their signal by >10,000 fold over conventional methods. MI with reperfusion surgery was performed on eleven female Wistar rats. Four sham animals were also prepared. Animals were given two hyperpolarized scans, of either [1-13C] or [2-13C] pyruvate, at 1, 6 and 22 weeks post-MI. [1-13C] or [2-13C] pyruvate were hyperpolarized and dissolved in a GE prototype polarizer. 1ml of 80mM hyperpolarized pyruvate was injected over 10s via a tail vein catheter into an anaesthetised rat positioned in a 7T MR scanner. Spectra were acquired every second for a 1min following injection, using a 5o RF excitation pulse. Signal was localised to the heart using a custom 13C RF surface coil. Metabolic alterations were correlated with ejection fraction (EF) assessed by echocardiography, at each timepoint to yield information on the interplay between cardiac function and mitochondrial metabolism. One week post-MI, there were no detectable alterations in in vivo cardiac mitochondrial metabolism over the range of EFs observed. This is an early adaptive phase post-MI, where scar formation and remodelling of the heart are occurring. Six weeks post-MI, a novel finding in this study was impaired in vivo mitochondrial Krebs cycle activity, in addition to decreased flux into acetylcarnitine, which correlated with the EF. These changes were seen in the absence of any alterations in pyruvate dehydrogenase (PDH) flux. Thus, in vivo alterations in Krebs cycle flux may indicate an early maladaptive phase in the metabolic derangement following MI. By 22 weeks post-MI, alterations were also seen in PDH flux, which positively correlated with EF, highlighting a reduction in glucose oxidation and Krebs cycle activity in the infarcted heart. At 22 weeks, biochemical analysis was performed on excised hearts, to further characterize the metabolic alterations accompanying MI. Enzyme activities of PDH, citrate synthase, isocitrate dehydrogenase and carnitine acetyltransferanse positively correlated with EF. Metabolomic analysis revealed reduced levels of Kerbs cycle intermediates. The correlation between function and metabolism raises an interesting paradox; is the reduction in PDH and Krebs cycle activity due to a reduction in contraction and therefore a reduced energy requirement, or does the altered PDH and Krebs cycle activity lead to reduced energy levels meaning cardiac contraction is impaired? This study highlights the importance of assessing metabolism at multiple timepoints in vivo, and demonstrates the potential of hyperpolarized MRS for investigating the metabolic effects of progressive diseases, potentially in a clinical setting. Figure 1

249 CARDIAC CHARACTERISATION OF A NEW RAT MODEL OF TYPE 2 DIABETES

Aims We aim to develop a type 2 diabetic rat model which represents closely the characteristics of the diseased state in type 2 diabetic humans such as hyperglycaemia, hyperinsulinaemia, dyslipidaemia and obesity. We based our model on the type 2 diabetes was proposed by Reed et al 1 , which combines high fat diet and streptozotocin (STZ) instead of relying on a genetic manipulation for disease development like many other rat models. We proposed to determine the optimal dose of streptozotocin (STZ) injection and to investigate cardiac-specific abnormalities in this new model of type 2 diabetes, to determine its suitability for studying diabetic cardiomyopathy. Methods Male Wistar rats were fed a high fat diet followed by an intraperitoneal injection of STZ at either 15, 20, 25 or 30 mg/kg body weight. Results We observed a dose-dependent increase in plasma glucose and non-esterified fatty acids with increasing concentration of STZ. There were dose-independent increases in cardiac and hepatic triglycerides, and decreases in cardiac and hepatic glycogen content in all diabetic rats. With increasing concentrations of STZ there were dose-dependent increases in cardiac UCP3, PDK4 and MCAD protein levels, and decreases in GLUT4 and GLUT1 protein levels. Consequently, the dose of 25 mg/kg STZ was chosen for further metabolic studies. These diabetic rats showed a 39% increase in fasted insulin concentrations and 73% increase in glucose concentrations. Isolated heart perfusion using 3H-glucose demonstrated that both insulin-independent and insulin-stimulated glycolytic rates were decreased by 56% and 43%, respectively, in diabetic hearts compared with controls, in the absence of any change in systolic function. Conclusions This demonstrates that high fat feeding combined with 25 mg/kg STZ induces a cardiac metabolic phenotype that resembles that found in type 2 diabetic patients

234 IMPAIRED METABOLIC AND FUNCTIONAL ADAPTATION TO HYPOXIA IN THE TYPE 2 DIABETIC HEART

Aims Cardiovascular disease is the leading cause of mortality in people with type 2 diabetes. Following a myocardial infarction, the heart must adapt to the decreased oxygen availability by switching to more oxygen-efficient metabolism. We hypothesise that adaptation to hypoxia is impaired in type 2 diabetes. Methods Type 2 diabetes was induced in rats by high fat feeding combined with a low dose of streptozotocin (25mg/kg i.p). Following induction of diabetes, control and diabetic rats were housed in a hypoxia chamber for three week at 11% oxygen, to study chronic adaptation to hypoxia. Subsequently, hearts were isolated and perfused for measurement of substrate metabolism, or mitochondria were isolated and respiration was measured. In a separate study, hearts from normoxic diabetic and control rats were perfused ex vivo with hypoxic buffer, to study the acute response to hypoxia. Results Under normoxic conditions, diabetic hearts had a 30% decrease in glycolysis, a 14% decrease in pyruvate oxidation and a 34% increase in fatty acid oxidation, compared with normoxic control rats. When control rats were housed in hypoxia they adapted their cardiac metabolism to be more oxygen efficient, by increasing anaerobic glycolysis by 18%, increasing glycogen reserves by 24%, decreasing fatty acid oxidation by 18% and decreasing mitochondrial respiration by 26%. In contrast, when diabetic rats were housed in hypoxia they were unable to adapt metabolism to the same extent, being significantly different to hypoxic control rats. Glycolytic rates and glycogen content was significantly lower in hypoxic diabetic hearts compared with hypoxic controls, similarly fatty acid oxidation rates and mitochondrial oxygen consumption were both significantly higher in hypoxic diabetic hearts compared with hypoxic control hearts. Metabolic rates and mitochondrial oxygen consumption from hypoxic diabetic rat hearts were comparable to those in normoxic control hearts. Normoxic control and diabetic rat hearts were perfused with hypoxic buffer to study the functional and metabolic response to acute hypoxia. During acute hypoxia, diabetic hearts had 58% lower heart rates and 68% higher end-diastolic pressures compared to control hearts, associated with 55% lower rate of hypoxia-induced glycolysis. During the subsequent reoxygenation period, diabetic hearts had a 34% decrease in recovery of cardiac function compared with control hearts. Conclusions Adaptation to chronic hypoxia is limited in type 2 diabetic rat hearts, with anaerobic metabolism lower and oxidative metabolism higher than control heart. This increased dependence on oxidative metabolism under oxygen-limited conditions was associated with contractile dysfunction in the diabetic heart. This may contribute to the decreased recovery of the diabetic heart following a myocardial infarction.

NORMOBARIC HYPOXIA IMPAIRS CARDIAC ENERGETICS IN NORMAL HUMAN VOLUNTEERS

In the first few days of hypoxic exposure, left ventricular dysfunction is consistently observed in the human heart, yet the cellular mechanisms underlying the dysfunction are poorly understood. Our hypothesis was that normobaric hypoxia impairs cardiac energetics, leading to cardiac dysfunction …

SI13 Noninvasive MRI Monitoring of Engraftment of Cultured Bone Marrow Mesenchymal Stem Cell into the Damaged Heart

Stem cells offer a promising approach to the treatment of myocardial infarction and prevention of heart failure. We have used iron‐labelling of bone marrow stromal cells (BMSCs) to noninvasively track cell location in the infarcted rat heart over 16 weeks using cine‐MRI and to isolate the BMSCs from the grafted hearts using the magnetic properties of the donor cells. BMSCs were isolated from rat bone marrow, characterised by flow cytometry, transduced with lentiviral vectors expressing GFP and labelled with iron particles. BMSCs were injected into the infarct periphery immediately following coronary artery ligation and rat hearts were imaged at 1, 4, 10 and 16 weeks post‐infarction. Signal voids caused by the iron particles in the BMSCs were detected in all rats at all time points. In mildly infarcted hearts, the volume of the signal void decreased over the 16 weeks, whilst the signal void volume did not decrease significantly in severely infarcted hearts. High resolution 3D MR microscopy identified hypointense regions at the same position as in vivo. Donor cells containing iron particles and expressing GFP were identified in MR‐targeted heart sections after magnetic cell separation from digested hearts. In conclusion, MRI can be used to track cells labelled with iron particles in damaged tissue for at least 16 weeks after injection and to guide tissue sectioning by accurately identifying regions of cell engraftment. The magnetic properties of the iron labelled donor cells can be used for their isolation from host tissue to enable further characterisation.