Josefina Adriana Maria Bastiaansen
Cardiac and skeletal muscle function relies on a continuous energy production via fatty acid metabolism and mitochondrial oxidation of pyruvate, with a fine balance between substrate delivery and utilization. Changes in metabolism are increasingly being implicated as playing an intrinsic role in many diseases, such as diabetes, cancer, and heart failure. Investigating and identifying fundamental metabolic processes are paramount to understanding pathologies. Beyond morphology and functional information, magnetic resonance can provide insights at a metabolic level using spectroscopic techniques such as 13C NMR. The low natural abundance and sensitivity of the 13C nucleus makes 13C NMR in biological systems challenging. In addressing this issue, hyperpolarized methods have emerged as a very promising tool, obtaining signal enhancements up to 10,000 fold. Spectra of hyperpolarized 13C labeled substrates and their downstream metabolic products offer insight into metabolic processes occurring in vivo within seconds after the injection. This thesis focused on the development of MR hyperpolarization methods and applications to study energy metabolism in cardiac and skeletal muscle in vivo. This ranged from development of the experimental frame work to mathematical tools to characterize the observed metabolic processes. Methods were developed to visualize 13C labeling kinetics of acetylcarnitine in vivo in resting skeletal muscle following the administration of hyperpolarized [1-13C]acetate. Two different, novel mathematical models were constructed to quantify the kinetic rate constants. Although separated by two enzymatic reactions, the conversion of acetate to acetylcarnitine was uniquely defined by the enzymatic activity of acetylCoA synthetase (ACS). A 13C MRS protocol was developed and implemented for hyperpolarized studies in the heart, which included selecting appropriate cardiac triggers to align the measurements with the cardiac phase. The 13C label propagation into acetylcarnitine and citrate could be measured in real time in the beating rat heart following the infusion of hyperpolarized [1-13C]acetate, using a newly constructed 13C RF coil which improved the detection sensitivity. The substantial spectral resolution at 9.4T and a triggered shimming and MR acquisition protocol allowed for the detection of citrate for the first time in vivo after injection of hyperpolarized [1-13C]acetate. Mathematical models were successfully extended to include mitochondrial oxidation and analytical expressions were derived to interpret the dynamic 13C labeling of citrate. Cardiac dysfunction is often associated with a shift in substrate preference, while diagnostic methods such as PET provide only information on substrate uptake. The potential of hyperpolarized 13C MRS to measure simultaneously lipid and carbohydrate oxidation was demonstrated in vivo, and the sensitivity of the method to a metabolic perturbation was assessed. Hyperpolarized [1-13C]butyrate and [1-13C]pyruvate were used as representatives of carbohydrate and lipid oxidation. Fasting led to significant changes in preference for the injected substrates. The appearance of a cohort of downstream metabolites (bicarbonate, lactate, alanine, glutamate, citrate, acetylcarnitine, β-hydroxybutyrate and acetoacetate) allowed the independent and simultaneous monitoring of myocardial oxidation of both fatty acid and carbohydrates in vivo and is a sensitive indicator of metabolic shift. Lactate is an important metabolic intermediate for mitochondrial oxidation. Carbohydrate metabolism in healthy rat skeletal muscle at rest was studied in different nutritional states using hyperpolarized [1-13C]lactate, which can be injected at physiological concentrations and leaves other oxidative processes undisturbed. A significant decrease in [1-13C]alanine and 13C bicarbonate were observed comparing both groups, attributed to a change in cellular alanine concentration and pyruvate dehydrogenase (PDH) flux. It was shown that lactate can be used to study carbohydrate oxidation in skeletal muscle at physiological levels and that the downstream metabolite signals are sensitive markers to probe metabolic changes. Since the detection of [5-13C]citrate and [5-13C]glutamate in the heart is hindered by the close proximity of the [1-13C]acetate resonance, acetylcarnitine could be an interesting substrate for hyperpolarized MR. Acetylcarnitine crosses the mitochondrial membrane easily, while skipping a few metabolic steps needed for acetate to cross the membrane. Moreover, it does not interfere with the detection of [5-13C]glutamate and [5-13C]citrate. [1-13C]Acetylcarnitine was successfully hyperpolarized and despite its short T1 it was possible to detect the formation of [5-13C]glutamate in the heart in vivo. Due to the absence of the [5-13C]citrate resonance, we hypothesized the existence of an intricate relationship between reaction, transport and relaxation rates in the choice of hyperpolarized substrates. Acetylcarnitine has relatively small poolsizes and has only been observed using hyperpolarized 13C MRS techniques. Therefore, it has not been possible to obtain reliable quantification of the metabolic turnover of acetylcarnitine, which is an essential intermediate in the metabolism of acetate. Methods were implemented to measure the oxidation of [2-13C]acetate locally with increased sensitivity at high field (14.1T), using a 1H-13C polarization transfer sequence. This allowed the observation of [2-13C]acetylcarnitine in vivo at 21.5 ppm, with a metabolic turnover time τ of 0.34 μmol/g/hr. The acetylcarnitine resonance assignment was confirmed by experiments infusing 13C labeled glucose. The measurement of the time courses of Glu C4 and C3 were also clearly observed, without lipid contamination. To conclude, this work presents novel metabolic information about skeletal and cardiac energy metabolism using developed methods in hyperpolarized 13C MRS. The use of hyperpolarized 13C substrates to measure absolute flux through metabolic pathways in vivo would not only revolutionize clinical diagnostic imaging but also serve as an important tool to better understand diseased metabolism and the metabolic effects of new drugs aimed to combat diseases.
EPFL2013
