|dc.description.abstract||Current heart donor procurement involves a period of cold storage during transport and rarely exceeds 6 hours. While this method reduces myocardial metabolism, it still results in ATP depletion, lactate accumulation, and myocyte damage. Hypothermic machine perfusion (HMP) has emerged as an alternative technique. Previous studies from our laboratory showed HMP maintains myocardial oxygen consumption, preserves ATP, reduces myocardial injury, minimizes lactate accumulation, and improves cardiac function after transplantation for storage intervals of over fourteen hours. Though HMP is more advantageous, myocardial metabolism and adjunctive protective strategies under these conditions are poorly understood. The purpose of this study was to 1) Determine myocardial substrate preferences during HMP 2) Evaluate the effect of metformin and insulin on substrate oxidation in the perfused heart 3) Demonstrate delivery of nanoparticles to the heart during HMP.
In Aim 1, I investigated myocardial substrate selection by perfusing isolated rat hearts octanoate, ketones, or acetate with and without an anaplerotic substrate. 13C magnetic resonance spectroscopy (MRS) was performed on myocardial extracts and substrate contributions to oxidative metabolism were assessed by isotopomer analysis. Additional samples were analyzed by gas chromatography/mass spectroscopy to determine substrate effects on tricarboxylic acid (TCA) cycle intermediates and isotopomer distributions. Aim 2 assessed the ability of metformin and insulin to alter myocardial substrate oxidation during normokalemic, hyperkalemic, and post-ischemic reperfusion using a rat normothermic Langendorff model. Substrate selection and oxidation rates were determined by 13C MRS and isotopomer analysis as in Aim 1. Cardiac function and efficiency were measured. For Aim 3, a nanoparticle delivery system was constructed, and a nanoparticle perfusion model was validated for future addition of nanoparticles to the perfusate to modify cardiac injury. Nanoparticles were characterized and then tested in three and six-hour perfusion models for their ability to localize in cells.
Data from Aim 1 demonstrated that octanoate and acetate were preferentially oxidized during HMP. Ketone oxidation remained a minor contributing substrate. TCA cycle intermediates were increased in all substrate containing groups compared to hearts immediately recovered or perfused without oxidizable substrate. An anaplerotic substrate was not required to achieve these results. In Aim 2, during normokalemia, insulin reduced ketone oxidation while the combination of insulin and metformin restored the control profile. Metformin reduced fatty acid oxidation in the cardioplegia model while neither drug influenced substrate selection during post-ischemic reperfusion. Cardiac function and efficiency were not altered in treatment groups. Lastly, Aim 3 results indicated that nanoparticles (diameter=376+/-98nm; polydispersity index=0.16+/-0.06; encapsulation efficiency=65+/-12%) showed successful uptake, reduced lactate, and increased high energy phosphate ratios in the 3-hour model.
Outcomes from these experiments demonstrate that myocardial substrate preferences are different during HMP compared to normothermia. Nanoparticle delivery to myocardium during HMP is possible and has the potential to modify myocardial injury by delivering therapeutic agents, miRNA (miRNA-499), or other gene products. This data is critical in designing preservation solutions for the machine perfused heart.||en