Abstract
Diabetes Mellitus (DM)-induced disease of the coronary microvessels contributes to the worldwide increase in cardiovascular morbidity and mortality in diabetic patients. These microvessels are vital to the regulation of regional blood flow, and facilitating oxygen and nutrient exchange within heart tissue. Whilst total coronary blood flow is readily measured, our limited ability to directly measure coronary microvascular perfusion has restricted our progress in understanding DM pathology. Therefore, I aimed to test the feasibility of adapting two techniques previously used in skeletal muscle, 1-methylxanthine metabolism and vascular casting, to measure coronary microvascular perfusion and assess how this is impaired in type 2 DM.
Isolated rat hearts were perfused under physiological conditions, with assessment of cardiac contractility and total coronary flow. This was combined with investigation of both a chemical and physical approach to assess microvascular perfusion. Firstly, metabolism of exogenous 1-methylxanthine (1-MX) by xanthine oxidase on the endothelium was investigated as a measure of capillary surface area. In addition, casts of the physiological vascular structure were produced using rapidly setting dental acrylic injected into the coronary vasculature, and visualised with micro-computerised tomography. To examine the feasibility of these microvascular measures, protocols were developed to induce known perfusion changes in male Sprague Dawley rat hearts; isoproterenol (1x10-8M, vasodilation) and angiotensin II (1x10-7M, vasoconstriction) were applied. Secondly, a pilot study was conducted applying the 1-MX and casting techniques to compare 20-week-old male type 2 DM Zucker Diabetic Fatty (ZDF) rats to their non-diabetic littermates.
In Sprague Dawley rats, isoproterenol significantly increased whilst, to a lesser extent, angiotensin II significantly decreased myocardial function and coronary flow (p≤0.05, n=6 and 7). Vascular casting produced promising results; a representative cast from the isoproterenol intervention displayed increased branching, and the angiotensin II intervention showed somewhat reduced branching of the microvessels relative to no intervention (n=1). However, 1-MX values did not reveal any changes between these interventions. Consequently, the 1-MX protocol was optimised to improve stability, before being applied in the type 2 DM pilot study. Under basal conditions, no significant difference was discerned between diabetic and non-diabetic rats in 1-MX disappearance (22.6±6.7nmol/min (n=5) vs. 23.4±3.9nmol/min (n=3); mean±SEM: n.s.), nor in measures of cardiac function. Likewise, no difference was discernible between a representative cast from the non-diabetic and diabetic group. However, a positive Spearman’s rank correlation was observed between coronary flow and 1-MX disappearance in the diabetic rats (rs=1, p≤0.05).
With this study I have set up the foundations of using 1-MX metabolism and vascular casting, as techniques to examine coronary microvascular perfusion in the isolated heart. Conclusions regarding DM-induced changes cannot be drawn at this stage. However, pilot data provide valuable information on how to further develop these techniques. Novel measures of coronary microvascular perfusion have the potential to enhance our understanding of coronary microvascular pathology, and eventually reduce DM-induced cardiovascular complications.