A¹³C NMR double-labeling method to quantitate local myocardial O₂consumption using frozen tissue samples

Abstract

Measurement of local myocardial O(2) consumption (VO(2)) has been problematic but is needed to investigate the heterogeneity of aerobic metabolism. The goal of the present investigation was to develop a method to measure local VO(2) using small frozen myocardial samples, suitable for determining VO(2) profiles. In 26 isolated rabbit hearts, 1.5 mmol/l [2-(13)C]acetate was infused for 4 min, followed by 1.5 min of [1,2-(13)C]acetate. The left ventricular (LV) free wall was then quickly frozen. High-resolution (13)C-NMR spectra were measured from extracts taken from 2- to 3-mm thick transmural layer samples. The multiplet intensities of glutamate were analyzed with a computer model allowing simultaneous estimation of the absolute flux through the tricarboxylic acid cycle and the fractional contribution of acetate to acetyl CoA formation from which local VO(2) was calculated. The (13)C-derived VO(2) in the LV free wall was linearly related to "gold standard" VO(2) from coronary venous O(2) electrode measurements in the same region (r = 0.932, n = 22, P < 0.0001, slope 1.05) for control and lowered metabolic rates. The ratio of subendocardial to subepicardial VO(2) was 1.52 +/- 0.19 (SE, significantly >1, P < 0.025). Local myocardial VO(2) can now be quantitated with this new (13)C method to determine profiles of aerobic energy metabolism.

Myocardial O2consumption in porcine left ventricle is heterogeneously distributed in parallel to heterogeneous O2delivery

“…The infusion of unlabeled acetate was then quickly switched to [2-13 C]acetate (at unchanged concentration) for exactly 4 min, after which another quick switch was made to the infusion of [1,2-13 C]acetate for 1.5 min. The second label increases parameter estimation accuracy (41,43). After exactly 5.5 min of [ 13 C]acetate infusion, a part of ϳ2 ϫ 2 cm of the anterior left ventricular free wall, immediately adjacent to the LAD, was quickly freeze-clamped in situ inside an aluminium clamp precooled to the temperature of liquid nitrogen, cut out of the heart, and immediately placed into liquid nitrogen.…”

“…The NMR multiplet peaks were analyzed with a previously published and validated computer model, schematically depicted in Fig. 2 (43). In brief, the model consists of 132 differential equations (for detailed description of the equations, see appendix to Ref.…”

“…In brief, the model consists of 132 differential equations (for detailed description of the equations, see appendix to Ref. 43), incorporating all possible combinations of 13 C and 12 C in 6 different molecular "pools" representing acetyl-CoA, the TCA cycle intermediates and associated amino acids (aspartate and glutamate). The isotope exchange reactions between these pools are incorporated in the differential equations.…”

“…From the NMR intensities, the TCA cycle flux is estimated using a computational model. The validity of this approach has been shown in isolated perfused rabbit hearts (43) and its feasibility on the in situ heart has been shown in the rabbit (42).…”

The mechanical and metabolic basis of myocardial blood flow heterogeneity

“…Sonntag et al (87) observed increased deoxyglucose uptake in high-flow regions. Likewise, van Beek et al (95) observed that the turnover of acetate was high in high-flow regions compared to lowflow regions; since this was the only substrate provided, they concluded that local flow must be nearly proportional to local O 2 consumption. Recently, Decking et al (31) using 13 Cpyruvate found a similar relationship.…”