Multinuclear NMR Spectroscopy of Myocardial Energetics and Substrate Utilization in Isolated Perfused Mouse Hearts

Abstract

In experimental cardiovascular physiology, non­ invasive and repeated measures of cardiac function, energetics, and metabolism provide a dynamic picture of myocardial performance during phys­ iological or pathological perturbations. All of these parameters can be assessed in one sample by combining isolated perfused heart experiments with a multinuclear nuclear magnetic resonance (NMR) spectroscopic approach. This method is advantageous because simultaneous measurements of left ventricular (LV) function, myocardial energetics, and substrate utilization are made in a contracting heart. NMR spectroscopy allows the user to measure metabolite content, as well as turnover rate, by assessing the content of a specific nucleus in biological samples [1,2]. For example, P NMR spectroscopy can be used to quantify phosphorous­ containing compounds in the sample. The most abundant phosphates in the cell are the high­energy phosphates phosphocreatine and ATP. Thus, P NMR spectroscopy of the heart can assess myocardial energetics in various studies of bioengineered mouse models under pathological conditions [3–6]. With this method, dynamic changes in phosphocreatine and ATP can be assessed with repetitive measurements during physiological and/or pathological pertur­ bations providing a detailed picture of energetic status. Assessment of myocardial substrate utilization presents a unique challenge in that, although carbon­ containing compounds are relatively abundant in the heart, the natural abundance of the NMR visible carbon isotope (C) is very low (∼1–3%). Therefore, isotopic enrichment of substrates delivered to the isolated perfused heart is required. The specific C­labeled substrates are chosen based on the C­labeling pattern of the acetyl CoAs that are ultimately yielded. Since glutamate is easily detected by C NMR spectroscopy and the enrichment pattern is similar to the TCA cycle intermediate, α­ketoglutarate under steady­state conditions, inferences of isotopic­enriched substrate entry into the tricarboxcylic acid (TCA) cycle can be made by examining the carbon labeling pattern of glutamate. As shown in Figure 41.1A, glucose with C carbons in the 1st and 6th position (1,6­C glucose) will yield two acetyl CoA molecules with the C carbon in the 2nd position (2­C acetyl CoA) once glycolysis and pyruvate decarboxylation are completed. Entry