Estimation of heart mitochondrial creatine kinase flux using magnetization transfer NMR spectroscopy

Abstract

In heart, both enzyme and substrate of the creatine kinase (CK) reaction are compartmentalized: 0-25% of total CK activity is associated with the mitochondrial CK isoenzyme (mito-CK); 2-30% of the total ATP pool is in the mitochondria. Because most ATP is produced by oxidative phosphorylation, this ATP may be the preferred substrate for the mito-CK reaction. Thus flux through the mito-CK reaction should increase in proportion to the amount of mito-CK, until the enzyme becomes saturated. We previously developed a model of saturation-transfer nuclear magnetic resonance (NMR) spectroscopy that permits calculation of mito-CK flux. Here, we test the model for consistency in two ways: 1) we compare fluxes in rabbit hearts with 0% mito-CK and with 6% mito-CK at two rates of ATP synthesis and 2) we analyze six groups of rat and rabbit hearts with differing amounts of mito-CK and differing work loads. Hearts with no detectable mito-CK activity do not increase their baseline low level of mito-CK flux in response to the increased demand of contraction, but mito-CK flux increases with increased work in hearts with measurable amounts of mito-CK. Furthermore, mito-CK flux increases monotonically with increasing ATP synthesis rates but increases and then saturates with increasing mito-CK activity. Calculated mito-CK flux is of the same order of magnitude as ATP synthesis rate, as would be expected from the coupling of the mito-CK reaction to adenine nucleotide translocase. Thus the model predicts the appropriate relationship between mito-CK activity and mito-CK flux.