ADP compartmentation analysis reveals coupling between pyruvate kinase and ATPases in heart muscle

Abstract

Cardiomyocytes have intracellular diffusion restrictions, which spatially compartmentalize ADP and ATP. However, the models that predict diffusion restrictions have used data sets generated in rat heart permeabilized fibers, where diffusion distances may be heterogeneous. This is avoided by using isolated, permeabilized cardiomyocytes. The aim of this work was to analyze the intracellular diffusion of ATP and ADP in rat permeabilized cardiomyocytes. To do this, we measured respiration rate, ATPase rate, and ADP concentration in the surrounding solution. The data were analyzed using mathematical models that reflect different levels of cell compartmentalization. In agreement with previous studies, we found significant diffusion restriction by the mitochondrial outer membrane and confirmed a functional coupling between mitochondria and a fraction of ATPases in the cell. In addition, our experimental data show that considerable activity of endogenous pyruvate kinase (PK) remains in the cardiomyocytes after permeabilization. A fraction of ATPases were inactive without ATP feedback by this endogenous PK. When analyzing the data, we were able to reproduce the measurements only with the mathematical models that include a tight coupling between the fraction of endogenous PK and ATPases. To our knowledge, this is the first time such a strong coupling of PK to ATPases has been demonstrated in permeabilized cardiomyocytes.

Figure 1

Models of permeabilized cardiomyocytes considered in this work. In the schemes, reactions are shown by curved arrows; exchange between compartments is indicated by straight arrows. The level of complexity increases from model 1 to models 3 and 4. All models have compartments representing solution, cytosol, and mitochondrial IMS. In models 3 and 4, an additional compartment was added (C4). The reactions considered in this work are ATP synthesis by mitochondria (ATPsyn), ATP consumption by ATPases (ATPase, ATPase1, ATPase2), and ATP synthesis by endogenous PK (PKend, PKend1, PKend2) and by exogenously added PK (PK). Compartment exchanges occurred between solution and cytosol (sol-cyt); IMS and cytosol through mitochondrial outer membrane (MoM); cytosol and C4 (cyt-C4); and IMS and C4 (IMS-C4).

Figure 2

Representative examples of individual experiments performed in this work. Vertical lines indicate the time moment when solution was changed by injecting ATP, ADP, PK, or PEP. (A and B) Respiration rates recorded during titration with ADP (A) and ATP (B). First, cardiomyocytes (CM) were added (moment of addition not shown) and then successive additions of ADP or ATP were performed to reach the concentrations indicated. (C and D) Inhibition by PK+PEP system. In C, 5 mM PEP was in solution before the experiment began. In D, the effects of addition of 5 mM PEP and 20 U/ml PK are demonstrated. (E and F) Mitochondrial respiration was inhibited by cyanide and oligomycin, as shown by ADP dependency of endogenous PK assessed spectrophotometrically (E) and ATPase activity of cardiomyocytes assessed by titration with ATP (F). In these experiments, NADH was sometimes replenished, leading to an increase in absorbance.

Figure 3

Experimental data (open circles, mean ± SD) are compared to the solutions using models 1 (dotted line) and 2–4 (lines with solid symbols). The fits obtained using the simplified versions of the models are indicated by the lines with the corresponding open symbols. The fitted experimental data were respiration rate recorded during titration with ADP (A; n = 10) or ATP (B; n = 8); endogenous ATPase (C; n = 9) and PK activities (D; n = 7) measured spectrophotometrically; and ADP concentration dependence on time in the presence (E; n = 5) or absence (F; n = 5) of oxidative phosphorylation. Note that in all cases, the lines corresponding to models 3 and 4 are very close to each other, leading to the formation of a single dashed line. In addition, since there is no endogenous PK activity in model 1, the rate of PK_end calculated by model 1 is zero in D.