Tight coupling of Na+/K+-ATPase with glycolysis demonstrated in permeabilized rat cardiomyocytes

Abstract

The effective integrated organization of processes in cardiac cells is achieved, in part, by the functional compartmentation of energy transfer processes. Earlier, using permeabilized cardiomyocytes, we demonstrated the existence of tight coupling between some of cardiomyocyte ATPases and glycolysis in rat. In this work, we studied contribution of two membrane ATPases and whether they are coupled to glycolysis--sarcoplasmic reticulum Ca2+ ATPase (SERCA) and plasmalemma Na+/K+-ATPase (NKA). While SERCA activity was minor in this preparation in the absence of calcium, major role of NKA was revealed accounting to ∼30% of the total ATPase activity which demonstrates that permeabilized cell preparation can be used to study this pump. To elucidate the contribution of NKA in the pool of ATPases, a series of kinetic measurements was performed in cells where NKA had been inhibited by 2 mM ouabain. In these cells, we recorded: ADP- and ATP-kinetics of respiration, competition for ADP between mitochondria and pyruvate kinase (PK), ADP-kinetics of endogenous PK, and ATP-kinetics of total ATPases. The experimental data was analyzed using a series of mathematical models with varying compartmentation levels. The results show that NKA is tightly coupled to glycolysis with undetectable flux of ATP between mitochondria and NKA. Such tight coupling of NKA to PK is in line with its increased importance in the pathological states of the heart when the substrate preference shifts to glucose.

Figure 1. Contribution of NKA to overall ATPase activity.

A: Representative example of respiration recording before and after NKA inhibition. In the plot, recorded respiration rate is shown after: cardiomyocyte (CM) addition giving the basal respiration rate, 2 mM ATP addition, and after addition of ouabain (OuB). Here, the vertical lines mark the time of ATP or inhibitor addition. B: Effect of NKA inhibition on total ATPase activity (n = 5) measured using PK+LDH system. Addition of 1 mM ouabain decreases ATPase activity by 25%, 2 mM ouabain and 4 mM ouabain reduced the total ATPase activity by 31% and 32% respectively. In these ATPase activity measurements, the ATP production by mitochondria is inhibited.

Figure 2. Representative examples of respiration experiments on NKA inhibited cells.

CM indicates the basal respiration after a cardiomyocyte (CM) suspension was introduced, vertical lines mark the time of metabolite or inhibitor addition. The top row shows an example of the ADP titration (left) and ATP titration (right), vertical lines mark the time of introduction of ADP or ATP at indicated millimolar concentrations into the solution. The bottom row demonstrates inhibition of respiration initiated by 2 mM ATP addition of 5 mM PEP and 20 U/ml PK into the system. Two cases were considered. On the bottom left, PEP was present in solution before addition of 2 mM ATP. On the bottom right, PEP and PK were added consecutively.

Figure 3. Experimental data recorded on NKA inhibited cells (open circles, mean ± STD) are compared to the calculated model solutions.

Obtained model solutions are shown by solid lines with filled symbols for models 1–4 and dashed lines with open symbols for the simplified versions of the models (2s, 3s and 4s). The experimental data from respiration experiments: oxygen consumption rate recorded during titration with ADP (A) or titration with ATP (B); data from spectrophotometric experiments: total ATPase activity (C), and endogenous PK activity (D). In addition, since there is no endogenous PK activity in model 1, the rate of PKend calculated by model 1 is zero in D. Note that all of the models that take into account endogenous PK (models 2–4) produce similar fits with no model fit being conclusively superior to the others.

Figure 4. Models of different spatial organization and reactions in permeabilized cardiomyocytes.

The compartments considered in different model versions: Solution, Cytosol, IMS, and an additional fourth compartment C4. The simplified description of ATP synthase taking place in IMS is justified by the experimental conditions with high concentrations of Pi, oxygen and substrates. The processes accounted for, noted with curved arrows are: ATP synthesis (ATPsyn), ATP consumption (ATPase_1,2), exogenous (PK) and endogenous (PK_end1,2) pyruvate kinase reaction; exchange of metabolites between compartments is marked with doubled-headed arrows.

Figure 5. ATPase activity in compartment C4 relative to total ATPase activity at given ATP concentration (A), or related to maximal ATP synthase rate (B).

Regardless of the used model, NKA inhibition reduces the relative activity in compartment C4. Here, the ATPase activities are calculated with inhibited respiration and regeneration of ATP by exogenous and endogenous PK, as in the experiments performed in spectrophotometer in this work.

Figure 6. Representative example of experimental recording showing the role of SERCA in mitochondrial respiration.

In both plots, vertical lines mark the introduction of a new agent indicated right of the line. First, the cardiomyocyte suspension (CM) is introduced, after a stable basal O₂ consumption is achieved, respiration is activated by 2 mM ATP via endogenous ATPases. In the first experiment (left), the respiration rate is unchanged after inhibition of SERCA by addition of 1 µM TG. In the second experiment (right), increasing the free Ca²⁺ to 600 nM gives approximately 50% rise in oxygen consumption. Subsequent addition of 1 µM TG reduces the achieved respiration rate less than 10%.

Figure 7. Scheme of NKA compartmentalization proposed on the basis of our analysis.

ATP from glycolytic origin is preferentially used by NKA over mitochondrially produced ATP and exogenously added ATP.