The Influence of Intracellular Acidification and Na+/H+ Exchanger Inhibition on LDH and PDH Activity in the Mouse Heart

Purpose: Disruption of acid base balance is linked to various diseases and conditions. In the heart, intracellular acidification is associated with heart failure, maladaptive cardiac hypertrophy and myocardial ischemia. Previously, we have reported that the ratio of the in-cell lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) activities is correlated with cardiac pH. To further characterize this correlation, these in-cell activities were investigated under induced intracellular acidification without and with Na+/H+ exchanger (NHE) inhibition (zoniporide).

Methods: Male mice hearts (n=30) were isolated and perfused retrogradely. Intracellular acidification was performed in two ways: 1) with the NH4Cl pre-pulse methodology, and 2) by combining the NH4Cl pre-pulse with zoniporide. 31P NMR spectroscopy was used to determine the cardiac pH. Polarization of [1-13C] pyruvate was performed using dissolution dynamic nuclear polarization. 13C NMR spectroscopy was used to monitor hyperpolarized [1-13C]pyruvate metabolism and determine enzyme activities in real-time at a temporal resolution of a few seconds.

Results: We found that NH4Cl pre-pulse led to intracellular acidification of 0.2 units (p=0.004), reduced LDH activity by 17±15% (p=0.04), and reduced PDH activity by 40±20% (p=0.001). Combining the NH4Cl pre-pulse with NHE inhibition led to further intracellular acidification of 0.4 units (p=0.001), reduced LDH activity by 30±25% (p=0.01), and surprisingly increased PDH activity by 40±60% (p=0.007).

Conclusions: Intracellular acidification with the NH4Cl pre-pulse alone led to an overall increase in the LDH-to-PDH ratio, in agreement with our previous results. However, combining acidification with NHE inhibition resulted with an expected reduction in LDH activity, but an unexpected elevation in PDH activity. NHEs are found both in the plasma membrane and (in an opposite orientation) in the inner mitochondrial membrane. As both LDH and PDH increase in activity from lower to higher physiological pH values, our findings suggest that treatment with zoniporide leads to increased pH in cardiac mitochondria.