Real-time influence of intracellular acidification and Na+ /H+ exchanger inhibition on in-cell pyruvate metabolism in the perfused mouse heart: A 31 P-NMR and hyperpolarized 13 C-NMR study

Abstract

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) to pyruvate dehydrogenase (PDH) activities is correlated with cardiac pH. To further characterize the basis for this correlation, these in-cell activities were investigated under induced intracellular acidification without and with Na⁺ /H⁺ exchanger (NHE1) inhibition by zoniporide. Male mouse hearts (n = 30) were isolated and perfused retrogradely. Intracellular acidification was performed in two ways: (1) with the NH₄ Cl prepulse methodology; and (2) by combining the NH₄ Cl prepulse with zoniporide. ³¹ P NMR spectroscopy was used to determine the intracellular cardiac pH and to quantify the adenosine triphosphate and phosphocreatine content. Hyperpolarized [1-¹³ C]pyruvate was obtained using dissolution dynamic nuclear polarization. ¹³ C NMR spectroscopy was used to monitor hyperpolarized [1-¹³ C]pyruvate metabolism and determine enzyme activities in real time at a temporal resolution of a few seconds using the product-selective saturating excitation approach. The intracellular acidification induced by the NH₄ Cl prepulse led to reduced LDH and PDH activities (-16% and -39%, respectively). This finding is in line with previous evidence of reduced myocardial contraction and therefore reduced metabolic activity upon intracellular acidification. Concomitantly, the LDH/PDH activity ratio increased with the reduction in pH, as previously reported. Combining the NH₄ Cl prepulse with zoniporide led to a greater reduction in LDH activity (-29%) and to increased PDH activity (+40%). These changes resulted in a surprising decrease in the LDH/PDH ratio, as opposed to previous predictions. Zoniporide alone (without intracellular acidification) did not change these enzyme activities. A possible explanation for the enzymatic changes observed during the combination of the NH₄ Cl prepulse and NHE1 inhibition may be related to mitochondrial NHE1 inhibition, which likely negates the mitochondrial matrix acidification. This effect, combined with the increased acidity in the cytosol, would result in an enhanced H⁺ gradient across the mitochondrial membrane and a temporarily higher pyruvate transport into the mitochondria, thereby increasing the PDH activity at the expense of the cytosolic LDH activity. These findings demonstrate the complexity of in-cell cardiac metabolism and its dependence on intracellular acidification. This study demonstrates the capabilities and limitations of hyperpolarized [1-¹³ C]pyruvate in the characterization of intracellular acidification as regards cardiac pathologies.

Keywords: NHE1; ammonium chloride; intracellular pH; lactate dehydrogenase; mitochondrial pH; pyruvate dehydrogenase; zoniporide.